Dhananjay Chitale

In human lung adenocarcinomas harboring EGFR mutations, a second-site point mutation that substitutes methionine for threonine at position 790 (T790M) is associated with approximately half of cases of acquired resistance to the EGFR kinase inhibitors, gefitinib and erlotinib. To identify other potential mechanisms that contribute to disease progression, we used array-based comparative genomic hybridization (aCGH) to compare genomic profiles of EGFR mutant tumors from untreated patients with those from patients with acquired resistance. Among three loci demonstrating recurrent copy number alterations (CNAs) specific to the acquired resistance set, one contained the MET proto-oncogene. Collectively, analysis of tumor samples from multiple independent patient cohorts revealed that MET was amplified in tumors from 9 of 43 (21%) patients with acquired resistance but in only two tumors from 62 untreated patients (3%) ( P = 0.007, Fisher's Exact test). Among 10 resistant tumors from the nine patients with MET amplification, 4 also harbored the EGFR T790M mutation. We also found that an existing EGFR mutant lung adenocarcinoma cell line, NCI-H820, harbors MET amplification in addition to a drug-sensitive EGFR mutation and the T790M change. Growth inhibition studies demonstrate that these cells are resistant to both erlotinib and an irreversible EGFR inhibitor (CL-387,785) but sensitive to a multikinase inhibitor (XL880) with potent activity against MET. Taken together, these data suggest that MET amplification occurs independently of EGFR T790M mutations and that MET may be a clinically relevant therapeutic target for some patients with acquired resistance to gefitinib or erlotinib.

ObjectiveTo establish evidence-based recommendations for the molecular analysis of lung cancers that are that are required to guide EGFR- and ALK-directed therapies, addressing which patients and samples should be tested, and when and how testing should be performed.ParticipantsThree cochairs without conflicts of interest were selected, one from each of the 3 sponsoring professional societies: College of American Pathologists, International Association for the Study of Lung Cancer, and Association for Molecular Pathology. Writing and advisory panels were constituted from additional experts from these societies.EvidenceThree unbiased literature searches of electronic databases were performed to capture articles published published from January 2004 through February 2012, yielding 1533 articles whose abstracts were screened to identify 521 pertinent articles that were then reviewed in detail for their relevance to the recommendations. Evidence was formally graded for each recommendation.Consensus ProcessInitial recommendations were formulated by the cochairs and panel members at a public meeting. Each guideline section was assigned to at least 2 panelists. Drafts were circulated to the writing panel (version 1), advisory panel (version 2), and the public (version 3) before submission (version 4).ConclusionsThe 37 guideline items address 14 subjects, including 15 recommendations (evidence grade A/B). The major recommendations are to use testing for EGFR mutations and ALK fusions to guide patient selection for therapy with an epidermal growth factor receptor (EGFR) or anaplastic lymphoma kinase (ALK) inhibitor, respectively, in all patients with advanced-stage adenocarcinoma, regardless of sex, race, smoking history, or other clinical risk factors, and to prioritize EGFR and ALK testing over other molecular predictive tests. As scientific discoveries and clinical practice outpace the completion of randomized clinical trials, evidence-based guidelines developed by expert practitioners are vital for communicating emerging clinical standards. Already, new treatments targeting genetic alterations in other, less common driver oncogenes are being evaluated in lung cancer, and testing for these may be addressed in future versions of these guidelines. To establish evidence-based recommendations for the molecular analysis of lung cancers that are that are required to guide EGFR- and ALK-directed therapies, addressing which patients and samples should be tested, and when and how testing should be performed. Three cochairs without conflicts of interest were selected, one from each of the 3 sponsoring professional societies: College of American Pathologists, International Association for the Study of Lung Cancer, and Association for Molecular Pathology. Writing and advisory panels were constituted from additional experts from these societies. Three unbiased literature searches of electronic databases were performed to capture articles published published from January 2004 through February 2012, yielding 1533 articles whose abstracts were screened to identify 521 pertinent articles that were then reviewed in detail for their relevance to the recommendations. Evidence was formally graded for each recommendation. Initial recommendations were formulated by the cochairs and panel members at a public meeting. Each guideline section was assigned to at least 2 panelists. Drafts were circulated to the writing panel (version 1), advisory panel (version 2), and the public (version 3) before submission (version 4). The 37 guideline items address 14 subjects, including 15 recommendations (evidence grade A/B). The major recommendations are to use testing for EGFR mutations and ALK fusions to guide patient selection for therapy with an epidermal growth factor receptor (EGFR) or anaplastic lymphoma kinase (ALK) inhibitor, respectively, in all patients with advanced-stage adenocarcinoma, regardless of sex, race, smoking history, or other clinical risk factors, and to prioritize EGFR and ALK testing over other molecular predictive tests. As scientific discoveries and clinical practice outpace the completion of randomized clinical trials, evidence-based guidelines developed by expert practitioners are vital for communicating emerging clinical standards. Already, new treatments targeting genetic alterations in other, less common driver oncogenes are being evaluated in lung cancer, and testing for these may be addressed in future versions of these guidelines.

KRAS mutations are found in approximately 25% of lung adenocarcinomas in Western countries and, as a group, have been strongly associated with cigarette smoking. These mutations are predictive of poor prognosis in resected disease as well as resistance to treatment with erlotinib or gefitinib.We determined the frequency and type of KRAS codon 12 and 13 mutations and characterized their association with cigarette smoking history in patients with lung adenocarcinomas.KRAS mutational analysis was done on 482 lung adenocarcinomas, 81 (17%) of which were obtained from patients who had never smoked cigarettes. KRAS mutations were found in 15% (12 of 81; 95% confidence intervals, 8-24%) of tumors from never smokers. Similarly, 22% (69 of 316; 95% confidence intervals, 17-27%) of tumors from former smokers, and 25% (21 of 85; 95% confidence intervals, 16-35%) of tumors from current smokers had KRAS mutations. The frequency of KRAS mutation was not associated with age, gender, or smoking history. The number of pack years of cigarette smoking did not predict an increased likelihood of KRAS mutations. Never smokers were significantly more likely than former or current smokers to have a transition mutation (G-->A) rather than the transversion mutations known to be smoking-related (G-->T or G-->C; P < 0.0001).Based on our data, KRAS mutations are not rare among never smokers with lung adenocarcinoma and such patients have a distinct KRAS mutation profile. The etiologic and biological heterogeneity of KRAS mutant lung adenocarcinomas is worthy of further study.

Abstract Patients with poorly differentiated thyroid cancers (PDTC), anaplastic thyroid cancers (ATC), and radioactive iodine-refractory (RAIR) differentiated thyroid cancers have a high mortality, particularly if positive on [18F]fluorodeoxyglucose (FDG)-positron emission tomography (PET). To obtain comprehensive genetic information on advanced thyroid cancers, we designed an assay panel for mass spectrometry genotyping encompassing the most significant oncogenes in this disease: 111 mutations in RET, BRAF, NRAS, HRAS, KRAS, PIK3CA, AKT1, and other related genes were surveyed in 31 cell lines, 52 primary tumors (34 PDTC and 18 ATC), and 55 RAIR, FDG-PET-positive recurrences and metastases (nodal and distant) from 42 patients. RAS mutations were more prevalent than BRAF (44 versus 12%; P = 0.002) in primary PDTC, whereas BRAF was more common than RAS (39 versus 13%; P = 0.04) in PET-positive metastatic PDTC. BRAF mutations were highly prevalent in ATC (44%) and in metastatic tumors from RAIR PTC patients (95%). Among patients with multiple metastases, 9 of 10 showed between-sample concordance for BRAF or RAS mutations. By contrast, 5 of 6 patients were discordant for mutations of PIK3CA or AKT1. AKT1_G49A was found in 9 specimens, exclusively in metastases. This is the first documentation of AKT1 mutation in thyroid cancer. Thus, RAIR, FDG-PET–positive metastases are enriched for BRAF mutations. If BRAF is mutated in the primary, it is likely that the metastases will harbor the defect. By contrast, absence of PIK3CA/AKT1 mutations in one specimen may not reflect the status at other sites because these mutations arise during progression, an important consideration for therapies directed at phosphoinositide 3-kinase effectors. [Cancer Res 2009;69(11):4885–93]

To establish evidence-based recommendations for the molecular analysis of lung cancers that are required to guide EGFR- and ALK-directed therapies, addressing which patients and samples should be tested, and when and how testing should be performed.Three cochairs without conflicts of interest were selected, one from each of the 3 sponsoring professional societies: College of American Pathologists, International Association for the Study of Lung Cancer, and Association for Molecular Pathology. Writing and advisory panels were constituted from additional experts from these societies.Three unbiased literature searches of electronic databases were performed to capture published articles from January 2004 through February 2012, yielding 1533 articles whose abstracts were screened to identify 521 pertinent articles that were then reviewed in detail for their relevance to the recommendations. EVIDENCE was formally graded for each recommendation.Initial recommendations were formulated by the cochairs and panel members at a public meeting. Each guideline section was assigned to at least 2 panelists. Drafts were circulated to the writing panel (version 1), advisory panel (version 2), and the public (version 3) before submission (version 4).The 37 guideline items address 14 subjects, including 15 recommendations (evidence grade A/B). The major recommendations are to use testing for EGFR mutations and ALK fusions to guide patient selection for therapy with an epidermal growth factor receptor (EGFR) or anaplastic lymphoma kinase (ALK) inhibitor, respectively, in all patients with advanced-stage adenocarcinoma, regardless of sex, race, smoking history, or other clinical risk factors, and to prioritize EGFR and ALK testing over other molecular predictive tests. As scientific discoveries and clinical practice outpace the completion of randomized clinical trials, evidence-based guidelines developed by expert practitioners are vital for communicating emerging clinical standards. Already, new treatments targeting genetic alterations in other, less common driver oncogenes are being evaluated in lung cancer, and testing for these may be addressed in future versions of these guidelines.

To establish evidence-based recommendations for the molecular analysis of lung cancers that are required to guide EGFR- and ALK-directed therapies, addressing which patients and samples should be tested, and when and how testing should be performed.Three cochairs without conflicts of interest were selected, one from each of the 3 sponsoring professional societies: College of American Pathologists, International Association for the Study of Lung Cancer, and Association for Molecular Pathology. Writing and advisory panels were constituted from additional experts from these societies.Three unbiased literature searches of electronic databases were performed to capture articles published from January 2004 through February 2012, yielding 1533 articles whose abstracts were screened to identify 521 pertinent articles that were then reviewed in detail for their relevance to the recommendations. Evidence was formally graded for each recommendation.Initial recommendations were formulated by the cochairs and panel members at a public meeting. Each guideline section was assigned to at least 2 panelists. Drafts were circulated to the writing panel (version 1), advisory panel (version 2), and the public (version 3) before submission (version 4).The 37 guideline items address 14 subjects, including 15 recommendations (evidence grade A/B). The major recommendations are to use testing for EGFR mutations and ALK fusions to guide patient selection for therapy with an epidermal growth factor receptor (EGFR) or anaplastic lymphoma kinase (ALK) inhibitor, respectively, in all patients with advanced-stage adenocarcinoma, regardless of sex, race, smoking history, or other clinical risk factors, and to prioritize EGFR and ALK testing over other molecular predictive tests. As scientific discoveries and clinical practice outpace the completion of randomized clinical trials, evidence-based guidelines developed by expert practitioners are vital for communicating emerging clinical standards. Already, new treatments targeting genetic alterations in other, less common driver oncogenes are being evaluated in lung cancer, and testing for these may be addressed in future versions of these guidelines.

Mutations in RAS proteins occur widely in human cancer. Prompted by the confirmation of KRAS mutation as a predictive biomarker of response to epidermal growth factor receptor (EGFR)-targeted therapies, limited clinical testing for RAS pathway mutations has recently been adopted. We performed a multiplatform genomic analysis to characterize, in a nonbiased manner, the biological, biochemical, and prognostic significance of Ras pathway alterations in colorectal tumors and other solid tumor malignancies. Mutations in exon 4 of KRAS were found to occur commonly and to predict for a more favorable clinical outcome in patients with colorectal cancer. Exon 4 KRAS mutations, all of which were identified at amino acid residues K117 and A146, were associated with lower levels of GTP-bound RAS in isogenic models. These same mutations were also often accompanied by conversion to homozygosity and increased gene copy number, in human tumors and tumor cell lines. Models harboring exon 4 KRAS mutations exhibited mitogen-activated protein/extracellular signal-regulated kinase kinase dependence and resistance to EGFR-targeted agents. Our findings suggest that RAS mutation is not a binary variable in tumors, and that the diversity in mutant alleles and variability in gene copy number may also contribute to the heterogeneity of clinical outcomes observed in cancer patients. These results also provide a rationale for broader KRAS testing beyond the most common hotspot alleles in exons 2 and 3.

BackgroundSomatic mutations in EGFR (exons 19 and 21) and KRAS (exon 2) are found in lung adenocarcinomas and have potential prognostic value in patients with advanced disease. These mutations also have therapeutic significance, as they predict for sensitivity and resistance, respectively, to EGFR tyrosine kinase inhibitor therapy. Whether EGFR and KRAS mutations also have an impact on survival in patients who undergo lung resection for curative intent in the absence of targeted therapy has not been established.MethodsWe analyzed the clinical characteristics and outcomes data for 296 patients who underwent resection at our institution for stage I–III lung adenocarcinoma. Tumors were assessed for both EGFR and KRAS mutations by established methods.ResultsEGFR and KRAS mutations were found in tumors from 40 (14%) and 50 (17%) patients, respectively. Patients with EGFR mutant tumors were more likely to be never smokers (48%), present with stage I disease (88%), and had a 90% (95% confidence interval [CI] 70–97%) 3-year overall survival, whereas patients with KRAS mutant tumors were more likely to be former/current smokers (92%), present with locally advanced disease (40%), and had a 66% (95% CI 48–79%) 3-year overall survival.ConclusionsEGFR and KRAS mutations define distinct molecular subsets of resected lung adenocarcinoma. Because EGFR and KRAS mutations also predict whether tumors are sensitive or resistant, respectively, to EGFR tyrosine kinase inhibitors, they can readily be used in clinical trials to help guide the administration of specific types of adjuvant therapy. Somatic mutations in EGFR (exons 19 and 21) and KRAS (exon 2) are found in lung adenocarcinomas and have potential prognostic value in patients with advanced disease. These mutations also have therapeutic significance, as they predict for sensitivity and resistance, respectively, to EGFR tyrosine kinase inhibitor therapy. Whether EGFR and KRAS mutations also have an impact on survival in patients who undergo lung resection for curative intent in the absence of targeted therapy has not been established. We analyzed the clinical characteristics and outcomes data for 296 patients who underwent resection at our institution for stage I–III lung adenocarcinoma. Tumors were assessed for both EGFR and KRAS mutations by established methods. EGFR and KRAS mutations were found in tumors from 40 (14%) and 50 (17%) patients, respectively. Patients with EGFR mutant tumors were more likely to be never smokers (48%), present with stage I disease (88%), and had a 90% (95% confidence interval [CI] 70–97%) 3-year overall survival, whereas patients with KRAS mutant tumors were more likely to be former/current smokers (92%), present with locally advanced disease (40%), and had a 66% (95% CI 48–79%) 3-year overall survival. EGFR and KRAS mutations define distinct molecular subsets of resected lung adenocarcinoma. Because EGFR and KRAS mutations also predict whether tumors are sensitive or resistant, respectively, to EGFR tyrosine kinase inhibitors, they can readily be used in clinical trials to help guide the administration of specific types of adjuvant therapy.

The emergence of castrate-resistant prostate cancer (CRPC) contributes to the high mortality of patients diagnosed with prostate cancer (PCa), which in part could be attributed to the existence and the emergence of cancer stem cells (CSCs). Recent studies have shown that deregulated expression of microRNAs (miRNAs) contributes to the initiation and progression of PCa. Among several known miRNAs, let-7 family appears to play a key role in the recurrence and progression of PCa by regulating CSCs; however, the mechanism by which let-7 family contributes to PCa aggressiveness is unclear. Enhancer of Zeste homolog 2 (EZH2), a putative target of let-7 family, was demonstrated to control stem cell function. In this study, we found loss of let-7 family with corresponding over-expression of EZH2 in human PCa tissue specimens, especially in higher Gleason grade tumors. Overexpression of let-7 by transfection of let-7 precursors decreased EZH2 expression and repressed clonogenic ability and sphere-forming capacity of PCa cells, which was consistent with inhibition of EZH2 3'UTR luciferase activity. We also found that the treatment of PCa cells with BR-DIM (formulated DIM: 3,3'-diindolylmethane by Bio Response, Boulder, CO, abbreviated as BR-DIM) up-regulated let-7 and down-regulated EZH2 expression, consistent with inhibition of self-renewal and clonogenic capacity. Moreover, BR-DIM intervention in our on-going phase II clinical trial in patients prior to radical prostatectomy showed upregulation of let-7 consistent with down-regulation of EZH2 expression in PCa tissue specimens after BR-DIM intervention. These results suggest that the loss of let-7 mediated increased expression of EZH2 contributes to PCa aggressiveness, which could be attenuated by BR-DIM treatment, and thus BR-DIM is likely to have clinical impact.

Women of sub-Saharan African descent have disproportionately higher incidence of triple-negative breast cancer (TNBC) and TNBC-specific mortality across all populations. Population studies show racial differences in TNBC biology, including higher prevalence of basal-like and quadruple-negative subtypes in African Americans (AA). However, previous investigations relied on self-reported race (SRR) of primarily U.S. populations. Due to heterogeneous genetic admixture and biological consequences of social determinants, the true association of African ancestry with TNBC biology is unclear. To address this, we conducted RNA sequencing on an international cohort of AAs, as well as West and East Africans with TNBC. Using comprehensive genetic ancestry estimation in this African-enriched cohort, we found expression of 613 genes associated with African ancestry and 2,000+ associated with regional African ancestry. A subset of African-associated genes also showed differences in normal breast tissue. Pathway enrichment and deconvolution of tumor cellular composition revealed that tumor-associated immunologic profiles are distinct in patients of African descent.Our comprehensive ancestry quantification process revealed that ancestry-associated gene expression profiles in TNBC include population-level distinctions in immunologic landscapes. These differences may explain some differences in race-group clinical outcomes. This study shows the first definitive link between African ancestry and the TNBC immunologic landscape, from an African-enriched international multiethnic cohort. See related commentary by Hamilton et al., p. 2496. This article is highlighted in the In This Issue feature, p. 2483.

Abstract Hyperactivated extracellular signal-regulated kinase (ERK) signaling is common in human cancer and is often the result of activating mutations in BRAF, RAS, and upstream receptor tyrosine kinases. To characterize the mitogen-activated protein kinase/ERK kinase (MEK)/ERK dependence of lung cancers harboring BRAF kinase domain mutations, we screened a large panel of human lung cancer cell lines (n = 87) and tumors (n = 916) for BRAF mutations. We found that non–small cell lung cancers (NSCLC) cells with both V600E and non-V600E BRAF mutations were selectively sensitive to MEK inhibition compared with those harboring mutations in epidermal growth factor receptor (EGFR), KRAS, or ALK and ROS kinase fusions. Supporting its classification as a “driver” mutation in the cells in which it is expressed, MEK inhibition in V600EBRAF NSCLC cells led to substantial induction of apoptosis, comparable with that seen with EGFR kinase inhibition in EGFR mutant NSCLC models. Despite high basal ERK phosphorylation, EGFR mutant cells were uniformly resistant to MEK inhibition. Conversely, BRAF mutant cell lines were resistant to EGFR inhibition. These data, together with the nonoverlapping pattern of EGFR and BRAF mutations in human lung cancer, suggest that these lesions define distinct clinical entities whose treatment should be guided by prospective real-time genotyping. To facilitate such an effort, we developed a mass spectrometry-based genotyping method for the detection of hotspot mutations in BRAF, KRAS, and EGFR. Using this assay, we confirmed that BRAF mutations can be identified in a minority of NSCLC tumors and that patients whose tumors harbor BRAF mutations have a distinct clinical profile compared with those whose tumors harbor kinase domain mutations in EGFR. [Cancer Res 2008;68(22):9375–83]

To address the biological heterogeneity of lung cancer, we studied 199 lung adenocarcinomas by integrating genome-wide data on copy number alterations and gene expression with full annotation for major known somatic mutations in this cancer. This showed non-random patterns of copy number alterations significantly linked to EGFR and KRAS mutation status and to distinct clinical outcomes, and led to the discovery of a striking association of EGFR mutations with underexpression of DUSP4, a gene within a broad region of frequent single-copy loss on 8p. DUSP4 is involved in negative feedback control of EGFR signaling, and we provide functional validation for its role as a growth suppressor in EGFR-mutant lung adenocarcinoma. DUSP4 loss also associates with p16/CDKN2A deletion and defines a distinct clinical subset of lung cancer patients. Another novel observation is that of a reciprocal relationship between EGFR and LKB1 mutations. These results highlight the power of integrated genomics to identify candidate driver genes within recurrent broad regions of copy number alteration and to delineate distinct oncogenetic pathways in genetically complex common epithelial cancers.

Genetic lesions affecting a number of kinases and other elements within the epidermal growth factor receptor (EGFR) signaling pathway have been implicated in the pathogenesis of human non-small-cell lung cancer (NSCLC). We performed mutational profiling of a large cohort of lung adenocarcinomas to uncover other potential somatic mutations in genes of this pathway that could contribute to lung tumorigenesis. We have identified in 2 of 207 primary lung tumors a somatic activating mutation in exon 2 of MEK1 (i.e., mitogen-activated protein kinase kinase 1 or MAP2K1) that substitutes asparagine for lysine at amino acid 57 (K57N) in the nonkinase portion of the kinase. Neither of these two tumors harbored known mutations in other genes encoding components of the EGFR signaling pathway (i.e., EGFR, HER2, KRAS, PIK3CA, and BRAF). Expression of mutant, but not wild-type, MEK1 leads to constitutive activity of extracellular signal-regulated kinase (ERK)-1/2 in human 293T cells and to growth factor-independent proliferation of murine Ba/F3 cells. A selective MEK inhibitor, AZD6244, inhibits mutant-induced ERK activity in 293T cells and growth of mutant-bearing Ba/F3 cells. We also screened 85 NSCLC cell lines for MEK1 exon 2 mutations; one line (NCI-H1437) harbors a Q56P substitution, a known transformation-competent allele of MEK1 originally identified in rat fibroblasts, and is sensitive to treatment with AZD6244. MEK1 mutants have not previously been reported in lung cancer and may provide a target for effective therapy in a small subset of patients with lung adenocarcinoma.

Abstract Purpose: A comprehensive comparison of biomarker expression between patients' primary breast carcinoma (PBC) and their metastatic breast carcinomas (MBC) has not been done. Experimental Design: We did rapid autopsies (postmortem intervals, 1-4 hours) on 10 consenting patients who died of MBC. We constructed single-patient tissue microarrays from the patients' archived PBC and multiple different MBCs harvested at autopsy, which were immunohistochemically labeled for multiple biomarkers. Methylation of multiple gene promoters was assessed quantitatively on dissected PBC and MBC samples. Results: Extensive heterogeneity was observed between PBC and their paired MBC, as well as among multiple MBC from the same patient. Estrogen and progesterone receptors tended to be uniformly down-regulated in metastases. E-cadherin was down-regulated in a subset of the MBC of one case. Variable overexpression in MBC compared with the PBC was observed for cyclooxygenase-2 (five cases), epidermal growth factor receptor (EGFR; four cases), MET (four cases), and mesothelin (four cases). No case strongly overexpressed HER-2/neu by immunohistochemistry, but eight cases showed variable protein expression ranging from negative to equivocal (2+) in different MBC. In one case, variable low-level HER-2/neu gene amplification was found. EGFR and MET overexpression were restricted to the four basal-type cancers. EGFR protein overexpression did not correlate with EGFR gene amplification. Multigene promoter hypermethylation of RASSF1a, HIN1, cyclin D2, Twist, estrogen receptor α, APC1, and RARβ was overall very similar in the PBC and all MBCs in all cases. Conclusions: Therapeutic targets identified in the PBC or even some MBC may not reflect targets present in all metastatic sites.

Lung adenocarcinomas responsive to epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors possess EGFR mutations and often increased EGFR copy number. We prospectively studied 334 clinical cases using polymerase chain reaction-based assays to detect deletions within exon 19 and the L858R mutation in exon 21, which together account for approximately 90% of EGFR mutations. Seventy-eight (23%) of these tumors had an EGFR mutation, with 55 (71%) exon 19 deletions and 23 (29%) exon 21 L858R mutations. We were able to compare mutant and normal EGFR alleles and found a preferential amplification of the mutant allele. The association of mutations with EGFR amplification (determined by chromogenic in situ hybridization) and EGFR expression (determined by immunohistochemistry) was further examined in a subset of 60 tumors. EGFR amplification (> or =5 EGFR signals per nucleus) was seen in 15 of 29 (52%) EGFR-mutated tumors but in only five of 31 (6%) non-mutated tumors (P = 0.006). EGFR overexpression was strongly associated with amplification but was statistically independent of EGFR mutation. Most patients with EGFR mutations (17 of 29, 59%) never smoked compared with 13% (four of 31) of patients lacking such mutations (P = 0.0003). The association of amplification with smoking status was marginal and was nonexistent with EGFR expression. Thus, these results indicate that EGFR amplification, preferentially of the mutant allele, often accompanies EGFR mutation, whereas EGFR immunohistochemical staining associates with amplification but cannot predict EGFR mutation status.

<h3>Introduction:</h3> The detection of mutations in the epidermal growth factor receptor (<i>EGFR</i>) gene, which predict sensitivity to treatment with <i>EGFR</i> tyrosine kinase inhibitors, represents a major advance in the treatment of lung adenocarcinoma. <i>KRAS</i> mutations confer resistance to <i>EGFR</i>-tyrosine kinase inhibitors. The prevalence of these mutations in African American patients has not been thoroughly investigated. <h3>Methods:</h3> We collected formalin-fixed, paraffin-embedded material from resected lung adenocarcinomas from African American patients at three institutions for DNA extraction. The frequencies of <i>EGFR</i> exon 19 deletions, exon 21 L858R substitutions, and <i>KRAS</i> mutations in tumor specimens from African American patients were compared with data in white patients (<i>n</i> = 476). <h3>Results:</h3> <i>EGFR</i> mutations were detected in 23 of the 121 specimens from African American patients (19%, 95% confidence interval [CI]: 13–27%), whereas <i>KRAS</i> mutations were found in 21 (17%, 95% CI: 12–25%). There was no significant difference between frequencies of <i>EGFR</i> mutations comparing African American and white patients, 19% versus 13% (61/476, 95% CI: 10–16%; <i>p</i> = 0.11). <i>KRAS</i> mutations were more likely among whites, 26% (125/476, 95% CI: 23–30%; <i>p</i> = 0.04). <h3>Conclusions:</h3> This is the largest study to date examining the frequency of mutations in lung adenocarcinomas in African Americans. Although <i>KRAS</i> mutations were somewhat less likely, there was no difference between the frequencies of <i>EGFR</i> mutations in African American patients, when compared with whites. These results suggest that all patients with advanced lung adenocarcinomas should undergo mutational analysis before initiation of therapy.

Earlier investigations have revealed that tumor cells undergo metabolic reprogramming and mainly derive their cellular energy from aerobic glycolysis rather than oxidative phosphorylation even in the presence of oxygen. However, recent studies have shown that certain cancer cells display increased oxidative phosphorylation or high metabolically active phenotype. Cellular bioenergetic profiling of 13 established and 12 patient derived ovarian cancer cell lines revealed significant bioenergetics diversity. The bioenergetics phenotype of ovarian cancer cell lines correlated with functional phenotypes of doubling time and oxidative stress. Interestingly, chemosensitive cancer cell lines (A2780 and PEO1) displayed a glycolytic phenotype while their chemoresistant counterparts (C200 and PEO4) exhibited a high metabolically active phenotype with the ability to switch between oxidative phosphorylation or glycolysis. The chemosensitive cancer cells could not survive glucose deprivation, while the chemoresistant cells displayed adaptability. In the patient derived ovarian cancer cells, a similar correlation was observed between a high metabolically active phenotype and chemoresistance. Thus, ovarian cancer cells seem to display heterogeneity in using glycolysis or oxidative phosphorylation as an energy source. The flexibility in using different energy pathways may indicate a survival adaptation to achieve a higher 'cellular fitness' that may be also associated with chemoresistance.

Abstract Background The detection of somatic mutations in cell-free DNA (cfDNA) from liquid biopsy has emerged as a noninvasive tool to monitor the follow-up of cancer patients. However, the significance of cfDNA clinical utility remains uncertain in patients with brain tumors, primarily because of the limited sensitivity cfDNA has to detect real tumor-specific somatic mutations. This unresolved challenge has prevented accurate follow-up of glioma patients with noninvasive approaches. Methods Genome-wide DNA methylation profiling of tumor tissue and serum cfDNA of glioma patients. Results Here, we developed a noninvasive approach to profile the DNA methylation status in the serum of patients with gliomas and identified a cfDNA-derived methylation signature that is associated with the presence of gliomas and related immune features. By testing the signature in an independent discovery and validation cohorts, we developed and verified a score metric (the “glioma-epigenetic liquid biopsy score” or GeLB) that optimally distinguished patients with or without glioma (sensitivity: 100%, specificity: 97.78%). Furthermore, we found that changes in GeLB score reflected clinicopathological changes during surveillance (eg, progression, pseudoprogression, and response to standard or experimental treatment). Conclusions Our results suggest that the GeLB score can be used as a complementary approach to diagnose and follow up patients with glioma.

Prostate cancer is the most frequently diagnosed and second most fatal nonskin cancer among men in the United States. African American men are two times more likely to develop and die of prostate cancer compared with men of other ancestries. Previous whole genome or exome tumor-sequencing studies of prostate cancer have primarily focused on men of European ancestry. In this study, we sequenced and characterized somatic mutations in aggressive (Gleason ≥7, stage ≥T2b) prostate tumors from 24 African American patients. We describe the locations and prevalence of small somatic mutations (up to 50 bases in length), copy number aberrations, and structural rearrangements in the tumor genomes compared with patient-matched normal genomes. We observed several mutation patterns consistent with previous studies, such as large copy number aberrations in chromosome 8 and complex rearrangement chains. However, TMPRSS2-ERG gene fusions and PTEN losses occurred in only 21% and 8% of the African American patients, respectively, far less common than in patients of European ancestry. We also identified mutations that appeared specific to or more common in African American patients, including a novel CDC27-OAT gene fusion occurring in 17% of patients. The genomic aberrations reported in this study warrant further investigation of their biologic significant role in the incidence and clinical outcomes of prostate cancer in African Americans. Cancer Res; 76(7); 1860-8. ©2016 AACR.

Amplification of chromosome 6p has been implicated in aggressive behavior in several cancers, but has not been characterized in renal cell carcinoma (RCC). We identified 9 renal tumors with amplification of chromosome 6p including the TFEB gene, 3 by fluorescence in situ hybridization, and 6 from the Cancer Genome Atlas (TCGA) databases. Patients’ ages were 28 to 78 years (median, 61 y). Most tumors were high stage (7/9 pT3a, 2/9 pN1). Using immunohistochemistry, 2/4 were positive for melanocytic markers and cathepsin K. Novel TFEB fusions were reported by TCGA in 2; however, due to a small composition of fusion transcripts compared with full-length transcripts (0.5/174 and 3.3/132 FPKM), we hypothesize that these represent secondary fusions due to amplification. Five specimens (4 TCGA, 1 fluorescence in situ hybridization) had concurrent chromosome 3p copy number loss or VHL deletion. However, these did not resemble clear cell RCC, had negative carbonic anhydrase IX labeling, lacked VHL mutation, and had papillary or unclassified histology (2/4 had gain of chromosome 7 or 17). One tumor each had somatic FH mutation and SMARCB1 mutation. Chromosome 6p amplification including TFEB is a previously unrecognized cytogenetic alteration in RCC, associated with heterogenous tubulopapillary eosinophilic and clear cell histology. The combined constellation of features does not fit cleanly into an existing tumor category (unclassified), most closely resembling papillary or translocation RCC. The tendency for high tumor stage, varied tubulopapillary morphology, and a subset with melanocytic marker positivity suggests the possibility of a unique tumor type, despite some variation in appearance and genetics.

Objective: To investigate subtype-specific risk of germline alleles associated with triple negative breast cancer (TNBC) in African ancestry populations. Background: Breast cancer (BC) mortality is higher in African American (AA) compared to White American (WA) women; this disparity is partly explained by 2-fold higher TNBC incidence. Methods: We used a surgically maintained biospecimen cohort of 2884 BC cases. Subsets of the total (760 AA; 962 WA; 910 West African/Ghanaian; 252 East African/Ethiopian) were analyzed for genotypes of candidate alleles. A subset of 417 healthy controls were also genotyped, to measure associations with overall BC risk and TNBC. Results: TNBC frequency was highest in Ghanaian and AA cases (49% and 44% respectively; P &lt; 0.0001) and lowest in Ethiopian and WA cases (17% and 24% respectively; P &lt; 0.0001). TNBC cases had higher West African ancestry than non-TNBC ( P &lt; 0.0001). Frequency of the Duffy-null allele (rs2814778; an African ancestral variant adopted under selective pressure as protection against malaria) was associated with TNBC-specific risk ( P &lt; 0.0001), quantified West African Ancestry ( P &lt; 0.0001) and was more common in AA, Ghanaians, and TNBC cases. Additionally, rs4849887 was significantly associated with overall BC risk, and both rs2363956 and rs13000023 were associated with TNBC-specific risk, although none as strongly as the Duffy-null variant. Conclusions: West African ancestry is strongly correlated with TNBC status, as well as germline variants related to BC risk. The Duffy-null allele was associated with TNBC risk in our cohort.

Tumor-specific immune response is an important aspect of disease prognosis and ultimately impacts treatment decisions for innovative immunotherapies. The atypical chemokine receptor 1 (ACKR1 or DARC) gene plays a pivotal role in immune regulation and harbors several single-nucleotide variants (SNV) that are specific to sub-Saharan African ancestry.Using computational The Cancer Genome Atlas (TCGA) analysis, case-control clinical cohort Luminex assays, and CIBERSORT deconvolution, we identified distinct immune cell profile-associated DARC/ACKR1 tumor expression and race with increased macrophage subtypes and regulatory T cells in DARC/ACKR1-high tumors.In this study, we report the clinical relevance of DARC/ACKR1 tumor expression in breast cancer, in the context of a tumor immune response that may be associated with sub-Saharan African ancestry. Briefly, we found that for infiltrating carcinomas, African Americans have a higher proportion of DARC/ACKR1-negative tumors compared with white Americans, and DARC/ACKR1 tumor expression is correlated with proinflammatory chemokines, CCL2/MCP-1 (P <0.0001) and anticorrelated with CXCL8/IL8 (P <0.0001). Sub-Saharan African-specific DARC/ACKR1 alleles likely drive these correlations. Relapse-free survival (RFS) and overall survival (OS) were significantly longer in individuals with DARC/ACKR1-high tumors (P <1.0 × 10-16 and P <2.2 × 10-6, respectively) across all molecular tumor subtypes.DARC/AKCR1 regulates immune responses in tumors, and its expression is associated with sub-Saharan African-specific alleles. DARC/ACKR1-positive tumors will have a distinct immune response compared with DARC/AKCR1-negative tumors.This study has high relevance in cancer management, as we introduce a functional regulator of inflammatory chemokines that can determine an infiltrating tumor immune cell landscape that is distinct among patients of African ancestry.

Inflammation characterized by the presence of T and B cells is often observed in prostate cancer, but it is unclear how T- and B-cell levels change during carcinogenesis and whether such changes influence disease progression.The study used a retrospective sample of 73 prostate cancer cases (45 whites and 28 African Americans) that underwent surgery as their primary treatment and had a benign prostate biopsy at least 1 year before diagnosis. CD3+, CD4+, and CD20+ lymphocytes were quantified by immunohistochemistry in paired pre- and post-diagnostic benign prostate biopsy and tumor surgical specimens, respectively. Clusters of similar trends of expression across two different timepoints and three distinct prostate regions-benign biopsy glands (BBG), tumor-adjacent benign glands (TAG), and malignant tumor glandular (MTG) regions-were identified using Time-series Anytime Density Peaks Clustering (TADPole). A Cox proportional hazards model was used to estimate the hazard ratio (HR) of time to biochemical recurrence associated with region-specific lymphocyte counts and regional trends.The risk of biochemical recurrence was significantly reduced in men with an elevated CD20+ count in TAG (HR = 0.81, p = 0.01) after adjusting for covariates. Four distinct patterns of expression change across the BBG-TAG-MTG regions were identified for each marker. For CD20+, men with low expression in BBG and higher expression in TAG compared to MTG had an adjusted HR of 3.06 (p = 0.03) compared to the reference group that had nominal differences in CD20+ expression across all three regions. The two CD3+ expression patterns that featured lower CD3+ expression in the BBG compared to the TAG and MTG regions had elevated HRs ranging from 3.03 to 4.82 but did not reach statistical significance.Longitudinal and spatial expression patterns of both CD3+ and CD20+ suggest that increased expression in benign glands during prostate carcinogenesis is associated with an aggressive disease course.

A benign diagnosis in a core needle biopsy (CNBx) of the breast performed for a clinically and/or radiologically suspicious abnormality is often due to a nonrepresentative sample. However, the discordance may not be recognized, resulting in a logistic delay in the diagnosis.Twenty-seven false-negative CNBxs were identified in 952 consecutive CNBxs of the breast (653 benign, 266 malignant, and 33 atypical) performed during a 1-year period. Biopsies were analyzed with respect to clinical and radiologic findings, biopsy type, type of malignancy, and interval between the original CNBx and final diagnosis. Four hundred thirty-eight (67%) of the patients with a benign CNBx diagnosis either underwent excision or had a minimum of 1-year follow-up (mean, 35.6 months; median, 36 months).The cancers missed on CNBx included 6 ductal carcinomas in situ, 17 invasive ductal carcinomas, 3 invasive lobular carcinomas, and 1 non-Hodgkin lymphoma. The overall false-negative rate was 9.1%. For palpable lesions, ultrasound-guided CNBx had a lower rate of missed cancer (3.6%) compared with CNBx without image guidance (13.3%). The false-negative rate for vacuum assisted CNBx biopsy was 7.6% (3.3% for the 11-gauge needle, 22.2% for the 14-gauge needle; 5.6% for nonpalpable mass lesions, 8.2% for microcalcifications). In all seven false-negative CNBxs performed by radiologists, the discordance between the radiologic and pathologic findings was promptly recognized due to their standard follow-up protocol. The discordance between the degree of clinical suspicion, radiologic impression, and the pathologic findings was not immediately recognized in 5 of 20 false-negative CNBxs performed by surgeons (4 without radiologic guidance and 1 with ultrasound guidance), resulting in a delay in the diagnosis ranging from 112-336 days.A false-negative diagnosis of breast carcinoma was found to be more common in CNBx performed without image guidance but occurred to a lesser degree in image-guided biopsies. A delay in diagnosis can be avoided by establishing a standard post-CNBx follow-up protocol.

<h3>Abstract</h3> Benign changes ranging from atrophy and inflammation to high-grade prostatic intraepithelial neoplasia (HGPIN) are common findings on prostate core needle biopsies. Although atrophy and inflammation may be precursors of prostate cancer, only HGPIN is currently recommended to be included in surgical pathology reports. To determine whether these benign findings increase prostate cancer risk, we conducted a case–control study nested within a historical cohort of 6692 men with a benign prostate specimen collected between 1990 and 2002. The analytic sample included 574 case–control pairs comprised of cases diagnosed with prostate cancer a minimum of 1 year after cohort entry and controls matched to cases on date and age at cohort entry, race, and type of specimen. The initial benign specimen was reviewed for presence of HGPIN, atrophy (simple, lobular, and partial) and inflammation (glandular and/or stromal). HGPIN significantly increased risk for prostate cancer (odds ratio (OR)=2.00; 95% confidence interval (CI)=1.25–3.20). Inflammation within the stromal compartment was associated with decreased risk (OR=0.66; CI=0.52–0.84), and diffuse stromal inflammation of severe grade had the strongest inverse association with risk (OR=0.21; CI=0.07–0.62). In a model adjusted for prostate-specific antigen (PSA) level at cohort entry and inflammation, simple atrophy was associated with a 33% increased prostate cancer risk that was marginally significant (<i>P</i>=0.03). Clinicians should consider patterns and extent of inflammation when managing high-risk patients with negative biopsy results. Identifying benign inflammatory processes that underlie high PSA levels would help to reduce the number of unnecessary repeated prostate biopsies.

Adenomyoepithelioma of the breast is an uncommon tumor characterized by dual differentiation into luminal cells and myoepithelial cells. A spectrum of histologic patterns is observed among these tumors and even in different areas of individual tumors. These lesions can be diagnostically challenging, especially when a core needle biopsy is performed, because of the heterogeneity of adenomyoepitheliomas. Recognition of the biphasic cellular elements and the characteristic overall architecture of the tumors in combination with immunohistochemistry are essential to establish the correct diagnosis. Although most tumors have a benign clinical course, local recurrences, malignant transformations, and distant metastases have been reported. All the reported malignant adenomyoepitheliomas with metastases have shown significant cytologic atypia and brisk mitotic rates. Therefore, adequate sampling of the tumor to identify these features is necessary. A complete excision with adequate margins would lower the chance of local recurrence or potential for metastasis.

Myc is up-regulated in almost all cancer types and is the subject of intense investigation because of its pleiotropic effects controlling a broad spectrum of cell functions. However, despite its recognition as a stand-alone molecular target, development of suitable strategies to block its function is hindered because of its nonenzymatic nature. We reported earlier that arachidonate 5-lipoxygenase (5-Lox) plays an important role in the survival and growth of prostate cancer cells, although details of the underlying mechanisms have yet to be characterized. By whole genome gene expression array, we observed that inhibition of 5-Lox severely down-regulates the expression of c-<i>Myc</i> oncogene in prostate cancer cells. Moreover, inhibition of 5-Lox dramatically decreases the protein level, nuclear accumulation, DNA binding, and transcriptional activities of c-Myc. Both the 5-Lox inhibition-induced down-regulation of c-Myc and induction of apoptosis are mitigated when the cells are treated with 5-oxoeicosatetraenoic acid, a metabolite of 5-Lox, confirming a role of 5-Lox in these processes. c-<i>Myc</i> is a transforming oncogene widely expressed in prostate cancer cells and maintains their transformed phenotype. Interestingly, MK591, a specific 5-Lox inhibitor, strongly affects the viability of Myc-overactivated prostate cancer cells and completely blocks their invasive and soft agar colony-forming abilities, but it spares nontransformed cells where expression of 5-Lox is undetectable. These findings indicate that the oncogenic function of c-Myc in prostate cancer cells is regulated by 5-Lox activity, revealing a novel mechanism of 5-Lox action and suggesting that the oncogenic function of c-Myc can be suppressed by suitable inhibitors of 5-Lox.

Abstract Recurrence of meningiomas is unpredictable by current invasive methods based on surgically removed specimens. Identification of patients likely to recur using noninvasive approaches could inform treatment strategy, whether intervention or monitoring. In this study, we analyze the DNA methylation levels in blood (serum and plasma) and tissue samples from 155 meningioma patients, compared to other central nervous system tumor and non-tumor entities. We discover DNA methylation markers unique to meningiomas and use artificial intelligence to create accurate and universal models for identifying and predicting meningioma recurrence, using either blood or tissue samples. Here we show that liquid biopsy is a potential noninvasive and reliable tool for diagnosing and predicting outcomes in meningioma patients. This approach can improve personalized management strategies for these patients.

Androgen Receptor (AR) signaling is critically important during the development and progression of prostate cancer (PCa). The AR signaling is also important in the development of castrate resistant prostate cancer (CRPC) where AR is functional even after androgen deprivation therapy (ADT); however, little is known regarding the transcriptional and functional regulation of AR in PCa. Moreover, treatment options for primary PCa for preventing the occurrence of CRPC is limited; therefore, novel strategy for direct inactivation of AR is urgently needed. In this study, we found loss of miR-34a, which targets AR, in PCa tissue specimens, especially in patients with higher Gleason grade tumors, consistent with increased expression of AR. Forced overexpression of miR-34a in PCa cell lines led to decreased expression of AR and prostate specific antigen (PSA) as well as the expression of Notch-1, another important target of miR-34a. Most importantly, BR-DIM intervention in PCa patients prior to radical prostatectomy showed re-expression of miR-34a, which was consistent with decreased expression of AR, PSA and Notch-1 in PCa tissue specimens. Moreover, BR-DIM intervention led to nuclear exclusion both in PCa cell lines and in tumor tissues. PCa cells treated with BR-DIM and 5-aza-dC resulted in the demethylation of miR-34a promoter concomitant with inhibition of AR and PSA expression in LNCaP and C4-2B cells. These results suggest, for the first time, epigenetic silencing of miR -34a in PCa, which could be reversed by BR-DIM treatment and, thus BR-DIM could be useful for the inactivation of AR in the treatment of PCa.

The objective of this work was to demonstrate that autoantibodies in breast cancer sera are not epiphenomena, and exhibit unique immunologic features resembling the rheumatic autoimmune diseases.We performed a comprehensive study of autoantibodies on a collection of sera from women with breast cancer or benign breast disease, undergoing annual screening mammography. All women in this study had suspicious mammography assessment and underwent a breast biopsy. We used indirect immunofluorescence, the crithidia assay for anti-dsDNA antibodies, and multiple ELISAs for extractable nuclear antigens.Autoantibodies were detected in virtually all patients with breast cancer, predominantly of the IgG1 and IgG3 isotypes. The profile detected in breast cancer sera showed distinctive features, such as antibodies targeting mitochondria, centrosomes, centromeres, nucleoli, cytoskeleton, and multiple nuclear dots. The majority of sera showing anti-mitochondrial antibodies did not react with the M2 component of pyruvate dehydrogenase, characteristic of primary biliary cirrhosis. Anti-centromere antibodies were mainly anti-CENP-B. ELISAs for extractable nuclear antigens and the assays for dsDNA were negative.The distinctive autoantibody profile detected in BC sera is the expression of tumor immunogenicity. Although some of these features resemble those in the rheumatic autoimmune diseases and primary biliary cirrhosis, the data suggest the involvement of an entirely different set of epithelial antigens in breast cancer. High titer autoantibodies targeting centrosomes, centromeres, and mitochondria were detected in a small group of healthy women with suspicious mammography assessment and no cancer by biopsy; this suggests that the process triggering autoantibody formation starts in the pre-malignant phase and that future studies using validated autoantibody panels may allow detection of breast cancer risk in asymptomatic women. Autoantibodies developing in breast cancer are not epiphenomena, but likely reflect an antigen-driven autoimmune response triggered by epitopes developing in the mammary gland during breast carcinogenesis. Our results support the validity of the multiple studies reporting association of autoantibodies with breast cancer. Results further suggest significant promise for the development of panels of breast cancer-specific, premalignant-phase autoantibodies, as well as studies on the autoantibody response to tumor associated antigens in the pathogenesis of cancer.

Triple-negative breast cancers (TNBCs) are more common among African-ancestry populations, such as African Americans and western, sub-Saharan Africans, compared with European-ancestry populations. This phenotype prevalence contributes to disparities in breast cancer outcomes between African Americans and White Americans. Breast cancer stem cells represent the tumor subpopulation involved in metastatic virulence, and ongoing research seeks to characterize the extent to which TNBC versus non-TNBC stem cells may differ. This review summarizes the existing literature regarding TNBCs and stem cells as they pertain to the burden of breast cancer among African-ancestry populations. Additional research related to variations in somatic tumor genomics between the African-American and White-American populations is also summarized. This review furthermore explores the history of insights regarding breast cancer disparities related to racial/ethnic identity, socioeconomic status, and tumor biology.

Summary Systemic AL amyloidosis (AL) is a disorder in which light chains form fibrillar deposits, leading to organ dysfunction and death. Rarely, AL has been associated with non‐Hodgkin's lymphoma (NHL), although this association has not been well characterized. We report a series of six patients with AL associated with NHL, primarily lymphoplasmacytic lymphoma. Organ involvement was variable, with frequent bulky lymphadenopathy and visceral cavity deposits, but no cardiac involvement. Positron emission tomography scans were negative. Bone marrow and lymph node biopsies showed a mixed population of CD20 + lymphoid and CD138 + plasma cells. Serum free light chains were elevated, and correlated with response to therapy. Immunoglobulin light chain variable region (Ig V L ) germline gene use was typical for AL, reflecting previously observed correlations between germline gene use and organ tropism. Five patients received rituximab‐based therapies with two responses. Two patients underwent autologous stem cell transplantation with one complete haematological response. Four patients survive at 10–132 months from diagnosis. AL with NHL has distinctive clinical features but employs the same Ig V L gene repertoire as AL with clonal plasma cell dyscrasias. Serial serum free light chain levels are useful for tracking response to therapy. Treatments aimed at both lymphoid and plasma cell components appear warranted.

Background: Primary cutaneous mucinous carcinoma (PCMC) is a rare malignancy with probable apocrine differentiation. It is important to differentiate it from metastatic mucinous carcinoma (MMC), especially from the breast. The histologic and immunohistochemical features overlap between PCMC and breast mucinous carcinomas. In this study, we introduce the presence of myoepithelial component in PCMC as a new morphologic parameter to distinguish it from MMC from either breast or sites elsewhere in the body. Materials and Methods: We studied 7 cases of PCMC. The possible in situ component in the tumor was assessed by the presence of a peripheral myoepithelial cell layer. Myoepithelial cell differentiation was confirmed with immunohistochemical stains for p63, CK 5/6, calponin, smooth muscle actin (SMA), HHF-35, and CD10. Estrogen and progesterone receptor (ER/PR), gross cystic disease fluid protein (GCDFP 15), CK7, CK20, and S-100 immunostains were also performed. Results: Histologically, multiple small monomorphic epithelial islands floating in multilocular pools of mucin characterized the tumor. Focally, epithelial islands were bordered by dermal connective tissue at the periphery of mucin pools. Secretory snouts were apparent in all cases providing evidence for apocrine differentiation. In 5 of the 7 cases, an in situ component was identified as epithelial islands being bounded by a myoepithelial layer, which was highlighted by p63, CK 5/6, calponin, SMA, and HHF-35. ER/PR and CK7 were positive in all the cases. GCDFP-15 and CD10 were focally positive in the tumor cells and myoepithelial cells, respectively. All 7 cases were negative for S-100 and CK 20. Conclusion: We conclude that an in situ component is frequently present in PCMC (5/7) and may help in distinguishing this entity from MMC, especially of breast origin. Furthermore, it may provide insight into the pathogenetic mechanism of mucinous carcinoma evolving from in situ carcinoma with luminal mucinous distention to cellular tumor with a little surrounding mucin.

The presence of the JAK2 V617F mutation is now part of clinical diagnostic algorithms, and JAK2 status is routinely assessed when BCR/ABL- chronic myeloproliferative neoplasms (MPNs) are suspected. The aim of this study was to evaluate performance of 3 screening and 1 quantitative method for JAK2 V617F detection. For the study, 43 samples (27 bone marrow aspirates and 16 peripheral blood samples) were selected. The screening assays were the JAK2 Activating Mutation Assay (InVivoScribe, San Diego, CA), JAK2 MutaScreen kit (Ipsogen, Luminy Biotech, Marseille, France), and a home-brew melting curve analysis method. Ipsogen's JAK2 MutaQuant assay was used for quantification of mutant and wild-type alleles. The limit of detection was 1% for the kit-based screening methods and 10% for the melting curve method. The JAK2 MutaQuant assay demonstrated analytic sensitivity of 0.01%. All 4 methods detected cases of BCR/ABL- MPNs and gave negative results with BCR/ABL+ chronic myelogenous leukemia, multiple myeloma, myelodysplastic syndrome, and normal cases.

Thyroid cancer is the most common endocrine cancer with 1,690 deaths each year. There are four main types of which the papillary and follicular types together account for >90% followed by medullary cancers with 3% to 5% and anaplastic carcinomas making up <3%. Epigenetic events of DNA hypermethylation are emerging as promising molecular targets for cancer detection. Our immediate and long term goal is to identify DNA methylation markers for early detection of thyroid cancer. This pilot study comprised of 21 patients to include 11 papillary thyroid cancers (PTC), 2 follicular thyroid cancers (FTC), 5 normal thyroid cases, and 3 hyperthyroid cases. Aberrant promoter methylation was examined in 24 tumor suppressor genes using the methylation specific multiplex ligation-dependent probe amplification (MS-MLPA) assay and in the NIS gene using methylation-specific PCR (MSP). The frequently methylated genes were CASP8 (17/21), RASSF1 (16/21) and NIS (9/21). In the normal samples, CASP8, RASSF1 and NIS were methylated in 5/5, 4/5 and 1/5 respectively. In the hyperthyroid samples, CASP8, RASSF1 and NIS were methylated in 3/3, 2/3 and 1/3 respectively. In the thyroid cancers, CASP8, RASSF1, and NIS were methylated in 9/13, 10/13, and 7/13 respectively. CASP8, RASSF1 and NIS were also methylated in concurrently present normal thyroid tissue in 3/11, 4/11 and 3/11 matched thyroid cancer cases (matched for presence of both normal thyroid tissue and thyroid cancer), respectively. Our data suggests that aberrant methylation of CASP8, RASSF1, and NIS maybe an early change in thyroid tumorigenesis regardless of cell type.

No AccessJournal of UrologyInvestigative Urology1 Jul 2013Methylation of the RARB Gene Increases Prostate Cancer Risk in Black Americans Deliang Tang, Oleksandr N. Kryvenko, Nicoleta Mitrache, Kieu C. Do, Michelle Jankowski, Dhananjay A. Chitale, Sheri Trudeau, Andrew Rundle, Steven A. Belinsky, and Benjamin A. Rybicki Deliang TangDeliang Tang Department of Environmental Health Sciences, Columbia University, New York, New York More articles by this author , Oleksandr N. KryvenkoOleksandr N. Kryvenko Department of Surgical Pathology, Henry Ford Health System, Detroit, Michigan More articles by this author , Nicoleta MitracheNicoleta Mitrache Department of Public Health Sciences, Henry Ford Health System, Detroit, Michigan More articles by this author , Kieu C. DoKieu C. Do Lung Cancer Division, Lovelace Respiratory Research Institute, Albuquerque, New Mexico More articles by this author , Michelle JankowskiMichelle Jankowski Department of Public Health Sciences, Henry Ford Health System, Detroit, Michigan More articles by this author , Dhananjay A. ChitaleDhananjay A. Chitale Department of Surgical Pathology, Henry Ford Health System, Detroit, Michigan More articles by this author , Sheri TrudeauSheri Trudeau Department of Public Health Sciences, Henry Ford Health System, Detroit, Michigan More articles by this author , Andrew RundleAndrew Rundle Department of Epidemiology, Columbia University, New York, New York Financial interest and/or other relationship with EHE International. More articles by this author , Steven A. BelinskySteven A. Belinsky Lung Cancer Division, Lovelace Respiratory Research Institute, Albuquerque, New Mexico More articles by this author , and Benjamin A. RybickiBenjamin A. Rybicki Department of Public Health Sciences, Henry Ford Health System, Detroit, Michigan More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2013.01.083AboutFull TextPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract Purpose: Gene promoter hypermethylation may be useful as a biomarker for cancer risk in histopathologically benign prostate specimens. Materials and Methods: We performed a nested case-control study of gene promoter methylation status for 5 genes (APC, RARB, CCND2, RASSF1 and MGMT) measured in benign biopsy specimens from 511 prostate cancer case-control pairs. We estimated the overall and race stratified risk of subsequent prostate cancer associated with methylation status. Results: On race stratified analysis RARB methylation was associated with a higher cancer risk in black American men (OR 2.18, 95% CI 1.39–3.44). APC methylation was associated with an increased risk of high grade tumors (OR 2.43, 95% CI 1.20–4.90), which was higher in black than in white men (OR 3.21 vs 2.04). In cases RARB and APC gene methylation in benign prostate samples persisted in matched malignant specimens. In black cases the combined risk associated with RARB and APC methylation (OR 3.04, 95% CI 1.44–6.42) was greater than the individual risk of each gene and significantly different from that in white cases (OR 1.14, 95% CI 0.56–2.30). Conclusions: RARB gene methylation in histopathologically benign prostate samples was associated with a statistically significant increased risk of subsequent prostate cancer in black men. Methylation data on additional genes may improve risk stratification and clinical decision making algorithms for cancer screening and diagnosis. References 1 : DNA methylation changes in prostate cancer: current developments and future clinical implementation. Expert Rev Mol Diagn2009; 9: 243. 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Google Scholar © 2013 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetailsCited byAndersson K (2018) This Month in Investigative UrologyJournal of Urology, VOL. 190, NO. 1, (6-7), Online publication date: 1-Jul-2013. Volume 190Issue 1July 2013Page: 317-324Supplementary Materials Advertisement Copyright & Permissions© 2013 by American Urological Association Education and Research, Inc.Keywordsprostateprostatic neoplasmsAfrican AmericansDNA methylationriskAcknowledgmentsTravis Wheeler microdissected prostate specimens. Loren Muirhead assisted with early manuscript drafts.MetricsAuthor Information Deliang Tang Department of Environmental Health Sciences, Columbia University, New York, New York More articles by this author Oleksandr N. Kryvenko Department of Surgical Pathology, Henry Ford Health System, Detroit, Michigan More articles by this author Nicoleta Mitrache Department of Public Health Sciences, Henry Ford Health System, Detroit, Michigan More articles by this author Kieu C. Do Lung Cancer Division, Lovelace Respiratory Research Institute, Albuquerque, New Mexico More articles by this author Michelle Jankowski Department of Public Health Sciences, Henry Ford Health System, Detroit, Michigan More articles by this author Dhananjay A. Chitale Department of Surgical Pathology, Henry Ford Health System, Detroit, Michigan More articles by this author Sheri Trudeau Department of Public Health Sciences, Henry Ford Health System, Detroit, Michigan More articles by this author Andrew Rundle Department of Epidemiology, Columbia University, New York, New York Financial interest and/or other relationship with EHE International. More articles by this author Steven A. Belinsky Lung Cancer Division, Lovelace Respiratory Research Institute, Albuquerque, New Mexico More articles by this author Benjamin A. Rybicki Department of Public Health Sciences, Henry Ford Health System, Detroit, Michigan More articles by this author Expand All Advertisement PDF downloadLoading ...

Sarcoidosis is a complex, multi-organ granulomatous disease with a likely genetic component. West African ancestry confers a higher risk for sarcoidosis than European ancestry. Admixture mapping provides the most direct method to locate genes that underlie such ethnic variation in disease risk. We sought to identify genetic risk variants within four previously-identified ancestry-associated regions—6p24.3–p12.1, 17p13.3–13.1, 2p13.3–q12.1, and 6q23.3–q25.2—in a sample of 2,727 African Americans. We used logistic regression fit by generalized estimating equations and the MIX score statistic to determine which variants within ancestry-associated regions were associated with risk and responsible for the admixture signal. Fine mapping was performed by imputation, based on a previous genome-wide association study; significant variants were validated by direct genotyping. Within the 6p24.3–p12.1 locus, the most significant ancestry-adjusted SNP was rs74318745 (p = 9.4*10−11), an intronic SNP within the HLA-DRA gene that did not solely explain the admixture signal, indicating the presence of more than a single risk variant within this well-established sarcoidosis risk region. The locus on chromosome 17p13.3–13.1 revealed a novel sarcoidosis risk SNP, rs6502976 (p = 9.5*10−6), within intron 5 of the gene X-linked Inhibitor of Apoptosis Associated Factor 1 (XAF1) that accounted for the majority of the admixture linkage signal. Immunohistochemical expression studies demonstrated lack of expression of XAF1 and a corresponding high level of expression of its downstream target, X-linked Inhibitor of Apoptosis (XIAP) in sarcoidosis granulomas. In conclusion, ancestry and association fine mapping revealed a novel sarcoidosis susceptibility gene, XAF1, which has not been identified by previous genome-wide association studies. Based on the known biology of the XIAP/XAF1 apoptosis pathway and the differential expression patterns of XAF1 and XIAP in sarcoidosis granulomas, we suggest that this pathway may play a role in the maintenance of sarcoidosis granulomas.

Clear cell renal cell carcinoma is by far the most common form of kidney cancer; however, a number of histologically similar tumors are now recognized and considered distinct entities. The Cancer Genome Atlas published data set was queried (http://cbioportal.org) for clear cell renal cell carcinoma tumors lacking VHL gene mutation and chromosome 3p loss, for which whole-slide images were reviewed. Of the 418 tumors in the published Cancer Genome Atlas clear cell renal cell carcinoma database, 387 had VHL mutation, copy number loss for chromosome 3p, or both (93%). Of the remaining, 27/31 had whole-slide images for review. One had 3p loss based on karyotype but not sequencing, and three demonstrated VHL promoter hypermethylation. Nine could be reclassified as distinct or emerging entities: translocation renal cell carcinoma (n=3), TCEB1 mutant renal cell carcinoma (n=3), papillary renal cell carcinoma (n=2), and clear cell papillary renal cell carcinoma (n=1). Of the remaining, 6 had other clear cell renal cell carcinoma-associated gene alterations (PBRM1, SMARCA4, BAP1, SETD2), leaving 11 specimens, including 2 high-grade or sarcomatoid renal cell carcinomas and 2 with prominent fibromuscular stroma (not TCEB1 mutant). One of the remaining tumors exhibited gain of chromosome 7 but lacked histological features of papillary renal cell carcinoma. Two tumors previously reported to harbor TFE3 gene fusions also exhibited VHL mutation, chromosome 3p loss, and morphology indistinguishable from clear cell renal cell carcinoma, the significance of which is uncertain. In summary, almost all clear cell renal cell carcinomas harbor VHL mutation, 3p copy number loss, or both. Of tumors with clear cell histology that lack these alterations, a subset can now be reclassified as other entities. Further study will determine whether additional entities exist, based on distinct genetic pathways that may have implications for treatment.

Metastasis of histologically benign chondroblastoma is a rare event. The authors report a new case, 12th in the literature, wherein multiple lung metastases appeared almost simultaneously with the primary lesion in the right talus bone.A histologic evaluation of the primary lesion in the talus and the pulmonary metastasis was performed, and an ultrastructural study of the latter was done. Published literature on metastasizing chondroblastoma was reviewed to identify any consistency in the pattern and the outcome.Metastasis of chondroblastoma is uncommon but well known. Although radiologic and histologic aggressive features have been sought, they do not necessarily correlate with the outcome.Metastasis in chondroblastoma has been insufficiently stressed in the literature, unlike metastasis in giant cell tumors. The purpose of this case report is not only to document this uncommon event (the 12th case of lung metastasis) but also to emphasize that patients with chondroblastoma may have metastasis at presentation. Hence, all patients need to be evaluated regularly from the onset for possible lung metastasis so that deposits can be detected early for total resection.

The authors analyzed 52 cases of female breast angiolipoma (AL). Age distribution was 25 to 80 years of age (56.81 ± 12.78). Most cases showed vascularity below 50%, and 14 cases had vascularity >50%. Cellular and low-vascularity ALs had different clinical and radiological presentations. The mean size was 7.00 ± 3.62 mm for cellular ALs and 19.61 ± 7.58 mm for low-vascularity ALs. In any paucicellular area, the authors could identify a cluster of at least 3 interconnected vessels. The endothelium was mostly flat with uniform, hyperchromatic nuclei, and mitoses and nucleoli were absent. Fibrin thrombi in proliferating capillaries were noted in 96% of cases. Low-vascularity AL can be reliably distinguished on needle core biopsy from other lipomatous and vascular tumors of the breast. Tortuosity and proliferation of capillaries with at least 3 interconnected capillary channels in 1 focus with associated fibrin thrombi constitute a very strong clue for the diagnosis of AL on a breast needle core biopsy. Definite diagnosis of cellular AL is not always feasible because of rare cases with mitotic activity and cellular atypia. Excision is often recommended for cellular AL.

Abstract Background Pan-cancer studies of somatic copy number alterations (SCNAs) have demonstrated common SCNA patterns across cancer types, but despite demonstrable differences in aggressiveness of some cancers by race, pan-cancer SCNA variation by race has not been explored. This study investigated a) racial differences in SCNAs in both breast and prostate cancer, b) the degree to which they are shared across cancers, and c) the impact of these shared, race-differentiated SCNAs on cancer survival. Methods Utilizing data from The Cancer Genome Atlas (TCGA), SCNAs were identified using GISTIC 2.0, and in each tumor type, differences in SCNA magnitude between African Americans (AA) and European Americans (EA) were tested using linear regression. Unsupervised hierarchical clustering of the copy number of genes residing in race-differentiated SCNAs shared between tumor types was used to identify SCNA-defined patient groups, and Cox proportional hazards regression was used to test for association between those groups and overall/progression-free survival (PFS). Results We identified SCNAs that differed by race in breast ( n = 58 SCNAs; permutation p &lt; 10 − 4 ) and prostate tumors ( n = 78 SCNAs; permutation p = 0.006). Six race-differentiated SCNAs common to breast and prostate found at chromosomes 5q11.2-q14.1, 5q15-q21.1, 8q21.11-q21.13, 8q21.3-q24.3, 11q22.3, and 13q12.3-q21.3 had consistent differences by race across both tumor types, and all six were of higher magnitude in AAs, with the chromosome 8q regions being the only amplifications. Higher magnitude copy number differences in AAs were also identified at two of these race-differentiated SCNAs in two additional hormonally-driven tumor types: endometrial (8q21.3-q24.3 and 13q12.3-q21.3) and ovarian (13q12.3-q21.3) cancers. Race differentiated SCNA-defined patient groups were significantly associated with survival differences in both cancer types, and these groups also differentiated within triple negative breast cancers based on PFS. While the frequency of the SCNA-defined patient groups differed by race, their effects on survival did not. Conclusions This study identified race-differentiated SCNAs shared by two related cancers. The association of SCNA-defined patient groups with survival demonstrates the clinical significance of combinations of these race-differentiated genomic aberrations, and the higher frequency of these alterations in AA relative to EA patients may explain racial disparities in risk of aggressive breast and prostate cancer.

Abstract Background: Telomere shortening is linked to aging and may be associated with increased risk for cancer. Most cancer studies have used telomere length in leukocytes rather than in the target tissue of cancer origin. Methods: A case–control study of 524 case–control pairs with a benign prostate biopsy nested within a historical cohort of 10,478 men was conducted to determine whether premalignant prostate telomere length (assessed using a modified qRT-PCR) is associated with prostate cancer risk. Results: Telomere lengths in benign prostate biopsies of cases versus controls were similar (1.46 ± 0.38 vs. 1.45 ± 0.42; P = 0.49). African American (AA) men had significantly shorter telomeres compared with White men (1.51 ± 0.38 vs. 1.63 ± 0.39; P &amp;lt; 0.0001). In race-stratified analyses, increasing telomere length was more strongly associated with prostate cancer risk in White men, wherein those with telomere length in the highest quartile had 1.9-fold greater adjusted risk of prostate cancer compared with men with prostate telomere lengths in the lowest quartile [OR = 1.90; 95% confidence interval (CI) = 1.08–3.36]. Men in the highest telomere length quartile also had a greater risk of aggressive prostate cancer compared with men with telomere lengths in the lowest quartile (OR = 2.78; 95% CI = 1.25–6.19). Conclusions: White men have longer telomeres in benign prostate tissue compared with AA men, and those with the longest telomeres may be at increased risk for prostate cancer, particularly the more aggressive form of the disease. Impact: Race-specific telomere length measures may be an early biomarker of aggressive prostate cancer.

Aurora-A is a mitotic kinase implicated in oncogenesis and is known to be overexpressed in B-cell lymphomas and plasma cell myeloma. The expression of Aurora-A kinase (henceforth referred to as Aurora-A) in T-cell lymphomas is not well characterized. In this study, we assessed Aurora-A expression by immunohistochemical analysis in 100 lymphomas encompassing a variety of T-cell lymphomas as categorized in the World Health Organization classification. Aurora-A expression was highest in anaplastic large-cell lymphomas and variably expressed in other types of T-cell lymphomas. In addition, the pattern of Aurora-A expression was predominantly cytoplasmic in ALK-positive anaplastic large-cell lymphoma and was nuclear in ALK-negative anaplastic large-cell lymphoma and other T-cell lymphomas, suggesting altered biochemical mechanisms of Aurora-A nuclear transport in ALK-positive anaplastic large-cell lymphoma. Reverse transcriptase-PCR analysis showed that Aurora-A is more highly expressed in ALK-positive anaplastic large-cell lymphoma than in ALK-negative anaplastic large-cell lymphoma, and is relatively lower in peripheral T-cell lymphomas. Using western blot analysis and the DEL cell line (derived from ALK-positive anaplastic large-cell lymphoma), we showed that Aurora-A expression is decreased after treatment with either MYC or MEK inhibitors, consistent with the MYC and MAP kinase signaling pathways being involved in driving Aurora-A expression; the greatest decrease was observed after MYC inhibition. These findings provide insights into the possible importance of Aurora-A overexpression in anaplastic large-cell lymphoma pathogenesis, and also suggest that Aurora-A inhibition could be a potential therapeutic approach for patients with anaplastic large-cell lymphoma.

Context.—Breast cancer treatment has greatly evolved from radical mastectomy to more cosmetically acceptable and less-debilitating surgeries. Nipple-sparing mastectomy is increasingly done for both cancer treatment and risk reduction. The frequency of terminal duct lobular units (TDLUs) and occult neoplastic epithelial proliferation in grossly/clinically unremarkable nipples (GUNs) is not well investigated. Objective.—To describe frequency of TDLUs and occult and overt neoplastic nipple involvement. Design.—Nipples from 105 consecutive specimens (90 therapeutic, 15 prophylactic) were studied. Sixty-five nipples were entirely submitted to evaluate frequency of TDLUs; the rest had 1 vertical section submitted. Results.—Terminal duct lobular unit was seen in 17 GUNs (26%). Six had TDLU in the base, 6 had it in the papilla, and 5 in both. Four GUNs showed lobular carcinoma in situ (1), Paget disease (1), and pagetoid extension of underlying malignancy (2). Grossly/clinically abnormal nipples had Paget disease (2), lymphovascular invasion (2), invasive carcinoma (4), and pagetoid extension (5). Involved nipples were closer to tumor (mean, 1.1 versus 3.2 cm, P &amp;lt; .001), had larger underlying tumors (mean, 4.3 versus 2.6 cm, P = .03) and of higher grade (P = .04), and more often had lymph node metastases (91% versus 44%, P = .007). No pathologic abnormalities were found in prophylactic mastectomy nipples. Conclusions.—Terminal duct lobular units were seen in 26% of nipples. They were frequently seen in the nipple papilla. Occult neoplastic epithelial proliferation was seen in 5% of grossly/clinically unremarkable therapeutic mastectomy nipples. Pagetoid extension was the dominant spread of underlying malignancy. Overall, the nipple was more often involved by larger and higher-grade tumors located closer to the nipple. All prophylactic mastectomies had unremarkable nipples. These findings should be considered while selecting patients for nipple-sparing mastectomy.

Abstract NF1 mutations predispose to neurofibromatosis type 1 (NF1) and women with NF1 have a moderately elevated risk for breast cancer, especially under age 50. Germline genomic analysis may better define the risk so screening and prevention can be applied to the individuals who benefit the most. Survey conducted in several neurofibromatosis clinics in the United States has demonstrated a 17.2% lifetime risk of breast cancer in women affected with NF1. Cumulated risk to age 50 is estimated to be 9.27%. For genomic profiling, fourteen women with NF1 and a history of breast cancer were recruited and underwent whole exome sequencing (WES), targeted genomic DNA based and RNA‐based analysis of the NF1 gene. Deleterious NF1 pathogenic variants were identified in each woman. Frameshift mutations because of deletion/duplication/complex rearrangement were found in 50% (7/14) of the cases, nonsense mutations in 21% (3/14), in‐frame splice mutations in 21% (3/14), and one case of missense mutation (7%, 1/14). No deleterious mutation was found in the following high/moderate‐penetrance breast cancer genes: ATM, BRCA1, BRCA2, BARD1, BRIP1, CDH1, CHEK2, FANCC, MRE11A, NBN, PALB2, PTEN, RAD50, RAD51C, TP53, and STK11 . Twenty‐five rare or common variants in cancer related genes were discovered and may have contributed to the breast cancers in these individuals. Breast cancer predisposition modifiers in women with NF1 may involve a great variety of molecular and cellular functions.

Thyroid cancer has the fastest rising incidence rates and is the fifth most common cancer in women.There are four main types of which the papillary and follicular types together account for >90%, followed by medullary cancers (3%-5%) and anaplastic carcinomas (<3%).For individuals who present with early stage disease of papillary and follicular cancers, there are no accurate markers to predict whether they will develop metastatic or recurrent disease.Our immediate goal is to molecularly differentiate follicular cancer subtypes for enhanced classification.Promoter methylation status of genes with reported associations in thyroid cancer (CASP8, CDKN2A, DAPK1, ESR1, NIS, RASSF1 and TIMP3) were examined in a cohort of follicular thyroid cancers comprising of 26 Hurthle and 27 Classic subtypes utilizing quantitative methylation-specific PCR.RASSF1 was differentially methylated in Classic tumor tissue compared to Hurthle (p<0.001).Methylation of RASSF1 pointed to racial group differences between African Americans and Caucasian Americans (p=0.05).Extra thyroidal extension was found to be associated with DAPK1 (p=0.014) and ESR1 (p=0.036)methylation.Late stage disease was associated with older age (p<0.001) and methylation of DAPK1 (p=0.034) and ESR1 (p=0.035).The methylation status of RASSF1, DAPK1 and ESR1 suggests the utility of methylation markers to molecularly differentiate thyroid cancer subtypes for enhanced classification and early detection of thyroid cancer.

Epidemiologic studies, primarily done in white men, suggest that a history of clinically-diagnosed prostatitis increases prostate cancer risk, but that histological prostate inflammation decreases risk. The relationship between a clinical history of prostatitis and histologic inflammation in terms of how these two manifestations of prostatic inflammation jointly contribute to prostate cancer risk and whether racial differences exist in this relationship is uncertain.Using a nested design within a cohort of men with benign prostate tissue specimens, we analyzed the data on both clinically-diagnosed prostatitis (NIH categories I-III) and histological inflammation in 574 prostate cancer case-control pairs (345 white, 229 African American).Clinical prostatitis was not associated with increased prostate cancer risk in the full sample, but showed a suggestive inverse association with prostate cancer in African Americans (odds ratio (OR)=0.47; 95% confidence interval (CI)=0.27-0.81). In whites, clinical prostatitis increased risk by 40%, but was only associated with a significant increased prostate cancer risk in the absence of evidence of histological inflammation (OR=3.56; 95% CI=1.15-10.99). Moreover, PSA velocity (P=0.008) and frequency of PSA testing (P=0.003) were significant modifiers of risk. Clinical prostatitis increased risk of prostate cancer almost three-fold (OR=2.97; 95% CI=1.40-6.30) in white men with low PSA velocity and about twofold in white men with more frequent PSA testing (OR=1.91; 95% CI=1.09-3.35).In our cohort of men with benign prostate specimens, race, and histological inflammation were important cofactors in the relationship between clinical prostatitis and prostate cancer. Clinical prostatitis was associated with a slightly decreased risk for prostate cancer in African American men. In white men, the relationship between clinical prostatitis and prostate cancer risk was modified by histological prostatic inflammation, PSA velocity, and frequency of PSA testing-suggesting a complex interplay between these indications of prostatic inflammation and prostate cancer detection.

Objectives/Hypothesis To obtain biological insight into keloid pathogenesis and treatment using pathway analysis of genome‐wide differentially methylated gene profiles between keloid and normal skin. Study Design Prospective cohort. Methods Genome‐wide profiling was previously done, with institutional review board approval, on six fresh keloid and six fresh normal skin tissue samples, using the Infinium HumanMethylation450 BeadChip kit. Statistically significant differentially methylated cytosine‐phosphodiester bond‐guanines (CpGs, n = 197) between keloid and normal tissue mapped to 152 genes. These genes were uploaded into Ingenuity Pathway Analysis (IPA) software to identify biological functions or regulatory networks interacting. The pathways (or “network”) with an enrichment probability value ≤ .01 were subjected to a heuristic filter of keywords associated with keloid pathogenesis. Results Of the 197 CpGs, 191 were found in the IPA database and mapped to 152 unique genes. The top 10 hypermethylated genes were ACTR3C , LRRC61 , PAQR4 , C1orf109 , SLCO2B1 , CMKLR1 , AHDC1 , FYCO1 , CCDC34 , and CACNB2 . The top 10 hypomethylated genes were GALNT3 , SCML4 , PPP1R13L , ANKRD11 , WIPF1 , MX2 , IFFO1 , DENND1C , CFH , and GHDC . IPA identified nine pathways with enrichment probability values ≤ .01, of which five (histidine degradation V1, phospholipase C signaling, colorectal cancer metastasis signaling, P2Y purinergic receptor signaling, and Gαi signaling) were associated with keloid keywords and contained “keloid genes” ( P &lt; .05). Conclusions Genes differentially methylated between keloid and normal skin reside in known bionetwork pathways involved in critical biological functioning and signaling events in the cell. This information could be used to refine screening processes for biological significance to better understand keloid pathogenesis and to develop molecular‐targeted therapy. Level of Evidence NA Laryngoscope , 127:70–78, 2017

Aims To evaluate the molecular underpinnings of the rare aggressive prostate cancer variants adenosquamous carcinoma, pleomorphic giant‐cell carcinoma, and sarcomatoid carcinoma. Methods and results We retrieved 19 tumours with one or more variant(s), and performed ERG immunohistochemistry, a next‐generation sequencing assay targeting recurrent gene fusions, and fluorescence in‐situ hybridisation (FISH) for ERG and BRAF . Divergent differentiation included: sarcomatoid carcinoma ( n = 10), adenosquamous carcinoma ( n = 7), and pleomorphic giant‐cell carcinoma ( n = 7). Five patients had more than one variant. Four had variants only in metastases. ERG rearrangement was detected in nine (47%, seven via sequencing, showing TMPRSS2 – ERG fusions and one GRHL2 – ERG fusion, and two via FISH, showing rearrangement via deletion). ERG was immunohistochemically positive in the adenocarcinoma in eight of nine (89%) patients, but was immunohistochemically positive in the variant in only five of nine patients (56%, typically decreased). One patient had a false‐positive ERG immunohistochemical result in the sarcomatoid component despite a negative FISH result. Two (11%) harboured BRAF fusions ( FAM131A – BRAF and SND1 – BRAF ). Conclusions ERG fusions are present in these rare prostate cancer variants with a frequency close to that in conventional prostate cancer (9/19, 47%). ERG immunohistochemistry usually detects rearrangement in the adenocarcinoma, but is less sensitive for the variant histology, with weak to negative staining. Adenosquamous and sarcomatoid variants can, particularly, occur together. Molecular assessment may be an additional tool in selected cases to confirm the prostatic origin of unusual tumours. The presence of two BRAF rearrangements suggests that this gene fusion may be enriched in this setting, as RAF kinase fusions have been previously reported in 1–2% of prostate cancers.

Background Perineuriomas of the gastrointestinal tract are benign neoplasms that commonly develop in the distal colon and are identified during screening colonoscopy; however, perineuriomas of the stomach are exceedingly rare and less frequently identified. Differentiating gastric perineuriomas from other more serious gastric neoplasms is critical to avoid unnecessarily aggressive treatments. Thus far, only six patients with gastric perineurioma have been described, and the molecular characterization of this entity is still lacking. Case Presentation We report a 52-year-old woman who presented with abdominal pain and gastric acid reflux and was found to have a 1.5 cm subepithelial gastric neoplasm composed of bland spindle cells displacing the gastric glands with no cytologic atypia or mitotic activity, suggesting a benign spindle cell neoplasm. Immunohistochemical analysis showed reactivity for perineurial markers glucose transporter-1 and epithelial membrane antigen, consistent with benign gastric perineurioma. DNA extracted from the tissue was used for a capture-based target sequence enrichment panel followed by Illumina next-generation sequencing and targeted bioinformatic analysis for oncogenic alterations within defined disease-associated target regions. No sequence variants in the BRAF gene were identified. Conclusions This rare case of gastric perineurioma helps solidify our understanding of how to discern various types of gastric neoplasms through traditional laboratory analysis alongside genetic sequencing approaches. Although extremely rare, gastric perineurioma should be kept in the differential diagnosis when assessing spindle cell gastric tumors to avoid unnecessary therapies, and physicians should understand the molecular characteristics of benign versus malignant tumors.

Abstract Background: FDA-approved enzalutamide is commonly prescribed for advanced prostate cancer. However, enzalutamide-resistant prostate cancer (ERPC) invariably develops, which leads to aggressive, lethal disease. Recently, we found that the Tribbles 2 (TRIB2) pseudokinase is overexpressed in ERPC cells and confers resistance to enzalutamide by promoting lineage plasticity to neuroendocrine differentiation. Though TRIB2 emerged as an excellent molecular target for ERPC, suitable inhibitors are not commercially available for effective targeting. Methods: Compounds were tested using a luciferase-tagged TRIB2 fusion protein-based assay system. Binding of drugs with TRIB2 protein was analyzed by thermal shift assays and by advanced computer-based homology modeling. Degradation of TRIB2 protein was measured by Western blot. Drug effects on re-sensitization of ERPC cells and synergy with enzalutamide were determined through cell viability and apoptosis assays. To gauge the in vivo effects of Daclatasvir (DCV), ERPC tumor xenograft-bearing mice were treated with varying doses of DCV via oral gavage. Tumor growth was calculated by measuring volumes and molecular markers in tumors were analyzed by immunohistochemistry. Results: By designing a luciferase-tagged TRIB2 fusion protein-based assay system, we screened a library of about 1,600 FDA-approved compounds and found that DCV, effectively inhibits the TRIB2-luciferase activity (more than 70% inhibition in 24 hours at 10 micromolar) but does not inhibit the activity of free luciferase. Notably, DCV directly binds to pure TRIB2 protein, leading to its destabilization and a reduction in the half-maximal melting temperature (Tm) from 41oC to 37oC, as confirmed by thermal shift assays. Interestingly, we found that DCV degrades TRIB2 proteins via activation of proteasomes and re-sensitizes ERPC cells to enzalutamide. DCV downregulates the master neuronal transcription factor, BRN2, and the stemness factor, SOX2, and synergizes with enzalutamide at lower, sub-lethal doses to decrease the viability of prostate cancer cells by inducing apoptosis. Finally, DCV was found to effectively inhibit the growth of ERPC tumors and decrease the protein level of TRIB2 in mice when delivered via oral gavage. Conclusion: These findings indicate that DCV effectively downregulates TRIB2 both in vitro and in vivo and suggest that further testing of DCV may help design an innovative therapeutic approach for management of enzalutamide-resistant, aggressive, lethal prostate cancer. Citation Format: Jitender Monga, Rohith Guddeti, Craig Rogers, Shirish Gadgeel, Dhananjay Chitale, Jagadananda Ghosh. The antiviral, Daclatasvir, downregulates Tribbles 2 pseudokinase and reverses enzalutamide resistance in prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4751.

Introduction Pancreatic tumors and cell lines derived from them exhibit elevated expression of 5-lipoxygenase (5-Lox), whereas non-tumor glands or normal cells do not exhibit this overexpression. Arachidonic acid stimulates pancreatic cancer cell growth via metabolic conversion through the 5-Lox pathway, and inhibition of 5-Lox activity decreases the viability of pancreatic cancer cells. However, the downstream signaling mechanisms through which 5-Lox exerts its effects on the survival of pancreatic cancer cells remain to be elucidated. Methods The effects of 5-Lox inhibition on cell proliferation, apoptosis, and invasive potential were investigated in pancreatic cancer cells. The protein expression was analyzed by Western blot. Apoptosis was analyzed by Annexin-V binding assay and by detecting the degradation of chromatin-DNA to nucleosomal fragments. The protein kinase C-epsilon (PKCε) activity was measured by an immunoprecipitation-kinase assay. The in vivo effects of MK591 were evaluated in pancreatic tumor xenograft model. Results MK591, a specific inhibitor of 5-Lox activity, killed pancreatic cancer cells via induction of apoptosis, involving externalization of phosphatidylserine, cleavage of PARP (poly-ADP ribose polymerase) and degradation of chromatin DNA to nucleosomes. MK591 effectively blocked in vitro invasion and soft-agar colony formation by pancreatic cancer cells and decreased pancreatic tumor growth in nude mice xenografts. Furthermore, inhibition of 5-Lox downregulated K-Ras and inhibited phosphorylation of c-Raf and ERKs. Interestingly, 5-Lox inhibition induced apoptosis in pancreatic cancer cells without the inhibition of Akt but the protein level of PKCε was dramatically downregulated. Furthermore, inhibition of 5-Lox decreased the phosphorylation of Stat3 at Serine-727. Pre-treatment of pancreatic cancer cells with peptide activators of PKCε prevented apoptosis induced by 5-Lox inhibition, suggesting that the mechanism by which 5-Lox inhibition causes cell death in pancreatic cancer involves downregulation of PKCε. The combination of low doses of MK591 and gemcitabine synergistically reduced the oncogenic phenotype and killed pancreatic cancer cells by inducing apoptosis. Discussion These findings indicate that inhibition of 5-Lox interrupts an Akt-independent, PKCε-dependent survival mechanism in pancreatic cancer cells and suggest that metabolism of arachidonic acid through the 5-Lox pathway plays an integral part in the survival of pancreatic cancer cells via signaling through PKCε, an oncogenic, pro-survival serine/threonine kinase.

<h2>Abstract</h2> We previously demonstrated a high specificity of immunohistochemistry using epidermal growth factor receptor (EGFR) mutation-specific antibodies in lung adenocarcinoma and correlation with EGFR mutation analysis. In this study, we assessed EGFR mutation status by immunohistochemistry in a variety of extrapulmonary malignancies, especially those that frequently show EGFR overexpression. Tissue microarrays containing triplicate cores of breast carcinomas (<i>n</i>=300), colorectal carcinomas (<i>n</i>=65), pancreatic adenocarcinoma (<i>n</i>=145), and uterine carcinosarcoma or malignant mixed müllerian tumors (<i>n</i>=25) were included in the study. Tissue microarray of lung adenocarcinoma with known EGFR mutation status was used as reference. Immunohistochemistry was performed using antibodies specific for the E746-A750del and L858R mutations. In pulmonary adenocarcinoma, a staining intensity of 2+ or 3+ correlates with mutation status and is therefore considered as positive. Out of 300 breast carcinomas, 293 (98%) scored 0, 5 (2%) had 1+ staining, 2 (1%) were 2+ for the L858R antibody. All breast carcinomas scored 0 with the E746-A750 antibody. All the colorectal, pancreatic carcinomas and malignant mixed müllerian tumors were negative (0) for both antibodies. Molecular analysis of the breast carcinomas that scored 2+ for L858R showed no mutation. Our results show that EGFR mutation-specific antibodies could be an additional tool distinguishing primary <i>versus</i> metastatic carcinomas in the lung. False-positivity can be seen in breast carcinoma but is extremely rare (1%).

We present the functional characterization of a pseudogene associated recurrent gene fusion in prostate cancer. The fusion gene KLK4-KLKP1 is formed by the fusion of the protein coding gene KLK4 with the noncoding pseudogene KLKP1. Screening of a cohort of 659 patients (380 Caucasian American; 250 African American, and 29 patients from other races) revealed that the KLK4-KLKP1 is expressed in about 32% of prostate cancer patients. Correlative analysis with other ETS gene fusions and SPINK1 revealed a concomitant expression pattern of KLK4-KLKP1 with ERG and a mutually exclusive expression pattern with SPINK1, ETV1, ETV4, and ETV5. Development of an antibody specific to KLK4-KLKP1 fusion protein confirmed the expression of the full-length KLK4-KLKP1 protein in prostate tissues. The in vitro and in vivo functional assays to study the oncogenic properties of KLK4-KLKP1 confirmed its role in cell proliferation, cell invasion, intravasation, and tumor formation. Presence of strong ERG and AR binding sites located at the fusion junction in KLK4-KLKP1 suggests that the fusion gene is regulated by ERG and AR. Correlative analysis of clinical data showed an association of KLK4-KLKP1 with lower preoperative PSA values and in young men (<50 years) with prostate cancer. Screening of patient urine samples showed that KLK4-KLKP1 can be detected noninvasively in urine. Taken together, we present KLK4-KLKP1 as a class of pseudogene associated fusion transcript in cancer with potential applications as a biomarker for routine screening of prostate cancer.

Abstract Background Expression profiles of erythroblast transformation‐specific (ETS)‐related gene fusions and serine protease inhibitor Kazal‐type 1 (SPINK1) in early onset prostate cancer have not been thoroughly explored. Methods We retrieved 151 radical prostatectomy specimens from young men with prostate cancer (&lt;55 years) and characterized the expression of ETS‐related gene (ERG), SPINK1, ETS Variant 1 ( ETV1 ), and ETV4 by dual immunohistochemistry and dual RNA in situ hybridization. Age, race, family history, preoperative prostate‐specific antigen, biochemical recurrence, and pathological variables using whole‐mount radical prostatectomy tissue were collected. Results A total of 313 tumor nodules from 151 men including 68 (45%) Caucasians and 61 (40%) African Americans were included in the analysis. Positive family history of prostate cancer was seen in 65 (43%) patients. Preoperative prostate‐specific antigen ranged from 0.3 to 52.7 ng/mL (mean = 7.04). The follow‐up period ranged from 1 to 123.7 months (mean = 30.3). Biochemical recurrence was encountered in 8 of 151 (5%). ERG overexpression was observed in 85 of 151 (56%) cases, followed by SPINK1 in 61 of 151 (40%), ETV1 in 9 of 149 (6%), and ETV4 in 4 of 141 (3%). There were 25 of 151 (17%) cases showing both ERG and SPINK1 overexpression within different regions of either the same tumor focus or different foci. Higher frequency of ERG overexpression was seen in younger patients (≤45 years old; 76% vs 49%, P = .002), Caucasian men (71% vs 41% P = .0007), organ‐confined tumors (64% vs 33%, P = .0008), and tumors of Gleason Grade groups 1 and 2 (62% vs 26%, P = .009). SPINK1 overexpression was more in African American men (68% vs 26%, P = .00008), in tumors with high tumor volume (&gt;20%) and with anterior located tumors. ETV1 and ETV4 demonstrated rare overexpression in these tumors, particularly in the higher‐grade tumors. Conclusion This study expands the knowledge of the clonal evolution of multifocal cancer in young patients and support differences in relation to racial background and genetics of prostate cancer.

Large-scale efforts to identify breast cancer (BC) risk alleles have historically taken place among women of European ancestry. Recently, there are new efforts to verify if these alleles increase risk in African American (AA) women as well. We investigated the effect of previously reported AA breast cancer and triple-negative breast cancer (TNBC) risk alleles in our African-enriched International Center for the Study of Breast Cancer Subtypes (ICSBCS) cohort. Using case-control, case-series and race-nested approaches, we report that the Duffy-null allele (rs2814778) is associated with TNBC risk (OR = 3.814, p = 0.001), specifically among AA individuals, after adjusting for self-indicated race and west African ancestry (OR = 3.368, p = 0.007). We have also validated the protective effect of the minor allele of the ANKLE1 missense variant rs2363956 among AA for TNBC (OR = 0.420, p = 0.005). Our results suggest that an ancestry-specific Duffy-null allele and differential prevalence of a polymorphic gene variant of ANKLE1 may play a role in TNBC breast cancer outcomes. These findings present opportunities for therapeutic potential and future studies to address race-specific differences in TNBC risk and disease outcome.

Differential classification of prostate cancer grade group (GG) 2 and 3 tumors remains challenging, likely because of the subjective quantification of the percentage of Gleason pattern 4 (%GP4). Artificial intelligence assessment of %GP4 may improve its accuracy and reproducibility and provide information for prognosis prediction. To investigate this potential, a convolutional neural network (CNN) model was trained to objectively identify and quantify Gleason pattern (GP) 3 and 4 areas, estimate %GP4, and assess whether CNN-predicted %GP4 is associated with biochemical recurrence (BCR) risk in intermediate-risk GG 2 and 3 tumors. The study was conducted in a radical prostatectomy cohort (1999-2012) of African American men from the Henry Ford Health System (Detroit, Michigan). A CNN model that could discriminate 4 tissue types (stroma, benign glands, GP3 glands, and GP4 glands) was developed using histopathologic images containing GG 1 (n = 45) and 4 (n = 20) tumor foci. The CNN model was applied to GG 2 (n = 153) and 3 (n = 62) tumors for %GP4 estimation, and Cox proportional hazard modeling was used to assess the association of %GP4 and BCR, accounting for other clinicopathologic features including GG. The CNN model achieved an overall accuracy of 86% in distinguishing the 4 tissue types. Furthermore, CNN-predicted %GP4 was significantly higher in GG 3 than in GG 2 tumors (P = 7.2 × 10-11). %GP4 was associated with an increased risk of BCR (adjusted hazard ratio, 1.09 per 10% increase in %GP4; P = .010) in GG 2 and 3 tumors. Within GG 2 tumors specifically, %GP4 was more strongly associated with BCR (adjusted hazard ratio, 1.12; P = .006). Our findings demonstrate the feasibility of CNN-predicted %GP4 estimation, which is associated with BCR risk. This objective approach could be added to the standard pathologic assessment for patients with GG 2 and 3 tumors and act as a surrogate for specialist genitourinary pathologist evaluation when such consultation is not available.

Cancer-testis (CT) antigens are expressed in a variety of malignant tumors, but in normal adult tissue, they are only expressed in testicular germ cells. Owing to this tumor-associated expression pattern, these antigens are of major interest as potential targets for immunotherapy and possibly for diagnostic purposes. This study was performed to analyze the expression of four CT antigens, NY-ESO-1, MAGE-A3, MAGE-A4, and CT7/MAGE-C1, in endometrial carcinoma using immunohistochemistry, and to correlate expression with histologic subtypes, grade, and expression of WT1 and p53. Formalin-fixed paraffin-embedded tissues of 130 endometrial carcinomas of the following types and grades were analyzed using a tissue microarray: 85 endometrioid carcinomas (FIGO grade 1, 39; grade 2, 11; and grade 3, 35), 18 papillary serous carcinomas, 12 clear cell carcinomas, 13 malignant mixed mullerian tumors, one mucinous adenocarcinoma, and one undifferentiated carcinoma. The following anti-CT monoclonal antibodies/antigens were studied by immunohistochemistry: monoclonal antibody ES121/NY-ESO-1, monoclonal antibody M3H67/MAGE-A3, monoclonal antibody 57B/MAGE-A4, and monoclonal antibody CT7-33/CT7. The CT expression data were compared to WT1 and p53 protein expression as analyzed in a previous study. Positive staining with anti-CT monoclonal antibodies was graded as follows: focal, <5% positive cells; 1+, 5-25% cells; 2+, 26-50% cells; 3+, 51-75%; and 4+, >75% cells. The 3+ and 4+ staining patterns were considered homogeneous patterns of potential clinical significance and were scored positive for statistical analysis. In low-grade tumors, the most immunoreactivity was seen with mAb M3H67 but little labeling was observed with the other monoclonal antibodies. In high-grade tumors, monoclonal antibodies M3H67 (25%), 57B (23%), and CT7-33 (20%) showed the highest reactivity, while ES121 showed the lowest immunoreactivity (6%). The staining pattern was mostly heterogeneous. Statistical significance was found solely for the correlation of monoclonal antibody 57B staining and p53 expression. No correlation was found for any anti-CT monoclonal antibody staining and clinical stage or for anti-CT staining and WT1 expression. CT antigens CT7, MAGE-A3 and MAGE-A4, but not NY-ESO-1, are expressed in high-grade endometrial carcinomas, and expression of MAGE-A4 is correlated with the presence of overexpressed p53.

Accurate and timely molecular test results play an important role in patient management; consequently, there is a customer expectation of short testing turnaround times. Baseline data analysis revealed that the greatest challenge to timely result generation occurred in the preanalytic phase of specimen collection and transport. Here, we describe our efforts to improve molecular testing turnaround times by focusing primarily on redesign of preanalytic processes using the principles of LEAN production. Our goal was to complete greater than 90% of the molecular tests in less than 3 days. The project required cooperation from different laboratory disciplines as well as individuals outside of the laboratory. The redesigned processes involved defining and standardizing the protocols and approaching blood and tissue specimens as analytes for molecular testing. The LEAN process resulted in fewer steps, approaching the ideal of a one-piece flow for specimens through collection/retrieval, transport, and different aspects of the testing process. The outcome of introducing the LEAN process has been a 44% reduction in molecular test turnaround time for tissue specimens, from an average of 2.7 to 1.5 days. In addition, extending LEAN work principles to the clinician suppliers has resulted in a markedly increased number of properly collected and shipped blood specimens (from 50 to 87%). These continuous quality improvements were accomplished by empowered workers in a blame-free environment and are now being sustained with minimal management involvement. Accurate and timely molecular test results play an important role in patient management; consequently, there is a customer expectation of short testing turnaround times. Baseline data analysis revealed that the greatest challenge to timely result generation occurred in the preanalytic phase of specimen collection and transport. Here, we describe our efforts to improve molecular testing turnaround times by focusing primarily on redesign of preanalytic processes using the principles of LEAN production. Our goal was to complete greater than 90% of the molecular tests in less than 3 days. The project required cooperation from different laboratory disciplines as well as individuals outside of the laboratory. The redesigned processes involved defining and standardizing the protocols and approaching blood and tissue specimens as analytes for molecular testing. The LEAN process resulted in fewer steps, approaching the ideal of a one-piece flow for specimens through collection/retrieval, transport, and different aspects of the testing process. The outcome of introducing the LEAN process has been a 44% reduction in molecular test turnaround time for tissue specimens, from an average of 2.7 to 1.5 days. In addition, extending LEAN work principles to the clinician suppliers has resulted in a markedly increased number of properly collected and shipped blood specimens (from 50 to 87%). These continuous quality improvements were accomplished by empowered workers in a blame-free environment and are now being sustained with minimal management involvement. Molecular diagnostic laboratories, just as for other areas of pathology, face challenges associated with increasing testing volumes, decreasing reimbursement, and maintaining and improving quality levels. Diagnostic accuracy is crucial in pathology; nucleic acid-based diagnostic test results are often important for subsequent therapeutic decision making. Accurate and timely molecular testing can add a great deal of value to total patient management. Specimen types such as peripheral blood, bone marrow aspirates, and formalin-fixed, paraffin-embedded (FFPE) tissue, are routinely evaluated using molecular techniques. For tissue-based nucleic acid assays to enter a clinical setting, nucleic acids must be obtainable through current practices of diagnostic pathology. This might involve dealing with individuals who are based at off-site locations, have different priorities, and often have very little understanding of molecular testing requirements. Finally, the isolation of nucleic acids from FFPE tissue, which makes it possible to bring molecular testing to surgical pathology, requires close collaboration between molecular and histology personnel. For accurate and reliable test results, FFPE tissue must be handled in a standardized fashion, similar to how blood and other body fluids are used in routine clinical assays. Furthermore, it is important for individuals doing molecular testing on blood samples collected at different locations to understand the factors outside of their laboratories or sphere of influence. All of these factors might require molecular laboratory personnel to collaborate and become intimately involved with the education of different customer and supplier groups involved in the preanalytic and sometimes postanalytic phases of the testing cycle. This way, roles and boundaries of responsibility pertaining to each group become well defined and the expertise of each group can be used in the most efficient way. Issues with the preanalytic phase of the testing cycle in particular are not unique to molecular laboratories. Other studies have shown that many laboratory errors occur during the preanalytic phase. These usually consist of all activities leading up to actual analysis of the specimen.1Bonini P Plebani M Ceriotti F Rubboli F Errors in laboratory medicine.Clin Chem. 2002; 48: 691-698PubMed Google Scholar2Lippi G Salvagno GL Montagnana M Franchini M Guidi GS Phlebotomy issues and quality improvement in results of laboratory testing.Clin Lab. 2006; 52: 217-230PubMed Google Scholar3Persoon TJ Zaleski S Frerichs J Improving preanalytic processes using the principles of lean production (Toyota Production System).Am J Clin Pathol. 2006; 125: 16-25Crossref PubMed Scopus (45) Google Scholar In 2006 Plebani4Plebani M Errors in clinical laboratories or errors in laboratory medicine?.Clin Chem Lab Med. 2006; 44: 750-759PubMed Google Scholar reported that defects in specimen adequacy occurred most often, with more than 60% of preanalytic errors involving inadequate quantity or unacceptable quality of specimen. Causes of unacceptable quality included collection in the wrong container, improper collection procedure, and improper storage and transportation techniques. Preanalytic factors during collection, processing, and storage of blood specimens may affect DNA and RNA quality and their subsequent use as biomarkers.5Holland NT Smith MT Eskenazi B Bastaki M Biological sample collection and processing for molecular epidemiological studies.Mutat Res. 2003; 543: 217-234Crossref PubMed Scopus (209) Google Scholar6Baechler EC Batliwalla FM Karypis G Gaffney PM Moser K Ortmann WA Espe KJ Balasubramanian S Hughes KM Chan JP Begovich A Chang S-YP Gregersen PK Behrens TW Expression levels for many genes in human peripheral blood cells are highly sensitive to ex vivo incubation.Genes Immun. 2004; 5: 347-353Crossref PubMed Scopus (130) Google Scholar7Pahl A Brune K Stabilization of gene expression profiles in blood after phlebotomy.Clin Chem. 2002; 48: 2251-2253PubMed Google Scholar In terms of FFPE tissues, factors such as fixation and storage can also affect quality of specimens,8Srinivasan M Sedmak D Jewell S Effect of fixatives and tissue processing on the content and integrity of nucleic acids.Am J Pathol. 2002; 161: 1961-1971Abstract Full Text Full Text PDF PubMed Scopus (943) Google Scholar as can preanalytic tissue processing.9Cronin M Pho M Dutta D Stephans JC Shark S Kiefer MC Esteban JM Baker JB Measurement of gene expression in archival paraffin-embedded tissues: development and performance of a 92-gene reverse transcriptase-polymerase chain reaction assay.Am J Pathol. 2004; 164: 35-42Abstract Full Text Full Text PDF PubMed Scopus (473) Google Scholar To streamline overall laboratory services at our institution a continuous quality improvement initiative was implemented in early 2006 as the Henry Ford Production System (HFPS).10Zarbo RJ D'Angelo R Transforming to a quality culture: the Henry Ford Production System.Am J Clin Pathol. 2006; 126: S21-S29Google Scholar This approach to quality improvement was initially adopted in the various sections of the surgical pathology laboratory at Henry Ford Hospital but now is practiced as LEAN management by more than 500 anatomical and clinical pathology employees at Henry Ford Health System. The encompassing goal is to streamline work processes of the pathology department so they are analogous to manufacturing processes (in the creation of value in its work product). Therefore, our pathology laboratory benchmarked the continuous process improvement disciplines of the very successful Toyota LEAN Production System11Ohno T Toyota Production System: Beyond Large Scale Production. Productivity Press, Portland1988Google Scholar as well as those of Henry Ford.12Ford H Today and Tomorrow. Doubleday, New York1926Google Scholar Our laboratory-based quality effort melded a cultural transformation of management's role and the employees' work approach to go beyond simple approaches of leaning out operations, with the aim of reducing commonly encountered defects and waste.13Zarbo RJ D'Angelo R The Henry Ford Production System: effective reduction of process defects and waste in surgical pathology.Am J Clin Pathol. 2007; 128: 1015-1022Crossref PubMed Scopus (53) Google Scholar,14D'Angelo R Zarbo RJ The Henry Ford Production System: measures of process defects and waste in surgical pathology as a basis for quality improvement initiatives.Am J Clin Pathol. 2007; 128: 423-429Crossref PubMed Scopus (51) Google Scholar The chief focus of LEAN is a continuous effort to eliminate process defects and waste while improving practice efficiency. Baseline data analysis in our laboratory revealed that the greatest challenge for timely molecular test result generation was defects that occurred during the preanalytic phase of specimen collection and transport. We have defined defects to measure waste and reworked imperfections in product requirements including discrepancies in flow or deficiencies in specimen collection and processing resulting in a bottleneck. These defects caused work to be delayed, stopped, or returned to the sender, but once defects and waste were so defined, processes were modified and standardized in accordance with our own HFPS principles. Two types of specimens are currently used in the molecular laboratory: formalin-fixed, paraffin-embedded tissue, which is processed and archived in histology, and blood and bone marrow aspirate specimens, which are collected at Henry Ford Hospital, associated hospitals, and regional medical centers. In this study we share our experience with the participation of the molecular pathology laboratory in the Henry Ford Production System as a framework for implementation of LEAN process redesign. Through a blameless work environment and contributions from all workers, we undertook the process of eliminating non-value-added waste and standardizing the process of electronic test ordering, of reporting, and of specimen collection, triaging, and transport, all of which contributed to an increase in overall efficiency and greatly reduced testing turnaround times (TATs). Before implementation of HFPS, different divisions and sections of pathology operated as independent units. No formal channels of communication were in place such as electronic test ordering or team meetings between departments. Any communication that did occur between groups tended to focus on fault finding, blame, and denial, rather than on using a team effort for reaching common goals successfully. Work performed in different sections lacked a method of standardization, which added to overall testing delays and waste. Our molecular laboratory is centrally located and is in close proximity to the histology laboratory. Despite being a part of the pathology department, the molecular laboratory remained isolated from the rest of the anatomical pathology laboratory because of its focus on nucleic acid-based testing rather than total tissue-based testing. Working in the molecular laboratory requires a highly specialized set of skills. Frequently, individuals who have mastered molecular biology techniques have little familiarity with important histology-based tissue processing techniques such as tissue fixation, paraffin embedding, sectioning on a microtome, and staining. To incorporate FFPE specimens into the molecular work flow, the choice was either to train molecular personnel in basic tissue processing or to rely on the expertise of histology personnel to prepare tissue sections. The histology laboratory is a large and very busy laboratory that already handles multiple requests for tissue processing. Having to take on the additional burden of training molecular laboratory personnel was seen as just creating more inefficiency, so the histology personnel reluctantly chose to take on the responsibility of providing tissue sections for molecular testing. At baseline, molecular testing was perceived by histology as “research” and was assigned a very low priority in regard to other forms of testing. The process for molecular testing went as follows. A pathologist would be sitting in his or her office (in a section approximately 500 feet from the molecular laboratory) reviewing and signing out cases. If gene rearrangement testing was needed, for example, the pathologist would have to stop his or her work and carry a written request to the molecular laboratory. A busy pathologist might have preferred to wait until the end of the day or the following morning to submit that request. Someone from the laboratory would then have to walk over to the histology area (approximately 100 feet away) and manually enter the request in histology's “recut log.” If the pathologist forgot to specify which tissue block was needed for testing, the molecular technologists then had to contact the pathologist to clarify the order, thus causing an additional delay. Once the correct request was entered in the recut log in histology, it took several days until someone “got around” to it. In addition, the tissue sections submitted by histology were not always appropriate for molecular testing, necessitating more walking back and forth. The testing process itself took 1 to 2 business days to complete, yet the total testing cycle, from request to results being available for signing out, might have taken as many as 6 business days. Blood and bone marrow specimens are routinely submitted to the molecular laboratory by clinician customers, primarily for hematology-based tests. A lack of standardization in this process was unveiled as well, as delays in specimen delivery to the laboratory occurred. At Henry Ford Hospital patients are routinely seen at the hospital's hematology/oncology clinic and at oncology clinics at several off-site locations. Phlebotomy is performed by nursing personnel, who at the baseline were uneducated in molecular testing. Most specimens would either be collected and shipped incorrectly or directed to wrong laboratory areas. Frequently blood was collected into a wrong collection tube or was transported and stored at a wrong temperature. Storage temperature has been shown to affect quality of extracted nucleic acids, especially RNA.15Kim SJ Dix DJ Thompson KE Murrell RN Schmid JE Gallagher JE Rockett JC Effects of storage. RNA extraction, genechip type, and donor sex on gene expression profiling of human whole blood.Clin Chem. 2007; 53: 1038-1045Crossref PubMed Scopus (46) Google Scholar,16Tanner MAS Berk LS Felten DL Blidy AD Bit SL Ruff DW Substantial changes in gene expression level due to the storage temperature and storage duration of human whole blood.Clin Lab Haematol. 2002; 24: 337-341Crossref PubMed Scopus (61) Google Scholar DNA is more stable, but storage at room temperature for more than 3 days affects specimen quality (unpublished data). Prolonged storage at room temperature occurred when specimens were directed to wrong laboratory sections. It generally took several days for them to be re-routed to the molecular laboratory. Suboptimal specimen quality necessitated repeating the tests or, when the quality was unacceptable, calling patients back to the clinic for recollection. Having to ask a patient to return to the clinic for re-collection because a blood specimen was rejected by the laboratory caused considerable frustration, cost, and inconvenience. The patient, who might have been in poor health to begin with, had to arrange for transportation, travel a distance of up to 100 miles round trip, endure another sample extraction, and spend time waiting at the clinic. Delayed test results further affected patient care either because of delayed diagnosis or delayed treatment. Customer-supplier meetings were introduced by HFPS team members as an effort to “hear the voice of the customer.” These meetings proved to be instrumental as collaborative intra- and interdepartmental efforts to assist in defining problems, increasing accountability, determining root causes, brainstorming solutions, and eventually implementing our efforts. Weekly customer-supplier meetings were undertaken with the mission of congregating workers to discuss their expectations and customer requirements as product was produced and passed from one work cell to another.10Zarbo RJ D'Angelo R Transforming to a quality culture: the Henry Ford Production System.Am J Clin Pathol. 2006; 126: S21-S29Google Scholar The goal was to create highly specified requirements to aid in direct handoffs between customers and suppliers so that the main types of waste in processes could be eliminated. Molecular pathology personnel were educated in HFPS principles in May 2006. The group also participated in a Saturday 5S exercise to reorganize the physical aspects of the laboratory.10Zarbo RJ D'Angelo R Transforming to a quality culture: the Henry Ford Production System.Am J Clin Pathol. 2006; 126: S21-S29Google Scholar This consisted of applying the 5S concepts of Sort, Stabilize, Shine, Standardize, and Sustain to clean, eliminate non-value added equipment and supplies, and organize and label what remained so that the changes could be sustained. This weekend event was the first opportunity for the individuals in the molecular laboratory to interact informally with personnel from other areas of pathology. The camaraderie generated during this informal exercise was subsequently carried over into the customer-supplier meetings and different phases of the LEAN process improvement, making the change less challenging. Although a minority of individuals viewed the process at first as “a lot of extra work” and change that distracted them away from their main goals (sample processing and testing), a gradual acceptance of the necessity of continuous improvement developed, as increases in efficiency became the most strategic, forward thinking plan. Focused communication between the histology and molecular groups brought to light reasons for delayed tissue sectioning. Histology suppliers explained that tissue sectioning for molecular testing (cutting tissue ribbons and cutting thicker sections on charged or uncharged slides) was different from the routine protocols of histology. Only a few histotechnologists felt comfortable doing these tasks, and each approached the process a bit differently. And because molecular requests were seen as “low priority,” there never seemed to be enough time for these highly sought after individuals to do “additional work.” Once the LEAN process had been implemented in histology, the entire team began thinking more creatively. Histology personnel became educated about molecular testing and its importance in patient care. That resulted in increased effort to process molecular section requests in a timely manner. It is well known that fixation and sectioning of the tissue block can impair the recovery of nucleic acids.8Srinivasan M Sedmak D Jewell S Effect of fixatives and tissue processing on the content and integrity of nucleic acids.Am J Pathol. 2002; 161: 1961-1971Abstract Full Text Full Text PDF PubMed Scopus (943) Google Scholar,17Hewitt SM Lewis FA Cao Y Conrad RC Cronin M Danenberg KD Goralski TJ Langmore JP Raja RG Williams PM Palma JF Warrington JA Tissue handling and specimen preparation in surgical pathology—issues concerning the recovery of nucleic acids from formalin-fixed, paraffin-embedded tissue.Arch Pathol Lab Med. 2008; 132: 1929-1935PubMed Google Scholar,18Medeiros F Rigl CT Anderson GG Becker SH Halling KC Tissue handling for genome-wide expression analysis: a review of the issues, evidence and opportunities.Arch Pathol Lab Med. 2007; 131: 1805-1816PubMed Google Scholar Furthermore, contamination of specimens with a different tissue source could occur and has been reported.19Bernsen MR Dijkman HB de Vries E Figdor CG Ruiter DJ Adema GJ van Muijen GN Identification of multiple mRNA and DNA sequences from small tissue samples isolated by laser-assisted microdissection.Lab Invest. 1998; 78: 1267-1273PubMed Google Scholar Education of histology personnel was undertaken to prevent misunderstandings and ensure correct processing of molecular requests. On a suggestion from the leaders of the histology group, the molecular laboratory director provided an informal lecture and a clear set of instructions on how FFPE tissue blocks needed to be handled to avoid contamination and maintain nuclease-free conditions. These efforts were so well received that over a period of 1 week a “Molecular Tissue Processing” procedure was implemented in histology and everyone in the laboratory was trained. For training purposes (and as a quick reference) the molecular laboratory also provided a chart listing all of the tests performed, names that would show up on the electronic log, and types of sections required for each test (Table 1). This chart is now posted in the general histology cutting area and can quickly be consulted as a visual aid for histology personnel when they are unsure about the electronic section requests (eg, 10 μm sections, 4 μm sections on uncharged slides, one set of sections for two tests, and others).Table 1Histology Protocol: Tissue Sectioning Requirements for Molecular TestingAP Molecular specimen cuttingTest-curls → 5–6 curls at 10 μm into tubes providedTest-slides → 5 blanks on uncharged slides (no oven) Bcell If both ordered, cut once only Tcell MGMT If both ordered, cut once only LOH EGFRvIII KRAS MLH1 EGFR MSI IDFinished specimens go to Molecular Lab or into Molecular box in Histology Lab Open table in a new tab Other process improvements were being implemented simultaneously throughout pathology,13Zarbo RJ D'Angelo R The Henry Ford Production System: effective reduction of process defects and waste in surgical pathology.Am J Clin Pathol. 2007; 128: 1015-1022Crossref PubMed Scopus (53) Google Scholar,14D'Angelo R Zarbo RJ The Henry Ford Production System: measures of process defects and waste in surgical pathology as a basis for quality improvement initiatives.Am J Clin Pathol. 2007; 128: 423-429Crossref PubMed Scopus (51) Google Scholar which had a “ripple” effect on the molecular testing process. A very significant improvement was made possible when the pathology informatics group developed a system for electronic test requests and result reporting. With some modifications, this system was expanded to cover the molecular laboratory. The newly created electronic pathways eliminated the need for the pathologists to walk to the molecular laboratory with a test request and for molecular personnel to walk to histology to manually log in the tissue sectioning request. Orders for sectioning of the blocks could be entered electronically by the pathologist while he or she was in the process of reviewing a case and thus the right block could be selected from the start. Implementation of the LEAN processes and standardization of tissue processing in the histology laboratory resulted in greater efficiency in that area, a dramatic reduction in tissue sectioning delays, and a better leveling of workload for all types of tissue specimens. As a result, “one piece” work flow was made possible, which resulted in molecular tissue sections being cut through the day. This meant that the molecular laboratory was now able to start nucleic acid extractions from tissue sections shortly after the request for testing was submitted or at the latest the following morning (if requests were received late in the day). The combination of these streamlined processes in the preanalytic phase significantly reduced the waiting, extra steps, and needs for recuts, which in the past might have accounted for 2 to 3 days of wasted time in the total testing cycle. The histology protocol for how FFPE tissue blocks need to be handled for molecular testing to achieve optimal DNA or RNA recovery is now very specific. For isolation of nucleic acids, fresh sections are cut from blocks with a new microtome blade after general cleaning of the microtome with sodium hypochlorite solution and 100% ethanol to avoid any potential issues with contamination. During tissue sectioning, the first section of the block is discarded and disposable microtome blades are replaced when different blocks are sectioned. When microdissection is not needed because the block contains mostly tumor tissue, paraffin sections are cut as ribbons and are placed directly into a microcentrifuge tube. When some form of microdissection is required because of focal involvement, the sections are applied to a solid support, such as a glass slide, typically by floating the tissue in a water bath to obtain an unwrinkled section. To avoid contamination, water baths must be emptied and cleaned between processing of samples from different individuals. Storage of unstained microscope slides can be problematic as precut sections can degrade over a relatively short period of time.20Fergenbaum JH Garcia-Closas M Hewitt SM Lissowska J Sakoda LC Sherman ME Loss of antigenicity in stored sections of breast cancer tissue microarrays.Cancer Epidemiol Biomarkers Prev. 2004; 13: 667-672PubMed Google Scholar,21Jacobs TW Prioleau JE Stillman IE Schnitt SJ Loss of tumor marker immunostaining intensity on stored paraffin slides of breast cancer.J Natl Cancer Inst. 1996; 88: 1054-1059Crossref PubMed Scopus (232) Google Scholar When microdissection is to be performed, sections are cut just before testing and immediately delivered to the molecular laboratory. As soon as the sections are received the molecular laboratory initiates isolation of DNA or RNA. Some tissue blocks are considered to be suboptimal from the start because of their very small size or extensive necrosis. Histotechnologists are the experts in tissue processing and can quickly judge how to approach cutting sections from very small specimens, such as skin biopsies, or from necrotic tissues. Occasionally tissue blocks from very small specimens need to be re-embedded in paraffin before sectioning, which is accomplished quickly and efficiently by a histotechnologist. When immunostains are ordered on the same block, it is a general practice to cut those sections first. More tissue is needed for most molecular tests (five to six sections at 10-μm thickness compared with 4-μm thickness for immunostains). When a small biopsy specimen is involved, the tissue block will be quickly depleted and the area of tumor involvement may even be lost. Yet there are instances for which molecular testing is deemed more valuable than immunostaining. When one is dealing with very small biopsy specimens, it is always a good idea to consult the case pathologist about establishing the cutting order. Total test times and individual steps were compared during three time periods (Figure 1). We listed individual steps and measured average processing times needed to complete the entire testing cycle (test ordering to result sign out) for each phase. Phase I (wasteful phase): Baseline measurements were defined and customer-supplier relationships were established. The time period was January to December 2005, and 158 specimens were included in the study (61 B-cell gene rearrangement and 98 T-cell gene rearrangement). Phase II (leaner phase): LEAN process improvements were implemented across different disciplines. Measurement of efficacy of the process was done for 3 months, June to August 2006, and 116 specimens were included in the study (40 B-cell gene rearrangement, 19 T-cell gene rearrangement, and 57 MGMT promoter methylation). Phase III (leanest phase): Several “Rapid Process Improvement” modifications were added to the initial LEAN work flow to further increase efficiency. Measurement of efficacy of the process was done from January to December 2007, and 338 specimens were included in the study (92 B-cell gene rearrangement, 145 T-cell gene rearrangement, and 101 MGMT promoter methylation). The total testing cycle at the baseline (time the pathologist realizes the testing is needed to the time the test results are generated in the molecular laboratory) took 33.5 to 145 hours (1.4 to 6.0 days), average 64.8 hours (2.7 days). Although the analytical phase itself remained consistent at 24 to 48 hours (1 to 2 days), the rest of the time was wasted on hand-offs between different sections of the laboratory (Figure 1, top row). In phase II electronic test ordering and reporting became available, and LEAN process improvement was implemented in histology (Figure 1, middle row). We estimate that just the use of an electronic ordering and reporting system alone cut hours and sometimes even 1 to 2 days from the total testing cycle. In phase III additional posted visual aids were created for histology personnel to reduce misunderstanding of te

Compelling evidence about the differences in the biology and behavior of invasive breast cancer between African-American (AA) and White-American (WA) women motivate inquiry into comparing the clinicopathology of non-invasive breast cancer (ductal carcinoma in situ, DCIS). AA and WA women diagnosed with their first primary DCIS between 1990 and 1999 were identified from the institutional tumor registry. Data on method of presentation, treatment, and patient characteristics were retrieved from electronic medical records. Patients were followed up through the medical records until the diagnosis of a subsequent cancer or the last day of contact with the institution. A total of 100 (29.6%) AAs and 236 (70.4%) WAs with the mean age of 60 (SD ± 13) and 57 (SD ± 12), respectively, contributed to this study. DCIS was detected during routine screening mammography for 81% (n = 81) of AAs and 88.4% (n = 206) of WAs (P = 0.073). Differences in the distributions of grade, margin status, necrosis, or treatment modalities were not statistically significant between AAs and WAs. Analysis of competing risks Cox proportional hazard multivariate modeling yielded a significant 8-year cumulative risk of a second cancer for AAs but only in the ipsilateral breast (HR = 3.96, 95% CI 1.42–11.04, P = 0.01). Despite comparable clinical presentation and treatment, 8 years after the initial treatment, AAs experienced a higher risk of second breast cancer in ipsilateral but not in the contralateral breast. The observed excess risk of a second cancer in the ipsilateral breast may suggest of intrinsic differences in the biology of cancer.

Formalin-fixed paraffin-embedded (FFPE) specimens, when used in conjunction with patient clinical data history, represent an invaluable resource for molecular studies of cancer. Even though nucleic acids extracted from archived FFPE tissues are degraded, their molecular analysis has become possible. In this study, we optimized a laboratory-based next-generation sequencing barcoded cDNA library preparation protocol for analysis of small RNAs recovered from archived FFPE tissues. Using matched fresh and FFPE specimens, we evaluated the robustness and reproducibility of our optimized approach, as well as its applicability to archived clinical specimens stored for up to 35 years. We then evaluated this cDNA library preparation protocol by performing a miRNA expression analysis of archived breast ductal carcinoma in situ (DCIS) specimens, selected for their relation to the risk of subsequent breast cancer development and obtained from six different institutions. Our analyses identified six miRNAs (miR-29a, miR-221, miR-375, miR-184, miR-363, miR-455-5p) differentially expressed between DCIS lesions from women who subsequently developed an invasive breast cancer (cases) and women who did not develop invasive breast cancer within the same time interval (control). Our thorough evaluation and application of this laboratory-based miRNA sequencing analysis indicates that the preparation of small RNA cDNA libraries can reliably be performed on older, archived, clinically-classified specimens.

<h3>Importance</h3> Compared with white American (WA) women, African American (AA) women have a 2-fold higher incidence of breast cancers that are negative for estrogen receptor, progesterone receptor, and<i>ERBB2</i>(triple-negative breast cancer [TNBC]). Triple-negative breast cancer, compared with non-TNBC, likely arises from different pathogenetic pathways, and benign breast disease (BBD) predicts future non-TNBC. <h3>Objective</h3> To determine whether AA identity remains associated with TNBC for women with a prior diagnosis of BBD. <h3>Design, Setting, and Participants</h3> This study is a retrospective analysis of data of a cohort of 2588 AA and 3566 WA women aged between 40 and 70 years with a biopsy-proven BBD diagnosis. The data—obtained from the Pathology Information System of Henry Ford Health System (HFHS), an integrated multihospital and multispecialty health care system headquartered in Detroit, Michigan—include specimens of biopsies performed between January 1, 1994, and December 31, 2005. Data analysis was performed from November 1, 2015, to June 15, 2016. <h3>Main Outcomes and Measures</h3> Subsequent breast cancer was stratified on the basis of combinations of hormone receptor and<i>ERBB2</i>expression. <h3>Results</h3> Case management, follow-up, and outcomes received or obtained by our cohort of 2588 AA and 3566 WA patients were similar, demonstrating that HFHS delivered care equitably. Subsequent breast cancers developed in 103 (4.1%) of AA patients (mean follow-up interval of 6.8 years) and 143 (4.0%) of WA patients (mean follow-up interval of 6.1 years). More than three-quarters of subsequent breast cancers in each subset were ductal carcinoma in situ or stage I. The 10-year probability estimate for developing TNBC was 0.56% (95% CI, 0.32%-1.0%) for AA patients and 0.25% (95% CI, 0.12%-0.53%) for WA patients. Among the 66 AA patients who developed subsequent invasive breast cancer, 16 (24.2%) developed TNBC compared with 7 (7.4%) of the 94 WA patients who developed subsequent invasive breast cancers and had complete biomarker data (<i>P</i> = .01). <h3>Conclusions and Relevance</h3> This study is the largest analysis to date of TNBC in the context of racial/ethnic identity and BBD as risk factors. The study found that AA identity persisted as a significant risk factor for TNBC. This finding suggests that AA identity is associated with inherent susceptibility for TNBC pathogenetic pathways.

Pseudosarcomatous myofibroblastic proliferations of the genitourinary tract have a debatable relationship with inflammatory myofibroblastic tumour (generally lacking ALK rearrangement); however, they share several overlapping features with nodular fasciitis of soft tissue. As rearrangement of the USP6 gene has been recently recognised as a recurrent alteration in soft tissue nodular fasciitis, and several other alternative gene fusions have been recently recognised in inflammatory myofibroblastic tumour, the aim of this study was to investigate whether USP6, ROS1 or ETV6 rearrangements were present in these lesions (12 cases).Fluorescence in-situ hybridisation analysis was performed by the use of bacterial artificial chromosome-derived break-apart probes against USP6, ROS1, and ETV6. Two cases with adequate genetic material from recent paraffin tissue blocks were also tested by use of a solid tumour gene fusion detection assay via next-generation sequencing, targeting >50 known genes involved in recurrent fusions. None of the genitourinary pseudosarcomatous myofibroblastic proliferations was found to harbour USP6 (0/12), ROS1 (0/8) or ETV6 (0/7) rearrangements, and no gene fusions were detected in two cases studied by sequencing.Despite overlap in histological and immunohistochemical features between pseudosarcomatous myofibroblastic proliferation and nodular fasciitis, these tumours lack the recently recognised USP6 rearrangements that occur in nodular fasciitis, as well as alternative fusions found in ALK-negative inflammatory myofibroblastic tumours. At present, this diagnosis remains based primarily on clinical, histological and immunohistochemical features.

Abstract M2 (tumor-supportive) macrophages may upregulate growth differentiation factor 15 (GDF15), which is highly expressed in prostate tumors, but the combined utility of these markers as prognostic biomarkers are unclear. We retrospectively studied 90 prostate cancer cases that underwent radical prostatectomy as their primary treatment and were followed for biochemical recurrence (BCR). These cases also had a benign prostate biopsy at least 1 year or more before their prostate cancer surgery. Using computer algorithms to analyze digitalized immunohistochemically stained slides, GDF15 expression and the presence of M2 macrophages based on the relative density of CD204- and CD68-positive macrophages were measured in prostate: (i) benign biopsy, (ii) cancer and (iii) tumor-adjacent benign (TAB) tissue. Both M2 macrophages (P = 0.0004) and GDF15 (P &amp;lt; 0.0001) showed significant inter-region expression differences. Based on a Cox proportional hazards model, GDF15 expression was not associated with BCR but, in men where GDF15 expression differences between cancer and TAB were highest, the risk of BCR was significantly reduced (hazard ratio = 0.26; 95% confidence interval = 0.09–0.94). In addition, cases with high levels of M2 macrophages in prostate cancer had almost a 5-fold increased risk of BCR (P = 0.01). Expression of GDF15 in prostate TAB was associated with M2 macrophage levels in both prostate cancer and TAB and appeared to moderate M2-macrophage-associated BCR risk. In summary, the relationship of GDF15 expression and CD204-positive M2 macrophage levels is different in a prostate tumor environment compared with an earlier benign biopsy and, collectively, these markers may predict aggressive disease.

ABSTRACT Growth and differentiation factor 15 (GDF‐15), also known as macrophage inhibitory cytokine 1 (MIC‐1), may act as both a tumor suppressor and promotor and, by regulating NF‐κB and macrophage signaling, promote early prostate carcinogenesis. To determine whether expression of these two inflammation‐related proteins affect prostate cancer susceptibility, dual immunostaining of benign prostate biopsies for GDF‐15 and NF‐κB was done in a study of 503 case‐control pairs matched on date, age, and race, nested within a historical cohort of 10,478 men. GDF‐15 and NF‐κB expression levels were positively correlated ( r = 0.39; p &lt; 0.0001), and both were significantly lower in African American (AA) compared with White men. In adjusted models that included both markers, the odds ratio (OR) for NF‐κB expression was statistically significant, OR =0.87; p = 0.03; 95% confidence interval (CI) =0.77–0.99, while GDF‐15 expression was associated with a nominally increased risk, OR =1.06; p = 0.27; 95% CI =0.96–1.17. When modeling expression levels by quartiles, the highest quartile of NF‐κB expression was associated with almost a fifty percent reduction in prostate cancer risk (OR =0.51; p = 0.03; 95% CI =0.29–0.92). In stratified models, NF‐κB had the strongest negative association with prostate cancer in non‐aggressive cases ( p = 0.03), older men ( p = 0.03), and in case‐control pairs with longer follow‐up ( p = 0.02). Risk associated with GDF‐15 expression was best fit using nonlinear regression modeling where both first ( p = 0.02) and second ( p = 0.03) order GDF‐15 risk terms were associated with significantly increased risk. This modeling approach also revealed significantly increased risk associated with GDF‐15 expression for subsamples defined by AA race, aggressive disease, younger age, and in case‐control pairs with longer follow‐up. Therefore, although positively correlated in benign prostatic biopsies, NF‐κB and GDF‐15 expression appear to exert opposite effects on risk of prostate tumor development.

Enzalutamide, a second-generation antiandrogen, is commonly prescribed for the therapy of advanced prostate cancer, but enzalutamide-resistant, lethal, or incurable disease invariably develops. To understand the molecular mechanism(s) behind enzalutamide resistance, here, we comprehensively analyzed a range of prostate tumors and clinically relevant models by gene expression array, immunohistochemistry, and Western blot, which revealed that enzalutamide-resistant prostate cancer cells and tumors overexpress the pseudokinase, Tribbles 2 (TRIB2). Inhibition of TRIB2 decreases the viability of enzalutamide-resistant prostate cancer cells, suggesting a critical role of TRIB2 in these cells. Moreover, the overexpression of TRIB2 confers resistance in prostate cancer cells to clinically relevant doses of enzalutamide, and this resistance is lost upon inhibition of TRIB2. Interestingly, we found that TRIB2 downregulates the luminal markers androgen receptor and cytokeratin 8 in prostate cancer cells but upregulates the neuronal transcription factor BRN2 (Brain-2) and the stemness factor SOX2 (SRY-box 2) to induce neuroendocrine characteristics. Finally, we show that inhibition of either TRIB2 or its downstream targets, BRN2 or SOX2, resensitizes resistant prostate cancer cells to enzalutamide. Thus, TRIB2 emerges as a potential new regulator of transdifferentiation that confers enzalutamide resistance in prostate cancer cells via a mechanism involving increased cellular plasticity and lineage switching.

In DNA from prostate tumors, methylation patterns in gene promoter regions can be a biomarker for disease progression. It remains unclear whether methylation patterns in benign prostate tissue--prior to malignant transformation--may provide similar prognostic information. To determine whether early methylation events predict prostate cancer outcomes, we evaluated histologically benign prostate specimens from 353 men who eventually developed prostate cancer and received "definitive" treatment [radical prostatectomy (58%) or radiation therapy (42%)]. Cases were drawn from a large hospital-based cohort of men with benign prostate biopsy specimens collected between 1990 and 2002. Risk of disease progression associated with methylation was estimated using time-to-event analyses. Average follow-up was over 5 years; biochemical recurrence (BCR) occurred in 91 cases (26%). In White men, methylation of the APC gene was associated with increased risk of BCR, even after adjusting for standard clinical risk factors for prostate cancer progression (adjusted hazard ratio (aHR) = 2.26; 95%CI 1.23-4.16). APC methylation was most strongly associated with a significant increased risk of BCR in White men with low prostate specific antigen at cohort entry (HR = 3.66; 95%CI 1.51-8.85). In additional stratified analyses, we found that methylation of the RARB gene significantly increased risk of BCR in African American cases who demonstrated methylation of at least one of the other four genes under study (HR = 3.80; 95%CI 1.07-13.53). These findings may have implications in the early identification of aggressive prostate cancer as well as reducing unnecessary medical procedures and emotional distress for men who present with markers of indolent disease.

Cadmium is a known carcinogen that has been implicated in prostate cancer, but how it affects prostate carcinogenesis in humans remains unclear. Evidence from basic science suggests that cadmium can bind to the androgen receptor causing endocrine disruption. The androgen receptor is required for normal prostate development and is the key driver of prostate cancer progression. In this study, we examined the association between cadmium content and androgen receptor protein expression in prostate cancer tissue of African American (N = 22) and European American (N = 30) men. Although neither overall tumor cadmium content (log transformed) nor androgen receptor protein expression level differed by race, we observed a race-cadmium interaction with regard to androgen receptor expression (P = 0.003) even after accounting for age at prostatectomy, smoking history, and Gleason score. African American men had a significant positive correlation between tumor tissue cadmium content and androgen receptor expression (Pearson correlation = 0.52, P = 0.013), while European Americans showed a non-significant negative correlation between the two (Pearson correlation = -0.19, P = 0.31). These results were unchanged after further accounting for tissue zinc content or dietary zinc or selenium intake. African American cases with high-cadmium content (>median) in tumor tissue had more than double the androgen receptor expression (0.021 vs. 0.008, P = 0.014) of African American men with low-cadmium level. No difference in androgen receptor expression was observed in European Americans by cadmium level (high 0.015 vs. low 0.011, P = 0.30). Larger studies are needed to confirm these results and if upheld, determine the biologic mechanism by which cadmium increases androgen receptor protein expression in a race-dependent manner. Our results suggest that cadmium may play a role in race disparities observed in prostate cancer.

Mucinous mammary carcinoma (MC) is a tumor type with relatively favorable prognosis. Unlike the circumstances surrounding conventional invasive duct carcinoma, data are limited regarding precursor lesions for MC. This study characterizes patterns of mucinous ductal carcinoma in situ (DCIS) as a precursor lesion for MC. All slides from 130 cases of MC encountered between 2000 and 2011 at Henry Ford Hospital, Detroit, MI were reviewed to subclassify MC, identify DCIS, and explore transition patterns from DCIS to MC. Calponin, p63, chromogranin, synaptophysin, CD56, and MIB-1 immunostaining analyses were performed in 65 cases. Among 106 cases of pure (71 type A, 35 type B) and 24 cases of mixed MC, DCIS appeared in 88 (68%) specimens, with all but 4 showing luminal mucin accumulation. Dominant patterns of mucinous DCIS were cribriform/solid (66), cribriform and papillary (7), papillary (5), micropapillary (3), and flat (3). Fifty-seven (68%) cases of mucinous DCIS demonstrated transitions from DCIS to MC. Luminal mucinous distention, focal flattening and attenuation of the epithelium, and disruption of the duct wall resulting in a mucocele-like extravasation of malignant epithelia with escaping mucin was a transition pattern seen with all architectures of DCIS and in all types of MC. This was the only pattern of transition to type A MC. The epithelial outpouching, formation of a cleft with accumulation of mucin around the epithelium, and transition into mucin pools with floating tumor cell clusters was the second transition pattern that went from cribriform/solid DCIS to type B and mixed MC. DCIS preceding aggressive phenotypes of MC (type B and mixed) more often had a cribriform/solid architecture, higher nuclear grade, and higher Ki-67-labeling index (all P<0.05). In summary, mucinous DCIS is a precursor to MC with distinctive features that link patterns of DCIS with aggressive MC phenotypes. The 2 observed transitions between mucinous DCIS and MC suggest that pathogenesis of different types of MC is different correlating with less or more aggressive behavior of the latter.

BACKGROUND Obesity is associated with risk of aggressive prostate cancer (PCa), but not with over‐all PCa risk. However, obese men have larger prostates which may lower biopsy accuracy and cause a systematic bias toward the null in epidemiologic studies of over‐all risk. METHODS Within a cohort of 6692 men followed‐up after a biopsy or transurethral resection of the prostate (TURP) with benign findings, a nested case‐control study was conducted of 495 prostate cancer cases and controls matched on age, race, follow‐up duration, biopsy versus TURP, and procedure date. Data on body mass index and prostate volume at the time of the initial procedure were abstracted from medical records. RESULTS Prior to consideration of differences in prostate volume, overweight (OR = 1.41; 95%CI 1.01, 1.97), and obese status (OR = 1.59; 95%CI 1.09, 2.33) at the time of the original benign biopsy or TURP were associated with PCa incidence during follow‐up. Prostate volume did not significantly moderate the association between body‐size and PCa, however it did act as an inverse confounder; adjustment for prostate volume increased the effect size for overweight by 22% (adjusted OR = 1.52; 95%CI 1.08, 2.14) and for obese status by 23% (adjusted OR = 1.77; 95%CI 1.20, 2.62). Larger prostate volume at the time of the original benign biopsy or TURP was inversely associated with PCa incidence during follow‐up (OR = 0.92 per 10 cc difference in volume; 95%CI 0.88, 0.97). In analyses that stratified case‐control pairs by tumor aggressiveness of the case, prostate volume acted as an inverse confounder in analyses of non‐aggressive PCa but not in analyses of aggressive PCa. CONCLUSIONS In studies of obesity and PCa, differences in prostate volume cause a bias toward the null, particularly in analyses of non‐aggressive PCa. A pervasive underestimation of the association between obesity and overall PCa risk may exist in the literature.

PR-10 Somatic mutations in exons encoding the tyrosine kinase domain of the epidermal growth factor receptor (EGFR) are found in about 10 and 25% of non-small cell lung cancers (NSCLCs) from the United States and east Asia, respectively. Nearly 90% of these mutations occur as either multi-nucleotide in-frame deletions in exon 19 that eliminate four amino acids (LREA), or as single missense mutations that result in substitution of arginine for leucine at position 858 (L858R). Both mutations are associated with increased sensitivity of lung adenocarcinomas to the selective EGFR kinase inhibitors, gefitinib and erlotinib. Unfortunately, patients with drug-sensitive EGFR mutations whose tumors initially respond to gefitinib or erlotinib eventually develop acquired resistance. In about half of cases of acquired resistance to the EGFR inhibitors, a second-site point mutation leading to substitution of methionine for threonine at position 790 (T790M) occurs. To identify additional or alternative genetic alterations that contribute to acquired resistance to EGFR inhibitors and to disease progression, we performed high-resolution genomic analysis (array-based comparative genomic hybridization; aCGH) of tissue samples from 12 patients who initially responded but subsequently progressed while on these drugs. Results were compared to those obtained from genomic analysis of EGFR mutant lung adenocarcinomas resected from 38 patients who were never treated with kinase inhibitors. Among three loci found to be differentially altered in the acquired resistance set, one containedthe MET proto-oncogene. Subsequent quantitative PCR analysis from three independent patient cohorts collectively revealed that MET was amplified in tumors from nine of 43 (21%) patients with acquired resistance vs two of 62 untreated patients (3%) (p = 0.007, Fisher’s Exact Test). Among 10 resistant tumors from the nine patients with MET amplification, four also harbored the EGFRT790M mutation. We also found that an existing EGFR mutant cell line, NCI-H820, harbors both a T790M mutation and MET activation. Growth inhibition studies with these cells demonstrate that they are resistant to erlotinib and an irreversible EGFR inhibitor (CL-387,785) but sensitive to a multi-kinase inhibitor with potent activity against MET (XL880). Furthermore, functional analysis reveals that ERBB3 and other downstream growth signalling is largely dependent on MET, and not EGFR, in these cells. Collectively, these data suggest that MET activation occurs independently of EGFRT790M mutations and that MET may be a clinically relevant therapeutic target for some patients with acquired resistance to gefitinib or erlotinib.
 This work was funded in part by the Thomas G. Labrecque Foundation.

Abstract Pigmented epithelioid melanocytoma (PEM) represents a group of rare, heavily pigmented melanocytic tumors encompassing lesions previously designated as “animal‐type melanomas” and “epithelioid blue nevi.” Despite the association of multiple such tumors in the setting of Carney complex, most cases of PEM occur spontaneously as solitary neoplasms in otherwise healthy patients. PEM may arise in both children and adults, and has a known propensity to spread to the regional lymph nodes. Despite this latter finding, recurrence at the biopsy site or spread beyond the lymph node basin is exceptionally uncommon. Although the molecular basis for PEM continues to be characterized, findings to date suggest that this category of melanocytic neoplasia has genetic alterations distinct from those seen in common nevi, dysplastic nevi, Spitz nevi, and melanoma. Herein, we present an in‐depth clinical, histopathologic, and molecular analysis of a case of PEM occurring on the scalp of a young African American girl found to have a novel NTRK3‐SCAPER gene fusion.

Consumption of cruciferous vegetables is associated with a decreased risk of developing prostate cancer. Antineoplastic effects of cruciferous vegetables are attributable to bioactive indoles, most prominently, 3, 3'-diindolylmethane (DIM). In addition to effects on proliferation and apoptosis, DIM acts as an antiandrogen in prostate cancer cell lines. This study characterized the effects of prostatic DIM on the androgen receptor (AR) in patients with prostate cancer. Men with localized prostate cancer were treated with a specially formulated DIM capsule designed for enhanced bioavailability (BR-DIM) at a dose of 225 mg orally twice daily for a minimum of 14 days. DIM levels and AR activity were assessed at the time of prostatectomy. Out of 28 evaluable patients, 26 (93%) had detectable prostatic DIM levels, with a mean concentration of 14.2 ng/gm. The mean DIM plasma level on BR-DIM therapy was 9.0 ng/mL; levels were undetectable at baseline and in follow-up samples. AR localization in the prostate was assessed with immunohistochemistry. After BR-DIM therapy, 96% of patients exhibited exclusion of the AR from the cell nucleus. In contrast, in prostate biopsy samples obtained prior to BR-DIM therapy, no patient exhibited AR nuclear exclusion. Declines in PSA were observed in a majority of patients (71%). Compliance was excellent and toxicity was minimal. In summary, BR-DIM treatment resulted in reliable prostatic DIM levels and anti-androgenic biologic effects at well tolerated doses. These results support further investigation of BR-DIM as a chemopreventive and therapeutic agent in prostate cancer.

In recent years, studies have suggested that promoter methylation in human papilloma virus (HPV) positive head and neck squamous cell carcinoma (HNSCC) has a mechanistic role and has the potential to improve patient survival. The present study aimed to replicate key molecular findings from previous analyses of the methylomes of HPV positive and HPV negative HNSCC in an independent cohort, to assess the reliability of differentially methylated markers in HPV-associated tumors. HPV was measured using real-time quantitative PCR and the biological significance of methylation differences was assessed by Ingenuity Pathway Analysis (IPA). Using an identical experimental design of a 450K methylation platform, 7 of the 11 genes were detected to be significantly differentially methylated and all 11 genes were either hypo- or hypermethylated, which was in agreement with the results of a previous study. IPA's enriched networks analysis identified one network with msh homeobox 2 (MSX2) as a central node. Locally dense interactions between genes in networks tend to reflect significant biology; therefore MSX2 was selected as an important gene. Sequestration in the top four canonical pathways was noted for 5-hydroxytryptamine receptor 1E (serotonin signaling), collapsin response mediator protein 1 (semaphorin signaling) and paired like homeodomain 2 (bone morphogenic protein and transforming growth factor-β signaling). Placement of 9 of the 11 genes in highly ranked pathways and bionetworks identified key biological processes to further emphasize differences between HNSCC HPV positive and negative pathogenesis.

Pleomorphic fibroma is a rare benign cutaneous neoplasm characterized by spindle‐shaped cells and multinucleated giant cells scattered throughout collagenous stroma. These morphologic features can lead to diagnostic confusion, including atypical lipomatous tumor as one consideration. In contrast to atypical lipomatous tumor, previous studies have found pleomorphic fibroma to be negative for MDM2 immunohistochemical staining and MDM2 gene amplification. Here, we present a case of pleomorphic fibroma of skin with nuclear MDM2 immunoreactivity in the absence of MDM2 gene amplification, underscoring the superiority of fluorescence in situ hybridization as a diagnostic test in this differential diagnosis. The RB1 locus is also explored for differential diagnosis with pleomorphic/spindle cell lipoma and related entities.

BACKGROUND.: Lymphatic invasion is necessary for regional lymph node (RLN) metastasis in breast cancer (BC), while systemic metastasis requires blood vessel (BV) invasion. The site of BV invasion could be at the primary BC site or through lymphovascular anastomoses. The vague pathologic term "lymphovascular invasion" (LVI) encourages the belief that peri/intratumoral BV invasion may be common. We investigated the relative contribution of RLN metastasis to systemic metastasis by studying the relationship among LVI and RLN and/or systemic metastasis in a population-based cohort of breast cancer patients. METHODS.: Fisher's exact test was done to assess global associations among LVI and RLN and/or systemic metastasis in a prospective database of breast cancer patients undergoing RLN biopsy. Logistic regression was used to determine multivariable contributions of LVI to metastasis when controlling for available demographic, radiologic, and pathologic variables. RESULTS.: Of 1668 patients evaluated 25.4% were RLN positive and 10.4% had LVI. RLN metastasis was predicted by tumor size (P < .0001), HER-2/neu overexpression (P = .0022) and the interaction between LVI positive and HER-2/neu positive tumors (< .0001). Patients with LVI/HER-2-neu positive were 3 times as likely to have positive RLNs compared with patients LVI/HER-2-neu negative. Systemic metastasis was significantly (P = .0013) associated with LVI/ RLN positive, but not with LVI positive/RLN negative patients (P = .137). CONCLUSIONS.: LVI predicted RLN metastasis. LVI also significantly predicted systemic metastasis, but only when the RLN was also positive. Since RLN requires lymphatic invasion, these data support the hypothesis that primary breast cancers often invade lymphatics to gain access to the systemic circulation.

Mutations in the KRAS gene are associated with poor response to epidermal growth factor receptor inhibitors used in the treatment of metastatic colorectal cancer. Factors influencing KRAS test results in tumor specimens include: tumor heterogeneity, sample handling, slide preparation, techniques for tumor enrichment, DNA preparation, assay design and sensitivity. We evaluated comparability and consistency of KRAS test results among five laboratories currently being used to determine KRAS mutation status of metastatic colorectal cancer specimens in a large, multi-center observational study.Twenty formalin-fixed paraffin-embedded human colorectal cancer samples from colon resections previously tested for KRAS mutations were selected based on mutation status (6 wild type, 8 codon 12 mutations, and 6 codon 13 mutations). We found good agreement across laboratories despite differences in mutation detection methods. Eighteen of twenty samples (90%) were concordant across all five labs. Discordant results are likely not due to laboratory error, but instead to tumor heterogeneity, contamination of the tumor sample with normal tissue, or analytic factors affecting assay sensitivity.Our results indicate commercial and academic laboratories provide reliable results for the common KRAS gene mutations at codons 12 and 13 when an adequate percentage of tumor cells is present in the sample.

Atypical lipomatous tumor/well-differentiated liposarcoma (ALT/WDL) and spindle cell lipoma are lipomatous tumors with distinct clinical, molecular, and prognostic features. Although histological and immunophenotypic features can overlap between ALT/WDL and spindle cell lipoma, the oncogenesis and clinical behavior are markedly different. In borderline cases, molecular analysis for MDM2 or CDK4 amplification can aid in distinguishing ALT/WDL from spindle cell lipoma. Although dedifferentiated liposarcoma has been reported to harbor both MDM2 amplification and loss of the RB1 region, we are not aware of a reported RB1 loss in well-differentiated ALT/WDL. In this article, we present a 69-year-old woman with a lipomatous tumor in the gluteal region that histologically, immunohistochemically, and molecularly mimicked spindle cell lipoma (with positive immunohistochemical staining for CD34 and loss of the RB1 gene region), yet harbored amplification of MDM2 and CDK4 confirmed by fluorescence in situ hybridization, supporting classification as ALT/WDL. This case strengthens the argument that in atypical clinical contexts, molecular studies for MDM2/CDK4 should be considered in tumors resembling spindle cell lipoma.

Systemic inflammation may increase risk for prostate cancer progression, but the role it plays in prostate cancer susceptibility is unknown. From a cohort of over 10,000 men who had either a prostate biopsy or transurethral resection that yielded a benign finding, we analyzed 517 incident prostate cancer cases identified during follow-up and 373 controls with one or more white blood cell tests during a follow-up period between one and 18 years. Multilevel, multivariable longitudinal models were fit to two measures of systemic inflammation, neutrophil-to-lymphocyte ratio (NLR) and monocyte-to-lymphocyte ratio (MLR), to determine NLR and MLR trajectories associated with increased risk for prostate cancer. For both measures, we found no significant differences in the trajectories by case/control status, however in modeling NLR trajectories there was a significant interaction between race (white or Black and case-control status. In race specific models, NLR and MLR values were consistently higher over time among white controls than white cases while case-control differences in NLR and MLR trajectories were not apparent among Black men. When cases were classified as aggressive as compared to non-aggressive, the case-control differences in NLR and MLR values over time among white men were most apparent for non-aggressive cases. For NLR among white men, significant case-control differences were observed for the entire duration of observation for men who had inflammation in their initial prostate specimen. It is possible that, among white men, monitoring of NLR and MLR trajectories after an initial negative biopsy may be useful in monitoring prostate cancer risk.

Prostate cancer is frequently multifocal. Although there may be morphological variation, the genetic underpinnings of each tumor are not clearly understood. To assess the inter and intra tumor molecular heterogeneity in prostate biopsy samples, we developed a combined immunohistochemistry and RNA in situ hybridization method for the simultaneous evaluation of ERG, SPINK1, ETV1, and ETV4. Screening of 601 biopsy cores from 120 consecutive patients revealed multiple alterations in a mutually exclusive manner in 37% of patients, suggesting multifocal tumors with considerable genetic differences. Furthermore, the incidence of molecular heterogeneity was higher in African Americans patients compared with Caucasian American patients. About 47% of the biopsy cores with discontinuous tumor foci showed clonal differences with distinct molecular aberrations. ERG positivity occurred in low-grade cancer, whereas ETV4 expression was observed mostly in high-grade cancer. Further studies revealed correlation between the incidence of molecular markers and clinical and pathologic findings, suggesting potential implications for diagnostic pathology practice, such as defining dominant tumor nodules and discriminating juxtaposed but molecularly different tumors of different grade patterns.

8006 Background: KRAS mutations are found in ∼ 25% of lung adenocarcinomas in Western countries and, as a group, are strongly associated with cigarette smoking. These mutations are predictive of poor prognosis in resected disease as well as resistance to treatment with erlotinib or gefitinib. In KRAS, transversions (substituting the purine G with a pyrimidine) are more common than transitions (substituting the purine G with A) and identify a molecular signature for the carcinogenic effects of cigarette smoke. Methods: We determined the frequency and type of KRAS codon 12 and 13 mutations and characterized their association with cigarette smoking history in patients with lung adenocarcinomas. Comparisons were made using Fisher’s exact test. Results: KRAS mutational analysis was performed on 482 lung adenocarcinomas, 81 (17%) of which were obtained from patients who had never smoked cigarettes. KRAS mutations were found in 15% (12/81; 95% CI 8%-24%) of tumors from never smokers. Twenty-two% (69/316; 95% CI 17%-27%) of tumors from former smokers, and 25% (21/85; 95% CI 16%-35%) of tumors from current smokers had KRAS mutations. The frequency of KRAS mutation was not associated with age, gender, or smoking history. The number of pack years of cigarette smoking did not predict an increased likelihood of KRAS mutations. When the type of KRAS mutation was examined, never smokers were significantly more likely than former or current smokers to have a transition mutation (G–>A) rather than the transversion mutations known to be smoking related (G–>T or G–>C; p<0.0001). Conclusions: Based upon our data, KRAS mutations are not rare among never smokers with lung adenocarcinoma and such patients have a distinct KRAS mutation profile. The etiologic and biological heterogeneity of KRAS mutant lung adenocarcinomas is worthy of further study. Mutation Nucleotide Former/current Never Total G12A GGT–>GCT 13 0 13 G12C GGT–>TGT 38 0 38 G12V GGT–>GTT 20 1 21 G13C GGC–>TGC 2 0 2 G13D GGC–>GAC 1 0 1 G12D GGT–>GAT 15 10 25 G12S GGT–>AGT 1 1 2 Total 90 12 No significant financial relationships to disclose.

SUMMARY Genome-wide DNA methylation profiling has shown that epigenetic abnormalities are biologically important in glioma and can be used to classify these tumors into distinct prognostic groups. Thus far, DNA profiling has required surgically resected glioma tissue; however, gliomas release tumoral material into biofluids, such as blood and cerebrospinal fluid, providing an opportunity for a minimally invasive testing. While prior studies have shown that genetic and epigenetic markers can be detected in blood or cerebrospinal fluid (e.g., liquid biopsy [LB]), there has been low sensitivity for tumor-specific markers. We hypothesize that the low sensitivity is due to the targeted assay methods. Therefore, we profiled the genome-wide CpG methylation levels in DNA of tumor tissue and cell-free DNA in serum of glioma patients, to identify non-invasive epigenetic LB (eLB) markers in the serum that reflect the characteristics of the tumor tissue. From the epigenetic profiles of serum from patients diagnosed with glioma (N=15 IDH mutant and N=7 IDH wildtype) and with epilepsy (N=3), we defined glioma-specific and IDH -specific eLB signatures (Glioma-eLB and IDH -eLB, respectively). The epigenetic profiles of the matched tissue demonstrate that these eLB signatures reflected the signature of the tumor. Through cross-validation we show that Glioma-eLB can accurately predict a patient’s glioma from those with other neoplasias (N=6 Colon; N=14 Pituitary; N=3 Breast; N=4 Lung), non-neoplastic immunological conditions (N=22 sepsis; N=9 pancreatic islet transplantation), and from healthy individuals (sensitivity: 98%; specificity: 99%). Finally, IDH -eLB includes promoter methylated markers associated with genes known to be involved in glioma tumorigenesis ( PVT1 and CXCR6 ). The application of the non-invasive eLB signature discovered in this study has the potential to complement the standard of care for patients harboring glioma.

Objectives/Hypothesis The human papillomavirus (HPV) is known to infect the tissues of the oropharynx as demonstrated in HPV‐positive oropharyngeal squamous cell carcinoma (OPSCC). HPV has also been shown to induce benign lymphoid hypertrophy. We sought to investigate an association between obstructive sleep apnea (OSA) and the presence of HPV in palatine and lingual tonsillar oropharyngeal tissue. Study Design Case series with chart review. Methods This retrospective laboratory‐based study of oropharyngeal tissue from patients with OSA included patients &gt;18 years old who underwent surgical treatment for OSA at a single institution between January 2012 and May 2014. Surgical specimens of adequate size were analyzed for HPV6, 11, and 16 using real‐time quantitative polymerase chain reaction from DNA extracted from formalin‐fixed paraffin‐embedded tissue blocks. Student t test, Pearson χ 2 test, and linear logistic regression were used to assess comparisons of body mass index (BMI), apnea‐hypopnea index (AHI), age, and gender between HPV‐positive and HPV‐negative groups. Results Of 99 cases included in the study, six were positive for HPV: two with HPV16 and four with HPV6. BMI, AHI, age, and gender showed no significant differences between the HPV‐positive and HPV‐negative groups. Logistic regression to predict HPV positivity accounting for each variable and multivariate analysis were not statistically significant. Conclusions Our study did not show HPV to have a statistically significant association with OSA. None of the covariates analyzed (BMI, AHI, gender, age) predicted HPV positivity in surgically resected oropharyngeal tissue from OSA patients. Level of Evidence 4 Laryngoscope , 127:1231–1234, 2017

HER2 (ERBB2) gene status serves as a strong predictive marker of response to HER2-targeted agents in invasive breast cancers, albeit with heterogeneous response. Our aim was to determine the distribution and prognosis of HER2 groups by fluorescent in situ hybridization (FISH) using the updated 2018 American Society of Clinical Oncology-College of American Pathologist (ASCO-CAP) guidelines. We identified 226 cases of equivocal or positive HER2 FISH invasive breast cancer (interpreted by ASCO-CAP guidelines at the time of reporting) who received HER2-targeted agents from 2006 to 2017. We subcategorized Group 1 further into three subgroups: low amplified (HER2/CEP17 ratio ≥ 2.0-2.99, mean HER2/cell 4.0-5.9), amplified (HER2/CEP17 ratio ≥ 2.0-2.99, mean HER2/cell ≥ 6), and excessive amplification (HER2/CEP17 ratio ≥ 3.0, mean HER2/cell ≥ 4.0). Outcomes studied were recurrence, metastasis, second breast primary, disease-specific survival (DSS), and overall survival (OS). Univariate analysis showed that the five categories of HER2 FISH were significantly associated with OS (p < 0.01), specifically higher HER2 amplification was associated with fewer deaths. HER2 FISH status also statistically significantly relates to DFS (p < 0.01) and metastasis (p = 0.01) but not with recurrence or second breast primary in our study. Tumor type and HER2 ISH Groups are independent predictors for both OS and DFS in our cohort. The proposed Group 1 subcategories were significantly associated with OS (p < 0.01) and DFS (p < 0.01), excessive HER2 amplification was associated with longer median survival. The Cox regression models showed better survival outcomes for the excessive amplification subgroup than the low amplified subgroup, with OS (hazard ratio = 0.63, 95% CI 0.42-0.93) and DFS (HR = 0.55, 95% CI 0.37-0.83). We demonstrated that in HER2 FISH Group 1 patients, high HER2 amplification was significantly associated with longer OS and DFS; these patients seem to benefit more from HER2-targeted regimens. We recommend reporting these Group 1 subcategories when assessing HER2 FISH.

Natural language processing (NLP) in pathology reports to extract biomarker information is an ongoing area of research. MetaMap is a natural language processing tool developed and funded by the National Library of Medicine to map biomedical text to the Unified Medical Language System Metathesaurus by applying specific tags to clinically relevant terms. Although results are useful without additional postprocessing, these tags lack important contextual information.Our novel method takes terminology-driven semantic tags and incorporates those into a semantic frame that is task-specific to add necessary context to MetaMap. We use important contextual information to capture biomarker results to support Community Health System's use of Precision Medicine treatments for patients with cancer. For each biomarker, the name, type, numeric quantifiers, non-numeric qualifiers, and the time frame are extracted. These fields then associate biomarkers with their context in the pathology report such as test type, probe intensity, copy-number changes, and even failed results. A selection of 6,713 relevant reports contained the following standard-of-care biomarkers for metastatic breast cancer: breast cancer gene 1 and 2, estrogen receptor, progesterone receptor, human epidermal growth factor receptor 2, and programmed death-ligand 1.The method was tested on pathology reports from the internal pathology laboratory at Henry Ford Health System. A certified tumor registrar reviewed 400 tests, which showed > 95% accuracy for all extracted biomarker types.Using this new method, it is possible to extract high-quality, contextual biomarker information, and this represents a significant advance in biomarker extraction.

Lymph node metastases (LNMets) are common problems for patients and those involved in their management. Patients with LNMets from various cancers are often at a higher risk for death from cancer than those whose lymph nodes (LNs) are free of cancer. Clinical and pathologic staging of cancer is dependent upon identification of LNMets. Understanding how tumor cells migrate to LNs is still evolving. The relationship among LNMets and systemic metastases are vigorously debated; those who believe that LNMets merely identify those tumors that have probably already metastasized systemically ("marker" hypothesis) and those who believe that systemic metastases are derived from passage through regional LNs and only very rarely by direct blood vessel invasion ("incubator" hypothesis). Diagnosis and treatment of LNMets has advanced significantly over the past century and it seems likely that further improvements will develop. This chapter is a brief introduction into the present understanding of LNMets.

NF1 germline mutation predisposes to breast cancer. NF1 mutations have also been proposed as oncogenic drivers in sporadic breast cancers. To understand the genomic and histologic characteristics of these breast cancers, we analyzed the tumors with NF1 germline mutations and also examined the genomic and proteomic profiles of unselected tumors. Among 14 breast cancer specimens from 13 women affected with neurofibromatosis type 1 (NF1), 9 samples (NF + BrCa) underwent genomic copy number (CN) and targeted sequencing analysis. Mutations of NF1 were identified in two samples and TP53 were in three. No mutation was detected in ATM, BARD1, BRCA1, BRCA2, BRIP1, CDH1, CHEK2, NBN, PALB2, PTEN, RAD50, and STK11 HER2 (ErbB2) overexpression was detected by IHC in 69.2% (9/13) of the tumors. CN gain/amplification of ERBB2 was detected in 4 of 9 with DNA analysis. By evaluating HER2 expression and NF1 alterations in unselected invasive breast cancers in TCGA datasets, we discovered that among samples with ERBB2 CN gain/amplification, the HER2 mRNA and protein expression were much more pronounced in NF1-mutated/deleted samples in comparison with NF1-unaltered samples. This finding suggests a synergistic interplay between these two genes, potentially driving the development of breast cancer harboring NF1 mutation and ERBB2 CN gain/amplification. NF1 gene loss of heterozygosity was observed in 4 of 9 NF + BrCa samples. CDK4 appeared to have more CN gain in NF + BrCa and exhibited increased mRNA expression in TCGA NF1--altered samples. Cancer Prev Res; 11(10); 655-64. ©2018 AACR.

We evaluated 328 patients (34.8% African American [AA]; 65.2% White American [WA]) with hormone receptor-positive, HER2/neu-negative breast cancer. Mean age (60 years); mean tumor size (1.6 and 1.7 cm for AA and WA, respectively) were similar, and mean BMI was higher for AA (33 vs 29.8; P = 0.001). Recurrence score (RS) distribution was similar- 8.3% AA and 5.9% WA with high RS (≥31). No significant differences were observed in delivery of chemotherapy stratified by score. With median follow-up 27.2 months for AA and 33.4 months for WA, distant recurrence occurred in 1.0% and 1.6%, respectively (P = 1). Our results suggest comparable RS utility in AA and WA patients.