James R. Eshleman

Somatic mutations have the potential to encode "non-self" immunogenic antigens. We hypothesized that tumors with a large number of somatic mutations due to mismatch-repair defects may be susceptible to immune checkpoint blockade.We conducted a phase 2 study to evaluate the clinical activity of pembrolizumab, an anti-programmed death 1 immune checkpoint inhibitor, in 41 patients with progressive metastatic carcinoma with or without mismatch-repair deficiency. Pembrolizumab was administered intravenously at a dose of 10 mg per kilogram of body weight every 14 days in patients with mismatch repair-deficient colorectal cancers, patients with mismatch repair-proficient colorectal cancers, and patients with mismatch repair-deficient cancers that were not colorectal. The coprimary end points were the immune-related objective response rate and the 20-week immune-related progression-free survival rate.The immune-related objective response rate and immune-related progression-free survival rate were 40% (4 of 10 patients) and 78% (7 of 9 patients), respectively, for mismatch repair-deficient colorectal cancers and 0% (0 of 18 patients) and 11% (2 of 18 patients) for mismatch repair-proficient colorectal cancers. The median progression-free survival and overall survival were not reached in the cohort with mismatch repair-deficient colorectal cancer but were 2.2 and 5.0 months, respectively, in the cohort with mismatch repair-proficient colorectal cancer (hazard ratio for disease progression or death, 0.10 [P<0.001], and hazard ratio for death, 0.22 [P=0.05]). Patients with mismatch repair-deficient noncolorectal cancer had responses similar to those of patients with mismatch repair-deficient colorectal cancer (immune-related objective response rate, 71% [5 of 7 patients]; immune-related progression-free survival rate, 67% [4 of 6 patients]). Whole-exome sequencing revealed a mean of 1782 somatic mutations per tumor in mismatch repair-deficient tumors, as compared with 73 in mismatch repair-proficient tumors (P=0.007), and high somatic mutation loads were associated with prolonged progression-free survival (P=0.02).This study showed that mismatch-repair status predicted clinical benefit of immune checkpoint blockade with pembrolizumab. (Funded by Johns Hopkins University and others; ClinicalTrials.gov number, NCT01876511.).

The genomes of cancers deficient in mismatch repair contain exceptionally high numbers of somatic mutations. In a proof-of-concept study, we previously showed that colorectal cancers with mismatch repair deficiency were sensitive to immune checkpoint blockade with antibodies to programmed death receptor-1 (PD-1). We have now expanded this study to evaluate the efficacy of PD-1 blockade in patients with advanced mismatch repair-deficient cancers across 12 different tumor types. Objective radiographic responses were observed in 53% of patients, and complete responses were achieved in 21% of patients. Responses were durable, with median progression-free survival and overall survival still not reached. Functional analysis in a responding patient demonstrated rapid in vivo expansion of neoantigen-specific T cell clones that were reactive to mutant neopeptides found in the tumor. These data support the hypothesis that the large proportion of mutant neoantigens in mismatch repair-deficient cancers make them sensitive to immune checkpoint blockade, regardless of the cancers' tissue of origin.

There are currently few therapeutic options for patients with pancreatic cancer, and new insights into the pathogenesis of this lethal disease are urgently needed. Toward this end, we performed a comprehensive genetic analysis of 24 pancreatic cancers. We first determined the sequences of 23,219 transcripts, representing 20,661 protein-coding genes, in these samples. Then, we searched for homozygous deletions and amplifications in the tumor DNA by using microarrays containing probes for ∼10 6 single-nucleotide polymorphisms. We found that pancreatic cancers contain an average of 63 genetic alterations, the majority of which are point mutations. These alterations defined a core set of 12 cellular signaling pathways and processes that were each genetically altered in 67 to 100% of the tumors. Analysis of these tumors' transcriptomes with next-generation sequencing-by-synthesis technologies provided independent evidence for the importance of these pathways and processes. Our data indicate that genetically altered core pathways and regulatory processes only become evident once the coding regions of the genome are analyzed in depth. Dysregulation of these core pathways and processes through mutation can explain the major features of pancreatic tumorigenesis.

In December 1997, the National Cancer Institute sponsored "The International Workshop on Microsatellite Instability and RER Phenotypes in Cancer Detection and Familial Predisposition," to review and unify the field. The following recommendations were endorsed at the workshop. (a) The form of genomic instability associated with defective DNA mismatch repair in tumors is to be called microsatellite instability (MSI). (b) A panel of five microsatellites has been validated and is recommended as a reference panel for future research in the field. Tumors may be characterized on the basis of: high-frequency MSI (MSI-H), if two or more of the five markers show instability (i.e., have insertion/deletion mutations), and low-frequency MSI (MSI-L), if only one of the five markers shows instability. The distinction between microsatellite stable (MSS) and low frequency MSI (MSI-L) can only be accomplished if a greater number of markers is utilized. (c) A unique clinical and pathological phenotype is identified for the MSI-H tumors, which comprise approximately 15% of colorectal cancers, whereas MSI-L and MSS tumors appear to be phenotypically similar. MSI-H colorectal tumors are found predominantly in the proximal colon, have unique histopathological features, and are associated with a less aggressive clinical course than are stage-matched MSI-L or MSS tumors. Preclinical models suggest the possibility that these tumors may be resistant to the cytotoxicity induced by certain chemotherapeutic agents. The implications for MSI-L are not yet clear. (d) MSI can be measured in fresh or fixed tumor specimens equally well; microdissection of pathological specimens is recommended to enrich for neoplastic tissue; and normal tissue is required to document the presence of MSI. (e) The "Bethesda guidelines," which were developed in 1996 to assist in the selection of tumors for microsatellite analysis, are endorsed. (f) The spectrum of microsatellite alterations in noncolonic tumors was reviewed, and it was concluded that the above recommendations apply only to colorectal neoplasms. (g) A research agenda was recommended.

Christine Iacobuzio-Donahue and colleagues use whole-genome exome sequencing to analyse primary pancreatic cancers and one or more metastases from the same patients, and find that tumours are composed of distinct subclones. The authors also determine the evolutionary maps by which metastatic cancer clones have evolved within the primary tumour, and estimate the timescales of tumour progression. On the basis of these data, they estimate a mean period of 11.8 years between the initiation of pancreatic tumorigenesis and the formation of the parental, non-metastatic tumour, and a further 6.8 years for the index metastasis clone to arise. These data point to a potentially large window of opportunity during which it might be possible to detect the cancer in a relatively early form. Peter Campbell and colleagues use next-generation sequencing to detect chromosomal rearrangements in 13 patients with pancreatic cancer. The results reveal considerable inter-patient heterogeneity and indicate ongoing genomic instability and evolution during the development of metastases. But for most of the patients studied, more than half of the genetic rearrangements found were present in all metastases and the primary tumour, making them potential targets for therapeutic intervention at early and late stages of the disease. Here, whole-genome sequencing has been used to analyse primary pancreatic tumours and one or more metastases from the same patients. The findings show that tumours are composed of several geographically distinct subclones, and allow maps to be produced showing how metastatic cancer clones evolve within the primary tumour. Moreover, a quantitative analysis of the timing of the genetic evolution of pancreatic cancer has been performed. Metastasis, the dissemination and growth of neoplastic cells in an organ distinct from that in which they originated1,2, is the most common cause of death in cancer patients. This is particularly true for pancreatic cancers, where most patients are diagnosed with metastatic disease and few show a sustained response to chemotherapy or radiation therapy3. Whether the dismal prognosis of patients with pancreatic cancer compared to patients with other types of cancer is a result of late diagnosis or early dissemination of disease to distant organs is not known. Here we rely on data generated by sequencing the genomes of seven pancreatic cancer metastases to evaluate the clonal relationships among primary and metastatic cancers. We find that clonal populations that give rise to distant metastases are represented within the primary carcinoma, but these clones are genetically evolved from the original parental, non-metastatic clone. Thus, genetic heterogeneity of metastases reflects that within the primary carcinoma. A quantitative analysis of the timing of the genetic evolution of pancreatic cancer was performed, indicating at least a decade between the occurrence of the initiating mutation and the birth of the parental, non-metastatic founder cell. At least five more years are required for the acquisition of metastatic ability and patients die an average of two years thereafter. These data provide novel insights into the genetic features underlying pancreatic cancer progression and define a broad time window of opportunity for early detection to prevent deaths from metastatic disease.

Pancreatic cancer remains one of the most lethal of malignancies and a major health burden. We performed whole-genome sequencing and copy number variation (CNV) analysis of 100 pancreatic ductal adenocarcinomas (PDACs). Chromosomal rearrangements leading to gene disruption were prevalent, affecting genes known to be important in pancreatic cancer (TP53, SMAD4, CDKN2A, ARID1A and ROBO2) and new candidate drivers of pancreatic carcinogenesis (KDM6A and PREX2). Patterns of structural variation (variation in chromosomal structure) classified PDACs into 4 subtypes with potential clinical utility: the subtypes were termed stable, locally rearranged, scattered and unstable. A significant proportion harboured focal amplifications, many of which contained druggable oncogenes (ERBB2, MET, FGFR1, CDK6, PIK3R3 and PIK3CA), but at low individual patient prevalence. Genomic instability co-segregated with inactivation of DNA maintenance genes (BRCA1, BRCA2 or PALB2) and a mutational signature of DNA damage repair deficiency. Of 8 patients who received platinum therapy, 4 of 5 individuals with these measures of defective DNA maintenance responded.

Purpose Contrary to the extensive data accumulated regarding pancreatic carcinogenesis, the clinical and molecular features characteristic of advanced stage (stage III and IV) disease are unknown. A comprehensive study of pancreatic cancers from patients who have succumbed to their disease has the potential to greatly expand our understanding of the most lethal stage of this disease and identify novel areas for intervention. Materials and Methods Rapid autopsies were performed on 76 patients with documented pancreatic cancer. The histologic features of end stage disease were determined and correlated to the stage at initial diagnosis, patterns of failure (locally destructive v metastatic disease) and the status of the KRAS2, TP53, and DPC4 genes. Results At autopsy, 30% of patients died with locally destructive pancreatic cancer, and 70% died with widespread metastatic disease. These divergent patterns of failure found at autopsy (locally destructive v metastatic) were unrelated to clinical stage at initial presentation, treatment history, or histopathologic features. However, Dpc4 immunolabeling status of carcinoma tissues harvested at autopsy, a sensitive marker of DPC4 genetic status, was highly correlated with the presence of widespread metastasis but not with locally destructive tumors (P = .007). Conclusion Pancreatic cancers are represented by distinct genetic subtypes with significantly different patterns of failure. Determinations of DPC4 status at initial diagnosis may be of value in stratifying patients into treatment regimens related to local control versus systemic therapy.

Through complete sequencing of the protein-coding genes in a patient with familial pancreatic cancer, we identified a germline, truncating mutation in PALB2 that appeared responsible for this patient's predisposition to the disease. Analysis of 96 additional patients with familial pancreatic cancer revealed three distinct protein-truncating mutations, thereby validating the role of PALB2 as a susceptibility gene for pancreatic cancer. PALB2 mutations have been previously reported in patients with familial breast cancer, and the PALB2 protein is a binding partner for BRCA2. These results illustrate that complete, unbiased sequencing of protein-coding genes can lead to the identification of a gene responsible for a hereditary disease.

More than 2% of the adult U.S. population harbors a pancreatic cyst. These often pose a difficult management problem because conventional criteria cannot always distinguish cysts with malignant potential from those that are innocuous. One of the most common cystic neoplasms of the pancreas, and a bona fide precursor to invasive adenocarcinoma, is called intraductal papillary mucinous neoplasm (IPMN). To help reveal the pathogenesis of these lesions, we purified the DNA from IPMN cyst fluids from 19 patients and searched for mutations in 169 genes commonly altered in human cancers. In addition to the expected KRAS mutations, we identified recurrent mutations at codon 201 of GNAS. A larger number (113) of additional IPMNs were then analyzed to determine the prevalence of KRAS and GNAS mutations. In total, we found that GNAS mutations were present in 66% of IPMNs and that either KRAS or GNAS mutations could be identified in 96%. In eight cases, we could investigate invasive adenocarcinomas that developed in association with IPMNs containing GNAS mutations. In seven of these eight cases, the GNAS mutations present in the IPMNs were also found in the invasive lesion. GNAS mutations were not found in other types of cystic neoplasms of the pancreas or in invasive adenocarcinomas not associated with IPMNs. In addition to defining a new pathway for pancreatic neoplasia, these data suggest that GNAS mutations can inform the diagnosis and management of patients with cystic pancreatic lesions.

Initial results by Le and colleagues, which were published in the June 25, 2015 issue of the New England Journal of Medicine, report significant responses of cancers with microsatellite instability (MSI) to anti-PD-1 inhibitors in patients who failed conventional therapy. This finding fits into a broader body of research associating somatic hypermutation and neoepitope formation with response to immunotherapy, with the added benefit of relying on a simple, widely used diagnostic test. This review surveys the pathogenesis and prognostic value of MSI, diagnostic guidelines for detecting it, and the frequency of MSI across tumors, with the goal of providing a reference for its use as a biomarker for PD-1 blockade. MSI usually arises from either germline mutations in components of the mismatch repair (MMR) machinery (MSH2, MSH6, MLH1, PMS2) in patients with Lynch syndrome or somatic hypermethylation of the MLH1 promoter. The result is a cancer with a 10- to 100-fold increase in mutations, associated in the colon with poor differentiation, an intense lymphocytic infiltrate, and a superior prognosis. Diagnostic approaches have evolved since the early 1990s, from relying exclusively on clinical criteria to incorporating pathologic features, PCR-based MSI testing, and immunohistochemistry for loss of MMR component expression. Tumor types can be grouped into categories based on the frequency of MSI, from colorectal (20%) and endometrial (22%-33%) to cervical (8%) and esophageal (7%) to skin and breast cancers (0%-2%). If initial results are validated, MSI testing could have an expanded role as a tool in the armamentarium of precision medicine.

More than 2% of adults harbor a pancreatic cyst, a subset of which progresses to invasive lesions with lethal consequences. To assess the genomic landscapes of neoplastic cysts of the pancreas, we determined the exomic sequences of DNA from the neoplastic epithelium of eight surgically resected cysts of each of the major neoplastic cyst types: serous cystadenomas (SCAs), intraductal papillary mucinous neoplasms (IPMNs), mucinous cystic neoplasms (MCNs), and solid pseudopapillary neoplasms (SPNs). SPNs are low-grade malignancies, and IPMNs and MCNs, but not SCAs, have the capacity to progress to cancer. We found that SCAs, IPMNs, MCNs, and SPNs contained 10 ± 4.6, 27 ± 12, 16 ± 7.6, and 2.9 ± 2.1 somatic mutations per tumor, respectively. Among the mutations identified, E3 ubiquitin ligase components were of particular note. Four of the eight SCAs contained mutations of the von Hippel-Lindau gene (VHL), a key component of the VHL ubiquitin ligase complex that has previously been associated with renal cell carcinomas, SCAs, and other neoplasms. Six of the eight IPMNs and three of the eight MCNs harbored mutations of RNF43, a gene coding for a protein with intrinsic E3 ubiquitin ligase activity that has not previously been found to be genetically altered in any human cancer. The preponderance of inactivating mutations in RNF43 unequivocally establish it as a suppressor of both IPMNs and MCNs. SPNs contained remarkably few genetic alterations but always contained mutations of CTNNB1, previously demonstrated to inhibit degradation of the encoded protein (β-catenin) by E3 ubiquitin ligases. These results highlight the essential role of ubiquitin ligases in these neoplasms and have important implications for the diagnosis and treatment of patients with cystic tumors.

Pancreatic cancer is the fifth leading cause of cancer death in the United States. We used cDNA microarrays to analyze global gene expression patterns in 14 pancreatic cancer cell lines, 17 resected infiltrating pancreatic cancer tissues, and 5 samples of normal pancreas to identify genes that are differentially expressed in pancreatic cancer. We found more than 400 cDNAs corresponding to genes that were differentially expressed in the pancreatic cancer tissues and cell lines as compared to normal pancreas. These genes that tended to be expressed at higher levels in pancreatic cancers were associated with a variety of processes, including cell-cell and cell-matrix interactions, cytoskeletal remodeling, proteolytic activity, and Ca(++) homeostasis. Two prominent clusters of genes were related to the high rates of cellular proliferation in pancreatic cancer cell lines and the host desmoplastic response in the resected pancreatic cancer tissues. Of 149 genes identified as more highly expressed in the pancreatic cancers compared with normal pancreas, 103 genes have not been previously reported in association with pancreatic cancer. The expression patterns of 14 of these highly expressed genes were validated by either immunohistochemistry or reverse transcriptase-polymerase chain reaction as being expressed in pancreatic cancer. The overexpression of one gene in particular, 14-3-3 sigma, was found to be associated with aberrant hypomethylation in the majority of pancreatic cancers analyzed. The genes and expressed sequence tags presented in this study provide clues to the pathobiology of pancreatic cancer and implicate a large number of potentially new molecular markers for the detection and treatment of pancreatic cancer.

Abstract Pancreatic cancers are the fourth most-common cause of cancer-related deaths in the Western world, with &amp;gt;200,000 cases reported in 2010. Although up to 10% of these cases occur in familial patterns, the hereditary basis for predisposition in the vast majority of affected families is unknown. We used next-generation sequencing, including whole-genome and whole-exome analyses, and identified heterozygous, constitutional, ataxia telangiectasia mutated (ATM) gene mutations in 2 kindreds with familial pancreatic cancer. Mutations segregated with disease in both kindreds and tumor analysis demonstrated LOH of the wild-type allele. By using sequence analysis of an additional 166 familial pancreatic cancer probands, we identified 4 additional patients with deleterious mutations in the ATM gene, whereas we identified no deleterious mutations in 190 spouse controls (P = 0.046). When we considered only the mostly severely affected families with 3 or more pancreatic cancer cases, 4 deleterious mutations were found in 87 families (P = 0.009). Our results indicate that inherited ATM mutations play an important role in familial pancreatic cancer predisposition. Significance: The genes responsible for the majority of cases of familial pancreatic ductal adenocarcinoma are unknown. We here identify ATM as a predisposition gene for pancreatic ductal adenocarcinoma. Our results have important implications for the management of patients in affected families and illustrate the power of genome-wide sequencing to identify the basis of familial cancer syndromes. Cancer Discovery; 2(1): 41–6. ©2011 AACR. Read the Commentary on this article by Bakker and de Winter, p. 14 This article is highlighted in the In This Issue feature, p. 1

Mutations in codons 12 and 13 of the KRAS oncogene are relatively common in colorectal and lung adenocarcinomas. Recent data indicate that these mutations result in resistance to anti-epidermal growth factor receptor therapy. Therefore, we assessed Sanger sequencing, pyrosequencing, and melting curve analysis for the detection of KRAS codon 12/13 mutations in formalin-fixed paraffin-embedded samples, including 58 primary and 42 metastatic colorectal adenocarcinomas, 63 primary and 17 metastatic lung adenocarcinomas, and 20 normal colon samples. Of 180 tumor samples, 62.2% were KRAS mutant positive, and 37.8% were negative. Melting curve analysis yielded no false positive or false negative results, but had 10% equivocal calls. Melting curve analysis also resulted in 4 cases with melting curves inconsistent with either wild-type or codon 12/13 mutations. These patterns were generated from samples with double mutants in codons 12/13 and with mutations outside of codons 12/13. Pyrosequencing yielded no false positive or false negative results as well. However, two samples from one patient yielded a pyrogram that was flagged as abnormal, but the mutation subtype could not be determined. Finally, using an electronic cutoff of 10%, Sanger sequencing showed 11.1% false positives and 6.1% false negatives. In our hands, the limit of detection for Sanger sequencing, pyrosequencing, and melting curve analysis was approximately 15 to 20%, 5%, and 10% mutant alleles, respectively. Mutations in codons 12 and 13 of the KRAS oncogene are relatively common in colorectal and lung adenocarcinomas. Recent data indicate that these mutations result in resistance to anti-epidermal growth factor receptor therapy. Therefore, we assessed Sanger sequencing, pyrosequencing, and melting curve analysis for the detection of KRAS codon 12/13 mutations in formalin-fixed paraffin-embedded samples, including 58 primary and 42 metastatic colorectal adenocarcinomas, 63 primary and 17 metastatic lung adenocarcinomas, and 20 normal colon samples. Of 180 tumor samples, 62.2% were KRAS mutant positive, and 37.8% were negative. Melting curve analysis yielded no false positive or false negative results, but had 10% equivocal calls. Melting curve analysis also resulted in 4 cases with melting curves inconsistent with either wild-type or codon 12/13 mutations. These patterns were generated from samples with double mutants in codons 12/13 and with mutations outside of codons 12/13. Pyrosequencing yielded no false positive or false negative results as well. However, two samples from one patient yielded a pyrogram that was flagged as abnormal, but the mutation subtype could not be determined. Finally, using an electronic cutoff of 10%, Sanger sequencing showed 11.1% false positives and 6.1% false negatives. In our hands, the limit of detection for Sanger sequencing, pyrosequencing, and melting curve analysis was approximately 15 to 20%, 5%, and 10% mutant alleles, respectively. The kirsten rat sarcoma viral oncogene homolog (K-ras), a GTPase binding protein encoded by the KRAS gene, serves as an intermediary signaling molecule in the epidermal growth factor receptor (EGFR) signaling pathway.1Ellis RW Defeo D Shih TY Gonda MA Young HA Tsuchida N Lowy DR Scolnick EM The p21 src genes of Harvey and Kirsten sarcoma viruses originate from divergent members of a family of normal vertebrate genes.Nature. 1981; 292: 506-511Crossref PubMed Scopus (463) Google Scholar KRAS mutations have been implicated in the pathogenesis of numerous tumors and are found in approximately 15 to 50% of lung adenocarcinomas, 30 to 60% of colorectal adenocarcinomas, and 90% of pancreatic adenocarcinomas.2Bos JL Fearon ER Hamilton SR Verlaan-de Vries M van Boom JH van der Eb AJ Vogelstein B Prevalence of ras gene mutations in human colorectal cancers.Nature. 1987; 327: 293-297Crossref PubMed Scopus (1603) Google Scholar, 3Minamoto T Mai M Ronai Z K-ras mutation: early detection in molecular diagnosis and risk assessment of colorectal, pancreas, and lung cancers–a review.Cancer Detect Prev. 2000; 24: 1-12PubMed Google Scholar Mutation of the KRAS oncogene leads to its constitutive signaling and subsequent dysregulated cellular proliferation.4Der CJ Cooper GM Altered gene products are associated with activation of cellular rasK genes in human lung and colon carcinomas.Cell. 1983; 32: 201-208Abstract Full Text PDF PubMed Scopus (109) Google Scholar KRAS mutations have been shown to occur relatively early in the development of colorectal adenocarcinoma and late in that of lung adenocarcinoma.5Sugio K Kishimoto Y Virmani AK Hung JY Gazdar AF K-ras mutations are a relatively late event in the pathogenesis of lung carcinomas.Cancer Res. 1994; 54: 5811-5815PubMed Google Scholar, 6Vogelstein B Fearon ER Hamilton SR Kern SE Preisinger AC Leppert M Nakamura Y White R Smits AM Bos JL Genetic alterations during colorectal-tumor development.N Engl J Med. 1988; 319: 525-532Crossref PubMed Scopus (5887) Google Scholar Anti-EGFR monoclonal antibody therapies such as cetuximab and panitumumab are approved by the Food and Drug Administration for use in patients with metastatic colon cancer. Anti-EGFR tyrosine kinase inhibitors such as Erlotinib are approved for use in patients with locally advanced or metastatic non-small cell lung cancer, after failure of at least one prior chemotherapeutic regimen. Recent data indicate that the presence of a codon 12, 13, or 61 KRAS mutation results in resistance to these anti-EGFR therapies.7Linardou H Dahabreh IJ Kanaloupiti D Siannis F Bafaloukos D Kosmidis P Papadimitriou CA Murray S Assessment of somatic k-RAS mutations as a mechanism associated with resistance to EGFR-targeted agents: a systematic review and meta-analysis of studies in advanced non-small-cell lung cancer and metastatic colorectal cancer.Lancet Oncol. 2008; 9: 962-972Abstract Full Text Full Text PDF PubMed Scopus (665) Google Scholar8Massarelli E Varella-Garcia M Tang X Xavier AC Ozburn NC Liu DD Bekele BN Herbst RS Wistuba II KRAS mutation is an important predictor of resistance to therapy with epidermal growth factor receptor tyrosine kinase inhibitors in non-small-cell lung cancer.Clin Cancer Res. 2007; 13: 2890-2896Crossref PubMed Scopus (551) Google Scholar9Loupakis F Ruzzo A Cremolini C Vincenzi B Salvatore L Santini D Masi G Stasi I Canestrari E Rulli E Floriani I Bencardino K Galluccio N Catalano V Tonini G Magnani M Fontanini G Basolo F Falcone A Graziano F KRAS codon 61, 146 and BRAF mutations predict resistance to cetuximab plus irinotecan in KRAS codon 12 and 13 wild-type metastatic colorectal cancer.Br J Cancer. 2009; 101: 715-721Crossref PubMed Scopus (476) Google Scholar10Karapetis CS Khambata-Ford S Jonker DJ O'Callaghan CJ Tu D Tebbutt NC Simes RJ Chalchal H Shapiro JD Robitaille S Price TJ Shepherd L Au HJ Langer C Moore MJ Zalcberg JR K-ras mutations and benefit from cetuximab in advanced colorectal cancer.N Engl J Med. 2008; 359: 1757-1765Crossref PubMed Scopus (3089) Google Scholar Current guidelines from the National Comprehensive Cancer Network recommend the use of anti-EGFR therapies only in patients with wild-type KRAS lung and colorectal adenocarcinomas. (NCCN Clinical Practice Guidelines in Oncology. Non Small Cell Lung Cancer (Version 2.2009, available at: http://www.nccn.org/professionals/physician_gls/PDF/ncl.pdf) and Colon Cancer (Version 3.2009, available at http://www.nccn.org/professionals/physician_gls/PDF/colon.pdf). Both accessed October 9, 2009) A variety of strategies can be used to assess KRAS mutation status in tumor samples including Sanger sequencing (SS), real-time PCR with or without melting curve analysis (MC), and pyrosequencing (PS). SS is considered by many to be the gold standard in mutation testing. SS can detect essentially all base substitutions, small insertions and deletions, but has a modest limit of detection, which can be highly variable depending on the exact sequence, and laboratory performing the test. Quantitative PCR with MC uses fluorescent probes to capitalize on differences in melting temperatures generated by genetic alterations. When a mutation is present in the target, the probe–target duplex is separated at a lower temperature as compared with a perfectly matching wild-type sequence. The differences in melting temperature (Tm) are detected by a loss of fluorescence as a function of increasing temperature. PS is a bioluminescence technique in which the pyrophosphate released during incorporation of a nucleotide into a growing DNA chain is converted to light through a series of enzymatic reactions. PS can identify individual bases or short stretches of nucleic acid sequence at predetermined positions. Here, we have compared these three platforms for the detection of KRAS codon 12 and 13 mutations in formalin-fixed paraffin-embedded (FFPE) lung and colorectal adenocarcinoma samples. This study was covered under Institutional Review Board approval NA 00002948 for the use of de-identified clinical samples to evaluate new methods/technologies. Two hundred FFPE tissue samples from 191 patients were obtained from the tissue archives at the Johns Hopkins Hospital. These samples included 180 tumor samples, including 58 primary and 42 metastatic colorectal adenocarcinomas, 63 primary and 17 metastatic lung adenocarcinomas, from 171 patients (84 males and 87 females). Nine of these patients each contributed 2 tumor samples to the study, including 6 patients where 2 different blocks from the same tumor were tested and 3 patients who had both primary and metastatic tumors tested. The mean age of the patients analyzed was 78 years (range, 31 to 98 years). In addition, we collected 20 histologically normal colon samples from 20 patients (10 males and 10 females) with diverticulosis or diverticulitis and no past medical history of adenomatous polyps or adenocarcinoma. The mean age for patients with diverticulitis or diverticulosis was 61 years (range, 38 to 71 years). H&E-stained slides from FFPE tumor and normal samples were reviewed by a pathologist and tumor tissue was selected for analysis. Corresponding tissue from five unstained, 10-μm-thick tissue sections was removed using Pinpoint reagents according the manufacturer's protocol (ZymoResearch, Orange, CA). DNA was purified from the sample using QIAmp DNA kit (Qiagen, Valencia, CA) and quantified by OD 260 nm. For our limit of detection study, we prepared dilutions of mutant KRAS cells from the LoVo cell line, which contains a heterozygous G13D mutation, into wild-type cells derived from a cell line of normal lymphoblasts. Total genomic DNA was isolated from cell pellets and purified with the Qiagen DNA-Mini kit. We analyzed each mixture for accuracy of the dilution using our clinical chimerism assay which employs the Identifiler reagents containing 15 short tandem repeat markers (Applied Biosystems, Foster City, CA). Reaction mixture consisted of LightCycler FastStart DNA Master Hybridization Probes Mix (Roche, Indianapolis Indiana), 500 nmol/L forward primer (5′-AAGGCCTGCTGAAAATGACTG-3′), 100 nmol/L reverse primer (5′-CCCTCCCCAGTCCTCATG-3′), 400 nmol/L sensor probe (5′-LC Red 640-TGCCTACGCCACCAGCTCCAA-phosphate-3′), and 200 nmol/L anchor probe (5′-CCACAAAATGATTCTGAATTAGCTGTATCGTCAAGGCACT-fluorescein-3′) in a final reaction volume of 20 μl. Reactions were thermal cycled in a LightCycler (Roche) as follows: 95°C for 10 minutes, followed by 40 cycles of 95°C for 10 seconds, 55°C for 10 seconds, and 72°C for 15 seconds. Melting curve analysis was performed through one cycle of 95°C for 20 seconds, 40°C for 30 seconds, and ramp to 85°C at 0.1°C/sec. Amplification and melting curves were generated using the LightCycler software. To validate the MC, we analyzed 29 pancreatic cancer samples that had previously been characterized for KRAS mutations (provided by Dr. Christine Iacobuzio-Donahue). Twenty-five of the 29 samples harbored one of four different KRAS mutations (G12D, G12V, G12R, G13D). The validation of lung and colorectal adenocarcinomas demonstrated three additional KRAS mutations (G12A, G12S, and G12C). We determined the Tm of wild-type alleles to be 71°C ± 2°C, while the seven most common KRAS mutations in lung and colorectal adenocarcinoma produced a Tm of 63°C ± 2°C. PCR amplification products generated by the Lightcycler PCR/melting curve analysis were purified using QiaQuick reagents (Qiagen) and were cycle sequenced using Big Dye v3.1 reagents (Applied Biosystems) according to the manufacturer's protocol. Sequencing products were purified with CleanSEQ Sequencing Purification System (Agencourt Bioscience Corp., Beverly, MA) and automated sequencing performed by capillary electrophoresis on an ABI3700 (Applied Biosystems). Sequences were aligned and examined by two separate approaches: electronically with a set threshold of 10% and by visual inspection of the electropherogram, using Sequencher software (Gene Codes Corp., Inc.). Samples were PCR amplified using the KRAS v2.0 kit (Qiagen) according to the manufacturer's protocol. Each reaction contained 1× PCR buffer, 1.5 mmol/L MgCl2, 0.2 mmol/L of each dNTP, 5 pmol of forward primer, and 5 pmol of reverse primer (biotinylated), 0.8 Units of HotStar TaqDNA polymerase (Qiagen), 10 ng of template DNA, and dH2O to 25 μl final volume. Cycling conditions were as follows: 95°C 15 minutes, 38× (95°C 20 seconds, 53°C 30 seconds, 72°C 20 seconds), 72°C 5 minutes, 8°C hold. Following amplification, 10 μl of biotinylated PCR product was immobilized on streptavidin-coated sepharose beads (streptavidin sepharose high performance, GE Health care Bio-Sciences Corp., Piscataway, NJ) and washed in 70% EtOH. The purified biotinylated PCR product was released into the PyroMark Q24 (Biotage, SE) with PyroMark Gold reagents (Qiagen) containing 0.3 μmol/L sequencing primer and annealing buffer. The nucleotide dispensation order for codons 12/13 was: 5′-TACGACTCAGATCGTAG-3′. Twenty non-tumor and 180 tumor samples were tested for mutations in codons 12 and 13 of KRAS by MC, SS, and PS. After reviewing our data, we decided to use PS as the gold standard for mutations in KRAS codons 12 and 13 because we felt that interpretation of the PS data were less subjective than for SS and MC. The 20 nontumor specimens all tested negative for KRAS mutations on all three platforms. Of the 180 tumor samples, 112 (62.2%) were KRAS mutant positive as determined. Our study deliberately included tumor samples known to harbor KRAS mutations and thus, our positive rate does not reflect the true incidence of KRAS mutations in our patient population. The mutations identified are summarized in Table 1. Consistent with the findings in the literature, mutations in codon 12 were far more common [98 of 112 (87.5%)] than in codon 13 [11 of 112 (9.8%)].11Andreyev HJ Norman AR Cunningham D Oates J Dix BR Iacopetta BJ Young J Walsh T Ward R Hawkins N Beranek M Jandik P Benamouzig R Jullian E Laurent-Puig P Olschwang S Muller O Hoffmann I Rabes HM Zietz C Troungos C Valavanis C Yuen ST Ho JW Croke CT O'Donoghue DP Giaretti W Rapallo A Russo A Bazan V Tanaka M Omura K Azuma T Ohkusa T Fujimori T Ono Y Pauly M Faber C Glaesener R de Goeij AF Arends JW Andersen SN Lövig T Breivik J Gaudernack G Clausen OP De Angelis PD Meling GI Rognum TO Smith R Goh HS Font A Rosell R Sun XF Zhang H Benhattar J Losi L Lee JQ Wang ST Clarke PA Bell S Quirke P Bubb VJ Piris J Cruickshank NR Morton D Fox JC Al-Mulla F Lees N Hall CN Snary D Wilkinson K Dillon D Costa J Pricolo VE Finkelstein SD Thebo JS Senagore AJ Halter SA Wadler S Malik S Krtolica K Urosevic N Kirsten ras mutations in patients with colorectal cancer: the ‘RASCAL II’ study.Br J Cancer. 2001; 85: 692-696Crossref PubMed Scopus (735) Google Scholar, 12Keohavong P DeMichele MA Melacrinos AC Landreneau RJ Weyant RJ Siegfried JM Detection of K-ras mutations in lung carcinomas: relationship to prognosis.Clin Cancer Res. 1996; 2: 411-418PubMed Google Scholar The G12D mutation was the most common mutation found in primary [13 of 56, (23%)] and metastatic [(10 of 42, (24%)] colorectal carcinoma. The G12C mutation was the most common mutation found in primary lung adenocarcinoma [14 of 63 (23%)], while the G12V mutation was the most common mutation found in metastatic lung adenocarcinoma [5 of 17 (29%)]. An example of a wild-type and mutant (G12D) result from all three platforms is demonstrated in Figure 1.Table 1KRAS Mutation Distribution by Body Site of Tested TumorKRAS mutationColorectal adenocarcinoma (primary)Colorectal adenocarcinoma (metastatic)Lung adenocarcinoma (primary)Lung adenocarcinoma (metastatic)TotalG12V767525G12A32218G12C1014419G12D13109133G12R20226G12S30003G12F11002G13D451111Q22K00101L19F01001G12C/G13D20002G12V/G13D10001Total37253614112Bold type highlights the most frequent mutation at each tumor site. Open table in a new tab Bold type highlights the most frequent mutation at each tumor site. A comparison of the results from all three platforms is summarized in Table 2. MC yielded 94 KRAS mutants in the 180 tumor samples (52.2%) with a Tm consistent with a codon 12/13 mutation, 4 (2.2%) KRAS mutants with a Tm not consistent with a codon 12/13 mutation, 18 (10%) equivocal, and 64 (35.6%) negative. Out of the 18 equivocal samples, 13 were positive, and 4 negative by both SS and PS.Table 2KRAS Mutation Testing Results by PlatformInterpretationSanger sequencing (visual inspection)Sanger sequencing (electronic 10% cutoff)Melt curve analysisPyrosequencingPositive11112098110Negative69606470Equivocal00180 Open table in a new tab SS yielded 100 of 180 (55.6%) KRAS mutants and 49 of 180 (27.2%) wild-type by both visual inspection and by automated interpretation with a 10% threshold. Eleven (6.1%) samples were interpreted as mutants by visual inspection but were not detected by the computer algorithm with a 10% threshold, and 20 cases (11.1%) were called positive using the automated interpretation, but were determined to be negative on visual inspection. Thus, the automated interpretation with a 10% threshold yielded both false positive and false negative mutation calls. PS identified 110 of 180 (61.1%) KRAS mutants and 70 (38.9%) wild-type. Two samples from the same patient yielded a PS pyrogram that was flagged as abnormal, but the pattern could not be definitively interpreted (See below). Five samples from three patients demonstrated two different mutations within codons 12/13. In one case, both a primary colon cancer and omental metastasis demonstrated mutations at each of the first two positions of codon 12. SS demonstrated G to T transversions at the first two positions of codon 12, indicating either a G12F mutation if the mutations occurred on the same allele, or G12C and G12V mutations if they occurred on different alleles (Figure 2A). MC demonstrated 1 mutant melt peak with a Tm of 55.9°C, which is significantly lower than that expected for a single mutation in either codon 12 or 13 (62 to 64.5°C), suggesting that the mutations were on the same allele (Figure 2B). PS yielded an error message in interpreting the data (Figure 2C). In light of the SS data, the PS data confirms that the two mutations were on the same allele, since the pyrogram would have yielded a different, distinct pattern had the mutations occurred on different alleles (Figure 2D). In a second case, two separate blocks from a primary colon adenocarcinoma demonstrated mutations in both codons 12 and 13. For these samples, SS and PS demonstrated G12C and G13D mutations. The MC assay demonstrated a single mutant melt peak with a Tm consistent with a codon 12 or 13 mutation, suggesting that the mutations were likely to occur on separate alleles, either on different alleles in the same cells, or in separate cell populations. Interestingly, although histological review indicated that both samples contained 75 to 80% tumor cells, the PS data demonstrated 26% G12C mutant alleles and 49% G13D mutant alleles in one specimen, and 8% G12C and 46% G13D mutant alleles in the other specimen. These results highlight intratumoral mutant heterogeneity that can exist between different tissue blocks from the same specimen. The third case demonstrating two KRAS mutations was from a primary colon cancer specimen. In this case, SS and PS demonstrated G12V and G13D mutations, and the MC assay demonstrated a single mutant melt peak with a Tm consistent with a codon 12 or 13 mutation, suggesting that the mutations were likely to occur on separate alleles. Pathology review of this specimen indicated that it contained 75% tumor cells. The PS data demonstrated 5% G12V mutant alleles and 17% G13D mutant alleles. Two samples demonstrated mutations outside of codons 12/13. SS detected a Q22K mutation in one sample and L19F mutation in the other. Both yielded MC results with mutant peaks with Tms that were significantly different from that expected from a codon 12/13 mutation (68.7°C and 66.96°C respectively). PS was programmed to detect mutations at codons 12 and 13 only, and thus did not identify these mutations. Mutations outside of codons 12 and 13 might be detected on this platform if the instrument were programmed to interrogate additional codons. To determine the reproducibility of each platform, eight samples were run at least three times on each platform. These samples included five wild-type, and one each of the G12C, G12V, and G13D mutants. Each platform yielded 100% reproducibility for qualitative interpretation of these samples. We also reviewed the quantitative reproducibility of the PS platform. Ten mutant positive samples, four G12C, four G12V, and two G13D, with percent mutant alleles ranging from 5% to 60%, were analyzed at least three times each. The data are summarized in Table 3. In addition, all 20 nontumor samples were run in duplicate. The average percent call at any base other than wild-type was 0.7, with a SD of 0.4 (data not shown).Table 3Reproducibility StudyMutation% Mutant per replicateAverageSDG12C11, 7, 10, 9, 9, 12101.8G12C42, 43, 40421.5G12C19, 17, 18181.0G12C8, 8, 9, 1091.0G12V7, 5, 661.0G12V59, 60, 59590.60G12V11, 5, 12, 6, 3, 573.6G12V7, 12, 892.7G13D46, 46, 48, 47471.0G13D53, 52, 51521.0SD, standard deviation. Open table in a new tab SD, standard deviation. Limit of detection studies were performed by mixing LoVo cells, which contain a heterozygous G13D mutation, with cells from a wild-type cell line. Short tandem repeat analysis of DNA isolated from the cell mixes indicated that the dilutions were accurate (Table 4). The undiluted mutant sample (100%) should have contained 50% mutant alleles if each cell contained one wild-type and one mutant allele. However, SS, PS, and MC all indicated that greater than 50% mutant alleles were present in the undiluted specimen (Table 4 and Figure 3). In fact, the percent mutant alleles identified by PS were consistent with LoVo cells carrying two mutant and one wild-type allele (Table 4). There are data in the literature indicating that some LoVo cell lines contain a gain of chromosome 12, which would be consistent with three copies of the KRAS gene.13Soulie P Poupon MF Remvikos Y Dutrillaux B Muleris M Distinct chromosomal alterations associated with TP53 status of LoVo cells under PALA selective pressure: a parallel with cytogenetic pathways of colorectal cancers.Oncogene. 1999; 18: 775-781Crossref PubMed Scopus (14) Google Scholar, 14Kleivi K Teixeira MR Eknaes M Diep CB Jakobsen KS Hamelin R Lothe RA Genome signatures of colon carcinoma cell lines.Cancer Genet Cytogenet. 2004; 155: 119-131Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar Cytogenetic analysis of our LoVo cell line demonstrated a gain of chromosome 12, which is consistent with this cell line having three copies of KRAS (data not shown). Thus, our predicted mutant alleles (Figure 3) are based on this cell line having one wild-type and two mutant copies of KRAS per cell (column 4 of Table 4).Table 4Limit of Detection StudyDilution (% mutant DNA)Identifiler (% mutant DNA)Expected % mutated alleles if 1 WT, 1 MT allele per cellExpected % mutated alleles if 1 WT, 2 MT allele per cellAverage % mutant alleles by PSSSMC100100506666PositivePositive5048254039PositivePositive2021101818PositivePositive10115109Not above baselineEquivocal562.555Not above baselineEquivocalWT, wild type; MT, mutant; PS, pyrosequencing; SS, Sanger sequencing; MC, melting curve analysis. Open table in a new tab WT, wild type; MT, mutant; PS, pyrosequencing; SS, Sanger sequencing; MC, melting curve analysis. PS was able to clearly identify a mixture containing 5% mutant alleles. Using these data in conjunction with the reproducibility data of the normal samples (above), we concluded that the limit of detection for PS is 5% mutant alleles. SS was able to clearly identify a mixture containing 18% mutant alleles. Determining the limit of detection for SS can be somewhat subjective. We considered the small mutant peak seen in the cell mixture containing 10% predicted mutant alleles not significantly above baseline variability, and thus, the limit of detection for SS to be 15 to 20% mutant alleles. It is important to note that the limit of detection for SS can be mutation specific, in part due to the difference in fluorescence of the fluorophores. While reviewing the data from our clinical specimens, we noted that SS tended to be less sensitive at detecting mutations at the first position of codon 12 (data not shown). The limit of detection for the MC assay is even more subjective. Interestingly, the mutant peak dropped off significantly from the 100% to the 50% cell mixture. At a mixture of 10% predicted mutant alleles, the melting curve became difficult to distinguish from wild-type, and we considered it equivocal. Thus, we consider the limit of detection for MC to be approximately 10% mutant alleles, with the understanding that some of these would be interpreted as equivocal, but could be confirmed by another method. Here, we have compared three platforms for the detection of KRAS codon 12 and 13 mutations. Each platform has unique strengths and challenges (summarized in Table 5). In our hands, MC had the shortest turnaround time and required the least amount of hands-on tech time. MC analysis is a closed system, which should reduce the risk for contamination with amplicons carried over from previous PCR reactions. MC is a qualitative assay and does not identify the specific mutation present. Currently, there are no significant data indicating that the specific mutation type impacts prognosis or therapeutic choices. Therefore, reporting the specific mutation in KRAS 12/13 is not clinically necessary. However, this may change as more data are accumulated. Confirmatory testing is probably required for samples yielding mutant melt curves with a Tm outside of the expected range for wild-type and codon 12/13 mutations. As shown in this study, these patterns can be generated both by mutations outside of codons 12 and 13, and by double mutations within codons 12 and 13. The subjective nature of peak interpretation presents additional challenges in analyzing data from this platform.Table 5Advantages and Disadvantages of Presented PlatformsPlatformAdvantagesDisadvantagesPSBest LOD (5%)Confirmation by another method occasionally requiredProvides sequence informationRelatively fast run-timeSSInterrogates the entire sequenceLOD 15−20%Longest run timeMost labor-intensiveMCClosed systemDoes not define specific mutationFastest run-timeConfirmation frequently requiredLeast labor-intensivePS, pyrosequencing; SS, Sanger sequencing; MC, melting curve analysis; LOD, limit of detection. Open table in a new tab PS, pyrosequencing; SS, Sanger sequencing; MC, melting curve analysis; LOD, limit of detection. SS had the longest turnaround time and most hands-on time of the three platforms analyzed. SS identifies specific mutations and can detect mutations outside of codons 12/13. Using an automated interpretation algorithm with a 10% threshold, SS yielded 11.1% false positives and 6.1% false negatives, highlighting the need for manual review of all SS data. The limit of detection for mutation detection by SS is subjective, and may depend on the experience level of the person interpreting the data. In addition, SS's limit of detection can be variable depending on the specific mutation. Our findings did not replicate the 95.5% sensitivity of SS recently described in FFPE colorectal cancer samples.15Tol J Dijkstra JR Vink-Börger ME Nagtegaal ID Punt CJ van Krieken JH Ligtenberg MJ High sensitivity of both sequencing and real-time PCR analysis of KRAS mutations in colorectal cancer tissue.J Cell Mol Med. 2009; https://doi.org/10.1111/j.1582-4934.2009.00788.xCrossref Scopus (70) Google Scholar One benefit of this platform is its ability to evaluate a relatively long gene sequence for all possible mutations, although the clinical significance of KRAS mutations outside of codons 12 and 13 is unclear. The most common mutations outside of 12/13 are missense mutations at codon 61, which occur in at least 1% and 2% of colon and lung adenocarcinomas respectively.16Rosell R Monzó M Pifarré A Ariza A Sánchez JJ Moreno I Maurel J López MP Abad A de Anta JM Molecular staging of non-small cell lung cancer according to K-ras genotypes.Clin Cancer Res. 1996; 2: 1083-1086PubMed Google Scholar17Rosell R Li S Skacel Z Mate JL Maestre J Canela M Tolosa E Armengol P Barnadas A Ariza A Prognostic impact of mutated K-ras gene in surgically resected non-small cell lung cancer patients.Oncogene. 1993; 8: 2407-2412PubMed Google Scholar18Winder T Mündlein A Rhomberg S Dirschmid K Hartmann BL Knauer M Drexel H Wenzl E De Vries A Lang A Different types of K-Ras mutations are conversely associated with overall survival in patients with colorectal cancer.Oncol Rep. 2009; 21: 1283-1287Crossref PubMed Scopus (47) Google Scholar19Oliveira C Westra JL Arango D Ollikainen M Domingo E Ferreira A Velho S Niessen R Lagerstedt K Alhopuro P Laiho P Veiga I Teixeira MR Ligtenberg M Kleibeuker JH Sijmons RH Plukker JT Imai K Lage P Hamelin R Albuquerque C Schwartz Jr, S Lindblom A Peltomaki P Yamamoto H Aaltonen LA Seruca R Hofstra RM Distinct patterns of KRAS mutations in colorectal carcinomas according to germline mismatch repair defects and hMLH1 methylation status.Hum Mol Genet. 2004; 13: 2303-2311Crossref PubMed Scopus (118) Google Scholar Recent data indicate that these mutations also predict resistance to anti-EGFR therapies, and should therefore be considered for clinical testing.9Loupakis F Ruzzo A Cremolini C Vincenzi B Salvatore L Santini D Masi G Stasi I Canestrari E Rulli E Floriani I Bencardino K Galluccio N Catalano V Tonini G Magnani M Fontanini G Basolo F Falcone A Graziano F KRAS codon 61, 146 and BRAF mutations predict resistance to cetuximab plus irinotecan in KRAS codon 12 and 13 wild-type metastatic colorectal cancer.Br J Cancer. 2009; 101: 715-721Crossref PubMed Scopus (476) Google Scholar For each of the three platforms, detection of codon 61 mutations requires an additional PCR amplification and analysis due to the genomic distance between codons 61 and 12/13. For mutations outside of 12, 13, and 61, the data indicating their relevance to patient management are limited, and therefore, clinical testing is not recommended at this time. PS detects specific mutations, but primarily at codons that it is programmed to analyze, typically codons 12/13. Our study confirmed a 5% limit of detection for mutant alleles as previously shown.20Ogino S Kawasaki T Brahmandam M Yan L Cantor M Namgyal C Mino-Kenudson M Lauwers GY Loda M Fuchs CS Sensitive sequencing method for KRAS mutation detection by Pyrosequencing.J Mol Diagn. 2005; 7: 413-421Abstract Full Text Full Text PDF PubMed Scopus (446) Google Scholar, 21Dufort S Richard MJ de Fraipont F Pyrosequencing method to detect KRAS mutation in formalin-fixed and paraffin-embedded tumor tissues.Anal Biochem. 2009; 391: 166-168Crossref PubMed Scopus (63) Google Scholar The limit of detection for different KRAS mutations appears to be more uniform than for SS, due to the fact that the detection at each position is the same (light emission), rather than different fluorophores. PS provided specific mutation data with an improved limit of detection over both SS and MC. PS and SS characterized three cases with two mutations within KRAS codons 12 and 13, which were identified as mutant by MC, but not as double mutants. The clinical significance of double mutations, if any, is unclear. These cases do, however, highlight the potential for tumor heterogeneity. Heterogeneity within the tumor cells, in conjunction with the fact that all specimens will contain some percentage of nontumor cells, may result in a relatively low percentage of mutated alleles within some specimens. In fact, we found a relatively poor correlation between the percentage of tumor cells in the population as estimated by a pathologist, and the percentage of mutant alleles by pyrosequencing. In our series, there were five samples with the percentage of tumor estimated to be between 40 and 80% by two separate pathologist (20% to 40% predicted mutant alleles), but with <10% KRAS mutant alleles identified by pyrosequencing. Thus, the discrepancy observed between the platforms due to differences in each platform's limit of detection could not solely be attributed to the tumor content of the samples. The clinical significance of low level mutants in relation to prognosis and therapeutic benefit has yet to be fully studied. Although limited data exist on the significance of KRAS mutations within a small subpopulation of a tumor, it is assumed that their identification will be clinically important. Interestingly, the percentage of KRAS mutant was not universally lower, but sometimes greater than that predicted based on the percent tumor in the specimen. We identified two samples in our series that had tumor percentages estimated to be ≤20% (≤10% predicted mutant alleles), but with >45% KRAS mutant alleles identified by pyrosequencing. This may be due to an underestimation of tumor by the pathologists, or individual tumor cells harboring extra copies of mutant KRAS alleles. In these cases, tumor percentage estimates, as determined independently by two pathologists, were concordant. For research purposes, the platforms evaluated here provide unique and complementary data. For example, to identify mutations outside of codons 12/13, detect low level (<10%) mutant alleles, and to determine whether double mutants reside on a single or multiple alleles, may require the use of all three platforms. When choosing a clinical testing platform, factors to consider include sensitivity (false negatives), specificity (false positives), reproducibility, limit of detection, turnaround time, ease of interpretation, and cost (including instrument, reagent, and tech time). In terms of cost, all three assays require an investment in instrumentation; SS, capillary electrophoresis, PS, pyrosequencer, and MC, real-time PCR instrument. In our experience, the reagent costs were higher, but comparable, for PS and SS, and lowest for MC. Not surprisingly, we found tech time to be the biggest factor for determining assay cost. SS was the most labor intensive, and MC was the least, which in our hands, made SS the most expensive, and MC the least expensive to perform. In our experience, the difference in turnaround time for the three platforms was insignificant when considering the time to process the sample (ie, fix, embedded, cut, review to identify tumor, and extract DNA), and that runs were batched and run twice weekly in our lab. We found PS to have the best limit of detection, which we found important given sample heterogeneity. Although PS can require confirmatory testing in rare instances, we found the interpretation of PS data overall to be the most straightforward.

Dyskeratosis congenita is a rare inherited disorder characterized by abnormal skin manifestations. Morbidity and mortality from this disease is usually due to bone marrow failure, but idiopathic pulmonary fibrosis and an increased cancer predisposition also occur. Families with autosomal dominant dyskeratosis congenita display anticipation and have mutations in the telomerase RNA gene. We identified a three-generation pedigree with autosomal dominant dyskeratosis congenita, anticipation, and telomere shortening. We show that a null mutation in motif D of the reverse transcriptase domain of the protein component of telomerase, hTERT, is associated with this phenotype. This mutation leads to haploinsufficiency of telomerase, and telomere shortening occurs despite the presence of telomerase. This finding emphasizes the importance of telomere maintenance and telomerase dosage for maintaining tissue proliferative capacity and has relevance for understanding mechanisms of age-related changes.

Effective development of targeted anticancer agents includes the definition of the optimal biological dose and biomarkers of drug activity. Currently available preclinical models are not optimal to this end. We aimed at generating a model for translational drug development using pancreatic cancer as a prototype. Resected pancreatic cancers from 14 patients were xenografted and expanded in successive groups of nude mice to develop cohorts of tumor-bearing mice suitable for drug therapy in simulated early clinical trials. The xenografted tumors maintain their fundamental genotypic features despite serial passages and recapitulate the genetic heterogeneity of pancreatic cancer. The in vivo platform is useful for integrating drug screening with biomarker discovery. Passages of tumors in successive cohorts of mice do not change their susceptibility to anticancer agents and represent a perpetual live bank, facilitating the application of new technologies that will result in the creation of an integrated stable database of tumor-drug response data and biomarkers.

Pancreatic adenocarcinoma has the worst mortality of any solid cancer. In this study, to evaluate the clinical implications of genomic alterations in this tumour type, we perform whole-exome analyses of 24 tumours, targeted genomic analyses of 77 tumours, and use non-invasive approaches to examine tumour-specific mutations in the circulation of these patients. These analyses reveal somatic mutations in chromatin-regulating genes MLL, MLL2, MLL3 and ARID1A in 20% of patients that are associated with improved survival. We observe alterations in genes with potential therapeutic utility in over a third of cases. Liquid biopsy analyses demonstrate that 43% of patients with localized disease have detectable circulating tumour DNA (ctDNA) at diagnosis. Detection of ctDNA after resection predicts clinical relapse and poor outcome, with recurrence by ctDNA detected 6.5 months earlier than with CT imaging. These observations provide genetic predictors of outcome in pancreatic cancer and have implications for new avenues of therapeutic intervention. Somatic mutations have been reported in pancreatic adenocarcinomas. Here, Sausen et al. identify further mutations and find that mutations in the chromatin modifying gene, MLL, are associated with increased survival, and that the presence of circulating tumour DNA in the serum of patients is associated with poor survival.

Background & AimsThe management of pancreatic cysts poses challenges to both patients and their physicians. We investigated whether a combination of molecular markers and clinical information could improve the classification of pancreatic cysts and management of patients.MethodsWe performed a multi-center, retrospective study of 130 patients with resected pancreatic cystic neoplasms (12 serous cystadenomas, 10 solid pseudopapillary neoplasms, 12 mucinous cystic neoplasms, and 96 intraductal papillary mucinous neoplasms). Cyst fluid was analyzed to identify subtle mutations in genes known to be mutated in pancreatic cysts (BRAF, CDKN2A, CTNNB1, GNAS, KRAS, NRAS, PIK3CA, RNF43, SMAD4, TP53, and VHL); to identify loss of heterozygozity at CDKN2A, RNF43, SMAD4, TP53, and VHL tumor suppressor loci; and to identify aneuploidy. The analyses were performed using specialized technologies for implementing and interpreting massively parallel sequencing data acquisition. An algorithm was used to select markers that could classify cyst type and grade. The accuracy of the molecular markers was compared with that of clinical markers and a combination of molecular and clinical markers.ResultsWe identified molecular markers and clinical features that classified cyst type with 90%−100% sensitivity and 92%−98% specificity. The molecular marker panel correctly identified 67 of the 74 patients who did not require surgery and could, therefore, reduce the number of unnecessary operations by 91%.ConclusionsWe identified a panel of molecular markers and clinical features that show promise for the accurate classification of cystic neoplasms of the pancreas and identification of cysts that require surgery. The management of pancreatic cysts poses challenges to both patients and their physicians. We investigated whether a combination of molecular markers and clinical information could improve the classification of pancreatic cysts and management of patients. We performed a multi-center, retrospective study of 130 patients with resected pancreatic cystic neoplasms (12 serous cystadenomas, 10 solid pseudopapillary neoplasms, 12 mucinous cystic neoplasms, and 96 intraductal papillary mucinous neoplasms). Cyst fluid was analyzed to identify subtle mutations in genes known to be mutated in pancreatic cysts (BRAF, CDKN2A, CTNNB1, GNAS, KRAS, NRAS, PIK3CA, RNF43, SMAD4, TP53, and VHL); to identify loss of heterozygozity at CDKN2A, RNF43, SMAD4, TP53, and VHL tumor suppressor loci; and to identify aneuploidy. The analyses were performed using specialized technologies for implementing and interpreting massively parallel sequencing data acquisition. An algorithm was used to select markers that could classify cyst type and grade. The accuracy of the molecular markers was compared with that of clinical markers and a combination of molecular and clinical markers. We identified molecular markers and clinical features that classified cyst type with 90%−100% sensitivity and 92%−98% specificity. The molecular marker panel correctly identified 67 of the 74 patients who did not require surgery and could, therefore, reduce the number of unnecessary operations by 91%. We identified a panel of molecular markers and clinical features that show promise for the accurate classification of cystic neoplasms of the pancreas and identification of cysts that require surgery.

Abstract Purpose: Recently, the majority of protein coding genes were sequenced in a collection of pancreatic cancers, providing an unprecedented opportunity to identify genetic markers of prognosis for patients with adenocarcinoma of the pancreas. Experimental Design: We previously sequenced more than 750 million base pairs of DNA from 23,219 transcripts in a series of 24 adenocarcinomas of the pancreas. In addition, 39 genes that were mutated in more than one of these 24 cancers were sequenced in a separate panel of 90 well-characterized adenocarcinomas of the pancreas. Of these 114 patients, 89 underwent pancreaticoduodenectomy, and the somatic mutations in these cancers were correlated with patient outcome. Results: When adjusted for age, lymph node status, margin status, and tumor size, SMAD4 gene inactivation was significantly associated with shorter overall survival (hazard ratio, 1.92; 95% confidence interval, 1.20-3.05; P = 0.006). Patients with SMAD4 gene inactivation survived a median of 11.5 months, compared with 14.2 months for patients without SMAD4 inactivation. By contrast, mutations in CDKN2A or TP53 or the presence of multiple (≥4) mutations or homozygous deletions among the 39 most frequently mutated genes were not associated with survival. Conclusions: SMAD4 gene inactivation is associated with poorer prognosis in patients with surgically resected adenocarcinoma of the pancreas.

Pancreatic cancer is projected to become the second leading cause of cancer-related death in the United States by 2020. A familial aggregation of pancreatic cancer has been established, but the cause of this aggregation in most families is unknown. To determine the genetic basis of susceptibility in these families, we sequenced the germline genomes of 638 patients with familial pancreatic cancer and the tumor exomes of 39 familial pancreatic adenocarcinomas. Our analyses support the role of previously identified familial pancreatic cancer susceptibility genes such as BRCA2, CDKN2A, and ATM, and identify novel candidate genes harboring rare, deleterious germline variants for further characterization. We also show how somatic point mutations that occur during hematopoiesis can affect the interpretation of genome-wide studies of hereditary traits. Our observations have important implications for the etiology of pancreatic cancer and for the identification of susceptibility genes in other common cancer types.The genetic basis of disease susceptibility in the majority of patients with familial pancreatic cancer is unknown. We whole genome sequenced 638 patients with familial pancreatic cancer and demonstrate that the genetic underpinning of inherited pancreatic cancer is highly heterogeneous. This has significant implications for the management of patients with familial pancreatic cancer.

Papanicolaou (Pap) smears have revolutionized the management of patients with cervical cancers by permitting the detection of early, surgically curable tumors and their precursors. In recent years, the traditional Pap smear has been replaced by a liquid-based method, which allows not only cytologic evaluation but also collection of DNA for detection of human papillomavirus, the causative agent of cervical cancer. We reasoned that this routinely collected DNA could be exploited to detect somatic mutations present in rare tumor cells that accumulate in the cervix once shed from endometrial or ovarian cancers. A panel of genes that are commonly mutated in endometrial and ovarian cancers was assembled with new whole-exome sequencing data from 22 endometrial cancers and previously published data on other tumor types. We used this panel to search for mutations in 24 endometrial and 22 ovarian cancers and identified mutations in all 46 samples. With a sensitive massively parallel sequencing method, we were able to identify the same mutations in the DNA from liquid Pap smear specimens in 100% of endometrial cancers (24 of 24) and in 41% of ovarian cancers (9 of 22). Prompted by these findings, we developed a sequence-based method to query mutations in 12 genes in a single liquid Pap smear specimen without previous knowledge of the tumor's genotype. When applied to 14 samples selected from the positive cases described above, the expected tumor-specific mutations were identified. These results demonstrate that DNA from most endometrial and a fraction of ovarian cancers can be detected in a standard liquid-based Pap smear specimen obtained during routine pelvic examination. Although improvements need to be made before applying this test in a routine clinical manner, it represents a promising step toward a broadly applicable screening methodology for the early detection of gynecologic malignancies.

Mutations in the chromatin remodeling gene ARID1A have recently been identified in the majority of ovarian clear cell carcinomas (OCCCs). To determine the prevalence of mutations in other tumor types, we evaluated 759 malignant neoplasms including those of the pancreas, breast, colon, stomach, lung, prostate, brain, and blood (leukemias). We identified truncating mutations in 6% of the neoplasms studied; nontruncating somatic mutations were identified in an additional 0.4% of neoplasms. Mutations were most commonly found in gastrointestinal samples with 12 of 119 (10%) colorectal and 10 of 100 (10%) gastric neoplasms, respectively, harboring changes. More than half of the mutated colorectal and gastric cancers displayed microsatellite instability (MSI) and the mutations in these tumors were out-of-frame insertions or deletions at mononucleotide repeats. Mutations were also identified in 2-8% of tumors of the pancreas, breast, brain (medulloblastomas), prostate, and lung, and none of these tumors displayed MSI. These findings suggest that the aberrant chromatin remodeling consequent to ARID1A inactivation contributes to a variety of different types of neoplasms.

Abstract Metastasis and drug resistance are the major causes of mortality in patients with pancreatic cancer. Once developed, the progression of pancreatic cancer metastasis is virtually unstoppable with current therapies. Here, we report the remarkable clinical outcome of a patient with advanced, gemcitabine-resistant, pancreatic cancer who was later treated with DNA damaging agents, on the basis of the observation of significant activity of this class of drugs against a personalized xenograft generated from the patient's surgically resected tumor. Mitomycin C treatment, selected on the basis of its robust preclinical activity in a personalized xenograft generated from the patient's tumor, resulted in long-lasting (36+ months) tumor response. Global genomic sequencing revealed biallelic inactivation of the gene encoding PalB2 protein in this patient's cancer; the mutation is predicted to disrupt BRCA1 and BRCA2 interactions critical to DNA double-strand break repair. This work suggests that inactivation of the PALB2 gene is a determinant of response to DNA damage in pancreatic cancer and a new target for personalizing cancer treatment. Integrating personalized xenografts with unbiased exomic sequencing led to customized therapy, tailored to the genetic environment of the patient's tumor, and identification of a new biomarker of drug response in a lethal cancer. Mol Cancer Ther; 10(1); 3–8. ©2010 AACR. Mol Cancer Ther; 10(1); 3–8. ©2010 AACR.

Pancreatic cancer is molecularly diverse, with few effective therapies. Increased mutation burden and defective DNA repair are associated with response to immune checkpoint inhibitors in several other cancer types. We interrogated 385 pancreatic cancer genomes to define hypermutation and its causes. Mutational signatures inferring defects in DNA repair were enriched in those with the highest mutation burdens. Mismatch repair deficiency was identified in 1% of tumors harboring different mechanisms of somatic inactivation of MLH1 and MSH2. Defining mutation load in individual pancreatic cancers and the optimal assay for patient selection may inform clinical trial design for immunotherapy in pancreatic cancer. Pancreatic cancer is molecularly diverse, with few effective therapies. Increased mutation burden and defective DNA repair are associated with response to immune checkpoint inhibitors in several other cancer types. We interrogated 385 pancreatic cancer genomes to define hypermutation and its causes. Mutational signatures inferring defects in DNA repair were enriched in those with the highest mutation burdens. Mismatch repair deficiency was identified in 1% of tumors harboring different mechanisms of somatic inactivation of MLH1 and MSH2. Defining mutation load in individual pancreatic cancers and the optimal assay for patient selection may inform clinical trial design for immunotherapy in pancreatic cancer. Pancreatic ductal adenocarcinoma has a 5-year survival of <5%, with therapies offering only incremental benefit,1Vogelzang N.J. et al.J Clin Oncol. 2012; 30: 88-109Crossref PubMed Scopus (85) Google Scholar potentially due to the diversity of its genomic landscape.2Bailey P. et al.Nature. 2016; 531: 47-52Crossref PubMed Scopus (1973) Google Scholar, 3Biankin A.V. et al.Nature. 2012; 491: 399-405Crossref PubMed Scopus (1379) Google Scholar, 4Waddell N. et al.Nature. 2015; 518: 495-501Crossref PubMed Scopus (1466) Google Scholar Recent reports link high mutation burden with response to immune checkpoint inhibitors in several cancer types.5Le D.T. et al.N Engl J Med. 2015; 372: 2509-2520Crossref PubMed Scopus (6099) Google Scholar Defining tumors that are hypermutated with an increased mutation burden and understanding the underlying mechanisms in pancreatic cancer has the potential to advance therapeutic development, particularly for immunotherapeutic strategies. Whole genome sequencing (WGS, n = 180) and whole exome sequencing (n = 205) of 385 unselected predominantly sporadic pancreatic ductal adenocarcinoma (Supplementary Table 1) defined a mean mutation load of 1.8 and 1.1 mutation per megabase (Mb), respectively (Supplementary Table 2). Outlier analysis identified 20 tumors with the highest mutation burden (5.2%, 15 WGS and 5 exome) (Table 1 and Supplementary Figure 1A), 5 of which were considered extreme outliers and classified as hypermutated as they contained ≥12 somatic mutations/Mb, the defined threshold for hypermutation in colorectal cancer.6Cancer Genome Atlas NetworkNature. 2012; 487: 330-337Crossref PubMed Scopus (5894) Google Scholar Immunohistochemistry for mismatch repair (MMR) proteins (MSH2, MSH6, MLH1, and PMS2) identified 4 MMR-deficient tumors, all of which were hypermutated (n = 180, Figure 1).Table 1Clinical and Histologic Features and Proposed Etiology for Highly Mutated Pancreatic Ductal Adenocarcinoma Tumors (n = 20)Sample IDPersonal and family history of malignancyHistologyMutation load, mutations/MbIHC resultMSIsensor scoreKRAS mutationPredominant mutation signature (mutations/Mb)SV subtype (no. of events)Proposed etiologyHypermutation (extreme outliers) ICGC_0076aSample sequenced by WGS, other samples by exome sequencing.NoneMixed signet ring, mucinous and papillary adenocarcinoma38.55Absent MLH1 and PMS228.3p.G12VMMR (18.3)Scattered (131)MMR deficiency: >280 kb somatic homozygous deletion over MSH2. ICGC_0297aSample sequenced by WGS, other samples by exome sequencing.NoneUndifferentiated adenocarcinoma60.62Absent MSH2 and MSH627.33WTMMR (33.4)Scattered (75)MMR deficiency: Somatic MLH1 promoter hypermethylation. ICGC_0548aSample sequenced by WGS, other samples by exome sequencing.NoneDuctal adenocarcinoma, moderately differentiated30.13Absent MSH2 and MSH617.47WTMMR (16.6)Stable (49)MMR deficiency: >27 kb somatic inversion rearrangement disrupting MSH2. ICGC_0328aSample sequenced by WGS, other samples by exome sequencing.NoneDuctal adenocarcinoma16.63Normal3.2p.G12DUnknown (11.9)Scattered (110)Cell line with signature: etiology unknown. ICGC_00901 FDR, father CRCDuctal adenocarcinoma, moderately differentiated12.9Absent MSH2 and MSH60.21p.G12CNANAMMR deficiency: somatic MSH2 splice site c.2006G>A.Highly mutated tumors ICGC_0054aSample sequenced by WGS, other samples by exome sequencing.NoneDuctal adenocarcinoma, poorly differentiated6.52Normal0.01p.G12VHR deficiency (1.3)Unstable (310)HR deficiency: no germline or somatic cause found. ICGC_0290aSample sequenced by WGS, other samples by exome sequencing.NoneDuctal adenocarcinoma, poorly differentiated6.54Not available0.07p.G12VHR deficiency (3.1)Unstable (558)HR deficiency: Germline BRCA2 mutation c.7180A>T, p.A2394*. Somatic CN-LOH. ICGC_0215aSample sequenced by WGS, other samples by exome sequencing.2 FDR lung cancer, 2 FDR prostate cancer. Previous CRC and melanomaDuctal adenocarcinoma, moderately differentiated6.27Normal0.01p.G12VHR deficiency (1.9)Scattered (111)HR deficiency: Germline ATM mutation c.7539_7540delAT, p.Y2514*. Somatic CN-LOH. ICGC_0324NoneDuctal adenocarcinoma, moderately differentiated6.24Normal0p.G12DNANAUndefined ICGC_0034aSample sequenced by WGS, other samples by exome sequencing.NoneDuctal adenocarcinoma, poorly differentiated6.09Normal4.02p.G12DHR deficiency (3.4)Unstable (366)HR deficiency: Germline BRCA2 mutation c.5237_5238insT, p.N1747*. Somatic CN-LOH. ICGC_0131aSample sequenced by WGS, other samples by exome sequencing.Lung cancer after PCDuctal adenocarcinoma, moderately differentiated5.63Normal0p.G12DT>G at TT sites (3.0)Focal (147)T>G at TT sites signature: etiology potentially associated with DNA oxidation ICGC_0006aSample sequenced by WGS, other samples by exome sequencing.1 FDR, father lung cancerAdenocarcinoma arising from IPMN, moderately differentiated5.29Normal0.01p.G12DHR deficiency (1.2)Unstable (211)HR deficiency: Somatic BRCA2 c.5351dupA, p.N1784KfsTer3. Somatic CN-LOH. ICGC_0321aSample sequenced by WGS, other samples by exome sequencing.2 FDR, mother and cousin breast cancerDuctal adenocarcinoma, poorly differentiated4.79Not available0p.G12DHR deficiency (2.1)Unstable (286)HR deficiency: Germline BRCA2 c.6699delT, p.F2234LfsTer7. Somatic CN loss- 1 copy. ICGC_0309aSample sequenced by WGS, other samples by exome sequencing.NoneAdenocarcinoma arising from IPMN, moderately differentiated4.74Normal0.03p.G12VT>G at TT sites (3.1)Unstable (232)T>G at TT sites signature: etiology potentially associated with DNA oxidation ICGC_0005aSample sequenced by WGS, other samples by exome sequencing.1 FDR, mother CRCDuctal adenocarcinoma, poorly differentiated4.72Not available1p.G12VHR deficiency (1.1)Focal (95)HR deficiency: No germline or somatic cause found. ICGC_0016aSample sequenced by WGS, other samples by exome sequencing.NoneDuctal adenocarcinoma, poorly differentiated4.61Normal3.03p.G12VHR deficiency (1.7)Unstable (447)HR deficiency: potentially linked to Somatic RPA1 c.273G>T, p.R91S ICGC_00461 FDR, brother PCDuctal adenocarcinoma, poorly differentiated4.3Normal0p.Q61HNANAUndefined GARV_0668aSample sequenced by WGS, other samples by exome sequencing.NoneDuctal adenocarcinoma, poorly differentiated4.3Not available2.19p.G12VHR deficiency (1.6)Unstable (464)HR deficiency: Germline BRCA2 c.7068_7069delTC, p.L2357VfsTer2. Somatic CN loss - 1 copy. ICGC_0291NoneDuctal adenocarcinoma, well differentiated3.84Not available0.03p.G12RNANAHR deficiency: Somatic BRCA2 c.7283T>A, p.L2428*. ICGC_0256NoneDuctal adenocarcinoma, poorly differentiated3.72Not available0.06p.G12DNANAUndefinedCRC, colorectal cancer; FDR, first-degree relative; IHC, immunohistochemistry; IPMN, intraductal papillary mucinous neoplasm; CN-LOH, copy neutral loss of heterozygosity; CN, copy number; PC, pancreatic cancer; NA, not applicable to exome data.a Sample sequenced by WGS, other samples by exome sequencing. Open table in a new tab CRC, colorectal cancer; FDR, first-degree relative; IHC, immunohistochemistry; IPMN, intraductal papillary mucinous neoplasm; CN-LOH, copy neutral loss of heterozygosity; CN, copy number; PC, pancreatic cancer; NA, not applicable to exome data. KRAS mutation status and histopathologic characteristics have been associated with MMR-deficient pancreatic tumors.7Goggins M. et al.Am J Pathol. 1998; 152: 1501-1507PubMed Google Scholar Of the 4 MMR-deficient tumors in our cohort, 2 were KRAS wild-type; 3 had undifferentiated to moderately differentiated histology and one had a signet-ring component. These features were not predictive of MMR deficiency in our cohort, as 11 additional non−MMR-deficient tumors had a signet-ring cell component or colloid morphology, and 131 of 347 assessable tumors had poorly or undifferentiated histology. Mutational signature analysis can detect MMR deficiency indirectly based on the pattern of somatic mutations.8Alexandrov L.B. et al.Nature. 2013; 500: 415-421Crossref PubMed Scopus (6213) Google Scholar An MMR-deficient signature dominated the MMR-deficient tumors (with WGS), and was minimal in MMR intact tumors (Supplementary Figure 1). In addition, microsatellite instability (MSI), a hallmark of MMR deficiency in colorectal cancer, was detected in all three MMR deficient tumors with WGS using MSIsensor9Niu B. Ye K. et al.Bioinformatics. 2014; 30: 1015-1016Crossref PubMed Scopus (294) Google Scholar (Supplementary Table 2). MSI was not identified for the fourth MMR deficient sample potentially due to the reduced number of microsatellite loci in exome data. The underlying causes of MMR deficiency in the 4 cases were private somatic events. For 2 cases, MSH2 was disrupted by different structural rearrangements, 1 case contained a missense MSH2 mutation and the last, methylation of the MLH1 promoter (Figure 1). The missense mutation caused an MSH2 splice acceptor site mutation that alters the same nucleotide results in a pathogenic skipping of exon 13 in germline studies.10Thompson B.A. et al.Nat Genet. 2014; 46: 107-115Crossref PubMed Scopus (346) Google Scholar Hypermethylation of the MLH1 promoter is the predominant mechanism of MSI in sporadic colon cancer.11Boland C.R. et al.Gastroenterology. 2010; 138: 2073-2087 e3Abstract Full Text Full Text PDF PubMed Scopus (1359) Google Scholar The remaining hypermutated tumor contained an intact MMR pathway, and was a cell line (ATCC, CRL-2551) with an unidentified mutational signature, therefore the high mutation burden in this sample may be the result of long-term cell culture. The 15 samples (11 WGS and 4 exome) identified in the outlier analysis with high mutation burden, but not hypermutated (∼4 to 12 mutations/Mb) contained no evidence of MMR deficiency. Mutational signature analysis of the WGS samples indicated homologous recombination (HR) repair deficiency as the most substantial (range, 1.0–3.4 mutations/Mb) contributor to the mutation burden for 8 WGS mutation load outlier tumors. In support of a HR defect4Waddell N. et al.Nature. 2015; 518: 495-501Crossref PubMed Scopus (1466) Google Scholar; 7 of these tumors contained high levels of genomic instability with >200 structural variants and mutations in genes involved in HR were present for 6 of 8 cases (Supplementary Table 2). In addition, 1 case that had undergone exome sequencing had a somatic BRCA2 nonsense mutation that likely contributed to HR deficiency in this case. A mutational signature associated with T>G mutations at TT sites previously described in other cancers, including esophageal cancer12Nones K. Waddell N. Wayte N. et al.Nat Commun. 2014; : 5Google Scholar was the major contributor (>3 mutations/Mb) in 2 samples. For these 2 and the remaining 4 cases, no potential causative event could be identified. Although germline defects in MMR genes are well reported in pancreatic cancer13Grant R.C. Selander I. et al.Gastroenterology. 2015; 148: 556-564Abstract Full Text Full Text PDF PubMed Scopus (211) Google Scholar in our cohort, they did not contribute to MMR deficiency even in those with familial pancreatic cancer or a personal or family history of Lynch-related tumors. A germline truncating variant was detected in PMS2 in 1 case, but did not have loss of the second allele, had normal immunohistochemistry staining and did not display a MMR mutational signature (Supplementary Table 2). MMR deficiency is important in the evolution in a small, but meaningful proportion of pancreatic cancers with a prevalence of 1% (4 of 385) in our cohort. This is consistent with recent studies using the Bethesda polymerase chain reaction panel,14Laghi L. et al.PLoS One. 2012; 7: e46002Crossref PubMed Scopus (55) Google Scholar and with previous estimates of MSI prevalence of 2%−3%.15Nakata B. et al.Clin Cancer Res. 2002; 8: 2536-2540PubMed Google Scholar However, in tumors with low epithelial content that underwent exome sequencing, the sensitivity of somatic mutation detection is reduced, which will affect mutation burden and signature analysis. While cognizant of small numbers, immunohistochemistry was the most accurate in defining MMR due to multiple genomic mechanisms of MMR gene inactivation. Multiple methods to define MMR deficiency may be required for clinical trials that aim to recruit MMR-deficient participants to assess the potential efficacy of checkpoint inhibitors or other therapies in pancreatic cancer. Homologous recombination-deficient tumors, and those with a novel signature seen in esophageal cancer had an increased mutation burden, and need further evaluation as potential patient selection markers for clinical trials of checkpoint inhibitor and other therapies that target tumors with a high mutation burden. The authors would like to thank Cathy Axford, Deborah Gwynne, Mary-Anne Brancato, Clare Watson, Michelle Thomas, Gerard Hammond, and Doug Stetner for central coordination of the Australian Pancreatic Cancer Genome Initiative, data management, and quality control; Mona Martyn-Smith, Lisa Braatvedt, Henry Tang, Virginia Papangelis, and Maria Beilin for biospecimen acquisition; and Sonia Grimaldi and Giada Bonizzato of the ARC-Net Biobank for biospecimen acquisition. For a full list of contributors see Australian Pancreatic Cancer Genome Initiative: http://www.pancreaticcancer.net.au/apgi/collaborators. The cohort consisted of 385 patients with histologically verified pancreatic exocrine carcinoma, prospectively recruited between 2006 and 2013 through the Australian Pancreatic Cancer Genome Initiative (www.pancreaticcancer.net.au) as part of the International Cancer Genome Consortium.1Hudson T.J. et al.Nature. 2010; 464: 993-998Crossref PubMed Scopus (1689) Google Scholar Ethical approval was granted at all treating institutions and individual patients provided informed consent upon entry to the study. The clinicopathologic information for the cohort is described in (Supplementary Table 1), and the global mutation profile has previously been reported for some of these tumors (Supplementary Table 2). Tumor and normal DNA were extracted after histologic review from fresh frozen tissue samples collected at the time of surgical resection or biopsy, as described previously.2Biankin A.V. et al.Nature. 2012; 491: 399-405Crossref PubMed Scopus (1513) Google Scholar Tumor cellularity was determined from single-nucleotide polymorphism array data using qpure.3Song S. et al.PLoS One. 2012; 7: e45835Crossref PubMed Scopus (85) Google Scholar Tumors with epithelial content ≥40% underwent WGS lower cellularity tumors underwent whole exome sequencing. DNA from patient-derived pancreas cell lines and matched normal was also extracted. Exome and WGS were performed using paired 100-bp reads on the Illumina HiSeq 2000, as described previously.2Biankin A.V. et al.Nature. 2012; 491: 399-405Crossref PubMed Scopus (1513) Google Scholar, 4Waddell N. et al.Nature. 2015; 518: 495-501Crossref PubMed Scopus (1686) Google Scholar Regions of germline and somatic copy number change were detected using Illumina SNP BeadChips with GAP.5Popova T. et al.Genome Biol. 2009; 10 (R128−R128)Crossref PubMed Scopus (151) Google Scholar Somatic structural variants were identified from WGS reads using the qSV tool.4Waddell N. et al.Nature. 2015; 518: 495-501Crossref PubMed Scopus (1686) Google Scholar, 6Patch A.M. et al.Nature. 2015; 521: 489-494Crossref PubMed Scopus (930) Google Scholar Single nucleotide variants were called using 2 variant callers: qSNP7Kassahn K.S. et al.PLoS One. 2013; 8: e74380Crossref PubMed Scopus (52) Google Scholar and GATK.8McKenna A. et al.Genome Res. 2010; 20: 1297-1303Crossref PubMed Scopus (14755) Google Scholar Mutations identified by both callers or, those that were unique to a caller but verified by an orthogonal sequencing approach, were considered high confidence and used in all subsequent analyses. Small indels (<200 bp) were identified using Pindel9Ye K. et al.Bioinformatics. 2009; 25: 2865-2871Crossref PubMed Scopus (1391) Google Scholar and each indel was visually inspected in the Integrative Genome Browser. The distribution of the total number of small somatic mutations (coding and noncoding single nucleotide and indel variants) identified per megabase for exome and WGS sequence data were analyzed separately. The group of samples with high mutation load, at the top of each distribution, were defined as the upper distribution outliers for mutations per megabase, that is, ≥75th centile + (1.5× interquartile range). The threshold for detecting outliers in the exome and WGS groups was 3.4 and 4.2 mutations/Mb, respectively. From within the highly mutated set of tumors, hypermutated samples were identified as those with a mutation rate exceeding the thresholds for extreme distribution outliers (≥75th centile + [5× interquartile range]) of 7.4 and 8.1 mutations/Mb for exome and WGS sequencing, respectively. MSIsensor was used to detect microsatellite instability by directly comparing microsatellite repeat lengths between paired normal and tumor sequencing data.10Niu B. et al.Bioinformatics. 2014; 30: 1015-1016Crossref PubMed Scopus (378) Google Scholar A MSIsensor score of >3.5% of somatic microsatellites with repeat length shifts was the detection threshold used to indicate microsatellite instability as published for endometrial cancer.10Niu B. et al.Bioinformatics. 2014; 30: 1015-1016Crossref PubMed Scopus (378) Google Scholar This correlated well with the 5 and 7 microsatellite panels recommended in the Bethesda guidelines.10Niu B. et al.Bioinformatics. 2014; 30: 1015-1016Crossref PubMed Scopus (378) Google Scholar, 11Umar A. et al.J Natl Cancer Inst. 2004; 96: 261-268Crossref PubMed Scopus (2461) Google Scholar Tissue microarrays were constructed using at least three 1-mm formalin-fixed, paraffin-embedded tumor cores. Immunohistochemistry for MSH6 and PMS2 proteins was performed on tissue microarray sections as a screen for MMR deficiency due to MMR proteins forming heterodimers with concordant mismatch repair loss (ie, loss of MLH1 and PMS2 or loss of MSH2 and MSH6).12Hall G. et al.Pathology. 2010; 42: 409-413Abstract Full Text PDF PubMed Scopus (98) Google Scholar Immunohistochemistry on full tumor sections for MSH2, MLH1, MSH6, and PMS2 was performed in those with abnormal staining in core sections. The immunohistochemistry was performed as described previously12Hall G. et al.Pathology. 2010; 42: 409-413Abstract Full Text PDF PubMed Scopus (98) Google Scholar and scored by a senior pathologist. Somatic mutational signatures were extracted from the whole genome sequenced samples using the framework described previously.13Alexandrov L.B. et al.Cell Rep. 2013; 3: 246-259Abstract Full Text Full Text PDF PubMed Scopus (734) Google Scholar High confidence somatic substitutions were classified by the substitution change and sequence context, that is, the type of immediately neighboring bases to the variant. The framework processes the counts of somatic mutations at each context within each sample using non-negative factorization to produce the different signature profiles that are present in the data. The profiles identified were matched against reported signatures from the Cancer of Somatic Mutations in Cancer (http://cancer.sanger.ac.uk/cosmic/signatures). The major contributory signatures, defined as the mutational signature with the highest number of contributing somatic substitution variants, is reported for highly mutated whole genome samples. Bisulfite-converted whole-genome amplified DNA was hybridized to Infinium Human Methylation 450K Beadchips according to the manufacturers protocol (Illumina). Methylation arrays were performed on DNA from 174 pancreatic ductal adenocarcinoma samples, which were compared to DNA from 29 adjacent nonmalignant pancreata. A subset of the methylation data has been published previously.14Nones K. et al.Int J Cancer. 2014; 135: 1110-1118Crossref PubMed Scopus (156) Google Scholar We examined the data for evidence of tumor-specific hypermethylation of the promoter region of MLH1 and MSH2 genes. The methylation array data have been deposited into the International Cancer Genome Consortium data portal (dcc.icgc.org, project PACA-AU). Download .xlsx (.08 MB) Help with xlsx files Supplementary Tables 1 and 2

AR-V7-expressing metastatic prostate cancer is an aggressive phenotype with poor progression-free survival (PFS) and overall survival (OS).Preliminary evidence suggests that AR-V7-positive tumors may be enriched for DNA-repair defects, perhaps rendering them more sensitive to immune-checkpoint blockade.We enrolled 15 metastatic prostate cancer patients with AR-V7-expressing circulating tumor cells into a prospective phase-2 trial.Patients received nivolumab 3 mg/kg plus ipilimumab 1 mg/kg every 3 weeks for four doses, then maintenance nivolumab 3 mg/kg every 2 weeks.Targeted next-generation sequencing was performed to determine DNArepair deficiency (DRD) status.Outcomes included PSA response rates, objective response rates (ORR), PSA progression-free survival (PSA-PFS), clinical/radiographic PFS and OS.Median age of participants was 65, median PSA was 115 ng/mL, 67% had visceral metastases, and 60% had ≥4 prior systemic therapies.Six of 15 men (40%) had DRD mutations (three in BRCA2, two in ATM, one in ERCC4; none had microsatellite instability).Overall, the PSA response rate was 2/15 (13%), ORR was 2/8 (25%) in those with measurable disease, median PSA-PFS was 3.0 (95%CI 2.1-NR) months, PFS was 3.7 (95%CI 2.8-7.5)months, and OS was 8.2 (95%CI 5.5-10.4)months.Outcomes appeared generally better in DRD+ vs. DRD-tumors with respect to PSA responses (33% vs. 0%; P=0.14, nonsignificant), ORR (40% vs. 0%; P=0.46, nonsignificant), PSA-PFS (HR 0.19; P<0.01, significant), PFS (HR 0.31; P=0.01, significant), and OS (HR 0.41; P=0.11, nonsignificant).There were no new safety concerns.Ipilimumab plus nivolumab demonstrated encouraging efficacy in AR-V7positive prostate cancers with DRD mutations, but not in the overall study population.www.oncotarget.com

In research settings, circulating tumor DNA (ctDNA) shows promise as a tumor-specific biomarker for pancreatic ductal adenocarcinoma (PDAC). This study aims to perform analytical and clinical validation of a KRAS ctDNA assay in a Clinical Laboratory Improvement Amendments (CLIA) and College of American Pathology-certified clinical laboratory.Digital-droplet PCR was used to detect the major PDAC-associated somatic KRAS mutations (G12D, G12V, G12R, and Q61H) in liquid biopsies. For clinical validation, 290 preoperative and longitudinal postoperative plasma samples were collected from 59 patients with PDAC. The utility of ctDNA status to predict PDAC recurrence during follow-up was assessed.ctDNA was detected preoperatively in 29 (49%) patients and was an independent predictor of decreased recurrence-free survival (RFS) and overall survival (OS). Patients who had neoadjuvant chemotherapy were less likely to have preoperative ctDNA than were chemo-naïve patients (21% vs. 69%; P < 0.001). ctDNA levels dropped significantly after tumor resection. Persistence of ctDNA in the immediate postoperative period was associated with a high rate of recurrence and poor median RFS (5 months). ctDNA detected during follow-up predicted clinical recurrence [sensitivity 90% (95% confidence interval (CI), 74%-98%), specificity 88% (95% CI, 62%-98%)] with a median lead time of 84 days (interquartile range, 25-146). Detection of ctDNA during postpancreatectomy follow-up was associated with a median OS of 17 months, while median OS was not yet reached at 30 months for patients without ctDNA (P = 0.011).Measurement of KRAS ctDNA in a CLIA laboratory setting can be used to predict recurrence and survival in patients with PDAC.

Microsatellite instability (the replication error phenotype) is a new molecular assay that identifies a substantial fraction of human cancers. The microsatellite instability in these cancers arises from alterations in the tumors of the number of mono-, di-, and trinucleotide repeats that compose the microsatellites. Microsatellite instability is typical of colon and endometrial tumors arising in members of Lynch syndrome cancer families. Microsatellite instability is also found in a substantial percentage of sporadic cases of colon, endometrial, and gastric cancer, as well as in additional sporadic cancers, such as lung cancer, not usually seen in Lynch kindreds. Thus far, four different mutant genes, all homologous to bacterial DNA repair genes, have been identified as inherited in Lynch kindreds, and therefore are associated with the replication error phenotype. Clinical implications of the replication error phenotype include the demonstration that resistance to some alkylating agents appears to be directly altered in tumors with this phenotype.

Microsatellite instability (MSI) analysis of colorectal cancers is clinically useful to identify patients with hereditary nonpolyposis colorectal cancer (HNPCC) caused by germline mutations of mismatch repair genes. MSI status may also predict cancer response/resistance to certain chemotherapies. We evaluated the MSI Analysis System (Promega Corp.; five mononucleotide and two pentanucleotide repeats) and compared the results to the Bethesda panel, which interrogates five microsatellite loci recommended by the 1997 National Cancer Institute-sponsored MSI workshop (three dinucleotide and two mononucleotide repeats). Thirty-four colorectal cancers were analyzed by both assays. The overall concordance between the two assays was 85% (29 of 34). There was complete concordance between the two assays for all of the MSI-high (11 of 11) and microsatellite stable (MSS; 18 of 18) cases. In the 11 MSI-high cases, all 5 of the mononucleotide loci in the MSI Analysis System demonstrated shifted alleles (100% sensitivity), and each shift resulted in products that were smaller in size than the germline alleles. All (5 of 5) of the cases interpreted as MSI-low by the Bethesda assay were interpreted as MSS by the MSI Analysis System. Our results suggest that the MSI Analysis System is generally superior and may help resolve cases of MSI-low into either MSI-high or MSS.

Mismatch repair defects are carcinogenic. This conclusion comes some 80 years after the original description of a type of familial colorectal cancer in which mismatch repair defects are involved, and from decades of dedicated basic science research into fundamental mechanisms cells use to repair their DNA. Mismatch repair (MMR) was described first in bacteria, later in yeast and finally in higher eukaryotes. In bacteria, one of its roles is the rapid repair of replicative errors thereby providing the genome with a 100-1000-fold level of protection against mutation. It also guards the genome by preventing recombination between non-homologous regions of DNA. The information gained from bacteria suddenly became relevant to human neoplasia in 1993 when the RER phenotype of microsatellite instability was discovered in human cancers and was rapidly shown to be due to defects in mismatch repair. Evidence supporting the role of MMR defects in carcinogenesis comes from a variety of independent sources including: (i) theoretical considerations of the requirement for a mutator phenotype as a step in multistage carcinogenesis; (ii) discovering that MMR defects cause a 'mutator phenotype' destabilizing endogenous expressed genes including those integral to carcinogenesis; (iii) finding MMR defects in the germline of HNPCC kindred members; (iv) finding that such defects behave as classic tumor suppressor genes in both familial and sporadic colorectal cancers; (v) discovering that MMR 'knockout' mice have an increased incidence of tumors; and (vi) discovering that genetic complementation of MMR defective cells stabilizes the MMR deficiency-associated microsatellite instability. Models of carcinogenesis now must integrate the concepts of a MMR defect induced mutator phenotype (Loeb) with the concepts of multistep colon carcinogenesis (Fearon and Vogelstein) and clonal heterogeneity/selection (Nowell).

The HIV Network for Prevention Trials (HIVNET) 012 trial showed that NVP resistance (NVPR) emerged in some women and children after the administration of single-dose nevirapine (SD-NVP). We tested whether K103N-containing human immunodeficiency virus (HIV)-1 variants persisted in women and infants 1 year or more after the administration of SD-NVP.We analyzed samples from 9 women and 5 infants in HIVNET 012 who had NVPR 6-8 weeks after the administration of SD-NVP. Samples were analyzed with the ViroSeq system and with 2 sensitive resistance assays, LigAmp and TyHRT.ViroSeq detected the K103N mutation in 8 of 9 women and in 2 of 5 infants. LigAmp detected the K103N mutation at low levels in 8 of 9 women and in 4 of 5 infants. K103N was not detected by ViroSeq 12-24 months after the administration of SD-NVP but was detected by LigAmp in 3 of 9 women and in 1 of 5 infants. K103N was also detected in those samples by use of the TyHRT assay.K103N-containing variants persist in some women and infants for 1 year or more after the administration of SD-NVP. Sensitive resistance assays may provide new insight into the impact of antiretroviral drug exposure on HIV-1 evolution.

A subset of hereditary and sporadic colorectal carcinomas is defined by microsatellite instability (MSI), but the spectra of gene mutations have not been characterized extensively. Thirty-nine hereditary nonpolyposis colorectal cancer syndrome carcinomas (HNPCCa) and 57 sporadic right-sided colonic carcinomas (SRSCCa) were evaluated. Of HNPCCa, 95% (37/39) were MSI-positive as contrasted with 31% (18/57) of SRSCCa (<i>P</i> < 0.000001), but instability tended to be more widespread in SRSCCa (<i>P</i> = 0.08). Absence of nuclear hMSH2 mismatch repair gene product by immunohistochemistry was associated with germline <i>hMSH2</i> mutation (<i>P</i> = 0.0007). The prevalence of K-<i>ras</i> proto-oncogene mutations was similar in HNPCCa and SRSCCa (30% (11/37) and 30% (16/54)), but no HNPCCa from patients with germline<i>hMSH2</i> mutation had codon 13 mutation (<i>P</i> = 0.02), and two other HNPCCa had multiple K-<i>ras</i> mutations attributable to subclones. 18q allelic deletion and p53 gene product overexpression were inversely related to MSI (<i>P</i> = 0.0004 and<i>P</i> = 0.0001, respectively). Frameshift mutation of the transforming growth factor β type II receptor gene was frequent in all MSI-positive cancers (85%, 46/54), but mutation of the <i>E2F-4</i> transcription factor gene was more common in HNPCCa of patients with germline <i>hMSH2</i>mutation than in those with germline <i>hMLH1</i> mutation (100% (8/8) <i>versus</i> 40% (2/5),<i>P</i> = 0.04), and mutation of the<i>Bax</i> proapoptotic gene was more frequent in HNPCCa than in MSI-positive SRSCCa (55% (17/31) <i>versus</i> 13% (2/15), <i>P</i> = 0.01). The most common combination of mutations occurred in only 23% (8/35) of evaluable MSI-positive cancers. Our findings suggest that the accumulation of specific genetic alterations in MSI-positive colorectal cancers is markedly heterogeneous, because the occurrence of some mutations (eg, <i>ras</i>,<i>E2F-4</i>, and <i>Bax</i> genes), but not others (eg, transforming growth factor β type II receptor gene), depends on the underlying basis of the mismatch repair deficiency. This genetic heterogeneity may contribute to the heterogeneous clinical and pathological features of MSI-positive cancers.

Pancreatic carcinomas with acinar differentiation, including acinar cell carcinoma, pancreatoblastoma and carcinomas with mixed differentiation, are distinct pancreatic neoplasms with poor prognosis. Although recent whole-exome sequencing analyses have defined the somatic mutations that characterize the other major neoplasms of the pancreas, the molecular alterations underlying pancreatic carcinomas with acinar differentiation remain largely unknown. In the current study, we sequenced the exomes of 23 surgically resected pancreatic carcinomas with acinar differentiation. These analyses revealed a relatively large number of genetic alterations at both the individual base pair and chromosomal levels. There was an average of 119 somatic mutations/carcinoma. When three outliers were excluded, there was an average of 64 somatic mutations/tumour (range 12-189). The mean fractional allelic loss (FAL) was 0.27 (range 0-0.89) and heterogeneity at the chromosome level was confirmed in selected cases using fluorescence in situ hybridization (FISH). No gene was mutated in >30% of the cancers. Genes altered in other neoplasms of the pancreas were occasionally targeted in carcinomas with acinar differentiation; SMAD4 was mutated in six tumours (26%), TP53 in three (13%), GNAS in two (9%), RNF43 in one (4%) and MEN1 in one (4%). Somatic mutations were identified in genes in which constitutional alterations are associated with familial pancreatic ductal adenocarcinoma, such as ATM, BRCA2 and PALB2 (one tumour each), as well as in genes altered in extra-pancreatic neoplasms, such as JAK1 in four tumours (17%), BRAF in three (13%), RB1 in three (13%), APC in two (9%), PTEN in two (9%), ARID1A in two (9%), MLL3 in two (9%) and BAP1 in one (4%). Perhaps most importantly, we found that more than one-third of these carcinomas have potentially targetable genetic alterations, including mutations in BRCA2, PALB2, ATM, BAP1, BRAF and JAK1.

Intraductal papillary mucinous neoplasms (IPMNs) are the most common cystic precursor lesions of invasive pancreatic cancer. The recent identification of activating GNAS mutations at codon 201 in IPMNs is a promising target for early detection and therapy. The purpose of this study was to explore clinicopathological correlates of GNAS mutational status in resected IPMNs.Clinical and pathologic characteristics were retrieved on 54 patients in whom GNAS codon 201 mutational status was previously reported ("historical group", Wu et al. Sci Transl Med 3:92ra66, 2011). In addition, a separate cohort of 32 patients (validation group) was included. After microdissection and DNA extraction, GNAS status was determined in the validation group by pyrosequencing.GNAS activating mutations were found in 64% of the 32 IPMNs included in the validation group, compared with a previously reported prevalence of 57% in the historical group. Overall, 52 of 86 (61%) of IPMNs demonstrated GNAS mutations in the two studies combined. Analysis of both groups confirmed that demographic characteristics, tumor location, ductal system involvement, focality, size, grade of dysplasia, presence of an associated cancer, and overall survival were not correlated with GNAS mutational status. Stratified by histological subtype, 100% of intestinal type IPMNs demonstrated GNAS mutations compared to 51% of gastric IPMN, 71% of pancreatobiliary IPMNs, and 0% of oncocytic IPMNs.GNAS activating mutations can be reliably detected in IPMNs by pyrosequencing. In terms of clinicopathological parameters, only histological subtype was correlated with mutational frequency, with the intestinal phenotype always associated with GNAS mutations.

Abstract Purpose: The goal of this study was to comprehensively identify CpG island methylation alterations between pancreatic cancers and normal pancreata and their associated gene expression alterations. Experimental Design: We employed methylated CpG island amplification followed by CpG island microarray, a method previously validated for its accuracy and reproducibility, to analyze the methylation profile of 27,800 CpG islands covering 21 MB of the human genome in nine pairs of pancreatic cancer versus normal pancreatic epithelial tissues and in three matched pairs of pancreatic cancer versus lymphoid tissues from the same individual. Results: This analysis identified 1,658 known loci that were commonly differentially methylated in pancreatic cancer compared with normal pancreas. By integrating the pancreatic DNA methylation status with the gene expression profiles of the same samples before and after treatment with the DNA methyltransferase inhibitor 5-aza-2′-deoxycytidine, and the histone deacetylase inhibitor, trichostatin A, we identified dozens of aberrantly methylated and differentially expressed genes in pancreatic cancers including a more comprehensive list of hypermethylated and silenced genes that have not been previously described as targets for aberrant methylation in cancer. Conclusion: We expected that the identification of aberrantly hypermethylated and silenced genes will have diagnostic, prognostic, and therapeutic applications. Clin Cancer Res; 17(13); 4341–54. ©2011 AACR.

<h3>Objective</h3> Pancreatic cysts are commonly detected in patients undergoing pancreatic imaging. Better approaches are needed to characterise these lesions. In this study we evaluated the utility of detecting mutant DNA in secretin-stimulated pancreatic juice. <h3>Design</h3> Secretin-stimulated pancreatic juice was collected from the duodenum of 291 subjects enrolled in Cancer of the Pancreas Screening trials at five US academic medical centres. The study population included subjects with a familial predisposition to pancreatic cancer who underwent pancreatic screening, and disease controls with normal pancreata, chronic pancreatitis, sporadic intraductal papillary mucinous neoplasm (IPMN) or other neoplasms. Somatic <i>GNAS</i> mutations (reported prevalence ∼66% of IPMNs) were measured using digital high-resolution melt-curve analysis and pyrosequencing. <h3>Results</h3> <i>GNAS</i> mutations were detected in secretin-stimulated pancreatic juice samples of 50 of 78 familial and sporadic cases of IPMN(s) (64.1%), 15 of 33 (45.5%) with only diminutive cysts (&lt;5 mm), but none of 57 disease controls. <i>GNAS</i> mutations were also detected in five of 123 screened subjects without a pancreatic cyst. Among 97 subjects who had serial pancreatic evaluations, <i>GNAS</i> mutations detected in baseline juice samples predicted subsequent emergence or increasing size of pancreatic cysts. <h3>Conclusion</h3> Duodenal collections of secretin-stimulated pancreatic juice from patients with IPMNs have a similar prevalence of mutant <i>GNAS</i> to primary IPMNs, indicating that these samples are an excellent source of mutant DNA from the pancreas. The detection of <i>GNAS</i> mutations before an IPMN is visible suggests that analysis of pancreatic juice has the potential to help in the risk stratification and surveillance of patients undergoing pancreatic screening.

Cancer biomarkers are currently the subject of intense research because of their potential utility for diagnosis, prognosis, and targeted therapy. In theory, the gene products resulting from somatic mutations are the ultimate protein biomarkers, being not simply associated with tumors but actually responsible for tumorigenesis. We show here that the altered protein products resulting from somatic mutations can be identified directly and quantified by mass spectrometry. The peptides expressed from normal and mutant alleles were detected by selected reaction monitoring (SRM) of their product ions using a triple-quadrupole mass spectrometer. As a prototypical example of this approach, we demonstrated that it is possible to quantify the number and fraction of mutant Ras protein present in cancer cell lines. There were an average of 1.3 million molecules of Ras protein per cell, and the ratio of mutant to normal Ras proteins ranged from 0.49 to 5.6. Similarly, we found that mutant Ras proteins could be detected and quantified in clinical specimens such as colorectal and pancreatic tumor tissues as well as in premalignant pancreatic cyst fluids. In addition to answering basic questions about the relative levels of genetically abnormal proteins in tumors, this approach could prove useful for diagnostic applications.

Context.—DNA sequencing is critical to identifying many human genetic disorders caused by DNA mutations, including cancer. Pyrosequencing is less complex, involves fewer steps, and has a superior limit of detection compared with Sanger sequencing. The fundamental basis of pyrosequencing is that pyrophosphate is released when a deoxyribonucleotide triphosphate is added to the end of a nascent strand of DNA. Because deoxyribonucleotide triphosphates are sequentially added to the reaction and because the pyrophosphate concentration is continuously monitored, the DNA sequence can be determined. Objective.—To demonstrate the fundamental principles of pyrosequencing. Data Sources.—Salient features of pyrosequencing are demonstrated using the free software program Pyromaker (http://pyromaker.pathology.jhmi.edu), through which users can input DNA sequences and other pyrosequencing parameters to generate the expected pyrosequencing results. Conclusions.—We demonstrate how mutant and wild-type DNA sequences result in different pyrograms. Using pyrograms of established mutations in tumors, we explain how to analyze the pyrogram peaks generated by different dispensation sequences. Further, we demonstrate some limitations of pyrosequencing, including how some complex mutations can be indistinguishable from single base mutations. Pyrosequencing is the basis of the Roche 454 next-generation sequencer and many of the same principles also apply to the Ion Torrent hydrogen ion-based next-generation sequencers.

As next-generation sequencing (NGS) becomes a major sequencing platform in clinical diagnostic laboratories, it is critical to identify artifacts that constitute baseline noise and may interfere with detection of low-level gene mutations. This is especially critical for applications requiring ultrasensitive detection, such as molecular relapse of solid tumors and early detection of cancer. We recently observed a ~10-fold higher frequency of C:G > T:A mutations than the background noise level in both wild-type peripheral blood and formalin-fixed paraffin-embedded samples. We hypothesized that these might represent cytosine deamination events, which have been seen using other platforms.To test this hypothesis, we pretreated samples with uracil N-glycosylase (UNG). Additionally, to test whether some of the cytosine deamination might be a laboratory artifact, we simulated the heat associated with polymerase chain reaction thermocycling by subjecting samples to thermocycling in the absence of polymerase. To test the safety of universal UNG pretreatment, we tested known positive samples treated with UNG.UNG pretreatment significantly reduced the frequencies of these mutations, consistent with a biologic source of cytosine deamination. The simulated thermocycling-heated samples demonstrated significantly increased frequencies of C:G > T:A mutations without other baseline base substitutions being affected. Samples with known mutations demonstrated no decrease in our ability to detect these after treatment with UNG.Baseline noise during NGS is mostly due to cytosine deamination, the source of which is likely to be both biologic and an artifact of thermocycling, and it can be reduced by UNG pretreatment.

Objectives:To validate next-generation sequencing (NGS) technology for clinical diagnosis and to determine appropriate read depth.

Despite advances in sequencing, structural variants (SVs) remain difficult to reliably detect due to the short read length (<300 bp) of 2nd generation sequencing. Not only do the reads (or paired-end reads) need to straddle a breakpoint, but repetitive elements often lead to ambiguities in the alignment of short reads. We propose to use the long-reads (up to 20 kb) possible with 3rd generation sequencing, specifically nanopore sequencing on the MinION. Nanopore sequencing relies on a similar concept to a Coulter counter, reading the DNA sequence from the change in electrical current resulting from a DNA strand being forced through a nanometer-sized pore embedded in a membrane. Though nanopore sequencing currently has a relatively high mismatch rate that precludes base substitution and small frameshift mutation detection, its accuracy is sufficient for SV detection because of its long reads. In fact, long reads in some cases may improve SV detection efficiency.We have tested nanopore sequencing to detect a series of well-characterized SVs, including large deletions, inversions, and translocations that inactivate the CDKN2A/p16 and SMAD4/DPC4 tumor suppressor genes in pancreatic cancer. Using PCR amplicon mixes, we have demonstrated that nanopore sequencing can detect large deletions, translocations and inversions at dilutions as low as 1:100, with as few as 500 reads per sample. Given the speed, small footprint, and low capital cost, nanopore sequencing could become the ideal tool for the low-level detection of cancer-associated SVs needed for molecular relapse, early detection, or therapeutic monitoring.

Abstract Purpose: Inactivation of mismatch repair (MMR) genes may predict sensitivity to immunotherapy in metastatic prostate cancers. We studied primary prostate tumors with MMR defects. Experimental Design: A total of 1,133 primary prostatic adenocarcinomas and 43 prostatic small cell carcinomas (NEPC) were screened by MSH2 immunohistochemistry with confirmation by next-generation sequencing (NGS). Microsatellite instability (MSI) was assessed by PCR and NGS (mSINGS). Results: Of primary adenocarcinomas and NEPC, 1.2% (14/1,176) had MSH2 loss. Overall, 8% (7/91) of adenocarcinomas with primary Gleason pattern 5 (Gleason score 9–10) had MSH2 loss compared with 0.4% (5/1,042) of tumors with any other scores (P &amp;lt; 0.05). Five percent (2/43) of NEPC had MSH2 loss. MSH2 was generally homogenously lost, suggesting it was an early/clonal event. NGS confirmed MSH2 loss-of-function alterations in all (12/12) samples, with biallelic inactivation in 83% (10/12) and hypermutation in 83% (10/12). Overall, 61% (8/13) and 58% (7/12) of patients had definite MSI by PCR and mSINGS, respectively. Three patients (25%) had germline mutations in MSH2. Tumors with MSH2 loss had a higher density of infiltrating CD8+ lymphocytes compared with grade-matched controls without MSH2 loss (390 vs. 76 cells/mm2; P = 0.008), and CD8+ density was correlated with mutation burden among cases with MSH2 loss (r = 0.72, P = 0.005). T-cell receptor sequencing on a subset revealed a trend toward higher clonality in cases versus controls. Conclusions: Loss of MSH2 protein is correlated with MSH2 inactivation, hypermutation, and higher tumor-infiltrating lymphocyte density, and appears most common among very high-grade primary tumors, for which routine screening may be warranted if validated in additional cohorts. Clin Cancer Res; 23(22); 6863–74. ©2017 AACR.

Cigarette smoking doubles the risk of pancreatic cancer, and smoking accounts for 20% to 25% of pancreatic cancers. The recent sequencing of the pancreatic cancer genome provides an unprecedented opportunity to identify mutational patterns associated with smoking. We previously sequenced >750 million bp DNA from 23,219 transcripts in 24 adenocarcinomas of the pancreas (discovery screen). In this previous study, the 39 genes that were mutated more than once in the discovery screen were sequenced in an additional 90 adenocarcinomas of the pancreas (validation screen). Here, we compared the somatic mutations in the cancers obtained from individuals who ever smoked cigarettes (n = 64) to the somatic mutations in the cancers obtained from individuals who never smoked cigarettes (n = 50). When adjusted for age and gender, analyses of the discovery screen revealed significantly more nonsynonymous mutations in the carcinomas obtained from ever smokers (mean, 53.1 mutations per tumor; SD, 27.9) than in the carcinomas obtained from never smokers (mean, 38.5; SD, 11.1; P = 0.04). The difference between smokers and nonsmokers was not driven by mutations in known driver genes in pancreatic cancer (KRAS, TP53, CDKN2A/p16, and SMAD4), but instead was predominantly observed in genes mutated at lower frequency. No differences were observed in mutations in carcinomas from the head versus tail of the gland. Pancreatic carcinomas from cigarette smokers harbor more mutations than do carcinomas from never smokers. The types and patterns of these mutations provide insight into the mechanisms by which cigarette smoking causes pancreatic cancer.

To examine the clinicopathologic features and clonal relationship of multifocal intraductal papillary mucinous neoplasms (IPMNs) of the pancreas.Intraductal papillary mucinous neoplasms are increasingly diagnosed cystic precursor lesions of pancreatic cancer. Intraductal papillary mucinous neoplasms can be multifocal and a potential cause of recurrence after partial pancreatectomy.Thirty four patients with histologically documented multifocal IPMNs were collected and their clinicopathologic features catalogued. In addition, thirty multifocal IPMNs arising in 13 patients from 3 hospitals were subjected to laser microdissection followed by KRAS pyrosequencing and loss of heterozygosity (LOH) analysis on chromosomes 6q and 17p. Finally, we sought to assess the clonal relationships among multifocal IPMNs.We identified 34 patients with histologically documented multifocal IPMNs. Synchronous IPMNs were present in 29 patients (85%), whereas 5 (15%) developed clinically significant metachronous IPMNs. Six patients (18%) had a history of familial pancreatic cancer. A majority of multifocal IPMNs (86% synchronous, 100% metachronous) were composed of branch duct lesions, and typically demonstrated a gastric-foveolar subtype epithelium with low or intermediate grades of dysplasia. Three synchronous IPMNs (10%) had an associated invasive cancer. Molecular analysis of multiple IPMNs from 13 patients demonstrated nonoverlapping KRAS gene mutations in 8 patients (62%) and discordant LOH profiles in 7 patients (54%); independent genetic alterations were established in 9 of the 13 patients (69%).The majority of multifocal IPMNs arise independently and exhibit a gastric-foveolar subtype, with low to intermediate dysplasia. These findings underscore the importance of life-long follow-up after resection for an IPMN.

For decades, hundreds of different human tumor type-specific cell lines have been used in experimental cancer research as models for their respective tumors. The veracity of experimental results for a specific tumor type relies on the correct derivation of the cell line. In a worldwide effort, we verified the authenticity of all available esophageal adenocarcinoma (EAC) cell lines. We proved that the frequently used cell lines SEG-1 and BIC-1 and the SK-GT-5 cell line are in fact cell lines from other tumor types. Experimental results based on these contaminated cell lines have led to ongoing clinical trials recruiting EAC patients, to more than 100 scientific publications, and to at least three National Institutes of Health cancer research grants and 11 US patents, which emphasizes the importance of our findings. Widespread use of contaminated cell lines threatens the development of treatment strategies for EAC.

Background & AimsLittle is known about mechanisms of perineural invasion (PNI) by pancreatic ductal adenocarcinomas (PDAs) or other tumors. Annexin A2 (ANXA2) regulates secretion of SEMA3D, an axon guidance molecule, which binds and activates the receptor PLXND1 to promote PDA invasion and metastasis. We investigated whether axon guidance molecules promote PNI and metastasis by PDA cells in mice.MethodsWe performed studies in a dorsal root ganglion (DRG) invasion system, wild-type C57BL/6 mice (controls), mice with peripheral sensory neuron-specific disruption of PlxnD1 (PLAC mice), LSL-KRASG12D/+;LSL-TP53R172H/+;PDX-1-CRE+/+ (KPC) mice, and KPC mice crossed with ANXA2-knockout mice (KPCA mice). PDA cells were isolated from KPC mice and DRG cells were isolated from control mice. Levels of SEMA3D or ANXA2 were knocked down in PDA cells with small hairpin and interfering RNAs and cells were analyzed by immunoblots in migration assays, with DRGs and with or without antibodies against PLXND1. PDA cells were injected into the pancreas of control and PLAC mice, growth of tumors was assessed, and tumor samples were analyzed by histology. DRG cells were incubated with SEMA3D and analyzed by live imaging. We measured levels of SEMA3D and PLXND1 in PDA specimens from patients with PNI and calculated distances between tumor cells and nerves.ResultsDRG cells increase the migration of PDC cells in invasion assays; knockdown of SEMA3D in PDA cells or antibody blockade of PLXND1 on DRG cells reduced this invasive activity. In mice, orthotopic tumors grown from PDA cells with knockdown of SEMA3D, and in PLAC mice, orthotopic tumors grown from PDA cells, had reduced innervation and formed fewer metastases than orthotopic tumors grown from PDA cells in control mice. Increased levels of SEMA3D and PLXND1 in human PDA specimens associated with PNI.ConclusionsDRG cells increase the migratory and invasive activities of pancreatic cancer cells, via secretion of SEMA3D by pancreatic cells and activation of PLXND1 on DRGs. Knockdown of SEMA3D and loss of neural PLXND1 reduces innervation of orthotopic PDAs and metastasis in mice. Increased levels of SEMA3D and PLXND1 in human PDA specimens associated with PNI. Strategies to disrupt the axon guidance pathway mediated by SEMA3D and PLXND1 might be developed to slow progression of PDA. Little is known about mechanisms of perineural invasion (PNI) by pancreatic ductal adenocarcinomas (PDAs) or other tumors. Annexin A2 (ANXA2) regulates secretion of SEMA3D, an axon guidance molecule, which binds and activates the receptor PLXND1 to promote PDA invasion and metastasis. We investigated whether axon guidance molecules promote PNI and metastasis by PDA cells in mice. We performed studies in a dorsal root ganglion (DRG) invasion system, wild-type C57BL/6 mice (controls), mice with peripheral sensory neuron-specific disruption of PlxnD1 (PLAC mice), LSL-KRASG12D/+;LSL-TP53R172H/+;PDX-1-CRE+/+ (KPC) mice, and KPC mice crossed with ANXA2-knockout mice (KPCA mice). PDA cells were isolated from KPC mice and DRG cells were isolated from control mice. Levels of SEMA3D or ANXA2 were knocked down in PDA cells with small hairpin and interfering RNAs and cells were analyzed by immunoblots in migration assays, with DRGs and with or without antibodies against PLXND1. PDA cells were injected into the pancreas of control and PLAC mice, growth of tumors was assessed, and tumor samples were analyzed by histology. DRG cells were incubated with SEMA3D and analyzed by live imaging. We measured levels of SEMA3D and PLXND1 in PDA specimens from patients with PNI and calculated distances between tumor cells and nerves. DRG cells increase the migration of PDC cells in invasion assays; knockdown of SEMA3D in PDA cells or antibody blockade of PLXND1 on DRG cells reduced this invasive activity. In mice, orthotopic tumors grown from PDA cells with knockdown of SEMA3D, and in PLAC mice, orthotopic tumors grown from PDA cells, had reduced innervation and formed fewer metastases than orthotopic tumors grown from PDA cells in control mice. Increased levels of SEMA3D and PLXND1 in human PDA specimens associated with PNI. DRG cells increase the migratory and invasive activities of pancreatic cancer cells, via secretion of SEMA3D by pancreatic cells and activation of PLXND1 on DRGs. Knockdown of SEMA3D and loss of neural PLXND1 reduces innervation of orthotopic PDAs and metastasis in mice. Increased levels of SEMA3D and PLXND1 in human PDA specimens associated with PNI. Strategies to disrupt the axon guidance pathway mediated by SEMA3D and PLXND1 might be developed to slow progression of PDA.

Selective BRAF inhibitors, vemurafenib and dabrafenib, and the MEK inhibitor, trametinib, have been approved for treatment of metastatic melanomas with a BRAF p.V600E mutation. The clinical significance of non-codon 600 mutations remains unclear, in part, due to variation of kinase activity for different mutants.In this study, we categorized BRAF mutations according to the reported mutant kinase activity. A total of 1027 lung cancer, colorectal cancer or melanoma specimens were submitted for clinical mutation detection by next generation sequencing.Non-codon 600 mutations were observed in 37% of BRAF-mutated tumors. Of all BRAF mutants, 75% were kinase-activated, 15% kinase-impaired and 10% kinase-unknown. The most common kinase-impaired mutant involves codon 594, specifically, p.D594G (c.1781A > G) and p.D594N (c.1780G > A). Lung cancers showed significantly higher incidences of kinase-impaired or kinase-unknown mutants. Kinase-impaired BRAF mutants showed a significant association with concomitant activating KRAS or NRAS mutations, but not PIK3CA mutations, supporting the reported interaction of these mutations.BRAF mutants with impaired or unknown kinase activity as well as concomitant kinase-impaired BRAF mutations and RAS mutations were detected in lung cancers, colorectal cancers and melanomas. Different therapeutic strategies based on the BRAF mutant kinase activity and the concomitant mutations may be worthwhile.

Alterations in genes encoding epigenetic regulators are common in myeloid malignancies, and several recent studies have demonstrated that these mutations are present at high frequencies within peripheral blood cells in approximately 10% of individuals over 60 years of age. Although the presence of these mutations carries an increased risk of subsequent hematologic malignancies, the vast majority of individuals do not progress clinically and the natural history of clonal hematopoiesis is unclear.1-3 Thus, the term Clonal Hematopoiesis of Indeterminate Potential (CHIP) was proposed.4

PDAC patients who undergo surgical resection and receive effective chemotherapy have the best chance of long-term survival. Unfortunately, we lack predictive biomarkers to guide optimal systemic treatment. Ex-vivo generation of PDO for pharmacotyping may serve as predictive biomarkers in PDAC. The goal of the current study was to demonstrate the clinical feasibility of a PDO-guided precision medicine framework of care.PDO cultures were established from surgical specimens and endoscopic biopsies, expanded in Matrigel, and used for high-throughput drug testing (pharmacotyping). Efficacy of standard-of-care chemotherapeutics was assessed by measuring cell viability after drug exposure.A framework for rapid pharmacotyping of PDOs was established across a multi-institutional consortium of academic medical centers. Specimens obtained remotely and shipped to a central biorepository maintain viability and allowed generation of PDOs with 77% success. Early cultures maintain the clonal heterogeneity seen in PDAC with similar phenotypes (cystic-solid). Late cultures exhibit a dominant clone with a pharmacotyping profile similar to early passages. The biomass required for accurate pharmacotyping can be minimized by leveraging a high-throughput technology. Twenty-nine cultures were pharmacotyped to derive a population distribution of chemotherapeutic sensitivity at our center. Pharmacotyping rapidly-expanded PDOs was completed in a median of 48 (range 18-102) days.Rapid development of PDOs from patients undergoing surgery for PDAC is eminently feasible within the perioperative recovery period, enabling the potential for pharmacotyping to guide postoperative adjuvant chemotherapeutic selection. Studies validating PDOs as a promising predictive biomarker are ongoing.

N-Linked glycosylation usually occurs at the sequon, Asn-X-Ser/Thr. In this sequon, the side chain of the hydroxy amino acid (Ser or Thr) may play a direct catalytic role in the enzymatic transfer of core oligosaccharides to the Asn residue. Using recombinant variants of rabies virus glycoprotein (RGP), we examined the influence of the hydroxy amino acid on core glycosylation efficiency. A variant of RGP containing a single Asn-X-Ser sequon at Asn37 was modified by site-directed mutagenesis to change the sequon to either Asn-X-Cys or Asn-X-Thr. The impact of these changes on core glycosylation efficiency was assessed by expressing the variants in a cell-free transcription/translation/glycosylation system and in transfected tissue culture cells. Substitution of Cys at position 39 blocks glycosylation, whereas substitution of Thr dramatically increases core glycosylation efficiency of Asn37 in both membrane-anchored and secreted forms of RGP. The substitution of Thr for Ser also dramatically enhances the level of expression and cell surface delivery of RGP when the sequon at Asn37 is the only sequon in the protein. Novel forms of membrane-anchored and secreted RGP which are fully glycosylated at all three sequons were also generated by substitution of Thr at position 39. N-Linked glycosylation usually occurs at the sequon, Asn-X-Ser/Thr. In this sequon, the side chain of the hydroxy amino acid (Ser or Thr) may play a direct catalytic role in the enzymatic transfer of core oligosaccharides to the Asn residue. Using recombinant variants of rabies virus glycoprotein (RGP), we examined the influence of the hydroxy amino acid on core glycosylation efficiency. A variant of RGP containing a single Asn-X-Ser sequon at Asn37 was modified by site-directed mutagenesis to change the sequon to either Asn-X-Cys or Asn-X-Thr. The impact of these changes on core glycosylation efficiency was assessed by expressing the variants in a cell-free transcription/translation/glycosylation system and in transfected tissue culture cells. Substitution of Cys at position 39 blocks glycosylation, whereas substitution of Thr dramatically increases core glycosylation efficiency of Asn37 in both membrane-anchored and secreted forms of RGP. The substitution of Thr for Ser also dramatically enhances the level of expression and cell surface delivery of RGP when the sequon at Asn37 is the only sequon in the protein. Novel forms of membrane-anchored and secreted RGP which are fully glycosylated at all three sequons were also generated by substitution of Thr at position 39.

We have created a clinical molecular diagnostic assay to test for microsatellite instability (MSI) at multiple loci simultaneously in paraffin-embedded surgical pathology colon resection specimens. This fluorescent multiplex polymerase chain reaction (PCR) assay analyzes the five primary microsatellite loci recommended at the 1997 National Cancer Institute-sponsored conference on MSI for the identification of MSI or replication errors in colorectal cancer: Bat-25, Bat-26, D2S123, D5S346, and D17S250. Amplicon detection is accomplished by capillary electrophoresis using the ABI 310 Genetic Analyzer. Assay validation compared 18 specimens previously assessed by radioactive PCR and polyacrylamide gel electrophoresis detection to results generated by the reported assay. Germline and tumor DNA samples were amplified in separate multiplex PCR reactions, sized in separate capillary electrophoresis runs, and compared directly to identify novel length alleles in tumor tissue. A concordance of 100% between the two modalities was achieved. The multiplex assay routinely detected a subpopulation of 10% tumor alleles in the presence of 90% normal alleles. A novel statistical model was generated that corroborates the validity of using results generated by analysis of five independent microsatellites to achieve a single overall MSI diagnosis. The assay presented is superior to standard radioactive monoplex PCR, polyacrylamide gel electrophoretic analysis, primarily due to the multiplex PCR format. We have created a clinical molecular diagnostic assay to test for microsatellite instability (MSI) at multiple loci simultaneously in paraffin-embedded surgical pathology colon resection specimens. This fluorescent multiplex polymerase chain reaction (PCR) assay analyzes the five primary microsatellite loci recommended at the 1997 National Cancer Institute-sponsored conference on MSI for the identification of MSI or replication errors in colorectal cancer: Bat-25, Bat-26, D2S123, D5S346, and D17S250. Amplicon detection is accomplished by capillary electrophoresis using the ABI 310 Genetic Analyzer. Assay validation compared 18 specimens previously assessed by radioactive PCR and polyacrylamide gel electrophoresis detection to results generated by the reported assay. Germline and tumor DNA samples were amplified in separate multiplex PCR reactions, sized in separate capillary electrophoresis runs, and compared directly to identify novel length alleles in tumor tissue. A concordance of 100% between the two modalities was achieved. The multiplex assay routinely detected a subpopulation of 10% tumor alleles in the presence of 90% normal alleles. A novel statistical model was generated that corroborates the validity of using results generated by analysis of five independent microsatellites to achieve a single overall MSI diagnosis. The assay presented is superior to standard radioactive monoplex PCR, polyacrylamide gel electrophoretic analysis, primarily due to the multiplex PCR format. Microsatellite instability (MSI), or replication error, is a manifestation of genomic instability arising in a variety of human neoplasms where tumor cells have a decreased overall ability to faithfully replicate DNA. This phenomenon has been shown to be a frequent, if not obligatory, surrogate marker of underlying functional inactivation of one of the human DNA mismatch repair (MMR) genes.1Parsons R Li G Longley M Fang W Papadopoulos N Jen J de la Chapelle A Kinzler K Vogelstein B Modrich P Hypermutability and mismatch repair deficiency in RER+ tumor cells.Cell. 1993; 75: 1227-1236Abstract Full Text PDF PubMed Scopus (987) Google Scholar, 2Leach F Nicolaides N Papadopoulos N Liu B Jen J Parsons R Peltomaki P Sistonen P Aaltonen L Nystrom-Lahti M Guan XY Zhang J Meltzer P Yu J Kao F Chen D Cerosaletti K Fournier R Todd S Lewis T Leach R Naylor S Weissenbach J Mecklin J Jarvinen H Petersen G Hamilton S Green J Jass J Watson P Lynch H Trent J de la Chapelle A Kinzler K Vogelstein B Mutations of a MutS homolog in hereditary non-polyposis colorectal cancer.Cell. 1993; 75: 1215-1225Abstract Full Text PDF PubMed Scopus (2136) Google Scholar, 3Fishel R Lescoe MK Rao MRS Copeland NG Jenkins NA Garber J Kane M Kolodner R The human mutator gene homolog MSH2 and its association with hereditary nonpolyposis colon cancer.Cell. 1993; 75: 1027-1038Abstract Full Text PDF PubMed Scopus (2657) Google Scholar, 4Papadopoulos N Nicolaides N Wei Y Ruben S Carter K Rosen C Haseltine W Fleischmann R Fraser C Adams M Venter J Hamilton S Petersen G Watson P Lynch H Peltomaki P Mecklin J de la Chapelle A Kinzler K Vogelstein B Mutations of a mutL homolog in hereditary colon cancer.Science. 1994; 263: 1625-1629Crossref PubMed Scopus (1803) Google Scholar, 5Bronner C Baker S Morrison P Warren G Smith L Lescoe M Kane M Earabino C Lipford J Lindblom A Tannergard P Bollag R Godwin A Ward D Nordenskjold M Fishel R Kolodner R Liskay R Mutation in the DNA mismatch repair gene homologue hMLH1 is associated with hereditary non-polyposis colon cancer.Nature. 1994; 368: 258-261Crossref PubMed Scopus (1960) Google Scholar, 6Nicolaides N Papadopoulos N Liu B Wel Y Carter K Ruben S Rosen C Haseltine W Fleischmann R Fraser C Adams M Venter J Dunlopo M Hamilton S Petersen G de la Chapelle A Vogelstein B Kinzler K Mutations of two PMS homologues in hereditary nonpolyposis colon cancer.Nature. 1994; 371: 75-80Crossref PubMed Scopus (1469) Google Scholar Functional loss of a MMR gene occurs due to biallelic inactivation via some combination of coding region mutation, loss of heterozygosity (LOH), and/or promoter methylation.7Herman JG Umar A Polyak K Graff JR Ahuja N Issa JP Markowitz S Willson JK Hamilton SR Kinzler KW Kane MF Kolodner RD Vogelstein B Kunkel TA Baylin SB Incidence and functional consequences of hMLH1 promoter hypermethylation in colorectal carcinoma.Proc Natl Acad Sci USA. 1998; 95: 6870-6875Crossref PubMed Scopus (1726) Google Scholar, 8Veigl ML Kasturi L Olechnowicz J Ma AH Lutterbaugh JD Periyasamy S Li GM Drummond J Modrich PL Sedwick WD Markowitz SD Biallelic inactivation of hMLH1 by epigenetic gene silencing, a novel mechanism causing human MSI cancers.Proc Natl Acad Sci USA. 1998; 95: 8698-8702Crossref PubMed Scopus (566) Google Scholar Germlinemutation of a MMR gene has been shown to be the autosomal dominant genetic defect in most hereditary nonpolyposis colon cancer (HNPCC) kindreds.9Marra G Boland CR Hereditary nonpolyposis colorectal cancer: the syndrome, the genes, and historical perspectives.J Natl Cancer Inst. 1995; 87: 1114-1125Crossref PubMed Scopus (483) Google Scholar, 10Eshleman JR Markowitz SD Microsatellite instability in inherited and sporadic neoplasms.Curr Opin Oncol. 1995; 7: 83-89Crossref PubMed Scopus (249) Google Scholar A second hit incurred by tumor cells in HNPCC individuals results in biallelic inactivation of the specific MMR gene, causing loss of faithful replication of microsatellite DNA in tumor.11Hemminki A Peltomaki P Mecklin JP Jarvinen H Salovaara R Nystrom-Lahti M de la Chapelle A Aaltonen LA Loss of the wild type MLH1 gene is a feature of hereditary nonpolyposis colorectal cancer.Nat Genet. 1994; 8: 405-410Crossref PubMed Scopus (284) Google Scholar MSI is thus a marker of an underlying DNA mismatch repair defect and, additionally, is associated with enhanced mutation rates in coding DNA.12Eshleman JR Lang EZ Bowerfind GK Parsons R Vogelstein B Willson JKV Veigl ML Sedwick WD Markowitz SD Increased mutation rate at the hprt locus accompanies microsatellite instability in colon cancer.Oncogene. 1995; 10: 33-37PubMed Google Scholar, 13Bhattacharyya NP Skandalis A Ganesh A Groden J Meuth M Mutator phenotypes in human colorectal carcinoma cell lines.Proc Natl Acad Sci USA. 1994; 91: 6319-6323Crossref PubMed Scopus (410) Google Scholar This mutator phenotype, which results from the MMR defect, causes both coding region base substitutions and frameshift mutations at direct repeats, each occurring at equal frequencies,14Eshleman JR Markowitz SD Donover PS Lang EZ Lutterbaugh JD Li GM Longley M Modrich P Veigl ML Sedwick WD Diverse hypermutability of multiple expressed sequence motifs present in a cancer with microsatellite instability.Oncogene. 1996; 12: 1425-1432PubMed Google Scholar in addition to resulting in MSI. Generation of MMR defects and the resultant mutator phenotype is thought to be an early event in tumorigenesis15Shibata D Peinado MA Ionov Y Malkhosyan S Perucho M Genomic instability in repeated sequences is an early somatic event in colorectal tumorigenesis that persists after transformation.Nat Genet. 1994; 6: 273-281Crossref PubMed Scopus (462) Google Scholar and has been suggested to occur as early as the aberrant crypt focus stage.16Augenlicht LH Richards C Corner G Pretlow TP Evidence for genomic instability in human colonic aberrant crypt foci.Oncogene. 1996; 12: 1767-1772PubMed Google Scholar Although implicating a germline defect in HNPCC families, MSI is also found in 15 to 20% of sporadic colorectal cancers,17Aaltonen L Peltomaki P Leach F Sistonen P Pylkkanen L Mecklin J Jarvinen H Powell S Jen J Hamilton S Petersen G Kinzler K Vogelstein B de la Chapelle A Clues to the pathogenesis of familial colorectal cancer.Science. 1993; 260: 812-816Crossref PubMed Scopus (2629) Google Scholar where the finding also reflects an overall increase in genomic instability. Several reports have associated the finding of MSI defects in tumors with a better prognosis in stage-for-stage matched tumors.18Thibodeau S Bren G Schaid D Microsatellite instability in cancer of the proximal colon.Science. 1993; 260: 816-819Crossref PubMed Scopus (2865) Google Scholar, 19Sankila R Aaltonen LA Jarvinen HJ Mecklin JP Better survival rates in patients with MLH1-associated hereditary colorectal cancer.Gastroenterology. 1996; 110: 682-687Abstract Full Text Full Text PDF PubMed Scopus (309) Google Scholar Thus, it may become important clinically to identify tumors with MSI not only to implicate germline MMR defects (HNPCC families), but also for prognostic stratification. While clinical (Bethesda guidelines20Rodriguez-Bigas M Boland CR Hamilton SR Henson DE Jass JR Khan PM Lynch H Perucho M Smyrk T Sobin L Srivastava S A National Cancer Institute workshop on hereditary nonpolyposis colorectal cancer syndrome: meeting highlights and Bethesda guidelines.J Natl Cancer Inst. 1997; 89: 1758-1762Crossref PubMed Scopus (958) Google Scholar) and histopathological features21Kim H Jen J Vogelstein B Hamilton SR Clinical and pathological characteristics of sporadic colorectal carcinomas with DNA replication errors in microsatellite sequences.Am J Pathol. 1994; 145: 148-156PubMed Google Scholar may raise the suspicion that a colorectal tumor is microsatellite-unstable and perhaps has arisen in an HNPCC family, clinicopathological features are insufficient to diagnose the presence of MSI; thus, direct molecular testing has importance in documenting the MSI status of a clinically suspicious tumor.22Boland CR Thibodeau SN Hamilton SR Sidransky D Eshleman JR Burt RW Meltzer SJ Fodde R Rodriguez-Bigas MA Fodde R Ranzani GN Srivastava S A National Cancer Institute workshop on microsatellite instability for cancer detection and familial predisposition: development of international criteria for the determination of microsatellite instability in colorectal cancer.Cancer Res. 1998; 58: 5248-5257PubMed Google Scholar Many different microsatellite markers or loci have been used by different investigators to identify MSI in tumors. Controversy has existed among experts as to which and how many loci should be analyzed to diagnose MSI. In an attempt to gain the most information from research trials and to provide uniformity in clinical diagnosis, the National Cancer Institute (NCI) held an international meeting of MSI investigators in 1997 to create more consistent parameters for defining MSI in colorectal cancer (CRC) and to recommend microsatellite markers for use in CRC MSI testing in both clinical and research settings.22Boland CR Thibodeau SN Hamilton SR Sidransky D Eshleman JR Burt RW Meltzer SJ Fodde R Rodriguez-Bigas MA Fodde R Ranzani GN Srivastava S A National Cancer Institute workshop on microsatellite instability for cancer detection and familial predisposition: development of international criteria for the determination of microsatellite instability in colorectal cancer.Cancer Res. 1998; 58: 5248-5257PubMed Google Scholar Performing MSI assays using the five primary loci identified at the NCI MSI conference by standard radioactive monoplex polymerase chain reaction (PCR) and polyacrylamide gel electrophoresis (RMo-PAGE) requires a total of 10 separate PCR reactions (1 tumor and 1 normal DNA sample for each case, 5 loci assessed for each DNA sample), in addition to labor-intensive sequencing gel detection. The logistic difficulty of RMo-PAGE analysis led us to create a fluorescent multiplex PCR-capillary electrophoresis (FM-CE) assay using the five primary loci identified by investigators at the NCI-sponsored consensus conference.22Boland CR Thibodeau SN Hamilton SR Sidransky D Eshleman JR Burt RW Meltzer SJ Fodde R Rodriguez-Bigas MA Fodde R Ranzani GN Srivastava S A National Cancer Institute workshop on microsatellite instability for cancer detection and familial predisposition: development of international criteria for the determination of microsatellite instability in colorectal cancer.Cancer Res. 1998; 58: 5248-5257PubMed Google Scholar This format permits simultaneous amplification of all five loci in a single PCR reaction, with subsequent detection of amplicon length alterations using the automated ABI 310 Genetic Analyzer. We have also examined the validity of this overall approach to clinical MSI diagnosis by creating and applying a novel statistical model to the problem. Using this model, we have examined the number of loci and degree of microsatellite informativity required to achieve sufficient MSI diagnostic sensitivity. Here, we report on the development of assay parameters and the theoretical and practical validation of the assay for clinical use. Lymphoid normal and tumor matched cell lines were generously provided by Dr. James Willson at Case Western Reserve University (Cleveland, OH). Vaco 670 is a colon cancer cell line known to have MSI due to a defect in the MMR gene, hMSH2. L670 is an Epstein-Barr virus-transformed lymphocyte cell line from the same patient as Vaco 670. Tumor and normal tissue blocks for the validation study were obtained from patients' colon resection specimens obtained from the files of Johns Hopkins Hospital Department of Pathology. These specimens had been previously established to possess or lack MSI using the traditional radioactively labeled PCR primer, monoplex PCR, polyacrylamide sequencing gel electrophoresis detection, autoradiography approach23Fujiwara TSJ Watanabe T Rashid A Longo P Eshleman JR Booker S Lynch HT Jass JR Green JS Kim H Jen J Vogelstein B Hamilton SR Accumulated clonal genetic alterations in familial and sporadic colorectal carcinomas with widespread instability in microsatellite sequences.Am J Pathol. 1998; 153: 1063-1078Abstract Full Text Full Text PDF PubMed Scopus (175) Google Scholar (Hamilton SR, manuscript in preparation). The presence or absence of MSI by RMo-PAGE was blinded during FM-CE analysis. Serial 5-μm histological sections of formalin-fixed, paraffin-embedded tissue blocks of normal (N) and tumor (T) were prepared using DNA histology precautions. The first and fifth levels were stained with hematoxylin and eosin (H&E); the sandwiched tissue levels 2, 3, and 4 were mounted unstained on slides. Histological diagnoses were verified by light microscopy for each block using levels 1 and 5, and the areas to be microdissected were marked. Normal or tumor tissue was microdissected from unstained slides for each case by overlaying the unstained slide onto the H&E-stained slide. Dissection of unstained slides was performed in a laminar flow tissue culture hood after UV irradiation. Cell line DNA was isolated by the standard sodium dodecyl sulfate/proteinase K digestion, organic extraction, and ethanol/salt precipitation technique.24Sambrook J Fritsch EF Maniatis T Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1989: 9.16-9.19Google Scholar Tumor and normal DNA from microdissected paraffin embedded tissue was crudely isolated using xylene/ethanol deparaffinization followed by Proteinase K digestion and heat inactivation at 95°C for 10 minutes. Isolated genomic DNA was kept in a dedicated room which was used only to assemble PCR. No PCR products or equipment used in post-PCR analysis ever entered this room. The 16-μl PCR fluorescent multiplex reaction mix pool for each reaction was composed of the following final constituents: 1× AmpliTaq Gold PCR buffer with 1.5 mmol/L MgCl2 (Perkin Elmer, Foster City, CA), 0.002 mmol/L dNTPs (Perkin Elmer), 0.3 units AmpliTaq Gold DNA polymerase (Perkin Elmer), and primer sets in the final molar amounts listed in Table 1, with phosphoramidite fluorescent labels as indicated. A 9-μl aliquot of the reaction pool mix was used for each individual PCR reaction, with 1 μl of normal or tumor sample DNA. Control reactions included a water control for exclusion of contamination and a mix of 90% L670 (N) DNA and 10% Vaco 670 (T) DNA. The 90%/10% cell line mixture served as a combined positive/negative and limit of detection control.Table 1Primers and Characteristics of Microsatellite Loci AnalyzedLocus (phosphoramidite label): Primer sequenceGDB/Genbank no.Product sizeMolar concentrationInformativityD2S123 (Tet) Forward: 5′-AAACAGGATGCCTGCCTTTA-3′187953197–2274.0 pmol/L0.67 Reverse: 5′-GGACTTTCCACCTATGGGAC-3′56.0 pmol/LD17S250 (Fam) Forward: 5′-GGAAGAATCAAATAGACAAT-3′177030151–1698.0 pmol/L0.56 Reverse: 5′-GCTGGCCATATATATATTTAAACC-3′X54562112 pmol/LD5S346 (Fam) Forward: 5′-ACTCACTCTAGTGATAAATCGGG-3′18117196–1220.80 pmol/L0.67 Reverse: 5′-AGCAGATAAGACAGTATTACTAGTT-3′M7354711.2 pmol/LBat-25 (Hex) Forward: 5′-TCGCCTCCAAGAATGTAAGT-3′98345081202.0 pmol/L0.73 Reverse: 5′-TCTGCATTTTAACTATGGCTC-3′U6383428.0 pmol/LBat-26 (Tet) Forward: 5′-TGACTACTTTTGACTTCAGCC-3′98345051163.06 pmol/L1.00 Reverse: 5′-AACCATTCAACATTTTTAACCC-3′L4757542.3 pmol/L Open table in a new tab PCR was performed using either a PE 9600 or PE 9700 (Perkin Elmer) thermocycler under the following cycling conditions: initial denaturation 95°C for 9 minutes, followed by 35 cycles of: 94°C for 45 seconds, 55°C for 45 seconds and 72°C for 1 minute, with a final 45 minute, 60°C extension to aid nontemplated adenine addition. Fluorescently labeled PCR products were detected using the ABI 310 Genetic Analyzer and GeneScan Collection software. The following CE run parameters were used: GeneScan Short Tandem Repeat Performance Optimized Polymer (GS STR POP) 4 (1 ml) C module, GS POP 4 polymer, 5- to 10-second injection time at a voltage of 15.0 kV, a 15-kV electrophoresis voltage with a resultant 7- to 9-ampere current at a column temperature of 60°C, and a 20-minute electrophoresis time. GeneScan data analysis parameters were: matrix C virtual filter, 2800–6000 analysis range, baselining, multicomponent data processing, light peak smoothing, a minimum peak detection limit of 50 relative fluorescent units (RFU), size call range of 50 to 350 bp using the local Southern size calling method, Tamara labeled GS 500 size standard, no split peak correction, and a minimum peak half-width of 3 points. For the radioactive monoplex PCR/PAGE assay, the loci analyzed were D18S55, D18S58, D18S61, D18S64, and D18S69. Forward and reverse primers (Research Genetics, Huntsville, AL) were used at a 1:1 molar ratio after end-labeling of the forward primer with γ-32P-dATP and T4 polynucleotide kinase (New England Biolabs, Beverly, MA). Amplicon detection used standard 7% polyacrylamide sequencing gel electrophoresis and autoradiography as previously described.25Jen J Kim H Piantadosi S Liu Z-F Levitt RC Sistonen P Kinzler KW Vogelstein B Hamilton SR Allelic loss of chromosome 18q and prognosis in colorectal cancer.N Engl J Med. 1994; 331: 213-221Crossref PubMed Scopus (686) Google Scholar The size standard was generated using Phi-X 174 digested with Hin fI (Gibco/BRL, Rockville, MD), α-32P-dATP, and Klenow (Gibco/BRL) per manufacturer's instructions. A statistical model was constructed to analyze the diagnostic situation in which multiple independent assays are combined to establish an overall diagnosis, in this case, MSI-High, MSI-Low, or MSS. The following equations were applied to evaluate a population of MMR-defective tumor cells: MSS = (1 − I)n, MSI-Low = I × (1 − I)(n − 1) × n, and MSI-High = 1.00 − (MSS + MSI-Low), where I is the constant informativity of each locus and n is the number of loci analyzed. These equations represent a special case of the more generalized binomial distribution equation: f(X)=(nx)px(1-p)(n-x). Identification of normal or tumor allele amplicon sizes was accomplished by examining the appropriate electropherogram (N or T) and determining the predominant amplicon size(s) for each allele at each locus (greatest peak height or heights). More than two peaks can be seen at each allele in tumor samples if a shift is present. Each true microsatellite peak should be surrounded by stutter peaks of lower intensity with the appropriate delta (1 base for mononucleotide repeats, 2 bases for dinucleotide repeats) from the dominant peak. Stutter peak sizes can be larger or smaller than the predominant peak, but are more frequently smaller. Those peaks without stutter are considered to be nonspecific amplicons and are disregarded. Stutter peaks are thus considered a signature of amplification of a repetitive element lying within a forward and reverse primer set. Stutter peaks from a shifted dinucleotide locus allele can overlap with the predominant amplicon peak height of the second allele at that locus, causing difficulty in separating the stutter pattern of the first allele from the true predominant amplicon sizing of the second allele. The mononucleotide repeat loci, Bat-25 and Bat-26, are also subject to stutter and, as recently reported for Bat-2626Samowitz WS Slattery ML Potter JD Leppert MF BAT-26 and BAT-40 instability in colorectal adenomas and carcinomas and germline polymorphisms.Am J Pathol. 1999; 154: 1637-1641Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar and observed by the authors for Bat-25 (unpublished data), may also have a low occurrence of germline allele polymorphisms. The amplicon length(s) for each locus are recorded, and a direct comparison of the amplicon lengths between normal and tumor is made. If the tumor specimen has novel amplicon lengths in relation to the patient's germline (normal) amplicon length(s) at a specific locus, this is considered a locus positive for MSI. Despite microdissection, some small degree of stromal tissue contamination of the tumor sample is essentially unavoidable; thus, both tumor and germline amplicons may (in fact, should) be present in the tumor specimen. Sufficient amplification needs to occur at each locus within the tumor sample to ensure that low level instability would be detected if present. The parameters used for the diagnosis of MSI using the loci recommended by the NCI have been previously described.22Boland CR Thibodeau SN Hamilton SR Sidransky D Eshleman JR Burt RW Meltzer SJ Fodde R Rodriguez-Bigas MA Fodde R Ranzani GN Srivastava S A National Cancer Institute workshop on microsatellite instability for cancer detection and familial predisposition: development of international criteria for the determination of microsatellite instability in colorectal cancer.Cancer Res. 1998; 58: 5248-5257PubMed Google Scholar In brief, MSI-High (MSI-H) requires shifting of two of the five NCI recommended loci (≥30 to 40% of loci tested if more than five loci are analyzed). MSI-Low (MSI-L) is identified by shifting of one of the five NCI-recommended loci (≤30 to 40% of loci tested if more than five loci are analyzed). The diagnosis of microsatellite stable (MSS) requires that none of the loci analyzed be shifted. Accurate data interpretation requires meeting specific run criteria to ensure validity. The water control must be free of extraneous peaks. The 90% normal/10% tumor positive control must have detectable normal allele amplicons for all five loci in the peak height range of 2000 to 8000 RFU, and the tumor alleles should be shifted relative to germline in all five loci and have detectable peak heights in the range of 200-2000 RFU. Assay optimization involved identification of parameters important to the maximization of diagnostic accuracy, sensitivity, informativeness, and precision. Fluorescent labels were chosen such that the potential amplicons for each locus were sufficiently separated in size to prevent any potential overlap between PCR products (shifted or germline amplicons). Changes in fluorochrome assignments were made during optimization to enhance amplicon detection. For example, D2S123 was initially labeled in yellow (Hex), but was later moved to the green fluorochrome (Tet) to enhance its relative detectability. Initial analysis verified that all five loci were amplified and detectable by fluorescent monoplex PCR and CE detection. The specificity of amplification was optimized by varying forward to reverse primer concentrations (presented below). After maximization of amplification specificity, the primer pair for Bat-26 was systematically mixed with additional loci, beginning with duplex PCR reactions and proceeding to full multiplex of all five loci. Finally, the relative molar amounts of the various locus primer sets were adjusted to produce simultaneous amplification where peak heights of the predominant amplicons varied by no more than threefold for all loci (Table 1). Electropherogram data generated by the described assay, as applied to the control cell lines, can be seen in Figure 1. In Figure 1A, we compare the radioactive format (left panel) to a fluorescent monoplex format (right panel), using normal and tumor cell lines assayed at the Bat-25 locus. In Figure 1B, the full multiplex electropherogram data are shown as applied to cell line samples Vaco670 and L670. Figure 1C shows the full multiplex assay result on tissue samples normal and tumor from validation sample 10. Each of the five loci produced amplicons within the appropriate size range, surrounded by stutter peaks of appropriate size, diminishing in intensity with increasing distance from the predominant peak (see Materials and Methods). Repeat analyses of the same control and validation specimens produced consistent amplicon sizing. Eighteen cases of colon cancer previously characterized by RMo-PAGE23Fujiwara TSJ Watanabe T Rashid A Longo P Eshleman JR Booker S Lynch HT Jass JR Green JS Kim H Jen J Vogelstein B Hamilton SR Accumulated clonal genetic alterations in familial and sporadic colorectal carcinomas with widespread instability in microsatellite sequences.Am J Pathol. 1998; 153: 1063-1078Abstract Full Text Full Text PDF PubMed Scopus (175) Google Scholar (SR Hamilton, submitted) for the presence (14 cases) or absence (4 cases) of MSI were analyzed using the technique reported herein. Of the 14 cases diagnosed as MSI by RMo-PAGE, all 14 were diagnosed as MSI-H by FM-CE. Of the 4 cases shown to be MSS by RMo-PAGE, all four were MSS by FM-CE. Thus, of the18 cases analyzed, 100% showed overall diagnostic concordance between RMo-PAGE analysis and the FM-CE method (P = 0.0003, Fisher's exact test, Table 2).Table 2Comparison of Radioactive versus Fluorescence AssaysSample numberRMo-PAGE resultFM-CE result1N, 1TMSI-HMSI-H2N, 2TMSI-HMSI-H3N, 3TMSI-HMSI-H4N, 4TMSI-HMSI-H5N, 5TMSSMSS7N, 7TMSI-HMSI-H8N, 8TMSI-HMSI-H9N, 9TMSSMSS10N, 10TMSI-HMSI-H11N, 11TMSI-HMSI-H12N, 12TMSI-HMSI-H13N, 13TMSI-HMSI-H14N, 14TMSI-HMSI-H15N, 15TMSI-HMSI-H16N, 16TMSI-HMSI-H17N, 17TMSI-HMSI-H18N, 18TMSSMSS19N, 19TMSSMSSTotal MSI-H1414Total MSI-L00Total MSS44 Open table in a new tab Serial titration of labeled forward primer to unlabeled reverse primer was performed to determine whether nonspecific amplification could be eliminated while maintaining specific amplification (Parsons R, personal communication). Serial dilutions revealed that a forward to reverse primer dilution of 1:15 was optimal in reducing nonspecific amplicons while maintaining adequate specific amplicon signal (Figure 2). At a forward to reverse primer ratio of 1:1 (Figure 2A), amplification was suboptimal due detection of multiple nonspecific products, whereas at a forward to reverse ratio of 1:100 (Figure 2D)there was not enough forward primer present to allow generation of detectable PCR product. The ratio of 1:15 forward labeled to reverse primer was chosen as this primer ratio yielded the greatest specific amplicon product without loss of signal. The limit of detection of tumor DNA mixed with normal DNA was tested. Tumor cell line DNA (Vaco 670) was serially diluted into germline DNA (L670). Using the criteria that amplicons from all five normal and tumor loci must be identified in the mixed sample, the consistent lower limit of detection for this assay is 10% tumor (shifted) alleles in 90% germline (unshifted) alleles (Figure 3). The lower limit of detection for the multiplex assay is improved to 2 to 5% tumor detectable mixed into normal, when fewer than five loci are required to be detected. Sensitivity can be further enhanced as clinically indicated by using monoplex fluorescent PCR reactions. We have developed a statistical model to examine some of the interpretive considerations when multiple independent assays are combined to produce a single overall assay r

Using microarrays, we have screened for genes reactivated by drugs that modify epigenetic mechanisms in pancreatic cancer cells. One of the genes identified was tissue factor pathway inhibitor 2 (TFPI-2), which encodes for a broad-spectrum serine proteinase inhibitor that negatively regulates the extracellular matrix degradation, an essential step in tumor invasion and metastasis. We therefore investigated the expression and methylation patterns of the TFPI-2 gene in pancreatic adenocarcinoma, and determined its role in tumor growth and invasion. In contrast to its abundant expression in normal pancreas, TFPI-2 mRNA was undetectable in a high fraction of pancreatic cancer cell lines and in primary pancreatic ductal neoplasms (IPMNs). Loss of TFPI-2 expression was associated with aberrant hypermethylation of its promoter CpG island. Treatment with the phorbol ester (PMA), known to stimulate the TFPI-2 promoter activity, augmented the TFPI-2 expression in cell lines with unmethylated or partially methylated TFPI-2, but failed to induce the expression in cell lines that harbored fully methylated TFPI-2. Aberrant methylation of TFPI-2 was also detected in 73% (102/140) of pancreatic cancer xenografts and primary pancreatic adenocarcinomas, was more likely in older patients with pancreatic cancer, and significantly correlated with progression of IPMNs (P=0.0002). Restored expression of the TFPI-2 gene in nonexpressing pancreatic cancer cells resulted in marked suppression in their proliferation, migration, and invasive potential in vitro. We thus conclude that epigenetic inactivation of TFPI-2 is a common mechanism that contributes to the aggressive phenotype of pancreatic ductal adenocarcinoma.

Fourteen colorectal cancer cell lines, categorized according to the presence or absence of microsatellite instability, were further analysed for chromosomal stability by karyotyping. NonRER (microsatellite stable) cell lines typically displayed highly aberrant karyotypes with alterations not only of chromosome number but also of chromosome structure including chromosomal deletions, inversions, and translocations. RER (microsatellite unstable) cell lines, in contrast, displayed significantly fewer alterations of chromosome number. Moreover, RER cell lines also displayed significantly fewer cytogenetically evident alterations of chromosome structure. Compared to NonRER colon cancers, RER colon cancers are significantly less likely to have undergone chromosomal gain, loss, or breakage. Characterization of p53 gene status by gene sequencing was performed in an attempt to determine if p53 gene status correlated with the chromosomal stability of the RER cancers. Gene mutations in p53 were present in all of the NonRER colon cancers. However, p53 gene mutations were also found present in four of nine of the RER colon cancers. Unexpectedly, RER colon cancers bearing mutant p53 demonstrated the same stability of chromosome number, and the same stability of chromosome structure, as the RER colon cancers with wild-type p53. Therefore, in RER colon cancers specific p53 independent mechanisms actively maintain the stability of both chromosome number and structure.

Pancreatic intraepithelial neoplasia (PanIN) is a precursor to invasive ductal adenocarcinoma of the pancreas. Observations made in genetically engineered mouse models suggest that the acinar/centroacinar compartment can give rise to ductal neoplasia. To integrate findings in mice and men, we examined human acinar cells, acinar-ductal metaplasia (ADM) lesions, and PanINs for KRAS2 gene mutations. Surgically resected pancreata were screened for foci of ADM with or without an associated PanIN lesion. Stromal cells, acinar cells, ADMs, and PanINs were separately isolated using laser capture microdissection. KRAS2 status was analyzed using genomic DNA isolated from the microdissected tissue. Twelve of these 31 foci of ADM occurred in isolation, whereas 19 were in the same lobules as a PanIN lesion. All 31 microdissected foci of acinar cells were KRAS2 gene wild-type, as were all 12 isolated ADM lesions lacking an associated PanIN. KRAS2 gene mutations were present in 14 of 19 (74%) PanIN lesions and in 12 of the 19 (63%) foci of ADM associated with these PanINs. All ADM lesions with a KRAS2 gene mutation harbored the identical KRAS2 gene mutation found in their associated PanIN lesions. Ductal neoplasms of the human pancreas, as defined by KRAS2 gene mutations, do not appear to arise from acinar cells. Isolated AMD lesions are genetically distinct from those associated with PanINs, and the latter may represent retrograde extension of the neoplastic PanIN cells or less likely are precursors to PanIN.

Esophageal adenocarcinoma (EAC) arises in the backdrop of reflux-induced metaplastic phenomenon known as Barrett esophagus. The prognosis of advanced EAC is dismal, and there is an urgent need for identifying molecular targets for therapy. Serial Analysis of Gene Expression (SAGE) was performed on metachronous mucosal biopsies from a patient who underwent progression to EAC during endoscopic surveillance. SAGE confirmed significant upregulation of Axl "tags" during the multistep progression of Barrett esophagus to EAC. In a cohort of 92 surgically resected EACs, Axl overexpression was associated with shortened median survival on both univariate (p &lt; 0.004) and multivariate (p &lt; 0.036) analysis. Genetic knockdown of Axl receptor tyrosine kinase (RTK) function was enabled in two EAC lines (OE33 and JH-EsoAd1) using lentiviral short hairpin RNA (shRNA). Genetic knockdown of Axl in EAC cell lines inhibited invasion, migration, and in vivo engraftment, which was accompanied by downregulation in the activity of the Ral GTPase proteins (RalA and RalB). Restoration of Ral activation rescued the transformed phenotype of EAC cell lines, suggesting a novel effector mechanism for Axl in cancer cells. Pharmacological inhibition of Axl was enabled using a small molecule antagonist, R428 (Rigel Pharmaceuticals). Pharmacological inhibition of Axl with R428 in EAC cell lines significantly reduced anchorage-independent growth, invasion and migration. Blockade of Axl function abrogated phosphorylation of ERBB2 (Her-2/neu) at the Tyr877 residue, indicative of receptor crosstalk. Axl RTK is an adverse prognostic factor in EAC. The availability of small molecule inhibitors of Axl function provides a tractable strategy for molecular therapy of established EAC.

Sarcina organisms were first observed in and recorded from the stomach contents of a patient suffering from vomiting by John Goodsir in 1842. Since that time, their fine structure, phylogenetic classification, and biochemical characteristics have been described. Although numerous cases of fatal disease have been attributed to this organism in the veterinary literature, only a few human cases have been documented. As a result, whether this organism causes disease in humans has not been definitively established. We report the clinicopathologic findings in a series of 5 patients with Sarcina-like organisms identified in upper gastrointestinal endoscopic biopsies with molecular confirmation. Our findings have shown that the organism is most commonly found in patients with a history of gastric outlet obstruction or delayed gastric emptying. Although many of the patients do not demonstrate direct mucosal injury from the organism, the presence of a concurrent gastric ulcer puts the patient at increased risk for complications such as emphysematous gastritis or perforation. The finding of Sarcina organisms should prompt further investigation for functional causes of gastric outlet obstruction and delayed gastric emptying, such as occult malignancy.

The incidence of intrahepatic cholangiocarcinoma is increasing worldwide. The prognosis of intrahepatic cholangiocarcinoma is poor, and a better understanding of intrahepatic cholangiocarcinoma tumor biology is needed to more accurately predict clinical outcome and to suggest potential targets for more effective therapies. v-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog (KRAS) and BRAF are frequently mutated oncogenes that promote carcinogenesis in a variety of tumor types. In this study, we analyze a large set of intrahepatic cholangiocarcinoma tumors (n = 54) for mutations in these genes and compare the clinical outcomes of wild type versus KRAS and BRAF mutant cases. Of 54 cases, 7.4% were mutant for KRAS, 7.4% were mutant for BRAF, and these were mutually exclusive. These mutant cases were associated with a higher tumor stage at time of resection and a greater likelihood of lymph node involvement. These cases were also associated with a worse long-term overall survival. Therefore, testing for KRAS and BRAF mutations could be a valuable adjunct in improving both prognosis and outcome stratification among patients with intrahepatic cholangiocarcinoma.

<h3>Background & Aims</h3> Pancreatic imaging can identify neoplastic cysts but not microscopic neoplasms. Mutation analysis of pancreatic fluid after secretin stimulation might identify microscopic neoplasias in the pancreatic duct system. We determined the prevalence of mutations in <i>KRAS</i> and guanine nucleotide-binding protein α-stimulating genes in pancreatic juice from subjects undergoing endoscopic ultrasound for suspected pancreatic intraepithelial neoplasia, intraductal papillary mucinous neoplasms, or pancreatic adenocarcinoma. <h3>Methods</h3> Secretin-stimulated juice samples were collected from the duodenum of 272 subjects enrolled in Cancer of the Pancreas Screening studies; 194 subjects were screened because of a family history of, or genetic predisposition to, pancreatic cancer, and 78 subjects were evaluated for pancreatic cancer (n = 30) or other disorders (controls: pancreatic cysts, pancreatitis, or normal pancreata, n = 48). Mutations were detected by digital high-resolution melt-curve analysis and pyrosequencing. The number of replicates containing a mutation determined the mutation score. <h3>Results</h3> <i>KRAS</i> mutations were detected in pancreatic juice from larger percentages of subjects with pancreatic cancer (73%) or undergoing cancer screening (50%) than controls (19%) (<i>P</i> = .0005). A greater proportion of patients with pancreatic cancer had at least 1 <i>KRAS</i> mutation detected 3 or more times (47%) than screened subjects (21%) or controls (6%, <i>P</i> = .002). Among screened subjects, mutations in <i>KRAS</i> (but not guanine nucleotide-binding protein α-stimulating) were found in similar percentages of patients with or without pancreatic cysts. However, a greater proportion of patients older than age 50 years had <i>KRAS</i> mutations (54.6%) than younger patients (36.3%) (<i>P</i> = .032); the older subjects also had more mutations in <i>KRAS</i> (<i>P</i> = .02). <h3>Conclusions</h3> Mutations in <i>KRAS</i> are detected in pancreatic juice from the duodenum of 73% of patients with pancreatic cancer, and 50% of asymptomatic individuals with a high risk for pancreatic cancer. However, <i>KRAS</i> mutations were detected in pancreatic juice from 19% of controls. Mutations detected in individuals without pancreatic abnormalities, based on imaging analyses, likely arise from small pancreatic intraepithelial neoplasia lesions. ClinicalTrials.gov no: NCT00438906 and NCT00714701.

Microsatellite instability (MSI) is caused by defective mismatch repair in 15-20% of colorectal cancers (CRCs). Higher mutation loads in tumors with mismatch repair deficiency can predict response to pembrolizumab, an anti-programmed death 1 (PD-1) immune checkpoint inhibitor. We analyzed the mutations in 113 CRCs without MSI (MSS) and 29 CRCs with MSI-High (MSI-H) using the 50-gene AmpliSeq cancer panel. Overall, MSI-H CRCs showed significantly higher mutations than MSS CRCs, including insertion/deletion mutations at repeat regions. MSI-H CRCs showed higher incidences of mutations in the BRAF, PIK3CA, and PTEN genes as well as mutations in the receptor tyrosine kinase families. While the increased mutations in BRAF and PTEN in MSI-H CRCs are well accepted, we also support findings of mutations in the mTOR pathway and receptor tyrosine kinase family genes. MSS CRCs showed higher incidences of mutations in the APC, KRAS and TP53 genes, confirming previous findings. NGS assays may be designed to detect driver mutations for targeted therapeutics and to identify tumors with high mutation loads for potential treatment with immune checkpoint blockade therapies. Further studies may be warranted to elucidate potential targeted therapeutics against mutations in the mTOR pathway and the receptor tyrosine kinase family in MSI-H CRCs as well as the benefit of anti-PD-1 immunotherapy in hypermutated MSS CRCs or other cancers.

Purpose Tumor genomic profiling for personalized oncology therapy is being widely applied in clinical practice even as it is being evaluated more formally in clinical trials. Given the complexities of genomic data and its application to clinical use, molecular tumor boards with diverse expertise can provide guidance to oncologists and patients seeking to implement personalized genetically targeted therapy in practice. Methods A multidisciplinary molecular tumor board reviewed tumor molecular profiling reports from consecutive referrals at the Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins over a 3-year period. The tumor board weighed evidence for actionability of genomic alterations identified by molecular profiling and provided recommendations including US Food and Drug Administration–approved drug therapy, clinical trials of matched targeted therapy, off-label use of such therapy, and additional tumor or germline genetic testing. Results One hundred fifty-five patients were reviewed. Actionable genomic alterations were identified in 132 patients (85%). Off-label therapies were recommended in 37 patients (24%). Eleven patients were treated off-label, and 13 patients were enrolled onto clinical trials of matched targeted therapies. Median progression-free survival of patients treated with matched therapies was 5 months ( 95% CI, 2.9 months to not reached), and the progression-free survival probability at 6 months was 43% (95% CI, 26% to 71%). Lack of locally available clinical trials was the major limitation on clinical actionability of tumor profiling reports. Conclusion The molecular tumor board recommended off-label targeted therapies for a quarter of all patients reviewed. Outcomes were heterogeneous, although 43% of patients receiving genomically matched therapy derived clinical benefit lasting at least 6 months. Until more data become available from precision oncology trials, molecular tumor boards can help guide appropriate use of tumor molecular testing to direct therapy.

To evaluate whether germline variants in genes encoding pancreatic secretory enzymes contribute to pancreatic cancer susceptibility, we sequenced the coding regions of CPB1 and other genes encoding pancreatic secretory enzymes and known pancreatitis susceptibility genes (PRSS1, CPA1, CTRC, and SPINK1) in a hospital series of pancreatic cancer cases and controls. Variants in CPB1, CPA1 (encoding carboxypeptidase B1 and A1), and CTRC were evaluated in a second set of cases with familial pancreatic cancer and controls. More deleterious CPB1 variants, defined as having impaired protein secretion and induction of endoplasmic reticulum (ER) stress in transfected HEK 293T cells, were found in the hospital series of pancreatic cancer cases (5/986, 0.5%) than in controls (0/1,045, P = 0.027). Among familial pancreatic cancer cases, ER stress-inducing CPB1 variants were found in 4 of 593 (0.67%) vs. 0 of 967 additional controls (P = 0.020), with a combined prevalence in pancreatic cancer cases of 9/1,579 vs. 0/2,012 controls (P < 0.01). More ER stress-inducing CPA1 variants were also found in the combined set of hospital and familial cases with pancreatic cancer than in controls [7/1,546 vs. 1/2,012; P = 0.025; odds ratio, 9.36 (95% CI, 1.15-76.02)]. Overall, 16 (1%) of 1,579 pancreatic cancer cases had an ER stress-inducing CPA1 or CPB1 variant, compared with 1 of 2,068 controls (P < 0.00001). No other candidate genes had statistically significant differences in variant prevalence between cases and controls. Our study indicates ER stress-inducing variants in CPB1 and CPA1 are associated with pancreatic cancer susceptibility and implicate ER stress in pancreatic acinar cells in pancreatic cancer development.

103 Background: Mismatch repair deficient (dMMR) colon cancers are densely infiltrated with CD8+T cells and regress when treated with anti-programmed death-1 (PD-1) antibodies. This anti-tumor response is thought to be potentiated by the thousands of somatic mutations that when expressed as proteins result in hundreds of potentially immunogenic neo-antigens that can be recognized by the patient’s immune system. Methods: We previously reported a phase 2 study to evaluate the activity of pembrolizumab (pembro), a PD-1 antibody in treatment refractory dMMR colon cancers (NEJM 2015). We are reporting the expanded trial and updated data for the mismatch repair deficient CRC (dMMR, cohort A) and mismatch repair proficient CRC (pMMR, cohort B) cohorts. Pembro was administered at 10 mg/kg every 14 days in patients with 2 or more prior therapies. The co-primary endpoints were response and progression-free survival rate at 20 weeks. Secondary endpoints included disease control rate (DCR = CR + PR + SD), progression free survival (PFS), overall survival (OS) and safety. Results: A total of 53 patients (Cohort A: n = 28, Cohort B: n = 25) were treated. Median follow up time is 8.7 months. RR and DCR were 50% (14/28, 95% confidence interval (CI): 31-69%) and 89% (25/28) for dMMR CRC and 0% (0/25, 95% CI: 0-14%) and 16% (4/25) for pMMR CRC, respectively. Twenty-one of 28 dMMR CRC patients remain on study. Median PFS was not reached (NR) for dMMR CRC and 2.4 months for pMMR CRC (HR = 0.135; 95% CI 0.043 to 0.191; p=<0.0001). Median OS was NR for dMMR vs. 6 months for pMMR (HR = 0.247; 95% CI 0.117 to 0.589; p=0.001). For dMMR CRC, the PFS rates was 61% at 24 months and the OS rate was 66% at 24 months. Among patients with an objective response (n=14), only 1 has developed secondary resistance to pembro with a solitary brain metastasis after 4.6 months of therapy. Conclusions: Patients with dMMR CRC receive durable clinical benefit with pembrolizumab. Clinical trial information: NCT01876511.

LBA100 The full, final text of this abstract will be available at abstracts.asco.org at 2:00 PM (EDT) on Friday, May 29, 2015, and in the Annual Meeting Proceedings online supplement to the June 20, 2015, issue of the Journal of Clinical Oncology. Onsite at the Meeting, this abstract will be printed in the Saturday edition of ASCO Daily News.

Analysis of melanomas for actionable mutations has become the standard of care. Recently, a classification scheme has been proposed that categorizes BRAF mutations based on their mechanisms for activation of the MAPK pathway. In this analysis BRAF, KIT, NRAS, and PIK3CA mutations were examined by next generation sequencing (NGS) in 446 melanomas in a clinical diagnostic setting. KRAS and HRAS were also analyzed to elucidate coexisting BRAF and RAS mutations. BRAF mutations were categorized into class-1 (kinase-activated, codon 600), class-2 (kinase-activated, non-codon 600) and class-3 (kinase-impaired), based on the newly proposed classification scheme. NGS demonstrated high analytic sensitivity. Among 355 mutations detected, variant allele frequencies were 2–5% in 21 (5.9%) mutations and 2–10% in 47 (13%) mutations. Mutations were detected in BRAF (42%), NRAS (25%), KIT (4.9%) and PIK3CA (2.7%). The incidence of class-1, class-2 and class-3 mutations were 33% (26% p.V600E and 6.1% p.V600K), 3.1 and 4.9% respectively. With a broader reportable range of NGS, class-1, class-2 and class-3 mutations accounted for 77, 7.4 and 12% of all BRAF mutations. Class-3 mutations, commonly affecting codons 594, 466 and 467, showed a higher incidence of coexisting RAS mutations, consistent with their RAS-dependent signaling. Significant association with old age and primary tumors of head/neck/upper back suggest chronic solar damage as a contributing factor for melanomas harboring BRAF p.V600K or class-3 mutations. This study categorizes the range, frequency, coexisting driver mutations and clinical characteristics of the three classes of BRAF mutations in a large cohort of melanomas in a clinical diagnostic setting. Further prospective studies are warranted to elucidate the clinical outcomes and benefits of newly developed targeted therapy in melanoma patients carrying each class of BRAF mutation.

Abstract Background AR‐V7‐positive metastatic prostate cancer is a lethal phenotype with few treatment options and poor survival. Methods The two‐cohort nonrandomized Phase 2 study of combined immune checkpoint blockade for AR‐V7–expressing metastatic castration‐resistant prostate cancer (STARVE‐PC) evaluated nivolumab (3 mg/kg) plus ipilimumab (1 mg/kg), without (Cohort 1) or with (Cohort 2) the anti‐androgen enzalutamide. Co‐primary endpoints were safety and prostate‐specific antigen (PSA) response rate. Secondary endpoints included time‐to‐PSA‐progression‐free survival (PSA‐PFS), time‐to‐clinical/radiographic‐PFS, objective response rate (ORR), PFS lasting greater than 24 weeks, and overall survival (OS). Results Thirty patients were treated with ipilimumab plus nivolumab ( N = 15, Cohort 1, previously reported), or ipilimumab plus nivolumab and enzalutamide ( N = 15, Cohort 2) in patients previously progressing on enzalutamide monotherapy. PSA response rate was 2/15 (13%) in cohort 1 and 0/15 in cohort 2, ORR was 2/8 (25%) in Cohort 1 and 0/9 in Cohort 2 in those with measureable disease, median PSA‐PFS was 3.0 (95% confidence interval [CI]: 2.1–NR) in cohort 1 and 2.7 (95% CI: 2.1–5.9) months in cohort 2, and median PFS was 3.7 (95% CI: 2.8–7.5) in cohort 1 and 2.9 (95% CI: 1.3–5.8) months in cohort 2. Three of 15 patients in cohort 1 (20%, 95% CI: 7.1%–45.2%) and 4/15 patients (26.7%, 95% CI: 10.5%–52.4%) in cohort 2 achieved a durable PFS lasting greater than 24 weeks. Median OS was 8.2 (95% CI: 5.5–10.4) in cohort 1 and 14.2 (95% CI: 8.5–NA) months in cohort 2. Efficacy results were not statistically different between cohorts. Grade‐3/4 adverse events occurred in 7/15 cohort 1 patients (46%) and 8/15 cohort 2 patients (53%). Combined cohort ( N = 30) baseline alkaline phosphatase and cytokine analysis suggested improved OS for patients with lower alkaline phosphatase (hazards ratio [HR], 0.30; 95% CI: 0.11–0.82), lower circulating interleukin‐7 (IL‐7) (HR, 0.24; 95% Cl: 0.06–0.93) and IL‐6 (HR, 0.13; 95% Cl: 0.03–0.52) levels, and higher circulating IL‐17 (HR, 4.53; 95% CI: 1.47–13.93) levels. There was a trend towards improved outcomes in men with low sPD‐L1 serum levels. Conclusion Nivolumab plus ipilimumab demonstrated only modest activity in patients with AR‐V7‐expressing prostate cancer, and was not sufficient to justify further exploration in unselected patients. Stratification by baseline alkaline phosphatase and cytokines (IL‐6, −7, and −17) may be prognostic for outcomes to immunotherapy.

We determined the prevalence and significance of finding B cells without surface immunoglobulin (SIg) light chain expression. The flow cytometry database at Johns Hopkins Medical Institutions was searched for cases in which immunoglobulin light chain staining was performed to rule out a B-cell malignant neoplasm between January 1994 and February 2000. We excluded plasma cell dyscrasias, precursor B-cell acute lymphoblastic leukemia/lymphomas, and hematogones. Cases with more than 25% of B cells lacking SIg light chain expression were retrieved. Polymerase chain reaction assays for immunoglobulin heavy chain gene rearrangements were performed in SIg-negative cases with available tissue blocks. We identified 36 cases; all represented lymphoma. Their diagnoses included diffuse large B-cell lymphoma (20), HIV-related lymphoma (5), follicular lymphoma (5), Burkitt lymphoma (2), monomorphic posttransplant lymphoproliferative disorder (1), chronic lymphocytic leukemia/small lymphocytic lymphoma (1), marginal zone B-cell lymphoma (1), and low grade B-cell lymphoma (1). Of the 17 SIg-negative cases with amplifiable DNAs, 12 (71%) showed a clonal immunoglobulin heavy chain gene rearrangement. SIg-negative B-cell lymphomas are rare. Complete absence of SIg light chain expression in a mature B cell proliferation can be used as a surrogate marker to help diagnose peripheral B-cell lymphoma.

Many eukaryotic proteins are modified by N-linked glycosylation, a process in which oligosaccharides are added to asparagine residues in the sequon Asn-X-Ser/Thr. However, not all such sequons are glycosylated. For example, rabies virus glycoprotein (RGP) contains three sequons, only two of which appear to be glycosylated in virions. To examine further the signals in proteins which regulate N-linked core glycosylation, the glycosylation efficiencies of each of the three sequons in the antigenic domain of RGP were compared. For these studies, mutants were generated in which one or more sequons were deleted by site-directed mutagenesis. Core glycosylation of these mutants was studied using two independent systems: 1) in vitro translation in rabbit reticulocyte lysate supplemented with dog pancreatic microsomes, and 2) transfection into glycosylation-deficient Chinese hamster ovary cells. Parallel results were obtained with both systems, demonstrating that the sequon at Asn37 is inefficiently glycosylated, the sequons at Asn247 and Asn319 are efficiently glycosylated, and the glycosylation efficiency of each sequon is not influenced by glycosylation at other sequons in this protein. High levels of cell surface expression of RGP in Chinese hamster ovary cells are seen with any mutant containing an intact sequon at Asn247 or Asn319, whereas low levels of cell surface expression are seen when the sequon at Asn37 is present alone; deletion of all three sequons completely blocks RGP cell surface expression. Thus, although core glycosylation at Asn37 is inefficient, it is still sufficient to support a biological function, cell surface expression. Future studies using mutagenesis of this model protein and its expression in these two well defined systems will aim to begin to unravel the rules governing core glycosylation of glycoproteins.

Little is known about the genetic and epigenetic changes that contribute to familial pancreatic cancers. The aim of this study was to compare the prevalence of common genetic and epigenetic alterations in sporadic and familial pancreatic ductal adenocarcinomas.DNA was isolated from the microdissected cancers of 39 patients with familial and 36 patients with sporadic pancreatic adenocarcinoma. KRAS2 mutations were detected by BstN1 digestion and/or cycle sequencing. TP53 and SMAD4 status were determined by immunohistochemistry on tissue microarrays of 23 archival familial pancreatic adenocarcinomas and in selected cases by cycle sequencing to identify TP53 gene mutations. Methylation-specific PCR analysis of seven genes (FoxE1, NPTX2, CLDN5, P16, TFPI-2, SPARC, ppENK) was done on a subset of fresh-frozen familial pancreatic adenocarcinomas.KRAS2 mutations were identified in 31 of 39 (80%) of the familial versus 28 of 36 (78%) of the sporadic pancreatic cancers. Positive immunolabeling for p53 was observed in 57% of the familial pancreatic cancers and loss of SMAD4 labeling was observed in 61% of the familial pancreatic cancers, rates similar to those observed in sporadic pancreatic cancers. The mean prevalence of aberrant methylation in the familial pancreatic cancers was 68.4%, which was not significantly different from that observed in sporadic pancreatic cancers.The prevalence of mutant KRAS2, inactivation of TP53 and SMAD4, and aberrant DNA methylation of a seven-gene panel is similar in familial pancreatic adenocarcinomas as in sporadic pancreatic adenocarcinomas. These findings support the use of markers of sporadic pancreatic adenocarcinomas to detect familial pancreatic adenocarcinomas.

KRAS2 gene mutations are found in 75-90% of infiltrating pancreatic ductal adenocarcinomas but can also be present with other nonneoplastic pancreatic diseases. We recently developed a novel sensitive assay for point mutation detection, called "LigAmp", which can detect one mutant molecule in the presence of 10,000 wild-type molecules and can quantify mutant DNA over a wide dynamic range. We analyzed KRAS2 mutations in surgically-collected pancreatic duct juice samples from patients with pancreatic adenocarcinoma (n = 27) and chronic pancreatitis(n = 9). DNA sequencing demonstrated that 17 of the 27 pancreatic cancers harbored KRAS2 mutations at codon 12, including G12D (GGT-->GAT), G12V (GTT), and G12R (CGT). We determined the relative amounts of each KRAS2 mutant by simultaneously quantifying wild-type and mutant KRAS2 DNA. For all pancreatic adenocarcinoma patients, the dominant KRAS2 mutation detected in the pancreatic juice corresponded to that found in the primary cancer. Mutation levels were substantially higher in patients with pancreatic cancer (0.05 to 82% of total KRAS2 molecules) compared to those with chronic pancreatitis (0 to 0.7%). Among patients with mutant KRAS2 positive cancers, all but one (94%) had mutant KRAS2 DNA concentrations of more than 0.5% in their pancreatic juice samples, whereas only 1 of 9(11%) pancreatic juice samples from patients with chronic pancreatitis had more than 0.5% mutant KRAS2 DNA, corresponding to a sensitivity of 94% and a specificity of 89%. LigAmp quantification of mutant KRAS2 in pancreatic juice differentiates pancreatic adenocarcinoma from chronic pancreatitis, and may be a useful early detection tool for pancreatic cancer.

Abstract Purpose: Pancreatic cancer is the fourth cause of death from cancer in the western world. Majority of patients present with advanced unresectable disease responding poorly to most chemotherapeutic agents. Chemotherapy for pancreatic cancer might be improved by adjusting it to individual genetic profiles. We attempt to identify genetic predictors of chemosensitivity to broad classes of anticancer drugs. Experimental Design: Using a panel of genetically defined human pancreatic cancer cell lines, we tested gemcitabine (antimetabolite), docetaxel (antimicrotubule), mitomycin C (MMC; alkylating), irinotecan (topoisomerase I inhibitor), cisplatin (crosslinking), KU0058948 (Parp1 inhibitor), triptolide (terpenoid drug), and artemisinin (control). Results: All pancreatic cancer cell lines were sensitive to triptolide and docetaxel. Most pancreatic cancer cells were also sensitive to gemcitabine and MMC. The vast majority of pancreatic cancer cell lines were insensitive to cisplatin, irinotecan, and a Parp1 inhibitor. However, individual cell lines were often sensitive to these compounds in unique ways. We found that DPC4/SMAD4 inactivation sensitized pancreatic cancer cells to cisplatin and irinotecan by 2- to 4-fold, but they were modestly less sensitive to gemcitabine. Pancreatic cancer cells were all sensitive to triptolide and 18% were sensitive to the Parp1 inhibitor. P16/CDKN2A-inactivated pancreatic cancer cells were 3- to 4-fold less sensitive to gemcitabine and MMC. Conclusions: Chemosensitivity of pancreatic cancer cells correlated with some specific genetic profiles. These results support the hypothesis that genetic subsets of pancreatic cancer exist, and these genetic backgrounds may permit one to personalize the chemotherapy of pancreatic cancer in the future. Further work will need to confirm these responses and determine their magnitude in vivo. Clin Cancer Res; 18(23); 6519–30. ©2012 AACR.

A better molecular characterization of intraductal papillary mucinous neoplasm (IPMN), the most frequent cystic precursor lesion of pancreatic adenocarcinoma, may have a pivotal role in its early detection and in the development of effective therapeutic strategies. BRG1, a central component of the chromatin remodeling complex SWI/SNF regulating transcription, is inactive in several malignancies. In this study, we evaluate the Brg1 expression in intraductal papillary mucinous neoplasm to better understand its role in the pancreatic carcinogenesis. Tissue microarrays of 66 surgically resected IPMNs were immunolabeled for the Brg1 protein. Expression patterns were then correlated with clinicopathologic parameters. Normal pancreatic epithelium strongly immunolabeled for Brg1. Reduced Brg1 expression was observed in 32 (53.3%) of the 60 evaluable IPMN lesions and occurred more frequently in high-grade IPMNs (13 of 17 showed loss; 76%) compared to intermediate-grade (15 of 29 showed loss; 52%) and low-grade IPMNs (4 of 14 showed loss; 28%) (P = .03). A complete loss of Brg1 expression was observed in 5 (8.3%) of the 60 lesions. Finally, a decrease in Brg1 protein expression was furthermore found in a low-passage noninvasive IPMN cell line by Western blot analysis. We did not observe correlation between Brg1 expression and IPMN subtype or with location of the cyst. We provide first evidence that Brg1 expression is lost in noninvasive cystic precursor lesions of pancreatic adenocarcinoma.

Background:Existing evidence is controversial regarding the association between BRAF mutation status and aggressive features of papillary thyroid cancer (PTC). Specifically, no study has incorporated multiple surgical practices performing routine central lymph node dissection (CLND) and thus has patients who are truly evaluable for the presence or absence of central lymph node metastases (CLNMs).

Pancreatic ductal adenocarcinoma (PDAC) is nearly uniformly lethal, with a median overall survival in 2014 of only 6 months. The genetic progression of sporadic PDAC (SPC) is well established, with common somatic alterations in KRAS, p16/CDKN2A, TP53, and SMAD4/DPC4. Up to 10 % of all PDAC cases occur in families with two or more affected first-degree relatives (familial pancreatic cancer, FPC), but these cases do not appear to present at an obviously earlier age of onset. This is unusual because most familial cancer syndrome patients present at a substantially younger age than that of corresponding sporadic cases. Here we collated the reported age of onset for FPC and SPC from the literature. We then used an integrated approach including whole exomic sequencing, whole genome sequencing, RNA sequencing, and high density SNP microarrays to study a cohort of FPC cell lines and corresponding germline samples. We show that the four major SPC driver genes are also consistently altered in FPC and that each of the four detection strategies was able to detect the mutations in these genes, with one exception. We conclude that FPC undergoes a similar somatic molecular pathogenesis as SPC, and that the same gene targets can be used for early detection and minimal residual disease testing in FPC patients.

Activating mutations in downstream genes of the epidermal growth factor receptor (EGFR) pathway may cause anti-EGFR resistance in patients with colorectal cancers. We present performance characteristics of a next-generation sequencing assay designed to detect such mutations. In this retrospective quality assessment study, we analyzed mutation detected in the KRAS, NRAS, BRAF, and PIK3CA genes by a clinically validated next-generation sequencing assay in 310 colorectal cancer specimens. Tumor cellularity and mutant allele frequency were analyzed to identify tumor heterogeneity and mutant allele-specific imbalance. Next-generation sequencing showed precise measurement of mutant allele frequencies and detected 23% of mutations with 2–20% mutant allele frequencies. Of the KRAS mutations detected, 17% were outside of codons 12 and 13. Among PIK3CA mutations, 48% were outside of codons 542, 545, and 1047. The percentage of tumors with predicted resistance to anti-EGFR therapy increased from 40% when testing for only mutations in KRAS exon 2 to 47% when testing for KRAS exons 2–4, 48% when testing for KRAS and NRAS exons 2–4, 58% when including BRAF codon 600 mutations, and 59% when adding PIK3CA exon 20 mutations. Right-sided colorectal cancers carried a higher risk of predicted anti-EGFR resistance. A concomitant KRAS mutation was detected in 51% of PIK3CA, 23% of NRAS, and 33% of kinase-impaired BRAF-mutated tumors. Lower than expected mutant allele frequency indicated tumor heterogeneity, while higher than expected mutant allele frequency indicated mutant allele-specific imbalance. Two paired neuroendocrine carcinomas and adjacent adenomas showed identical KRAS mutations, but only PIK3CA mutations in neuroendocrine carcinomas. Next-generation sequencing is a robust tool for mutation detection in clinical laboratories. It demonstrates high analytic sensitivity and broad reportable range, and it provides simultaneous detection of concomitant mutations and a quantitative measurement of mutant allele frequencies to predict tumor heterogeneity and mutant allele-specific imbalance.

Activating point mutations of GNAS at codon 201 have been detected in approximately two thirds of intraductal papillary mucinous neoplasms (IPMNs) of the pancreas. Intraductal papillary neoplasms of the bile ducts (IPNBs) morphologically resemble pancreatic IPMNs. This study sought to assess the mutational status of GNAS at codon 201 in IPNBs.Thirty-four patients were included. DNA from microdissected IPNBs was subjected to a polymerase chain reaction and ligation method for the detection of GNAS mutations at codon 201 and of KRAS mutations at codon 12. Mutational status was compared with clinical and pathologic data.The IPNBs had a median diameter of 3.5 cm and were located intrahepatically (n= 6), extrahepatically (n= 13), both intra- and extrahepatically (n= 4) or in the gallbladder (intracystic papillary neoplasms, n= 11). Most exhibited pancreatobiliary differentiation (n= 20), high-grade dysplasia (n= 26) and an associated adenocarcinoma (n= 20). Analysis of GNAS codon 201 identified only one mutant sample in a multifocal intestinal subtype intrahepatic IPNB with high-grade dysplasia. Six lesions harboured a KRAS codon 12 mutation.GNAS codon 201 mutations are uncommon in IPNBs, by contrast with pancreatic IPMNs. More comprehensive molecular profiling is needed to uncover the pathways involved in IPNB development.

Studies have demonstrated an association of the BRAF(V600E) mutation and microRNA (miR) expression with aggressive clinicopathologic features in papillary thyroid cancer (PTC). Analysis of BRAF(V600E) mutations with miR expression data may improve perioperative decision making for patients with PTC, specifically in identifying patients harboring central lymph node metastases (CLNM).Between January 2012 and June 2013, 237 consecutive patients underwent total thyroidectomy and prophylactic central lymph node dissection (CLND) at four endocrine surgery centers. All tumors were tested for the presence of the BRAF(V600E) mutation and miR-21, miR-146b-3p, miR-146b-5p, miR-204, miR-221, miR-222, and miR-375 expression. Bivariate and multivariable analyses were performed to examine associations between molecular markers and aggressive clinicopathologic features of PTC.Multivariable logistic regression analysis of all clinicopathologic features found miR-146b-3p and miR-146b-5p to be independent predictors of CLNM, while the presence of BRAF(V600E) almost reached significance. Multivariable logistic regression analysis limited to only predictors available preoperatively (molecular markers, age, sex, and tumor size) found miR-146b-3p, miR-146b-5p, miR-222, and BRAF(V600E) mutation to predict CLNM independently. While BRAF(V600E) was found to be associated with CLNM (48% mutated in node-positive cases vs. 28% mutated in node-negative cases), its positive and negative predictive values (48% and 72%, respectively) limit its clinical utility as a stand-alone marker. In the subgroup analysis focusing on only classical variant of PTC cases (CVPTC), undergoing prophylactic lymph node dissection, multivariable logistic regression analysis found only miR-146b-5p and miR-222 to be independent predictors of CLNM, while BRAF(V600E) was not significantly associated with CLNM.In the patients undergoing prophylactic CLNDs, miR-146b-3p, miR-146b-5p, and miR-222 were found to be predictive of CLNM preoperatively. However, there was significant overlap in expression of these miRs in the two outcome groups. The BRAF(V600E) mutation, while being a marker of CLNM when considering only preoperative variables among all histological subtypes, is likely not a useful stand-alone marker clinically because the difference between node-positive and node-negative cases was small. Furthermore, it lost significance when examining only CVPTC. Overall, our results speak to the concept and interpretation of statistical significance versus actual applicability of molecular markers, raising questions about their clinical usefulness as individual prognostic markers.

Abstract PD-L1 expression in melanoma correlates with response to PD-1 pathway–blocking antibodies. Aberrant tumor-cell PD-L1 expression may be oncogene driven and/or induced by IFNγ. Melanomas express PD-L1 in association with tumor-infiltrating lymphocytes (TIL), but the potential contribution of the BRAF V600E mutation (BRAFmut) to induced PD-L1 expression has not been determined. Fifty-two archival melanocytic lesions were assessed for PD-L1 expression, TIL infiltration, and BRAFmut simultaneously. IFNγ-induced PD-L1 expression in cultured melanomas was assessed in parallel according to BRAF status. Melanocyte PD-L1 expression was observed in 40% of specimens, and BRAFmut was observed in 42% of specimens, but no significant concordance was found between these variables. Almost all melanocytes displaying PD-L1 expression were observed to be adjacent to TILs, irrespective of BRAF status. TIL− lesions were not more likely to be associated with BRAFmut, when compared with TIL+ lesions. Baseline expression of PD-L1 by melanoma cell lines was virtually nil, regardless of BRAFmut status, and the intensity of IFN-induced PD-L1 expression in melanoma cell lines likewise did not correlate with BRAF mutational status. PD-L1 expression in melanocytic lesions does not correlate with the BRAFmut. Thus, distinct populations of melanoma patients will likely benefit from BRAF inhibitors versus PD-1 pathway blockade. Cancer Immunol Res; 3(2); 110–5. ©2014 AACR.

Next-generation sequencing shows great promise by allowing rapid mutational analysis of multiple genes in human cancers. Recently, we implemented the multiplex PCR-based Ion AmpliSeq Cancer Hotspot Panel (>200 amplicons in 50 genes) to evaluate EGFR, KRAS, and BRAF in lung and colorectal adenocarcinomas. In 10% of samples, automated analysis identified a novel G873R substitution mutation in EGFR. By examining reads individually, we found this mutation in >5% of reads in 50 of 291 samples and also found similar events in 18 additional amplicons. These apparent mutations are present only in short reads and within 10 bases of either end of the read. We therefore hypothesized that these were from panel primers promiscuously binding to nearly complementary sequences of nontargeted amplicons. Sequences around the mutations matched primer binding sites in the panel in 18 of 19 cases, thus likely corresponding to panel primers. Furthermore, because most primers did not show this effect, we demonstrated that next-generation sequencing may be used to better design multiplex PCR primers through iterative elimination of offending primers to minimize mispriming. Our results indicate the need for careful sequence analysis to avoid false-positive mutations that can arise in multiplex PCR panels. The AmpliSeq Cancer panel is a valuable tool for clinical diagnostics, provided awareness of potential artifacts.

To assess the performance of a next-generation sequencing (NGS) platform for the clinical detection of BRAF mutations.In this retrospective quality assessment of an NGS assay, we analyzed BRAF mutations within parts of exons 11 and 15 in 835 neoplastic tissues submitted to our molecular diagnostics laboratory.The NGS assays detected a BRAF mutation in 5.9% of lung adenocarcinomas, 13% of colorectal cancers, and 44% of melanomas. Mutant allele frequencies were less than 20% in 28% of 88 BRAF-mutated specimens. Two lymph node specimens with subcapsular or infiltrative metastasis showed 1% to 2% mutant alleles. There were 26 unique BRAF mutations in exons 11 and 15, including three novel mutations. Mutations were located outside codon 600 in 39% of BRAF-mutated tumors. Lung adenocarcinomas showed significantly higher non-p.V600E mutations (86%) than did colorectal cancers (23%) and melanomas (34%). The three most common BRAF mutations in lung cancers accounted for only 41% of the observed BRAF mutations (p.D594G [18%], p.V600E [14%], and p.G469A [9%]).The NGS assay demonstrated a high analytic sensitivity and a broad reportable range for clinical detection of BRAF mutations. Elucidating the spectrum of non-p. V600E BRAF mutations in different malignancies is a first step toward understanding their clinical significance.

Patients with castration-resistant prostate cancer (CRPC) often are treated with drugs that target the androgen receptor (AR) ligand-binding domain. Constitutively active AR splice variant 7 (AR-V7) lacks the ligand-binding domain and, if detected in circulating tumor cells, may be associated with resistance to these agents. We validated an AR-V7 assay in a Clinical Laboratory Improvement Amendments (CLIA)-certified laboratory. Circulating tumor cells were isolated, and mRNA was reverse-transcribed into cDNA. Real-time quantitative PCR amplification of reference transcripts (beta-actin and glyceraldehyde-3-phosphate dehydrogenase), prostate-specific transcripts (prostate-specific membrane antigen, prostate-specific antigen, and AR-full length), and AR-V7 was performed. Specimens for validation included an AR-V7 expressing prostate cancer (LNCaP95), 38 peripheral blood controls, and 21 blood samples from CRPC patients. The assay detected as few as five LNCaP95 cells spiked into peripheral blood, showing high analytical sensitivity. Multiple inter-run and intrarun replicates of LNCaP95 cell line experiments yielded similar cycle threshold values for all genes, showing high analytical precision (AR-V7 cycle threshold CV of 0.67%). All 38 healthy control samples were negative for AR-V7, showing high diagnostic specificity (100%). The diagnostic accuracy was confirmed by concurrent testing of 21 CRPC samples in the research laboratory and the clinical diagnostic laboratory: concordance in AR-V7 status was achieved in all cases (positive in 4, negative in 17) (100% accuracy). This first validated clinical assay detects the AR-V7 with high analytical sensitivity, precision, specificity, and accuracy.

Purpose A splice variant of the androgen receptor, AR-V7, confers resistance to AR-targeted therapies (ATTs) but not taxane chemotherapies in patients with metastatic castration-resistant prostate cancer. Since August 2015, a clinical-grade assay to detect AR-V7 messenger RNA expression in circulating tumors cells (CTCs) has been available to providers through a Clinical Laboratory Improvement Amendments–certified laboratory at Johns Hopkins University. Methods We contacted ordering providers of the first 150 consecutive tests by using a questionnaire-based survey to determine how the results of AR-V7 testing were used to influence clinical practice. Results In all, 142 (95%) of 150 questionnaires were completed by 38 providers from 29 sites across the United States and Canada. AR-V7 test results were reported either as CTC– (28%), CTC+/AR-V7– (30%), or CTC+/AR-V7+ (42%). Prevalence of AR-V7 detection increased with prior exposure to ATTs (abiraterone and enzalutamide naïve, 22%; after abiraterone or enzalutamide, 35%; after abiraterone and enzalutamide, 43%). Overall, management was affected by AR-V7 testing in 53% of the patients and even more often with CTC+/AR-V7+ results. AR-V7+ patients were commonly switched from ATT to taxane chemotherapy (43%) or were offered a clinical trial (43%); management remained unchanged in only 14% of these patients. Overall, patients who had a change in management on the basis of AR-V7 testing were significantly more likely to achieve a physician-reported 50% decline in prostate-specific antigen response on next-line therapy than those who did not change treatment (54% v 31%; P = .015). Conclusion Providers used AR-V7 testing to influence clinical decision making more often than not. Physicians reported that men with AR-V7+ results had the most treatment changes, and such men were preferentially managed with taxane therapy or offered a clinical trial, which may have improved outcomes.

195 Background: Mismatch repair (MMR) deficient tumors harbor hundreds to thousands of mutations that may produce neoantigens that can be recognized and targeted by T cells. We have shown that MMR deficiency can serve as a predictive biomarker for selection of tumors across tumor histologies that may respond to programmed death-1 (PD-1) blockade. MMR deficiency is present in 15% of colorectal (CRC) however is also detected in 2-20% of gastric, small bowel, and hepatobiliary cancers. Methods: We conducted a phase II study to evaluate anti-PD-1, pembrolizumab, in patients with previously-treated, progressive, advanced cancer. Twenty-one patients with MMR deficient tumors were enrolled onto the non-CRC cohort with an additional 50 patient expansion underway. The pembrolizumab dose is 10mg/kg intravenously every 2 weeks. Objective response rate (ORR), progression-free survival (PFS), and overall survival (OS) will be reported in at least the first 17 patients with non-CRC gastrointestinal (GI) cancers. Results: As of September 18, 2015, 17 patients with non-CRC GI cancers have been enrolled on the protocol with additional patients identified. The diseases represented are ampullary (N=4), pancreas (N=4), biliary (N=3), small bowel (N=3), and gastric (N=3) cancers. For the 10 evaluable patients at the time of abstract admission, ORR is 50 % (N=5/10), disease control rate is 70% (N=7/10), OS is 21 months and the median duration of response (range 5.5-17+ months) and PFS have not been reached. The median follow up duration is 7.6 months. Conclusions: PD-1 blockade shows promising activity in mismatch repair deficient GI cancers. Clinical trial information: NCT01876511.

Genomic biomarkers that predict response to anti-PD1 therapy in prostate cancer are needed. Frameshift mutations are predicted to generate more neoantigens than missense mutations; therefore, we hypothesized that the number or proportion of tumor frameshift mutations would correlate with response to anti-PD1 therapy in prostate cancer.To enrich for response to anti-PD1 therapy, we assembled a multicenter cohort of 65 men with mismatch repair-deficient (dMMR) prostate cancer. Patient characteristics and outcomes were determined by retrospective chart review. Clinical somatic DNA sequencing was used to determine tumor mutational burden (TMB), frameshift mutation burden, and frameshift mutation proportion (FSP), which were correlated to outcomes on anti-PD1 treatment. We subsequently used data from a clinical trial of pembrolizumab in patients with nonprostatic dMMR cancers of various histologies as a biomarker validation cohort.Nineteen of 65 patients with dMMR metastatic castration-resistant prostate cancer were treated with anti-PD1 therapy. The PSA50 response rate was 65%, and the median progression-free survival (PFS) was 24 (95% confidence interval 16-54) weeks. Tumor FSP, more than overall TMB, correlated most strongly with prolonged PFS and overall survival (OS) on anti-PD1 treatment and with density of CD8+ tumor-infiltrating lymphocytes. High FSP similarly identified patients with longer PFS as well as OS on anti-PD1 therapy in a validation cohort.Tumor FSP correlated with prolonged efficacy of anti-PD1 treatment among patients with dMMR cancers and may represent a new biomarker of immune checkpoint inhibitor sensitivity.Given the modest efficacy of immune checkpoint inhibition (ICI) in unselected patients with advanced prostate cancer, biomarkers of ICI sensitivity are needed. To facilitate biomarker discovery, a cohort of patients with DNA mismatch repair-deficient (dMMR) prostate cancer was assembled, as these patients are enriched for responses to ICI. A high response rate to anti-PD1 therapy in these patients was observed; however, these responses were not durable in most patients. Notably, tumor frameshift mutation proportion (FSP) was identified as a novel biomarker that was associated with prolonged response to anti-PD1 therapy in this cohort. This finding was validated in a separate cohort of patients with nonprostatic dMMR cancers of various primary histologies. This works suggests that FSP predicts response to anti-PD1 therapy in dMMR cancers, which should be validated prospectively in larger independent cohorts.

Levels of carcinoembryonic antigen (CEA), carbohydrate antigen 19-9 (CA19-9), and cancer antigen 125 (CA-125) in blood are used as markers to determine the response of patients with cancer to therapy, but are not used to identify patients with pancreatic cancer.We obtained blood samples from 504 patients undergoing pancreatic surveillance from 2002 through 2018 who did not develop pancreatic cancer and measured levels of the tumor markers CA19-9, CEA, CA-125, and thrombospondin-2. Single-nucleotide polymorphisms (SNPs) in FUT3, FUT2, ABO, and GAL3ST2 that have been associated with levels of tumor markers were used to establish SNP-defined ranges for each tumor marker. We also tested the association between additional SNPs (in FUT6, MUC16, B3GNT3, FAM3B, and THBS2) with levels of tumor markers. To calculate the diagnostic specificity of each SNP-defined range, we assigned the patients under surveillance into training and validation sets. After determining the SNP-defined ranges, we determined the sensitivity of SNP-adjusted tests for the tumor markers, measuring levels in blood samples from 245 patients who underwent resection for pancreatic ductal adenocarcinoma (PDAC) from 2010 through 2017.A level of CA19-9 that identified patients with PDAC with 99% specificity had 52.7% sensitivity. When we set the cut-off levels of CA19-9 based on each SNP, the test for CA19-9 identified patients with PDAC with 60.8% sensitivity and 98.8% specificity. Among patients with FUT3 alleles that encode a functional protein, levels of CA19-9 greater than the SNP-determined cut-off values identified 66.4% of patients with PDAC, with 99.3% specificity. In the validation set, levels of CEA varied among patients with vs without SNP in FUT2, by blood group, and among smokers vs nonsmokers; levels of CA-125 varied among patients with vs without the SNP in GAL3ST2. The use of the SNPs to define the ranges of CEA and CA-125 did not significantly increase the diagnostic accuracy of the assays for these proteins. Combining data on levels of CA19-9 and CEA, CA19-9 and CA-125, or CA19-9 and thrombospondin-2 increased the sensitivity of detection of PDAC, but slightly reduced specificity.Including information on SNPs associated with levels of CA19-9, CEA, and CA-125 can improve the diagnostic accuracy of assays for these tumor markers in the identification of patients with PDAC. Clinicaltrials.gov no: NCT02000089.

Abstract Intraductal papillary mucinous neoplasms (IPMNs) are commonly identified non‐invasive cyst‐forming pancreatic neoplasms with the potential to progress into invasive pancreatic adenocarcinoma. There are few in vitro models with which to study the biology of IPMNs and their progression to invasive carcinoma. Therefore, we generated a living biobank of organoids from seven normal pancreatic ducts and ten IPMNs. We characterized eight IPMN organoid samples using whole genome sequencing and characterized five IPMN organoids and seven normal pancreatic duct organoids using transcriptome sequencing. We identified an average of 11,344 somatic mutations in the genomes of organoids derived from IPMNs, with one sample harboring 61,537 somatic mutations enriched for T→C transitions and T→A transversions. Recurrent coding somatic mutations were identified in 15 genes, including KRAS , GNAS , RNF43 , PHF3 , and RBM10 . The most frequently mutated genes were KRAS , GNAS , and RNF43 , with somatic mutations identified in six (75%), four (50%), and three (37.5%) IPMN organoid samples, respectively. On average, we identified 36 structural variants in IPMN derived organoids, and none had an unstable phenotype (&gt; 200 structural variants). Transcriptome sequencing identified 28 genes differentially expressed between normal pancreatic duct organoid and IPMN organoid samples. The most significantly upregulated and downregulated genes were CLDN18 and FOXA1 . Immunohistochemical analysis of FOXA1 expression in 112 IPMNs, 113 mucinous cystic neoplasms, and 145 pancreatic ductal adenocarcinomas demonstrated statistically significant loss of expression in low‐grade IPMNs ( p &lt; 0.0016), mucinous cystic neoplasms ( p &lt; 0.0001), and pancreatic ductal adenocarcinoma of any histologic grade ( p &lt; 0.0001) compared to normal pancreatic ducts. These data indicate that FOXA1 loss of expression occurs early in pancreatic tumorigenesis. Our study highlights the utility of organoid culture to study the genetics and biology of normal pancreatic duct and IPMNs. © 2020 The Pathological Society of Great Britain and Ireland. Published by John Wiley &amp; Sons, Ltd.

A 19-year-old woman with an aggressive lymphoma received a bone marrow transplant from her sister. Three and a half years later subcutaneous panniculitic T-cell lymphoma was diagnosed in the donor; the same rare tumor appeared in the recipient four and a half years after transplantation. The tumors had identical molecular features.

Somatic mutations of mitochondrial DNA (mtDNA) are common in many human cancers. We have described an oligonucleotide microarray ("MitoChip") for rapid sequencing of the entire mitochondrial genome (Zhou et al, J Mol Diagn 2006), facilitating the analysis of mtDNA mutations in preneoplastic lesions. We examined 14 precancerous lesions, including seven Barrett esophagus biopsies, with or without associated dysplasia; four colorectal adenomas; and three inflammatory colitis-associated dysplasia specimens. In all cases, matched normal tissues from the corresponding site were obtained as germline control. MitoChip analysis was performed on DNA obtained from cryostat-embedded specimens.A total of 513,639 bases of mtDNA were sequenced in the 14 samples, with 490,224 bases (95.4%) bases assigned by the automated genotyping software. All preneoplastic lesions examined demonstrated at least one somatic mtDNA sequence alteration. Of the 100 somatic mtDNA alterations observed in the 14 cases, 27 were non-synonymous coding region mutations (i.e., resulting in an amino acid change), 36 were synonymous, and 37 involved non-coding mtDNA. Overall, somatic alterations most commonly involved the COI, ND4 and ND5 genes. Notably, somatic mtDNA alterations were observed in preneoplastic lesions of the gastrointestinal tract even in the absence of histopathologic evidence of dysplasia, suggesting that the mitochondrial genome is susceptible at the earliest stages of multistep cancer progression.Our findings further substantiate the rationale for exploring the mitochondrial genome as a biomarker for the early diagnosis of cancer, and confirm the utility of a high-throughput array-based platform for this purpose from a clinical applicability standpoint.

AbstractBackground: It has recently been suggested that over expression of palladin in sporadic pancreatic cancer may contribute to pancreatic cancer’s invasive and migratory abilities. This hypothesis was based on reverse transcriptase-polymerase chain reaction analyses of bulk pancreatic tissue, yet pancreatic cancer is a complex admixture of neoplastic epithelial cells and desmoplastic stroma.Design: Immunohistochemical labeling of tissue microarrays was used to define the patterns of palladin protein expression in 177 ductal adenocarcinomas of the pancreas. Western blot analysis was used to determine the epitope(s) of palladin recognized by the antibody as well as the relative levels of palladin expression in short-term cultures of stromal fibroblasts, non-neoplastic ductal cells and pancreatic cancer cell lines.Results: Immunolabeling revealed that the palladin protein was strongly over expressed in non-neoplastic stromal cells in 171 (96.6%) of the 177 evaluable pancreatic cancers. By contrast, the over expression of palladin protein by the neoplastic epithelial cells relative to normal pancreatic epithelium was observed in only 22 (12.4%) of the 177 cancers. Western blot analysis confirmed that the antibody recognizes the ~90 kDa isoform of palladin, and demonstrated that fibroblast cell lines had higher expression of palladin than pancreatic cancer cell lines.Conclusions: The over expression of palladin relative to normal pancreas in the majority of pancreatic cancers is limited to non-neoplastic stromal cells. This observation highlights the limitations of relying on bulk tissues when analyzing gene expression. Since palladin is not over expressed in most pancreatic cancer cells, the over expression of palladin is not likely to be responsible for pancreatic cancer cells invasive and migratory abilities.

The exquisite specificity of proteins is a key feature driving their application to anticancer therapies. The therapeutic potential of another fundamental property of proteins, their ability to be regulated by molecular cues in their environment, is unknown. Here, we describe a synthetic biology strategy for designing protein therapeutics that autonomously activate a therapeutic function in response to a specific cancer marker of choice. We demonstrate this approach by creating a prodrug-activating enzyme that selectively kills human cancer cells that accumulate the marker hypoxia-inducible factor 1α. This property arises primarily through increased cellular accumulation of the enzyme in the presence of the marker. Our strategy offers a platform for the development of inherently selective protein therapeutics for cancer and other diseases.

McCune-Albright Syndrome (MAS) is a rare sporadic syndrome caused by post-zygotic mutations in the GNAS oncogene, leading to constitutional mosaicism for these alterations. Somatic activating GNAS mutations also commonly occur in several gastrointestinal and pancreatic neoplasms, but the spectrum of abnormalities in these organs in patients with MAS has yet to be systematically described. We report comprehensive characterization of the upper gastrointestinal tract in seven patients with MAS and identify several different types of polyps, including gastric heterotopia/metaplasia (7/7), gastric hyperplastic polyps (5/7), fundic gland polyps (2/7), and a hamartomatous polyp (1/7). In addition, one patient had an unusual adenomatous lesion at the gastroesophageal junction with high-grade dysplasia. In the pancreas, all patients had endoscopic ultrasound findings suggestive of intraductal papillary mucinous neoplasm (IPMN), but only two patients met the criteria for surgical intervention. Both of these patients had IPMNs at resection, one with low-grade dysplasia and one with high-grade dysplasia. GNAS mutations were identified in the majority of lesions analyzed, including both IPMNs and the adenomatous lesion from the gastroesophageal junction. These studies suggest that there is a broad spectrum of abnormalities in the gastrointestinal tract and pancreas in patients with MAS and that patients with MAS should be evaluated for gastrointestinal pathology, some of which may warrant clinical intervention due to advanced dysplasia.

Context Detection of KRAS mutation is mandatory to predict response to anti–epidermal growth factor receptor monoclonal antibodies in patients with metastatic colorectal cancers. Objective To demonstrate challenges posed to pathologists in the clinical detection of KRAS mutations in colorectal cancers. Design In this retrospective analysis for quality assessment of the pyrosequencing assay, we survey the characteristics of 463 formalin-fixed, paraffin-embedded neoplastic tissues submitted for KRAS mutation detection during a 26-month period. Results The KRAS mutation was detected in 39.2% of tumors. This included 2 tumors with complex pyrograms (GGT&amp;gt;GAG at codon 12 and GGC&amp;gt;GTT at codon 13, as resolved by a Pyromaker software program) and 3 tumors with an indeterminate percentage of mutant alleles (defined as 4% to 5% and confirmed by a next-generation sequencing platform). Among the 25 specimens (5.5%) with fewer than 20% tumor cells, 22 were resected after chemotherapy/radiation. Significant depletion of tumor cells was observed in rectal cancers resected after neoadjuvant therapy (31.0%) versus those without previous treatment (0%) (P = .01). We also explore other specimens with low tumor cellularity and potential causes of discrepancy between the estimated tumor cell percentage and detected mutant allele frequency, such as intratumor heterogeneity of KRAS mutation. Conclusions Neoadjuvant therapy may deplete tumor cells and confound the molecular diagnosis of KRAS mutations. Accurate detection of specimens with poor tumor cellularity requires the appropriate selection of neoplastic tissues, evaluation of tumor cellularity, use of assays with high sensitivity, and prospective quality assessment.

Detection of BRAF mutations is an established standard of care to predict small-molecule inhibitor (vemurafenib) response in metastatic melanoma. Molecular assays should be designed to detect not only the most common p.V600E mutation, but also p.V600K and other non-p.V600E mutations.The purpose of this study was to assess if tumor cellularity can function as a quality assurance (QA) measure in molecular diagnostics. Potential causes of discrepancy between the observed and predicted mutant allele percentage were also explored.We correlated pathologist-generated estimates of tumor cellularity versus mutant allele percentage via pyrosequencing as a QA measure for BRAF mutation detection in formalin-fixed, paraffin-embedded melanoma specimens.BRAF mutations were seen in 27/62 (44 %) specimens, with 93 % p.V600E and 7 % non-p.V600E. Correlation between p.V600E mutant percentage and tumor cellularity was poor-moderate (r = -0.02; p = 0.8), primarily because six samples showed a low p.V600E signal despite high tumor cellularity. A QA investigation revealed that our initial pyrosequencing assay showed a false positive, weak p.V600E signal in specimens with a p.V600K mutation. A redesigned assay detected BRAF mutations in 50/131 (38 %) specimens, including 30 % non-p.V600E. This revised assay showed strong correlation between p.V600E BRAF mutant percentage and tumor cellularity (r = 0.76; p ≤ 0.01). Re-evaluation of the previously discordant samples by the revised assay confirmed a high level of p.V600K mutation in five specimens.Pathologists play important roles in molecular diagnostics, beyond identification of correct cells for testing. Accurate evaluation of tumor cellularity not only ensures sufficient material for required analytic sensitivity, but also provides an independent QA measure of the molecular assays.

BACKGROUND Bone is a common metastatic site for solid tumors and is often the only source for molecular testing. Current routine decalcification protocols for the processing of bone specimens damage nucleic acids, leading to a high failure rate. METHODS In this retrospective quality‐assessment analysis, preanalytic factors that contributed to the failure of mutational profiling in metastatic bone specimens were evaluated using a next‐generation sequencing assay. RESULTS Mutational profiling was conducted in 33 formalin‐fixed, paraffin‐embedded bone lesions that were submitted to a clinical laboratory. Adequate depth of coverage was obtained in 21 specimens, of which, 16 had mutations detected. “No results” were reported in 12 specimens because the NGS assay failed. There was a significantly higher failure rate in bone specimens compared with nonbone specimens (36% vs 2.3%, respectively). Although nonbone specimens had a higher failure rate in biopsy/fine‐needle aspiration (FNA) specimens, in‐house bone biopsy/FNA specimens with or without short‐duration surface decalcification had a lower failure rate than resected bone specimens (11% vs 60%, respectively). The high failure rate in resected metastatic bone specimens was associated with regular decalcification but not with low DNA input. CONCLUSIONS Next‐generation sequencing assays demonstrated clinical utility in metastatic bone specimens. FNA (smear and cell block) and core‐biopsy specimens provided adequate resources of nucleic acids for molecular profiling of metastatic bone lesions. The current findings suggest the need for developing specific tissue procurement and processing protocols for bone metastases and greater use of small biopsy and FNA specimens. Cancer Cytopathol 2016;124:744–53 . © 2016 American Cancer Society .

The publication of the "Pan-Cancer Atlas" by the Pan-Cancer Analysis of Whole Genomes Consortium, a partnership formed by The Cancer Genome Atlas (TCGA) and International Cancer Genome Consortium (ICGC), provides a wonderful opportunity to reflect on where we stand in our understanding of the genetics of pancreatic cancer, as well as on the opportunities to translate this understanding to patient care. From germline variants that predispose to the development of pancreatic cancer, to somatic mutations that are therapeutically targetable, genetics is now providing hope, where there once was no hope, for those diagnosed with pancreatic cancer.

Mismatch repair deficiency (dMMR) predicts response to immune checkpoint inhibitor therapy in solid tumors. Long mononucleotide repeat (LMR) markers may improve the interpretation of microsatellite instability (MSI) assays. Our cohorts included mismatch repair (MMR) proficient and dMMR colorectal cancer (CRC) samples, MMR proficient and dMMR endometrial cancer (EC) samples, dMMR prostate cancer samples, MSI-high (MSI-H) samples of other cancer types, and MSI-low (MSI-L) samples of various cancer types. MMR status was determined by immunohistochemical staining and/or MSI Analysis System Version 1.2 (V1.2). The sensitivity and specificity of the LMR MSI panel for dMMR detection were both 100% in CRC. The sensitivity values of the MSI V1.2 and LMR MSI panels in EC were 88% and 98%, respectively, and the specificity values were both 100%. The sensitivity of the LMR panel was 75% in dMMR prostate cancer detected by immunohistochemistry. The 22 samples of other cancer types that were previously classified as MSI-H were also classified as MSI-H using the LMR MSI panel. For the 12 samples that were previously classified as MSI-L, 1 sample was classified as microsatellite stable using the LMR MSI panel, 8 as MSI-L, and 3 as MSI-H. The LMR MSI panel showed high concordance to the MSI V1.2 panel in CRC and greater sensitivity in EC. The LMR MSI panel improves dMMR detection in noncolorectal cancers.

Pancreatic ductal adenocarcinoma (PDAC) frequently presents with metastasis, but the molecular programs in human PDAC cells that drive invasion are not well understood. Using an experimental pipeline enabling PDAC organoid isolation and collection based on invasive phenotype, we assessed the transcriptomic programs associated with invasion in our organoid model. We identified differentially expressed genes in invasive organoids compared with matched noninvasive organoids from the same patients, and we confirmed that the encoded proteins were enhanced in organoid invasive protrusions. We identified 3 distinct transcriptomic groups in invasive organoids, 2 of which correlated directly with the morphological invasion patterns and were characterized by distinct upregulated pathways. Leveraging publicly available single-cell RNA-sequencing data, we mapped our transcriptomic groups onto human PDAC tissue samples, highlighting differences in the tumor microenvironment between transcriptomic groups and suggesting that non-neoplastic cells in the tumor microenvironment can modulate tumor cell invasion. To further address this possibility, we performed computational ligand-receptor analysis and validated the impact of multiple ligands (TGF-β1, IL-6, CXCL12, MMP9) on invasion and gene expression in an independent cohort of fresh human PDAC organoids. Our results identify molecular programs driving morphologically defined invasion patterns and highlight the tumor microenvironment as a potential modulator of these programs.