Jamie K. Teer

Identifying tumor-associated T cell epitopes can be laborious. Robbins et al. now report that they used whole-exome sequence data to identify mutated peptides from tumor antigens that are recognized by tumor-infiltrating T cells from patients with melanoma who experienced therapy-associated tumor regressions. The method will contribute to the identification of new tumor-specific epitopes that may further the development of effective T cell therapies for the treatment of patients with melanoma as well as a variety of additional malignancies. Substantial regressions of metastatic lesions have been observed in up to 70% of patients with melanoma who received adoptively transferred autologous tumor-infiltrating lymphocytes (TILs) in phase 2 clinical trials1,2. In addition, 40% of patients treated in a recent trial experienced complete regressions of all measurable lesions for at least 5 years following TIL treatment3. To evaluate the potential association between the ability of TILs to mediate durable regressions and their ability to recognize potent antigens that presumably include mutated gene products, we developed a new screening approach involving mining whole-exome sequence data to identify mutated proteins expressed in patient tumors. We then synthesized and evaluated candidate mutated T cell epitopes that were identified using a major histocompatibility complex–binding algorithm4 for recognition by TILs. Using this approach, we identified mutated antigens expressed on autologous tumor cells that were recognized by three bulk TIL lines from three individuals with melanoma that were associated with objective tumor regressions following adoptive transfer. This simplified approach for identifying mutated antigens recognized by T cells avoids the need to generate and laboriously screen cDNA libraries from tumors and may represent a generally applicable method for identifying mutated antigens expressed in a variety of tumor types.

The Proteus syndrome is characterized by the overgrowth of skin, connective tissue, brain, and other tissues. It has been hypothesized that the syndrome is caused by somatic mosaicism for a mutation that is lethal in the nonmosaic state.We performed exome sequencing of DNA from biopsy samples obtained from patients with the Proteus syndrome and compared the resultant DNA sequences with those of unaffected tissues obtained from the same patients. We confirmed and extended an observed association, using a custom restriction-enzyme assay to analyze the DNA in 158 samples from 29 patients with the Proteus syndrome. We then assayed activation of the AKT protein in affected tissues, using phosphorylation-specific antibodies on Western blots.Of 29 patients with the Proteus syndrome, 26 had a somatic activating mutation (c.49G→A, p.Glu17Lys) in the oncogene AKT1, encoding the AKT1 kinase, an enzyme known to mediate processes such as cell proliferation and apoptosis. Tissues and cell lines from patients with the Proteus syndrome harbored admixtures of mutant alleles that ranged from 1% to approximately 50%. Mutant cell lines showed greater AKT phosphorylation than did control cell lines. A pair of single-cell clones that were established from the same starting culture and differed with respect to their mutation status had different levels of AKT phosphorylation.The Proteus syndrome is caused by a somatic activating mutation in AKT1, proving the hypothesis of somatic mosaicism and implicating activation of the PI3K-AKT pathway in the characteristic clinical findings of overgrowth and tumor susceptibility in this disorder. (Funded by the Intramural Research Program of the National Human Genome Research Institute.).

The incidence of melanoma is increasing more than any other cancer, and knowledge of its genetic alterations is limited. To systematically analyze such alterations, we performed whole-exome sequencing of 14 matched normal and metastatic tumor DNAs. Using stringent criteria, we identified 68 genes that appeared to be somatically mutated at elevated frequency, many of which are not known to be genetically altered in tumors. Most importantly, we discovered that TRRAP harbored a recurrent mutation that clustered in one position (p. Ser722Phe) in 6 out of 167 affected individuals (∼4%), as well as a previously unidentified gene, GRIN2A, which was mutated in 33% of melanoma samples. The nature, pattern and functional evaluation of the TRRAP recurrent mutation suggest that TRRAP functions as an oncogene. Our study provides, to our knowledge, the most comprehensive map of genetic alterations in melanoma to date and suggests that the glutamate signaling pathway is involved in this disease.

The development of massively parallel sequencing technologies, coupled with new massively parallel DNA enrichment technologies (genomic capture), has allowed the sequencing of targeted regions of the human genome in rapidly increasing numbers of samples. Genomic capture can target specific areas in the genome, including genes of interest and linkage regions, but this limits the study to what is already known. Exome capture allows an unbiased investigation of the complete protein-coding regions in the genome. Researchers can use exome capture to focus on a critical part of the human genome, allowing larger numbers of samples than are currently practical with whole-genome sequencing. In this review, we briefly describe some of the methodologies currently used for genomic and exome capture and highlight recent applications of this technology.

While mutations in the KRAS oncogene are among the most prevalent in human cancer, there are few successful treatments to target these tumors. It is also likely that heterogeneity in KRAS-mutant tumor biology significantly contributes to the response to therapy. We hypothesized that the presence of commonly co-occurring mutations in STK11 and TP53 tumor suppressors may represent a significant source of heterogeneity in KRAS-mutant tumors. To address this, we utilized a large cohort of resected tumors from 442 lung adenocarcinoma patients with data including annotation of prevalent driver mutations (KRAS and EGFR) and tumor suppressor mutations (STK11 and TP53), microarray-based gene expression and clinical covariates, including overall survival (OS). Specifically, we determined impact of STK11 and TP53 mutations on a new KRAS mutation-associated gene expression signature as well as previously defined signatures of tumor cell proliferation and immune surveillance responses. Interestingly, STK11, but not TP53 mutations, were associated with highly elevated expression of KRAS mutation-associated genes. Mutations in TP53 and STK11 also impacted tumor biology regardless of KRAS status, with TP53 strongly associated with enhanced proliferation and STK11 with suppression of immune surveillance. These findings illustrate the remarkably distinct ways through which tumor suppressor mutations may contribute to heterogeneity in KRAS-mutant tumor biology. In addition, these studies point to novel associations between gene mutations and immune surveillance that could impact the response to immunotherapy.

ClinSeq is a pilot project to investigate the use of whole-genome sequencing as a tool for clinical research. By piloting the acquisition of large amounts of DNA sequence data from individual human subjects, we are fostering the development of hypothesis-generating approaches for performing research in genomic medicine, including the exploration of issues related to the genetic architecture of disease, implementation of genomic technology, informed consent, disclosure of genetic information, and archiving, analyzing, and displaying sequence data. In the initial phase of ClinSeq, we are enrolling roughly 1000 participants; the evaluation of each includes obtaining a detailed family and medical history, as well as a clinical evaluation. The participants are being consented broadly for research on many traits and for whole-genome sequencing. Initially, Sanger-based sequencing of 300-400 genes thought to be relevant to atherosclerosis is being performed, with the resulting data analyzed for rare, high-penetrance variants associated with specific clinical traits. The participants are also being consented to allow the contact of family members for additional studies of sequence variants to explore their potential association with specific phenotypes. Here, we present the general considerations in designing ClinSeq, preliminary results based on the generation of an initial 826 Mb of sequence data, the findings for several genes that serve as positive controls for the project, and our views about the potential implications of ClinSeq. The early experiences with ClinSeq illustrate how large-scale medical sequencing can be a practical, productive, and critical component of research in genomic medicine.

Genome- and exome-sequencing costs are continuing to fall, and many individuals are undergoing these assessments as research participants and patients. The issue of secondary (so-called incidental) findings in exome analysis is controversial, and data are needed on methods of detection and their frequency. We piloted secondary variant detection by analyzing exomes for mutations in cancer-susceptibility syndromes in subjects ascertained for atherosclerosis phenotypes. We performed exome sequencing on 572 ClinSeq participants, and in 37 genes, we interpreted variants that cause high-penetrance cancer syndromes by using an algorithm that filtered results on the basis of mutation type, quality, and frequency and that filtered mutation-database entries on the basis of defined categories of causation. We identified 454 sequence variants that differed from the human reference. Exclusions were made on the basis of sequence quality (26 variants) and high frequency in the cohort (77 variants) or dbSNP (17 variants), leaving 334 variants of potential clinical importance. These were further filtered on the basis of curation of literature reports. Seven participants, four of whom were of Ashkenazi Jewish descent and three of whom did not meet family-history-based referral criteria, had deleterious BRCA1 or BRCA2 mutations. One participant had a deleterious SDHC mutation, which causes paragangliomas. Exome sequencing, coupled with multidisciplinary interpretation, detected clinically important mutations in cancer-susceptibility genes; four of such mutations were in individuals without a significant family history of disease. We conclude that secondary variants of high clinical importance will be detected at an appreciable frequency in exomes, and we suggest that priority be given to the development of more efficient modes of interpretation with trials in larger patient groups. Genome- and exome-sequencing costs are continuing to fall, and many individuals are undergoing these assessments as research participants and patients. The issue of secondary (so-called incidental) findings in exome analysis is controversial, and data are needed on methods of detection and their frequency. We piloted secondary variant detection by analyzing exomes for mutations in cancer-susceptibility syndromes in subjects ascertained for atherosclerosis phenotypes. We performed exome sequencing on 572 ClinSeq participants, and in 37 genes, we interpreted variants that cause high-penetrance cancer syndromes by using an algorithm that filtered results on the basis of mutation type, quality, and frequency and that filtered mutation-database entries on the basis of defined categories of causation. We identified 454 sequence variants that differed from the human reference. Exclusions were made on the basis of sequence quality (26 variants) and high frequency in the cohort (77 variants) or dbSNP (17 variants), leaving 334 variants of potential clinical importance. These were further filtered on the basis of curation of literature reports. Seven participants, four of whom were of Ashkenazi Jewish descent and three of whom did not meet family-history-based referral criteria, had deleterious BRCA1 or BRCA2 mutations. One participant had a deleterious SDHC mutation, which causes paragangliomas. Exome sequencing, coupled with multidisciplinary interpretation, detected clinically important mutations in cancer-susceptibility genes; four of such mutations were in individuals without a significant family history of disease. We conclude that secondary variants of high clinical importance will be detected at an appreciable frequency in exomes, and we suggest that priority be given to the development of more efficient modes of interpretation with trials in larger patient groups.

Adoptive cell therapy using tumor-infiltrating lymphocytes (TILs) has shown activity in melanoma, but has not been previously evaluated in metastatic non-small cell lung cancer. We conducted a single-arm open-label phase 1 trial ( NCT03215810 ) of TILs administered with nivolumab in 20 patients with advanced non-small cell lung cancer following initial progression on nivolumab monotherapy. The primary end point was safety and secondary end points included objective response rate, duration of response and T cell persistence. Autologous TILs were expanded ex vivo from minced tumors cultured with interleukin-2. Patients received cyclophosphamide and fludarabine lymphodepletion, TIL infusion and interleukin-2, followed by maintenance nivolumab. The end point of safety was met according to the prespecified criteria of ≤17% rate of severe toxicity (95% confidence interval, 3-29%). Of 13 evaluable patients, 3 had confirmed responses and 11 had reduction in tumor burden, with a median best change of 35%. Two patients achieved complete responses that were ongoing 1.5 years later. In exploratory analyses, we found T cells recognizing multiple types of cancer mutations were detected after TIL treatment and were enriched in responding patients. Neoantigen-reactive T cell clonotypes increased and persisted in peripheral blood after treatment. Cell therapy with autologous TILs is generally safe and clinically active and may constitute a new treatment strategy in metastatic lung cancer.

Abstract Colorectal cancer (CRC) is a highly heterogeneous disease, for which prognosis has been relegated to clinicopathologic staging for decades. There is a need to stratify subpopulations of CRC on a molecular basis to better predict outcome and assign therapies. Here we report targeted exome-sequencing of 1,321 cancer-related genes on 468 tumour specimens, which identified a subset of 17 genes that best classify CRC, with APC playing a central role in predicting overall survival. APC may assume 0, 1 or 2 truncating mutations, each with a striking differential impact on survival. Tumours lacking any APC mutation carry a worse prognosis than single APC mutation tumours; however, two APC mutation tumours with mutant KRAS and TP53 confer the poorest survival among all the subgroups examined. Our study demonstrates a prognostic role for APC and suggests that sequencing of APC may have clinical utility in the routine staging and potential therapeutic assignment for CRC.

PURPOSE Outcomes for patients with newly diagnosed multiple myeloma (NDMM) are heterogenous, with overall survival (OS) ranging from months to over 10 years. METHODS To decipher and predict the molecular and clinical heterogeneity of NDMM, we assembled a series of 1,933 patients with available clinical, genomic, and therapeutic data. RESULTS Leveraging a comprehensive catalog of genomic drivers, we identified 12 groups, expanding on previous gene expression–based molecular classifications. To build a model predicting individualized risk in NDMM (IRMMa), we integrated clinical, genomic, and treatment variables. To correct for time-dependent variables, including high-dose melphalan followed by autologous stem-cell transplantation (HDM-ASCT), and maintenance therapy, a multi-state model was designed. The IRMMa model accuracy was significantly higher than all comparator prognostic models, with a c-index for OS of 0.726, compared with International Staging System (ISS; 0.61), revised-ISS (0.572), and R2-ISS (0.625). Integral to model accuracy was 20 genomic features, including 1q21 gain/amp, del 1p, TP53 loss, NSD2 translocations, APOBEC mutational signatures, and copy-number signatures (reflecting the complex structural variant chromothripsis). IRMMa accuracy and superiority compared with other prognostic models were validated on 256 patients enrolled in the GMMG-HD6 (ClinicalTrials.gov identifier: NCT02495922 ) clinical trial. Individualized patient risks were significantly affected across the 12 genomic groups by different treatment strategies (ie, treatment variance), which was used to identify patients for whom HDM-ASCT is particularly effective versus patients for whom the impact is limited. CONCLUSION Integrating clinical, demographic, genomic, and therapeutic data, to our knowledge, we have developed the first individualized risk-prediction model enabling personally tailored therapeutic decisions for patients with NDMM.

Massively parallel DNA sequencing technologies have greatly increased our ability to generate large amounts of sequencing data at a rapid pace. Several methods have been developed to enrich for genomic regions of interest for targeted sequencing. We have compared three of these methods: Molecular Inversion Probes (MIP), Solution Hybrid Selection (SHS), and Microarray-based Genomic Selection (MGS). Using HapMap DNA samples, we compared each of these methods with respect to their ability to capture an identical set of exons and evolutionarily conserved regions associated with 528 genes (2.61 Mb). For sequence analysis, we developed and used a novel Bayesian genotype-assigning algorithm, Most Probable Genotype (MPG). All three capture methods were effective, but sensitivities (percentage of targeted bases associated with high-quality genotypes) varied for an equivalent amount of pass-filtered sequence: for example, 70% (MIP), 84% (SHS), and 91% (MGS) for 400 Mb. In contrast, all methods yielded similar accuracies of >99.84% when compared to Infinium 1M SNP BeadChip-derived genotypes and >99.998% when compared to 30-fold coverage whole-genome shotgun sequencing data. We also observed a low false-positive rate with all three methods; of the heterozygous positions identified by each of the capture methods, >99.57% agreed with 1M SNP BeadChip, and >98.840% agreed with the whole-genome shotgun data. In addition, we successfully piloted the genomic enrichment of a set of 12 pooled samples via the MGS method using molecular bar codes. We find that these three genomic enrichment methods are highly accurate and practical, with sensitivities comparable to that of 30-fold coverage whole-genome shotgun data.

We report an early onset spastic ataxia-neuropathy syndrome in two brothers of a consanguineous family characterized clinically by lower extremity spasticity, peripheral neuropathy, ptosis, oculomotor apraxia, dystonia, cerebellar atrophy, and progressive myoclonic epilepsy. Whole-exome sequencing identified a homozygous missense mutation (c.1847G>A; p.Y616C) in AFG3L2, encoding a subunit of an m-AAA protease. m-AAA proteases reside in the mitochondrial inner membrane and are responsible for removal of damaged or misfolded proteins and proteolytic activation of essential mitochondrial proteins. AFG3L2 forms either a homo-oligomeric isoenzyme or a hetero-oligomeric complex with paraplegin, a homologous protein mutated in hereditary spastic paraplegia type 7 (SPG7). Heterozygous loss-of-function mutations in AFG3L2 cause autosomal-dominant spinocerebellar ataxia type 28 (SCA28), a disorder whose phenotype is strikingly different from that of our patients. As defined in yeast complementation assays, the AFG3L2(Y616C) gene product is a hypomorphic variant that exhibited oligomerization defects in yeast as well as in patient fibroblasts. Specifically, the formation of AFG3L2(Y616C) complexes was impaired, both with itself and to a greater extent with paraplegin. This produced an early-onset clinical syndrome that combines the severe phenotypes of SPG7 and SCA28, in additional to other "mitochondrial" features such as oculomotor apraxia, extrapyramidal dysfunction, and myoclonic epilepsy. These findings expand the phenotype associated with AFG3L2 mutations and suggest that AFG3L2-related disease should be considered in the differential diagnosis of spastic ataxias.

Synonymous mutations, which do not alter the protein sequence, have been shown to affect protein function [Sauna ZE, Kimchi-Sarfaty C (2011) Nat Rev Genet 12(10):683-691]. However, synonymous mutations are rarely investigated in the cancer genomics field. We used whole-genome and -exome sequencing to identify somatic mutations in 29 melanoma samples. Validation of one synonymous somatic mutation in BCL2L12 in 285 samples identified 12 cases that harbored the recurrent F17F mutation. This mutation led to increased BCL2L12 mRNA and protein levels because of differential targeting of WT and mutant BCL2L12 by hsa-miR-671-5p. Protein made from mutant BCL2L12 transcript bound p53, inhibited UV-induced apoptosis more efficiently than WT BCL2L12, and reduced endogenous p53 target gene transcription. This report shows selection of a recurrent somatic synonymous mutation in cancer. Our data indicate that silent alterations have a role to play in human cancer, emphasizing the importance of their investigation in future cancer genome studies.

Phosphatidylinositol glycan class A (PIGA) is involved in the first step of glycosylphosphatidylinositol (GPI) biosynthesis. Many proteins, including CD55 and CD59, are anchored to the cell by GPI. Loss of CD55 and CD59 on erythrocytes causes complement-mediated lysis in paroxysmal nocturnal hemoglobinuria (PNH), a disease that manifests after clonal expansion of hematopoietic cells with somatic <i>PIGA</i> mutations. Although somatic <i>PIGA</i> mutations have been identified in many PNH patients, it has been proposed that germline mutations are lethal. We report a family with an X-linked lethal disorder involving cleft palate, neonatal seizures, contractures, central nervous system (CNS) structural malformations, and other anomalies. An X chromosome exome next-generation sequencing screen identified a single nonsense <i>PIGA</i> mutation, c.1234C>T, which predicts p.Arg412^∗. This variant segregated with disease and carrier status in the family, is similar to mutations known to cause PNH as a result of PIGA dysfunction, and was absent in 409 controls. <i>PIGA</i>-null mutations are thought to be embryonic lethal, suggesting that p.Arg412^∗ PIGA has residual function. Transfection of a mutant p.Arg412^∗ PIGA construct into PIGA-null cells showed partial restoration of GPI-anchored proteins. The genetic data show that the c.1234C>T (p.Arg412^∗) mutation is present in an affected child, is linked to the affected chromosome in this family, is rare in the population, and results in reduced, but not absent, biosynthesis of GPI anchors. We conclude that c.1234C>T in <i>PIGA</i> results in the lethal X-linked phenotype recognized in the reported family.

Micrognathia, glossoptosis, and cleft palate comprise one of the most common malformation sequences, Robin sequence. It is a component of the TARP syndrome, talipes equinovarus, atrial septal defect, Robin sequence, and persistent left superior vena cava. This disorder is X-linked and severe, with apparently 100% pre- or postnatal lethality in affected males. Here we characterize a second family with TARP syndrome, confirm linkage to Xp11.23-q13.3, perform massively parallel sequencing of X chromosome exons, filter the results via a number of criteria including the linkage region, use a unique algorithm to characterize sequence changes, and show that TARP syndrome is caused by mutations in the RBM10 gene, which encodes RNA binding motif 10. We further show that this previously uncharacterized gene is expressed in midgestation mouse embryos in the branchial arches and limbs, consistent with the human phenotype. We conclude that massively parallel sequencing is useful to characterize large candidate linkage intervals and that it can be used successfully to allow identification of disease-causing gene mutations.

G protein-coupled receptors (GPCRs), the largest human gene family, are important regulators of signaling pathways. However, knowledge of their genetic alterations is limited. In this study, we used exon capture and massively parallel sequencing methods to analyze the mutational status of 734 GPCRs in melanoma. This investigation revealed that one family member, GRM3, was frequently mutated and that one of its mutations clustered within one position. Biochemical analysis of GRM3 alterations revealed that mutant GRM3 selectively regulated the phosphorylation of MEK, leading to increased anchorage-independent growth and migration. Melanoma cells expressing mutant GRM3 had reduced cell growth and cellular migration after short hairpin RNA-mediated knockdown of GRM3 or treatment with a selective MEK inhibitor, AZD-6244, which is currently being used in phase 2 clinical trials. Our study yields the most comprehensive map of genetic alterations in the GPCR gene family.

VarSifter is a graphical software tool for desktop computers that allows investigators of varying computational skills to easily and quickly sort, filter, and sift through sequence variation data. A variety of filters and a custom query framework allow filtering based on any combination of sample and annotation information. By simplifying visualization and analyses of exome-scale sequence variation data, this program will help bring the power and promise of massively-parallel DNA sequencing to a broader group of researchers.VarSifter is written in Java, and is freely available in source and binary versions, along with a User Guide, at http://research.nhgri.nih.gov/software/VarSifter/.

The antigenicity of cells is demarcated by the peptides bound by their Human Leucocyte Antigen (HLA) molecules. Through this antigen presentation, T cell specificity response is controlled. As a fraction of the expressed mutated peptides is presented on the HLA, these neo-epitopes could be immunogenic. Such neo-antigens have recently been identified through screening for predicted mutated peptides, using synthetic peptides or ones expressed from minigenes, combined with screening of patient tumor-infiltrating lymphocytes (TILs). Here we present a time and cost-effective method that combines whole-exome sequencing analysis with HLA peptidome mass spectrometry, to identify neo-antigens in a melanoma patient. Of the 1,019 amino acid changes identified through exome sequencing, two were confirmed by mass spectrometry to be presented by the cells. We then synthesized peptides and evaluated the two mutated neo-antigens for reactivity with autologous bulk TILs, and found that one yielded mutant-specific T-cell response. Our results demonstrate that this method can be used for immune response prediction and promise to provide an alternative approach for identifying immunogenic neo-epitopes in cancer.

Abstract Malignant peripheral nerve sheath tumor (MPNST) is an aggressive soft tissue sarcoma. To more fully characterize the genomic landscape of this tumor type, we performed next generation sequencing studies for mutational and copy number analysis. We analyzed whole exome sequencing data from 12 MPNST and SNP arrays for a subset of these. We additionally conducted a literature review of prior next generation sequencing studies in this disease and compared to the current study. We report recurrent mutations in NF1 , SUZ12 , EED , TP53 and CDKN2A in our study cohort. Combined with prior studies, we calculate the disease specific incidence of mutation in these genes to be: NF1 (56/64 = 87.5%). SUZ12 (69/123 = 56.1%), EED (40/123 = 32.5%), TP53 (29/72 = 40.3%), and CDKN2A (54/72 = 75.0%). Notably, we also identified frequent Ras pathway activating somatic mutations outside of these previously reported recurrently mutated genes. Five of the 12 MPNST in our cohort (42%) contained such a mutation. In conclusion, our study adds to the growing understanding of the genomic complexity of MPNST. We report a previously underappreciated frequency and variety of secondary or tertiary Ras pathway activating mutations, though not highly recurrent in a single gene.

The homeobox A (HOXA) region of protein-coding genes impacts female reproductive system embryogenesis and ovarian carcinogenesis. The 5-prime end of HOXA includes three long non-coding RNAs (lncRNAs) (HOXA10-AS, HOXA11-AS, and HOTTIP) that are underexplored in epithelial ovarian cancer (EOC). We evaluated whether common genetic variants in these lncRNAs are associated with EOC risk and/or have functional roles in EOC development. Using genome-wide association study data from 1,201 serous EOC cases and 2,009 controls, an exonic variant within HOXA11-AS, rs17427875 (A>T), was marginally associated with reduced serous EOC risk (OR = 0.88 (95% CI: 0.78-1.01, p = 0.06). Functional studies of ectopic expression of HOXA11-AS minor allele T in EOC cells showed decreased survival, proliferation, migration, and invasion compared to common allele A expression. Additionally, stable expression of HOXA11-AS minor allele T reduced primary tumor growth in mouse xenograft models to a greater extent than common allele A. Furthermore, HOXA11-AS expression levels were significantly lower in human EOC tumors than normal ovarian tissues (p < 0.05), suggesting that HOXA11-AS has a tumor suppressor function in EOC which may be enhanced by the T allele. These findings demonstrate for the first time a role for HOXA11-AS in EOC with effects that could be modified by germline variants.

Abstract How genomic and transcriptomic alterations affect the functional proteome in lung cancer is not fully understood. Here, we integrate DNA copy number, somatic mutations, RNA-sequencing, and expression proteomics in a cohort of 108 squamous cell lung cancer (SCC) patients. We identify three proteomic subtypes, two of which (Inflamed, Redox) comprise 87% of tumors. The Inflamed subtype is enriched with neutrophils, B-cells, and monocytes and expresses more PD-1 . Redox tumours are enriched for oxidation-reduction and glutathione pathways and harbor more NFE2L2/KEAP1 alterations and copy gain in the 3q2 locus. Proteomic subtypes are not associated with patient survival. However, B-cell-rich tertiary lymph node structures, more common in Inflamed, are associated with better survival. We identify metabolic vulnerabilities ( TP63 , PSAT1 , and TFRC ) in Redox. Our work provides a powerful resource for lung SCC biology and suggests therapeutic opportunities based on redox metabolism and immune cell infiltrates.

The increasing practicality of genomic sequencing technology has led to its incorporation into routine clinical practice. Successful identification and targeting of driver genomic alterations that provide proliferative and survival advantages to tumor cells have led to approval and ongoing development of several targeted cancer therapies. Within many major cancer centers, molecular tumor boards are constituted to shepherd precision medicine into clinical practice.In July 2014, the Clinical Genomics Action Committee (CGAC) was established as the molecular tumor board companion to the Personalized Medicine Clinical Service (PMCS) at Moffitt Cancer Center in Tampa, Florida. The processes and outcomes of the program were assessed in order to help others move into the practice of precision medicine.Through the establishment and initial 1,400 patients of the PMCS and its associated molecular tumor board at a major cancer center, five practical lessons of broad applicability have been learned: transdisciplinary engagement, the use of the molecular report as an aid to clinical management, clinical actionability, getting therapeutic options to patients, and financial considerations. Value to patients includes access to cutting-edge practice merged with individualized preferences in treatment and care.Genomic-driven cancer medicine is increasingly becoming a part of routine clinical practice. For successful implementation of precision cancer medicine, strategically organized molecular tumor boards are critical to provide objective evidence-based translation of observed molecular alterations into patient-centered clinical action. Molecular tumor board implementation models along with clinical and economic outcomes will define future treatment standards. The Oncologist 2017;22:144-151Implications for Practice: It is clear that the increasing practicality of genetic tumor sequencing technology has led to its incorporation as part of routine clinical practice. Subsequently, many cancer centers are seeking to develop a personalized medicine services and/or molecular tumor board to shepherd precision medicine into clinical practice. This article discusses the key lessons learned through the establishment and development of a molecular tumor board and personalized medicine clinical service. This article highlights practical issues and can serve as an important guide to other centers as they conceive and develop their own personalized medicine services and molecular tumor boards.

Abstract Purpose: Patients with head and neck squamous cell carcinoma (HNSCC) who actively smoke during treatment have worse survival compared with never-smokers and former-smokers. We hypothesize the poor prognosis in tobacco smokers with HNSCC is, at least in part, due to ongoing suppression of immune response. We characterized the tumor immune microenvironment (TIM) of HNSCC in a retrospective cohort of 177 current, former, and never smokers. Experimental Design: Tumor specimens were subjected to analysis of CD3, CD8, FOXP3, PD-1, PD-L1, and pancytokeratin by multiplex immunofluorescence, whole-exome sequencing, and RNA sequencing. Immune markers were measured in tumor core, tumor margin, and stroma. Results: Our data indicate that current smokers have significantly lower numbers of CD8+ cytotoxic T cells and PD-L1+ cells in the TIM compared with never- and former-smokers. While tumor mutation burden and mutant allele tumor heterogeneity score do not associate with smoking status, gene-set enrichment analyses reveal significant suppression of IFNα and IFNγ response pathways in current smokers. Gene expression of canonical IFN response chemokines, CXCL9, CXCL10, and CXCL11, are lower in current smokers than in former smokers, suggesting a mechanism for the decreased immune cell migration to tumor sites. Conclusions: These results suggest active tobacco use in HNSCC has an immunosuppressive effect through inhibition of tumor infiltration of cytotoxic T cells, likely as a result of suppression of IFN response pathways. Our study highlights the importance of understanding the interaction between smoking and TIM in light of emerging immune modulators for cancer management.

Acral melanoma is a rare subtype of melanoma that arises on the non-hair-bearing skin of the palms, soles, and nail beds. In this study, we used single-cell RNA sequencing (scRNA-seq) to map the transcriptional landscape of acral melanoma and identify novel immunotherapeutic targets.We performed scRNA-seq on nine clinical specimens (five primary, four metastases) of acral melanoma. Detailed cell type curation was performed, the immune landscapes were mapped, and key results were validated by analysis of The Cancer Genome Atlas (TCGA) and single-cell datasets. Cell-cell interactions were inferred and compared with those in nonacral cutaneous melanoma.Multiple phenotypic subsets of T cells, natural killer (NK) cells, B cells, macrophages, and dendritic cells with varying levels of activation/exhaustion were identified. A comparison between primary and metastatic acral melanoma identified gene signatures associated with changes in immune responses and metabolism. Acral melanoma was characterized by a lower overall immune infiltrate, fewer effector CD8 T cells and NK cells, and a near-complete absence of γδ T cells compared with nonacral cutaneous melanomas. Immune cells associated with acral melanoma exhibited expression of multiple checkpoints including PD-1, LAG-3, CTLA-4, V-domain immunoglobin suppressor of T cell activation (VISTA), TIGIT, and the Adenosine A2A receptor (ADORA2). VISTA was expressed in 58.3% of myeloid cells and TIGIT was expressed in 22.3% of T/NK cells.Acral melanoma has a suppressed immune environment compared with that of cutaneous melanoma from nonacral skin. Expression of multiple, therapeutically tractable immune checkpoints were observed, offering new options for clinical translation.

Current methods for detecting mutations in Fanconi anemia (FA)-suspected patients are inefficient and often miss mutations. We have applied recent advances in DNA sequencing and genomic capture to the diagnosis of FA. Specifically, we used custom molecular inversion probes or TruSeq-enrichment oligos to capture and sequence FA and related genes, including introns, from 27 samples from the International Fanconi Anemia Registry at The Rockefeller University. DNA sequencing was complemented with custom array comparative genomic hybridization (aCGH) and RNA sequencing (RNA-seq) analysis. aCGH identified deletions/duplications in 4 different FA genes. RNA-seq analysis revealed lack of allele specific expression associated with a deletion and splicing defects caused by missense, synonymous, and deep-in-intron variants. The combination of TruSeq-targeted capture, aCGH, and RNA-seq enabled us to identify the complementation group and biallelic germline mutations in all 27 families: FANCA (7), FANCB (3), FANCC (3), FANCD1 (1), FANCD2 (3), FANCF (2), FANCG (2), FANCI (1), FANCJ (2), and FANCL (3). FANCC mutations are often the cause of FA in patients of Ashkenazi Jewish (AJ) ancestry, and we identified 2 novel FANCC mutations in 2 patients of AJ ancestry. We describe here a strategy for efficient molecular diagnosis of FA.

Malignant hyperthermia susceptibility (MHS) is a life-threatening, inherited disorder of muscle calcium metabolism, triggered by anesthetics and depolarizing muscle relaxants. An unselected cohort was screened for MHS mutations using exome sequencing. The aim of this study was to pilot a strategy for the RYR1 and CACNA1S genes.Exome sequencing was performed on 870 volunteers not ascertained for MHS. Variants in RYR1 and CACNA1S were annotated using an algorithm that filtered results based on mutation type, frequency, and information in mutation databases. Variants were scored on a six-point pathogenicity scale. Medical histories and pedigrees were reviewed for malignant hyperthermia and related disorders.The authors identified 70 RYR1 and 53 CACNA1S variants among 870 exomes. Sixty-three RYR1 and 41 CACNA1S variants passed the quality and frequency metrics but the authors excluded synonymous variants. In RYR1, the authors identified 65 missense mutations, one nonsense, two that affected splicing, and one non-frameshift indel. In CACNA1S, 48 missense, one frameshift deletion, one splicing, and one non-frameshift indel were identified. RYR1 variants predicted to be pathogenic for MHS were found in three participants without medical or family histories of MHS. Numerous variants, previously described as pathogenic in mutation databases, were reclassified by the authors as being of unknown pathogenicity.Exome sequencing can identify asymptomatic patients at risk for MHS, although the interpretation of exome variants can be challenging. The use of exome sequencing in unselected cohorts is an important tool to understand the prevalence and penetrance of MHS, a critical challenge for the field.

Background— Massively parallel sequencing to identify rare variants is widely practiced in medical research and in the clinic. Genome and exome sequencing can identify the genetic cause of a disease (primary results), but it can also identify pathogenic variants underlying diseases that are not being sought (secondary or incidental results). A major controversy has developed surrounding the return of secondary results to research participants. We have piloted a method to analyze exomes to identify participants at risk for cardiac arrhythmias, cardiomyopathies, or sudden death. Methods and Results— Exome sequencing was performed on 870 participants not selected for arrhythmia, cardiomyopathy, or a family history of sudden death. Exome data from 22 cardiac arrhythmia- and 41 cardiomyopathy-associated genes were analyzed using an algorithm that filtered results on genotype quality, frequency, and database information. We identified 1367 variants in the cardiomyopathy genes and 360 variants in the arrhythmia genes. Six participants had pathogenic variants associated with dilated cardiomyopathy (n=1), hypertrophic cardiomyopathy (n=2), left ventricular noncompaction (n=1), or long-QT syndrome (n=2). Two of these participants had evidence of cardiomyopathy and 1 had left ventricular noncompaction on echocardiogram. Three participants with likely pathogenic variants had prolonged QTc. Family history included unexplained sudden death among relatives. Conclusions— Approximately 0.5% of participants in this study had pathogenic variants in known cardiomyopathy or arrhythmia genes. This high frequency may be due to self-selection, false positives, or underestimation of the prevalence of these conditions. We conclude that clinically important cardiomyopathy and dysrhythmia secondary variants can be identified in unselected exomes.

The cyclin-dependent kinase inhibitor 3 (CDKN3) has been perceived as a tumour suppressor. Paradoxically, CDKN3 is often overexpressed in human cancer. It was unclear if CDKN3 overexpression is linked to alternative splicing variants or mutations that produce dominant-negative CDKN3.We analysed CDKN3 expression and its association with patient survival in three cohorts of lung adenocarcinoma. We also examined CDKN3 mutations in the Cancer Genome Atlas (TCGA) and the Moffitt Cancer Center's Total Cancer Care (TCC) projects. CDKN3 transcripts were further analysed in a panel of cell lines and lung adenocarcinoma tissues. CDKN3 mRNA and protein levels in different cell cycle phases were examined.CDKN3 is overexpressed in non small cell lung cancer. High CDKN3 expression is associated with poor overall survival in lung adenocarcinoma. Two CDKN3 transcripts were detected in all samples. These CDKN3 transcripts represent the full length CDKN3 mRNA and a normal transcript lacking exon 2, which encodes an out of frame 23-amino acid peptide with little homology to CDKN3. CDKN3 mutations were found to be very rare. CDKN3 mRNA and protein were elevated during the mitosis phase of cell cycle.CDKN3 overexpression is prognostic of poor overall survival in lung adenocarcinoma. CDKN3 overexpression in lung adenocarcinoma is not attributed to alternative splicing or mutation but is likely due to increased mitotic activity, arguing against CDKN3 as a tumour suppressor.

Background: Gastric cancer (GC) is one of the most common types of malignancy and is associated with high morbidity and mortality rates around the world.With poor clinical outcomes, potential biomarkers for diagnosis and prognosis are important to investigate.Objective: The aim of this study is to investigate the gene expression module of GC and to identify potential diagnostic and prognostic biomarkers.Method: Microarray data (GSE13911, GSE29272, GSE54129, and GSE79973), including 293 stomach tumor tissues and 196 normal tissues, were analyzed to identify differentially expressed genes (DEGs).DEGs were identified in four profiles by intersecting four overlapping subsets, including 90 downregulated and 45 upregulated DEGs in common.Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes pathway analyses have been showed that extracellular matrix was the most enriched signal pathway.Furthermore, hub genes were analyzed by protein-protein interaction network and clinical outcomes were assessed by Kaplan-Meier survival analysis.Two independent datasets were used to validate the differential expression of two hub genes: Serpin Family E Member 1 (SERPINE1) and Secreted Protein Acidic and Cysteine Rich (SPARC).Results: Validation of independent datasets indicated that SERPINE1 and SPARC expression were drastically increased in gastric tumor tissues and associated with poor outcomes in GC patients.The expression of SERPINE1 was related to race (Asian and White) (P , 0.05).Conclusion: SERPINE1 and SPARC were significantly upregulated in gastric tissues and associated with poor outcomes.The investigations of SERPINE1 and SPARC may promote their predictive and prognostic value in GC.

Abstract Large-scale genome-wide association studies (GWAS) have identified approximately 35 loci associated with epithelial ovarian cancer (EOC) risk. The majority of GWAS-identified disease susceptibility variants are located in noncoding regions, and causal genes underlying these associations remain largely unknown. Here, we performed a transcriptome-wide association study to search for novel genetic loci and plausible causal genes at known GWAS loci. We used RNA sequencing data (68 normal ovarian tissue samples from 68 individuals and 6,124 cross-tissue samples from 369 individuals) and high-density genotyping data from European descendants of the Genotype-Tissue Expression (GTEx V6) project to build ovarian and cross-tissue models of genetically regulated expression using elastic net methods. We evaluated 17,121 genes for their cis-predicted gene expression in relation to EOC risk using summary statistics data from GWAS of 97,898 women, including 29,396 EOC cases. With a Bonferroni-corrected significance level of P &amp;lt; 2.2 × 10−6, we identified 35 genes, including FZD4 at 11q14.2 (Z = 5.08, P = 3.83 × 10−7, the cross-tissue model; 1 Mb away from any GWAS-identified EOC risk variant), a potential novel locus for EOC risk. All other 34 significantly associated genes were located within 1 Mb of known GWAS-identified loci, including 23 genes at 6 loci not previously linked to EOC risk. Upon conditioning on nearby known EOC GWAS-identified variants, the associations for 31 genes disappeared and three genes remained (P &amp;lt; 1.47 × 10−3). These data identify one novel locus (FZD4) and 34 genes at 13 known EOC risk loci associated with EOC risk, providing new insights into EOC carcinogenesis. Significance: Transcriptomic analysis of a large cohort confirms earlier GWAS loci and reveals FZD4 as a novel locus associated with EOC risk. Cancer Res; 78(18); 5419–30. ©2018 AACR.

Abstract Recent work points to a lack of diversity in genomics studies from genome-wide association studies to somatic (tumor) genome analyses. Yet, population-specific genetic variation has been shown to contribute to health disparities in cancer risk and outcomes. Immortalized cancer cell lines are widely used in cancer research, from mechanistic studies to drug screening. Larger collections of cancer cell lines better represent the genomic heterogeneity found in primary tumors. Yet, the genetic ancestral origin of cancer cell lines is rarely acknowledged and often unknown. Using genome-wide genotyping data from 1,393 cancer cell lines from the Catalogue of Somatic Mutations in Cancer (COSMIC) and Cancer Cell Line Encyclopedia (CCLE), we estimated the genetic ancestral origin for each cell line. Our data indicate that cancer cell line collections are not representative of the diverse ancestry and admixture characterizing human populations. We discuss the implications of genetic ancestry and diversity of cellular models for cancer research and present an interactive tool, Estimated Cell Line Ancestry (ECLA), where ancestry can be visualized with reference populations of the 1000 Genomes Project. Cancer researchers can use this resource to identify cell line models for their studies by taking ancestral origins into consideration.

Cancer sequencing efforts have revealed that cancer is the most complex and heterogeneous disease that affects humans. However, radiation therapy (RT), one of the most common cancer treatments, is prescribed on the basis of an empirical one-size-fits-all approach. We propose that the field of radiation oncology is operating under an outdated null hypothesis: that all patients are biologically similar and should uniformly respond to the same dose of radiation.We have previously developed the genomic-adjusted radiation dose, a method that accounts for biological heterogeneity and can be used to predict optimal RT dose for an individual patient. In this article, we use genomic-adjusted radiation dose to characterize the biological imprecision of one-size-fits-all RT dosing schemes that result in both over- and under-dosing for most patients treated with RT. To elucidate this inefficiency, and therefore the opportunity for improvement using a personalized dosing scheme, we develop a patient-specific competing hazards style mathematical model combining the canonical equations for tumor control probability and normal tissue complication probability. This model simultaneously optimizes tumor control and toxicity by personalizing RT dose using patient-specific genomics.Using data from two prospectively collected cohorts of patients with NSCLC, we validate the competing hazards model by revealing that it predicts the results of RTOG 0617. We report how the failure of RTOG 0617 can be explained by the biological imprecision of empirical uniform dose escalation which results in 80% of patients being overexposed to normal tissue toxicity without potential tumor control benefit.Our data reveal a tapestry of radiosensitivity heterogeneity, provide a biological framework that explains the failure of empirical RT dose escalation, and quantify the opportunity to improve clinical outcomes in lung cancer by incorporating genomics into RT.

Pancreatic cancer (PaCa) is the third leading cause of cancer-related deaths in the United States. There is an unmet need to develop strategies to detect PaCa at an early, operable stage and prevent its progression. Intraductal papillary mucinous neoplasms (IPMNs) are cystic PaCa precursors that comprise nearly 50% of pancreatic cysts detected incidentally via cross-sectional imaging. Since IPMNs can progress from low- and moderate-grade dysplasia to high-grade dysplasia and invasion, the study of these lesions offers a prime opportunity to develop early detection and prevention strategies. Organoids are an ideal preclinical platform to study IPMNs, and the objective of the current investigation was to establish a living biobank of patient-derived organoids (PDO) from IPMNs. IPMN tumors and adjacent normal pancreatic tissues were successfully harvested from 15 patients with IPMNs undergoing pancreatic surgical resection at Moffitt Cancer Center & Research Institute (Tampa, FL) between May of 2017 and March of 2019. Organoid cultures were also generated from cryopreserved tissues. Organoid count and size were determined over time by both Image-Pro Premier 3D Version 9.1 digital platform and Matlab application of a Circular Hough Transform algorithm, and histologic and genomic characterization of a subset of the organoids was performed using immunohistochemistry and targeted sequencing, respectively. The success rates for organoid generation from IPMN tumor and adjacent normal pancreatic tissues were 81% and 87%, respectively. IPMN organoids derived from different epithelial subtypes showed different morphologies in vitro, and organoids recapitulated histologic and genomic characteristics of the parental IPMN tumor. In summary, this preclinical model has the potential to provide new opportunities to unveil mechanisms of IPMN progression to invasion and to shed insight into novel biomarkers for early detection and targets for chemoprevention. The authors report the feasibility of generating, expanding, and enumerating viable patient-derived intraductal papillary mucinous neoplasm (IPMN) tumor and normal pancreatic organoids from fresh and cryopreserved resected tissue. Organoids were characterized histologically and genomically and recapitulated the morphological and mutational profiles of resected IPMNs, suggesting promise of organoids for translational efforts.

The origin recognition complex (ORC) is involved in formation of prereplicative complexes (pre-RCs) on replication origins in the G 1 phase.At the G 1 /S transition, elevated cyclin E-CDK2 activity triggers DNA replication to enter S phase.The CDK cycle works as an engine that drives progression of cell cycle events by successive activation of different types of cyclin-CDK.However, how the CDK cycle is coordinated with replication initiation remains elusive.Here we report that acute depletion of ORC2 by RNA interference (RNAi) arrests cells with low cyclin E-CDK2 activity.This result suggests that loss of a replication initiation protein prevents progression of the CDK cycle in G 1 .p27 and p21 proteins accumulate following ORC2 RNAi and are required for the CDK2 inhibition.Restoration of CDK activity by co-depletion of p27 and p21 allows many ORC2-depleted cells to enter S phase and go on to mitosis.However, in some cells the release of the CDK2 block caused catastrophic events like apoptosis.Therefore, the CDK2 inhibition observed following ORC2 RNAi seems to protect cells from premature S phase entry and crisis in DNA replication.These results demonstrate an unexpected role of ORC2 in CDK2 activation, a linkage that could be important for maintaining genomic stability.

Cancer-associated protein tyrosine kinase (PTK) mutations usually are gain-of-function (GOF) mutations that drive tumor growth and metastasis. We have found 50 JAK1 truncating mutations in 36 of 635 gynecologic tumors in the Total Cancer Care® (TCC®) tumor bank. Among cancer cell lines containing JAK1 truncating mutations in the Cancer Cell Line Encyclopedia databank, 68% are gynecologic cancer cells. Within JAK1 the K142, P430, and K860 frame-shift mutations were identified as hot spot mutation sites. Sanger sequencing of cancer cell lines, primary tumors, and matched normal tissues confirmed the JAK1 mutations and showed that these mutations are somatic. JAK1 mediates interferon (IFN)-γ-regulated tumor immune surveillance. Functional assays show that JAK1 deficient cancer cells are defective in IFN-γ-induced LMP2 and TAP1 expression, loss of which inhibits presentation of tumor antigens. These findings identify recurrent JAK1 truncating mutations that could contribute to tumor immune evasion in gynecologic cancers, especially in endometrial cancer.

Background. Recent data have suggested that regular aspirin use improves overall and cancer-specific survival in the subset of colorectal cancer (CRC) patients harboring PIK3CA mutations. However, the number of PIK3CA-mutated CRC patients examined in these studies was modest. Our collaborative study aims to validate the association between regular aspirin use and survival in patients with PIK3CA-mutated CRC.

Observations of recurrent somatic mutations in tumors have led to identification and definition of signaling and other pathways that are important for cancer progression and therapeutic targeting. As tumor cells contain both an individual's inherited genetic variants and somatic mutations, challenges arise in distinguishing these events in massively parallel sequencing datasets. Typically, both a tumor sample and a "normal" sample from the same individual are sequenced and compared; variants observed only in the tumor are considered to be somatic mutations. However, this approach requires two samples for each individual. We evaluate a method of detecting somatic mutations in tumor samples for which only a subset of normal samples are available. We describe tuning of the method for detection of mutations in tumors, filtering to remove inherited variants, and comparison of detected mutations to several matched tumor/normal analysis methods. Filtering steps include the use of population variation datasets to remove inherited variants as well a subset of normal samples to remove technical artifacts. We then directly compare mutation detection with tumor-only and tumor-normal approaches using the same sets of samples. Comparisons are performed using an internal targeted gene sequencing dataset (n = 3380) as well as whole exome sequencing data from The Cancer Genome Atlas project (n = 250). Tumor-only mutation detection shows similar recall (43–60%) but lesser precision (20–21%) to current matched tumor/normal approaches (recall 43–73%, precision 30–82%) when compared to a "gold-standard" tumor/normal approach. The inclusion of a small pool of normal samples improves precision, although many variants are still uniquely detected in the tumor-only analysis. A detailed method for somatic mutation detection without matched normal samples enables study of larger numbers of tumor samples, as well as tumor samples for which a matched normal is not available. As sensitivity/recall is similar to tumor/normal mutation detection but precision is lower, tumor-only detection is more appropriate for classification of samples based on known mutations. Although matched tumor-normal analysis is preferred due to higher precision, we demonstrate that mutation detection without matched normal samples is possible for certain applications.

The incidence and outcomes of patients with colorectal cancer (CRC) varies by age. Younger patients tend to have sporadic cancers that are not detected by screening and worse survival. To understand whether genetic differences exist between age cohorts, the authors sought to characterize unique genetic alterations in patients with CRC.In total, 283 patients who were diagnosed with sporadic CRC between 1998 and 2010 were identified and divided by age into 2 cohorts-ages ≤45 years (the younger cohort) and ≥65 years (the older cohort)-and targeted exome sequencing was performed. The Fisher exact test was used to detect differences in mutation frequencies between the 2 groups. Whole exome sequencing was performed on 21 additional younger patient samples for validation. Findings were confirmed in The Cancer Genome Atlas CRC data set.In total, 246 samples were included for final analysis (195 from the older cohort and 51 from the younger cohort). Mutations in the FBXW7 gene were more common in the younger cohort (27.5% vs 9.7%; P = .0022) as were mutations in the proofreading domain of polymerase ε catalytic subunit (POLE) (9.8% vs 1%; P = .0048). There were similar mutation rates between cohorts with regard to TP53 (64.7% vs 61.5%), KRAS (43.1% vs 46.2%), and APC (60.8% vs 73.8%). BRAF mutations were numerically more common in the older cohort, although the difference did not reach statistical significance (2% vs 9.7%; P = .082).In this retrospective study, a unique genetic profile was identified for younger patients who have CRC compared with patients who are diagnosed at an older age. These findings should be validated in a larger study and could have an impact on future screening and treatment modalities for younger patients with CRC. Cancer 2016. © 2016 American Cancer Society. Cancer 2016;122:2828-2835. © 2016 American Cancer Society.

Abstract Melanoma is one of the most highly mutated cancer types. To identify functional drivers of melanoma, we searched for cross-species conserved mutations utilizing a mouse melanoma model driven by loss of PTEN and CDKN2A, and identified mutations in Kras, Erbb3, and Ptpn11. PTPN11 encodes the SHP2 protein tyrosine phosphatase that activates the RAS/RAF/MAPK pathway. Although PTPN11 is an oncogene in leukemia, lung, and breast cancers, its roles in melanoma are not clear. In this study, we found that PTPN11 is frequently activated in human melanoma specimens and cell lines and is required for full RAS/RAF/MAPK signaling activation in BRAF wild-type (either NRAS mutant or wild-type) melanoma cells. PTPN11 played oncogenic roles in melanoma by driving anchorage-independent colony formation and tumor growth. In Pten- and Cdkn2a-null mice, tet-inducible and melanocyte-specific PTPN11E76K expression significantly enhanced melanoma tumorigenesis. Melanoma cells derived from this mouse model showed doxycycline-dependent tumor growth in nude mice. Silencing PTPN11E76K expression by doxycycline withdrawal caused regression of established tumors by induction of apoptosis and senescence, and suppression of proliferation. Moreover, the PTPN11 inhibitor (SHP099) also caused regression of NRASQ61K-mutant melanoma. Using a quantitative tyrosine phosphoproteomics approach, we identified GSK3α/β as one of the key substrates that were differentially tyrosine-phosphorylated in these experiments modulating PTPN11. This study demonstrates that PTPN11 plays oncogenic roles in melanoma and regulates RAS and GSK3β signaling pathways. Implications: This study identifies PTPN11 as an oncogenic driver and a novel and actionable therapeutic target for BRAF wild-type melanoma.

Acral melanomas arise on the non-hair bearing skin of the palms, soles and in the nail beds. These rare tumors comprise 2–3 % of all melanomas, are not linked to UV-exposure, and represent the most frequent subtype of melanomas in patients of Asian, African and Hispanic origin. Although recent work has revealed candidate molecular events that underlie acral melanoma development, this knowledge is not yet been translated into efficacious local, regional, or systemic therapies. In the current review, we describe the clinical characteristics of acral melanoma and outline the genetic basis of acral melanoma development. Further discussion is given to the current status of systemic therapy for acral melanoma with a focus on ongoing developments in both immunotherapy and targeted therapy for the treatment of advanced disease.

Next-generation sequencing (NGS) is rapidly expanding into routine oncology practice. Genetic variations in both the cancer and inherited genomes are informative for hereditary cancer risk, prognosis, and treatment strategies. Herein, we focus on the clinical perspective of integrating NGS results into patient care to assist with therapeutic decision making. Five key considerations are addressed for operationalization of NGS testing and application of results to patient care as follows: (1) NGS test ordering and workflow design; (2) result reporting, curation, and storage; (3) clinical consultation services that provide test interpretations and identify opportunities for molecularly guided therapy; (4) presentation of genetic information within the electronic health record; and (5) education of providers and patients. Several of these key considerations center on informatics tools that support NGS test ordering and referencing back to the results for therapeutic purposes. Clinical decision support tools embedded within the electronic health record can assist with NGS test utilization and identifying opportunities for targeted therapy including clinical trial eligibility. Challenges for project and change management in operationalizing NGS-supported, evidence-based patient care in the context of current information technology systems with appropriate clinical data standards are discussed, and solutions for overcoming barriers are provided.

Summary A clearer understanding of the tumor immune microenvironment (TIME) in metastatic clear cell renal cell carcinoma (ccRCC) may help to inform precision treatment strategies. We sought to identify clinically meaningful TIME signatures in ccRCC. We studied tumors from 39 patients with metastatic ccRCC using quantitative multiplexed immunofluorescence and relevant immune marker panels. Cell densities were analyzed in three regions of interest (ROIs): tumor core, tumor–stroma interface and stroma. Patients were stratified into low- and high-marker density groups using median values as thresholds. Log-rank and Cox regression analyses while controlling for clinical variables were used to compare survival outcomes to patterns of immune cell distributions. There were significant associations with increased macrophage (CD68+CD163+CD206+) density and poor outcomes across multiple ROIs in primary and metastatic tumors. In primary tumors, T-bet+ T helper type 1 (Th1) cell density was highest at the tumor–stromal interface (P = 0·0021), and increased co-expression of CD3 and T-bet was associated with improved overall survival (P = 0·015) and survival after immunotherapy (P = 0·014). In metastatic tumor samples, decreased forkhead box protein 3 (FoxP3)+ T regulatory cell density correlated with improved survival after immunotherapy (P = 0·016). Increased macrophage markers and decreased Th1 T cell markers within the TIME correlated with poor overall survival and treatment outcomes. Immune markers such as FoxP3 showed consistent levels across the TIME, whereas others, such as T-bet, demonstrated significant variance across the distinct ROIs. These findings suggest that TIME profiling outside the tumor core may identify clinically relevant associations for patients with metastatic ccRCC.

Clear cell renal cell carcinoma (ccRCC) tumors have low frequencies of genetic alterations compared with other malignancies, but very high levels of immune cell infiltration and favorable response rates to immunotherapy. Currently, the interplay between specific ccRCC somatic mutations and immune infiltration pattern is unclear.To analyze the associations between common ccRCC somatic mutations and immune cell infiltration patterns within the tumor immune microenvironment (TIME).The study included tumor samples (24 primary and 24 metastatic) from 48 patients with stage IV ccRCC. Targeted sequencing was performed for well-characterized recurrent somatic mutations in ccRCC, with the analysis focusing on the six most common ones: VHL, BAP1, PBRM1, SETD2, TP53, and KDM5C. For each sample, multiplex immunofluorescence (IF) was performed in lymphoid and myeloid panels, for seven regions of interest in three zones (tumor core, stroma, and tumor-stroma interface). IF-derived cellular densities were compared across patients, stratified by their somatic mutation status, using a linear mixed-model analysis. External validation was pursued using RNA-seq enrichment scoring from three large external data sources.Tumors with SETD2 mutations demonstrated significantly decreased levels of FOXP3+ T cells in the tumor core, stroma, and tumor-stroma interface. PBRM1 mutations were associated with decreased FOXP3+ T cells in the tumor core. Primary KDM5C mutations were associated with significantly increased CD206+ macrophage tumor infiltration in the tumor core. A computational method estimating immune cell types in the TIME using bulk RNA-seq data, xCell scoring, failed to validate associations from the IF analysis in large external data sets. A major limitation of the study is the relatively small patient population studied.This study provides evidence that common somatic mutations in ccRCC, such as SETD2, PBRM1, and KDM5C, are associated with distinct immune infiltration patterns within the TIME.In this study, we analyzed tumor samples from patients with metastatic kidney cancer to determine whether common genetic mutations that arise from the cancer cells are associated with the density of immune cells found within those tumors. We found several distinct immune cell patterns that were associated with specific genetic mutations. These findings provide insight into the interaction between cancer genetics and the immune system in kidney cancer.

Alternative mRNA splicing can be dysregulated in cancer, resulting in the generation of aberrant splice variants (SVs). Given the paucity of actionable genomic mutations in clear cell renal cell carcinoma (ccRCC), aberrant SVs may be an avenue to novel mechanisms of pathogenesis. To identify and characterize aberrant SVs enriched in ccRCC. Using RNA-seq data from the Cancer Cell Line Encyclopedia, we identified neojunctions uniquely expressed in ccRCC. Candidate SVs were then checked for expression across normal tissue in the Genotype-Tissue Expression Project and primary tumor tissue from The Cancer Genome Atlas (TCGA), Clinical Proteomic Tumor Analysis Consortium (CPTAC), and our institutional Total Cancer Care database. Clinicopathologic, genomic, and survival data were available for all cohorts. Epigenetic data were available for the TCGA and CPTAC cohorts. Proteomic data were available for the CPTAC cohort. The association of aberrant SV expression with these variables was examined using the Kruskal-Wallis test, pairwise t test, Spearman correlation test, and Cox regression analysis. Our pipeline identified 16 ccRCC-enriched SVs. EGFR, HPCAL1-SV and RNASET2-SV expression was negatively correlated with gene-specific CpG methylation. We derived a survival risk score based primarily on the expression of five SVs (RNASET2, FGD1, PDZD2, COBLL1, and PTPN14), which was consistent and applicable across multiple cohorts on multivariate analysis. The splicing factor RBM4, which modulates splicing of HIF-1α, exhibited significantly lower expression at the protein level in the high-risk group, as defined by our SV-based score. We describe 16 aberrant SVs enriched in ccRCC, many of which are associated with disease biology and/or clinical outcomes. This study provides a novel strategy for identifying and characterizing disease-specific aberrant SVs. We describe a method to identify disease targets and biomarkers using transcriptomic analysis beyond somatic mutations or gene expression. Kidney tumors express unique splice variants that may provide additional prognostic information following surgery.

In clear cell renal cell carcinoma (ccRCC), tumor-associated macrophage (TAM) induction of CD8+T cells into a terminally exhausted state has been implicated as a major mechanism of immunotherapy resistance, but a deeper biological understanding is necessary.Primary ccRCC tumor samples were obtained from 97 patients between 2004 and 2018. Multiplex immunofluorescence using lymphoid and myeloid markers was performed in seven regions of interest per patient across three predefined zones, and geospatial analysis was performed using Ripley's K analysis, a methodology adapted from ecology.Clustering of CD163+M2 like TAMs into the stromal compartment at the tumor-stroma interface was associated with worse clinical stage (tumor/CD163+nK(75): stage I/II: 4.4 (IQR -0.5 to 5.1); stage III: 1.4 (IQR -0.3 to 3.5); stage IV: 0.6 (IQR -2.1 to 2.1); p=0.04 between stage I/II and stage IV), and worse overall survival (OS) and cancer-specific survival (CSS) (tumor/CD163+nK(75): median OS-hi=149 months, lo=86 months, false-discovery rate (FDR)-adj. Cox p<0.001; median CSS-hi=174 months, lo=85 months; FDR-adj. Cox p<0.001). An RNA-seq differential gene expression score was developed using this geospatial metric, and was externally validated in multiple independent cohorts of patients with ccRCC including: TCGA KIRC, and the IMmotion151, IMmotion150, and JAVELIN Renal 101 clinical trials. In addition, this CD163+ geospatial pattern was found to be associated with a higher TIM-3+ proportion of CD8+T cells, indicative of terminal exhaustion (tumor-core: 0.07 (IQR 0.04-0.14) vs 0.40 (IQR 0.15-0.66), p=0.05).Geospatial clustering of CD163+M2 like TAMs into the stromal compartment at the tumor-stromal interface was associated with poor clinical outcomes and CD8+T cell terminal exhaustion.

Background Adoptive cell therapy (ACT) with tumor-infiltrating lymphocytes (TILs) is a promising immunotherapeutic approach for patients with advanced solid tumors. While numerous advances have been made, the contribution of neoantigen-specific CD4 + T cells within TIL infusion products remains underexplored and therefore offers a significant opportunity for progress. Methods We analyzed infused TIL products from metastatic melanoma patients previously treated with ACT for the presence of neoantigen-specific T cells. TILs were enriched on reactivity to neoantigen peptides derived and prioritized from patient sample-directed mutanome analysis. Enriched TILs were further investigated to establish the clonal neoantigen response with respect to function, transcriptomics, and persistence following ACT. Results We discovered that neoantigen-specific TIL clones were predominantly CD4 + T cells and were present in both therapeutic responders and non-responders. CD4 + TIL demonstrated an effector T cell response with cytotoxicity toward autologous tumor in a major histocompatibility complex class II-dependent manner. These results were validated by paired TCR and single cell RNA sequencing, which elucidated transcriptomic profiles distinct to neoantigen-specific CD4 + TIL. Conclusions Despite methods which often focus on CD8+T cells, our study supports the importance of prospective identification of neoantigen-specific CD4 + T cells within TIL products as they are a potent source of tumor-specific effectors. We further advocate for the inclusion of neoantigen-specific CD4 + TIL in future ACT protocols as a strategy to improve antitumor immunity.

Abstract DDR2 mutations occur in approximately 4% of lung squamous cell cancer (SCC) where the tyrosine kinase inhibitor dasatinib has emerged as a new therapeutic option. We found that ERK and AKT phosphorylation was weakly inhibited by dasatinib in DDR2-mutant lung SCC cells, suggesting that dasatinib inhibits survival signals distinct from other oncogenic receptor tyrosine kinases (RTK) and/or compensatory signals exist that dampen dasatinib activity. To gain better insight into dasatinib's action in these cells, we assessed altered global tyrosine phosphorylation (pY) after dasatinib exposure using a mass spectrometry-based quantitative phosphoproteomics approach. Overlaying protein–protein interaction relationships upon this dasatinib-regulated pY network revealed decreased phosphorylation of Src family kinases and their targets. Conversely, dasatinib enhanced tyrosine phosphorylation in a panel of RTK and their signaling adaptor complexes, including EGFR, MET/GAB1, and IGF1R/IRS2, implicating a RTK-driven adaptive response associated with dasatinib. To address the significance of this observation, these results were further integrated with results from a small-molecule chemical library screen. We found that dasatinib combined with MET and insulin-like growth factor receptor (IGF1R) inhibitors had a synergistic effect, and ligand stimulation of EGFR and MET rescued DDR2-mutant lung SCC cells from dasatinib-induced loss of cell viability. Importantly, we observed high levels of tyrosine-phosphorylated EGFR and MET in a panel of human lung SCC tissues harboring DDR2 mutations. Our results highlight potential RTK-driven adaptive-resistant mechanisms upon DDR2 targeting, and they suggest new, rationale cotargeting strategies for DDR2-mutant lung SCC. Cancer Res; 74(24); 7217–28. ©2014 AACR.

Introduction Metastasis is thought to be a clonal event whereby a single cell initiates the development of a new tumor at a distant site. However the degree to which primary and metastatic tumors differ on a molecular level remains unclear. To further evaluate these concepts, we used next generation sequencing (NGS) to assess the molecular composition of paired primary and metastatic colorectal cancer tissue specimens. Methods 468 colorectal tumor samples from a large personalized medicine initiative were assessed by targeted gene sequencing of 1,321 individual genes. Eighteen patients produced genomic profiles for 17 paired primary:metastatic (and 2 metastatic:metastatic) specimens. Results An average of 33.3 mutations/tumor were concordant (shared) between matched samples, including common well-known genes (APC, KRAS, TP53). An average of 2.3 mutations/tumor were discordant (unshared) among paired sites. KRAS mutational status was always concordant. The overall concordance rate for mutations was 93.5%; however, nearly all (18/19 (94.7%)) paired tumors showed at least one mutational discordance. Mutations were seen in: TTN, the largest gene (5 discordant pairs), ADAMTS20, APC, MACF1, RASA1, TP53, and WNT2 (2 discordant pairs), SMAD2, SMAD3, SMAD4, FBXW7, and 66 others (1 discordant pair). Conclusions Whereas primary and metastatic tumors displayed little variance overall, co-evolution produced incremental mutations in both. These results suggest that while biopsy of the primary tumor alone is likely sufficient in the chemotherapy-naïve patient, additional biopsies of primary or metastatic disease may be necessary to precisely tailor therapy following chemotherapy resistance or insensitivity in order to adequately account for tumor evolution.

Epithelial ovarian cancer is a leading cause of death in gynecological cancers. While several systematic studies have revealed the mutation landscape of serous epithelial ovarian cancer, other non-serous subtypes of the disease have not been explored as extensively. Here we conduct exome sequencing of nine non-serous epithelial ovarian tumors (six endometrioid and three mucinous) and their corresponding normal DNA as well as a tumor-only granulosa cell sample. We integrated the exome data with targeted gene sequencing for 1,321 genes selected for their involvement in cancer from additional 28 non-serous ovarian tumors and compared our results to TCGA ovarian serous cystadenocarcinoma and uterine corpus endometrial carcinomas. Prevalence of TP53 mutations in non-serous was much lower than in serous epithelial OC, whereas the prevalence of PIK3CA, PIK3R1, PTEN, CTNNB1, ARID1A, and KRAS was higher. We confirmed the high prevalence of FOXL2 and KRAS mutations in granulosa cell tumors and in mucinous tumors, respectively. We also identified POLE proofreading domain mutations in three endometrioid ovarian tumors. These results highlight mutational differences between serous and non-serous ovarian cancers, and further distinguish different non-serous subtypes.

Immune escape is a hallmark of cancer. In human lung cancer, we have identified a unique microRNA (miR)-based pathway employed by tumor cells to repress detection by immune cells via the NKG2D-MICA/B receptor-ligand system. MICA/B is readily induced by cell transformation and serves as a danger signal and ligand to alert NK and activated CD8+ T cells. However, immunohistochemical analysis indicated that human lung adenocarcinoma and squamous cell carcinoma specimens express little MICA/B while high levels of miR-183 were detected in both tumor types in a TCGA database. Human lung tumor cell lines confirmed the reverse relationship in expression of MICA/B and miR-183. Importantly, a miR-183 binding site was identified on the 3ʹuntranslated region (UTR) of both MICA and MICB, suggesting its role in MICA/B regulation. Luciferase reporter constructs bearing the 3ʹUTR of MICA or MICB in 293 cells supported the function of miR-183 in repressing MICA/B expression. Additionally, anti-sense miR-183 transfection into H1355 or H1299 tumor cells caused the upregulation of MICA/B. Abundant miR-183 expression in tumor cells was traced to transforming growth factor-beta (TGFβ), as evidenced by antisense TGFβ transfection into H1355 or H1299 tumor cells which subsequently lost miR-183 expression accompanied by MICA/B upregulation. Most significantly, anti-sense miR-183 transfected tumor cells became more sensitive to lysis by activated CD8+ T cells that express high levels of NKG2D. Thus, high miR-183 triggered by TGFβ expressed in lung tumor cells can target MICA/B expression to circumvent detection by NKG2D on immune cells.

It has recently been described that alternative oncogenic drivers may be found in KRAS wild-type (KRASWT) pancreatic cancers. This study aimed to determine the incidence of targetable gene fusions present in KRASWT pancreatic adenocarcinoma and response to targeted therapy.One hundred consecutive patients with pancreatic adenocarcinoma who underwent targeted next-generation sequencing using DNA sequencing with RNA sequencing (n = 47) or without RNA sequencing (n = 53) at a single institution were included in the study. The frequency and landscape of targetable fusions in KRASWT pancreatic adenocarcinoma was characterized and compared with the frequency of fusions in KRAS-mutated (KRASMUT) pancreatic adenocarcinoma. Results were validated in two independent cohorts using data from AACR GENIE (n = 1,252) and TCGA (n = 150). The clinical history of fusion-positive patients who received targeted treatment is described.Pancreatic cancers from 13 of 100 patients (13%) were found to be KRASWT. Targetable fusions were identified in 4/13 (31%) KRASWT tumors compared with 0/87 (0%) KRASMUT pancreatic adenocarcinomas (P = .0002). One patient with a novel MET fusion had a complete response to targeted therapy with crizotinib that is ongoing at 12+ months of treatment. In the validation cohorts, gene fusions were identified in 18/97 (19%) and 2/10 (20%) KRASWT tumors reported in the AACR GENIE and TCGA cohorts, respectively.Oncogene fusions are present in KRASWT pancreatic adenocarcinomas at an increased frequency when compared with KRASMUT pancreatic adenocarcinomas. As these fusions may be susceptible to targeted therapy, molecular analyses for the detection of fusions in KRASWT pancreatic adenocarcinomas may warrant increased consideration.

Immune-modulating systemic therapies are often used to treat advanced cancer such as metastatic clear cell renal cell carcinoma (ccRCC). Used alone, sequence-based biomarkers neither accurately capture patient dynamics nor the tumor immune microenvironment. To better understand the tumor ecology of this immune microenvironment, we quantified tumor infiltration across three distinct ccRCC patient tumor cohorts using complementarity determining region-3 (CDR3) sequence recovery counts in tumor-infiltrating lymphocytes and a generalized diversity index (GDI) for CDR3 sequence distributions. GDI can be understood as a curve over a continuum of diversity scales that allows sensitive characterization of distributions to capture sample richness, evenness, and subsampling uncertainty, along with other important metrics that characterize tumor heterogeneity. For example, richness quantified the total unique sequence count, while evenness quantified similarities across sequence frequencies. Significant differences in receptor sequence diversity across gender and race revealed that patients with larger and more clinically aggressive tumors had increased richness of recovered tumoral CDR3 sequences, specifically in those from T-cell receptor alpha and B-cell immunoglobulin lambda light chain. The GDI inflection point (IP) allowed for a novel and robust measure of distribution evenness. High IP values were associated with improved overall survival, suggesting that normal-like sequence distributions lead to better outcomes. These results propose a new quantitative tool that can be used to better characterize patient-specific differences related to immune cell infiltration, and to identify unique characteristics of tumor-infiltrating lymphocyte heterogeneity in ccRCC and other malignancies.Assessment of tumor-infiltrating T-cell and B-cell diversity in renal cell carcinoma advances the understanding of tumor-immune system interactions, linking tumor immune ecology with tumor burden, aggressiveness, and patient survival. See related commentary by Krishna and Hakimi, p. 764.

Cancer testis antigens have been of interest as possible targets for cancer immunotherapies. To better understand the opportunities for the use of such immunotherapy targets, we used a chemical complementarity scoring algorithm and an original web tool to establish aspects of electrostatic complementarity of the CTAs, MAGEA3 and MAGEA6, with melanoma specimen resident, T-cell receptor (TCR) complementarity determining region 3 (CDR3) amino acid sequences. Greater electrostatic complementarity between T-cell receptor CDR3 and tumor CTAs MAGEA3/6 was associated with a greater probability of overall survival, for both the cancer genome atlas and Moffitt Cancer Center samples; and was associated with high levels of T-cell cytotoxicity-related gene expression. Most importantly, this approach allowed for the highly efficient screening of specific segments of the MAGEA3/6 antigens which indicated that certain MAGE segments would have either more or less risk of auto-reactivity. In sum, the chemical complementarity algorithm, and its efficient application via the web tool, adaptivematch.com, offers a convenient opportunity to identify likely parameters important for immunotherapy considerations and melanoma patient risk stratifications.

Multiple myeloma (MM) incidence, mortality, and survival vary by race and ethnicity, but the causes of differences remain unclear. We investigated demographic, clinical, and molecular features of diverse MM patients to elucidate mechanisms driving clinical disparities. This study included 495 MM patients (self-reported Hispanic, n = 45; non-Hispanic Black, n = 52; non-Hispanic White, n = 398). Hispanic and non-Hispanic Black individuals had an earlier age of onset than non-Hispanic White individuals (53 and 57 vs 63 years, respectively, P < .001). There were no differences in treatment by race and ethnicity groups, but non-Hispanic Black patients had a longer time to hematopoietic cell transplant than non-Hispanic White patients (376 days vs 248 days; P = .01). Overall survival (OS) was improved for non-Hispanic Black compared with non-Hispanic White patients (HR, 0.50; 95% CI, 0.31-0.81; P = .005), although this association was attenuated after adjusting for clinical features (HR, 0.62; 95% CI, 0.37-1.03; P = .06). Tumor mutations in IRF4 were most common in Hispanic patients, and mutations in SP140, AUTS2, and SETD2 were most common in non-Hispanic Black patients. Differences in tumor expression of BCL7A, SPEF2, and ANKRD26 by race and ethnicity were observed. Clonal hematopoiesis was detected in 12% of patients and associated with inferior OS in non-Hispanic Black patients compared with patients without clonal hematopoiesis (HR, 4.36; 95% CI, 1.36-14.00). This study provides insight into differences in molecular features that may drive clinical disparities in MM patients receiving comparable treatment, with the novel inclusion of Hispanic individuals.

To utilize high-throughput sequencing to determine the etiology of juvenile-onset neurodegeneration in a 19-year-old woman with progressive motor and cognitive decline.Exome sequencing identified an initial list of 133,555 variants in the proband's family, which were filtered using segregation analysis, presence in dbSNP, and an empirically derived gene exclusion list. The filtered list comprised 52 genes: 21 homozygous variants and 31 compound heterozygous variants. These variants were subsequently scrutinized with predicted pathogenicity programs and for association with appropriate clinical syndromes.Exome sequencing data identified 2 GLB1 variants (c.602G>A, p.R201H; c.785G>T, p.G262V). β-Galactosidase enzyme analysis prior to our evaluation was reported as normal; however, subsequent testing was consistent with juvenile-onset GM1-gangliosidosis. Urine oligosaccharide analysis was positive for multiple oligosaccharides with terminal galactose residues.We describe a patient with juvenile-onset neurodegeneration that had eluded diagnosis for over a decade. GM1-gangliosidosis had previously been excluded from consideration, but was subsequently identified as the correct diagnosis using exome sequencing. Exome sequencing can evaluate genes not previously associated with neurodegeneration, as well as most known neurodegeneration-associated genes. Our results demonstrate the utility of "agnostic" exome sequencing to evaluate patients with undiagnosed disorders, without prejudice from prior testing results.

Genomic technologies, such as whole-exome sequencing, are a powerful tool in genetic research. Such testing yields a great deal of incidental medical information, or medical information not related to the primary research target. We describe the management of incidental medical information derived from whole-exome sequencing in the research context. We performed whole-exome sequencing on a monozygotic twin pair in which only 1 child was affected with congenital anomalies and applied an institutional review board-approved algorithm to determine what genetic information would be returned. Whole-exome sequencing identified 79525 genetic variants in the twins. Here, we focus on novel variants. After filtering artifacts and excluding known single nucleotide polymorphisms and variants not predicted to be pathogenic, the twins had 32 novel variants in 32 genes that were felt to be likely to be associated with human disease. Eighteen of these novel variants were associated with recessive disease and 18 were associated with dominantly manifesting conditions (variants in some genes were potentially associated with both recessive and dominant conditions), but only 1 variant ultimately met our institutional review board-approved criteria for return of information to the research participants.

Purpose Quantitative MS assays for I gs are compared with existing clinical methods in samples from patients with plasma cell dyscrasias, for example, multiple myeloma ( MM ). Experimental design Using LC ‐ MS/MS data, Ig constant region peptides, and transitions were selected for LC ‐ MRM MS . Quantitative assays were used to assess I gs in serum from 83 patients. RNA sequencing and peptide‐based LC‐MRM are used to define peptides for quantification of the disease‐specific Ig. Results LC ‐ MRM assays quantify serum levels of Igs and their isoforms (IgG1–4, IgA1–2, IgM, IgD, and IgE, as well as kappa (κ) and lambda (λ) light chains). LC ‐ MRM quantification has been applied to single samples from a patient cohort and a longitudinal study of an IgE patient undergoing treatment, to enable comparison with existing clinical methods. Proof‐of‐concept data for defining and monitoring variable region peptides are provided using the H929 MM cell line and two MM patients. Conclusions and clinical relevance LC ‐ MRM assays targeting constant region peptides determine the type and isoform of the involved Ig and quantify its expression; the LC ‐ MRM approach has improved sensitivity compared with the current clinical method, but slightly higher inter‐assay variability. Detection of variable region peptides is a promising way to improve Ig quantification, which could produce a dramatic increase in sensitivity over existing methods, and could further complement current clinical techniques.

Coronary artery calcification (CAC) is a heritable and definitive morphologic marker of atherosclerosis that strongly predicts risk for future cardiovascular events. To search for genes involved in CAC, we used an integrative transcriptomic, genomic, and protein expression strategy by using next-generation DNA sequencing in the discovery phase with follow-up studies using traditional molecular biology and histopathology techniques. RNA sequencing of peripheral blood from a discovery set of CAC cases and controls was used to identify dysregulated genes, which were validated by ClinSeq and Framingham Heart Study data. Only a single gene, TREML4, was upregulated in CAC cases in both studies. Further examination showed that rs2803496 was a TREML4 cis-eQTL and that the minor allele at this locus conferred up to a 6.5-fold increased relative risk of CAC. We characterized human TREML4 and demonstrated by immunohistochemical techniques that it is localized in macrophages surrounding the necrotic core of coronary plaques complicated by calcification (but not in arteries with less advanced disease). Finally, we determined by von Kossa staining that TREML4 colocalizes with areas of microcalcification within coronary plaques. Overall, we present integrative RNA, DNA, and protein evidence implicating TREML4 in coronary artery calcification. Our findings connect multimodal genomics data with a commonly used clinical marker of cardiovascular disease. Coronary artery calcification (CAC) is a heritable and definitive morphologic marker of atherosclerosis that strongly predicts risk for future cardiovascular events. To search for genes involved in CAC, we used an integrative transcriptomic, genomic, and protein expression strategy by using next-generation DNA sequencing in the discovery phase with follow-up studies using traditional molecular biology and histopathology techniques. RNA sequencing of peripheral blood from a discovery set of CAC cases and controls was used to identify dysregulated genes, which were validated by ClinSeq and Framingham Heart Study data. Only a single gene, TREML4, was upregulated in CAC cases in both studies. Further examination showed that rs2803496 was a TREML4 cis-eQTL and that the minor allele at this locus conferred up to a 6.5-fold increased relative risk of CAC. We characterized human TREML4 and demonstrated by immunohistochemical techniques that it is localized in macrophages surrounding the necrotic core of coronary plaques complicated by calcification (but not in arteries with less advanced disease). Finally, we determined by von Kossa staining that TREML4 colocalizes with areas of microcalcification within coronary plaques. Overall, we present integrative RNA, DNA, and protein evidence implicating TREML4 in coronary artery calcification. Our findings connect multimodal genomics data with a commonly used clinical marker of cardiovascular disease.

TO THE EDITOR: Population-level germ line heterogeneity, rare single nucleotide polymorphisms (SNPs), and sequence alignment to a single reference genome make it challenging for variant-calling algorithms to distinguish germ line variants from somatically acquired mutations with high throughput

Abstract HOXB13, a homeodomain transcription factor, is linked to recurrence following radical prostatectomy. While HOXB13 regulates Androgen Receptor (AR) functions in a context dependent manner, its critical effectors in prostate cancer (PC) metastasis remain largely unknown. To identify HOXB13 transcriptional targets in metastatic PCs, we performed integrative bioinformatics analysis of differentially expressed genes (DEGs) in the proximity of the human prostate tumor-specific AR binding sites. Unsupervised Principal Component Analysis (PCA) led to a focused core HOXB13 target gene-set referred to as HOTPAM9 ( HO XB13 T argets separating P rimary A nd M etastatic PCs). HOTPAM9 comprised 7 mitotic kinase genes overexpressed in metastatic PCs, TRPM8 , and the heat shock protein HSPB8 , whose levels were significantly lower in metastatic PCs compared to the primary disease. The expression of a two-gene set, CIT and HSPB8 with an overall balanced accuracy of 98.8% and a threshold value of 0.2347, was sufficient to classify metastasis. HSPB8 mRNA expression was significantly increased following HOXB13 depletion in multiple metastatic CRPC models. Increased expression of HSPB8 by the microtubule inhibitor Colchicine or by exogenous means suppressed migration of mCRPC cells. Collectively, our results indicate that HOXB13 promotes metastasis of PCs by coordinated regulation of mitotic kinases and blockade of a putative tumor suppressor gene.

Undifferentiated embryonal sarcoma of the liver (UESL) is a rare and aggressive malignancy. Though the molecular underpinnings of this cancer have been largely unexplored, recurrent chromosomal breakpoints affecting a noncoding region on chr19q13, which includes the chromosome 19 microRNA cluster (C19MC), have been reported in several cases. We performed comprehensive molecular profiling on samples from 14 patients diagnosed with UESL. Congruent with prior reports, we identified structural variants in chr19q13 in 10 of 13 evaluable tumors. From whole transcriptome sequencing, we observed striking expressional activity of the entire C19MC region. Concordantly, in 7 of 7 samples undergoing miRNAseq, we observed hyperexpression of the miRNAs within this cluster to levels >100 fold compared to matched normal tissue or a non-C19MC amplified cancer cell line. Concurrent TP53 mutation or copy number loss was identified in all evaluable tumors with evidence of C19MC overexpression. We find that C19MC miRNAs exhibit significant negative correlation to TP53 regulatory miRNAs and K-Ras regulatory miRNAs. Using RNA-seq we identified that pathways relevant to cellular differentiation as well as mRNA translation machinery are transcriptionally enriched in UESL. In summary, utilizing a combination of next-generation sequencing and high-density arrays we identify the combination of C19MC hyperexpression via chromosomal structural event with TP53 mutation or loss as highly recurrent genomic features of UESL.

Radiotherapy efficacy is the result of radiation-mediated cytotoxicity coupled with stimulation of antitumor immune responses. We develop an in silico 3-dimensional agent-based model of diverse tumor-immune ecosystems (TIES) represented as anti- or pro-tumor immune phenotypes. We validate the model in 10,469 patients across 31 tumor types by demonstrating that clinically detected tumors have pro-tumor TIES. We then quantify the likelihood radiation induces antitumor TIES shifts toward immune-mediated tumor elimination by developing the individual Radiation Immune Score (iRIS). We show iRIS distribution across 31 tumor types is consistent with the clinical effectiveness of radiotherapy, and in combination with a molecular radiosensitivity index (RSI) combines to predict pan-cancer radiocurability. We show that iRIS correlates with local control and survival in a separate cohort of 59 lung cancer patients treated with radiation. In combination, iRIS and RSI predict radiation-induced TIES shifts in individual patients and identify candidates for radiation de-escalation and treatment escalation. This is the first clinically and biologically validated computational model to simulate and predict pan-cancer response and outcomes via the perturbation of the TIES by radiotherapy.

Radiation therapy (RT) is a mainstay of cancer care, and accumulating evidence suggests the potential for synergism with components of the immune response. However, few data describe the tumor immune contexture in relation to RT sensitivity. To address this challenge, we used the radiation sensitivity index (RSI) gene signature to estimate the RT sensitivity of >10,000 primary tumors and characterized their immune microenvironments in relation to the RSI.We analyzed gene expression profiles of 10,469 primary tumors (31 types) within a prospective tissue collection protocol. The RT sensitivity of each tumor was estimated by the RSI and respective distributions were characterized. The tumor biology measured by the RSI was evaluated by differentially expressed genes combined with single sample gene set enrichment analysis. Differences in the expression of immune regulatory molecules were assessed and deconvolution algorithms were used to estimate immune cell infiltrates in relation to the RSI. A subset (n = 2368) of tumors underwent DNA sequencing for mutational frequency characterization.We identified a wide range of RSI values within and across various tumor types, with several demonstrating nonunimodal distributions (eg, colon, renal, lung, prostate, esophagus, pancreas, and PAM50 breast subtypes; P < .05). Across all tumor types, stratifying RSI at a tumor type-specific median identified 7148 differentially expressed genes, of which 146 were coordinate in direction. Network topology analysis demonstrates RSI measures a coordinated STAT1, IRF1, and CCL4/MIP-1β transcriptional network. Tumors with an estimated high sensitivity to RT demonstrated distinct enrichment of interferon-associated signaling pathways and immune cell infiltrates (eg, CD8+ T cells, activated natural killer cells, M1-macrophages; q < 0.05), which was in the context of diverse expression patterns of various immunoregulatory molecules.This analysis describes the immune microenvironments of patient tumors in relation to the RSI gene expression signature.

Metastasis is characterized by spreading of neoplastic cells to an organ other than where they originated and is the predominant cause of death among cancer patients. This holds true for melanoma, whose incidence is increasing more rapidly than any other cancer and once disseminated has few therapeutic options. Here we performed whole exome sequencing of two sets of matched normal and metastatic tumor DNAs.Using stringent criteria, we evaluated the similarities and differences between the lesions. We find that in both cases, 96% of the single nucleotide variants are shared between the two metastases indicating that clonal populations gave rise to the distant metastases. Analysis of copy number variation patterns of both metastatic sets revealed a trend similar to that seen with our single nucleotide variants. Analysis of pathway enrichment on tumor sets shows commonly mutated pathways enriched between individual sets of metastases and all metastases combined.These data provide a proof-of-concept suggesting that individual metastases may have sufficient similarity for successful targeting of driver mutations.

Based on KRAS testing, the subset of patients with metastatic colorectal cancer (CRC) that could benefit from anti-EGFR therapy can be better delineated. Though KRAS testing has become significantly more prevalent over the last few years, methods for testing remain heterogeneous and discordance has been reported between methods.In this study, we examined a CRC patient population and compared KRAS testing done in Clinical Laboratory Improvement Amendments (CLIA) approved laboratories as part of standard clinical care and by next-generation sequencing (NGS) using the Illumina platform. Discordances were further evaluated with manual review of the NGS testing.Out of 468 CRC patient samples, 77 had KRAS testing done by both CLIA assay and NGS. There were concordant results between testing methodologies in 74 out of 77 patients, or 96% (95% CI 89% to 99%). There were three patient samples that showed discordant results between the two methods of testing. Upon further investigation of the NGS results for the three discordant cases, one sample showed a low level of the mutation seen in the standard testing, one sample showed low tumour fraction and a third did not show any evidence of the mutation that was found with the standard assay. Five patients had KRAS mutations not typically tested with standard testing.Overall there was a high concordance rate between NGS and standard testing for KRAS. However, NGS revealed mutations that are not tested for with standard KRAS assays that might have clinical impact with regards to the role for anti-EGFR therapy.

Differences in response to cancer treatments have been observed among racially and ethnically diverse gastric cancer (GC) patient populations. In the era of targeted therapy, mutation profiling of cancer is a crucial aspect of making therapeutic decisions. Mapping driver gene mutations for the GC patient population as a whole has significant potential to advance precision therapy. GC patients with sequencing data (N = 473) were obtained from The Cancer Genome Atlas (TCGA; n = 295), Moffitt Cancer Center Total Cancer Care™ (TCC; n = 33), and three published studies (n = 145). In addition, relevant somatic mutation frequency data were obtained from cBioPortal, the TCC database, and an in-house analysis tool, as well as relevant publications. We found that the somatic mutation rates of several driver genes vary significantly between GC patients of Asian and Caucasian descent, with substantial variation across different geographic regions. Non-parametric statistical tests were performed to examine the significant differences in protein-altering somatic mutations between Asian and Caucasian GC patient groups. The frequencies of somatic mutations of five genes were: APC (Asian: Caucasian 6.06 vs. 14.40%, p = 0.0076), ARIDIA (20.7 vs. 32.1%, p = 0.01), KMT2A (4.04 vs. 12.35%, p = 0.003), PIK3CA (9.6 vs. 18.52%, p = 0.01), and PTEN (2.52 vs. 9.05%, p = 0.008), showing significant differences between Asian and Caucasian GC patients. Our study found significant differences in protein-altering somatic mutation frequencies in diverse geographic populations. In particular, we found that the somatic patterns may offer better insight and important opportunities for both targeted drug development and precision therapeutic strategies between Asian and Caucasian GC patients.

Inactivation of Ras GTPase activating proteins (RasGAPs) can activate Ras, increasing the risk for tumor development. Utilizing a melanoma whole genome sequencing (WGS) data from 13 patients, we identified two novel, clustered somatic missense mutations (Y472H and L481F) in RASA1 (RAS p21 protein activator 1, also called p120RasGAP). We have shown that wild type RASA1, but not identified mutants, suppresses soft agar colony formation and tumor growth of BRAF mutated melanoma cell lines via its RasGAP activity toward R-Ras (related RAS viral (r-ras) oncogene homolog) isoform. Moreover, R-Ras increased and RASA1 suppressed Ral-A activation among Ras downstream effectors. In addition to mutations, loss of RASA1 expression was frequently observed in metastatic melanoma samples on melanoma tissue microarray (TMA) and a low level of RASA1 mRNA expression was associated with decreased overall survival in melanoma patients with BRAF mutations. Thus, these data support that RASA1 is inactivated by mutation or by suppressed expression in melanoma and that RASA1 plays a tumor suppressive role by inhibiting R-Ras, a previously less appreciated member of the Ras small GTPases.

The "conditionally reprogrammed cells" (CRC) method, using a Rho kinase inhibitor and irradiated mouse fibroblast cells has been described for the efficient growth of cells from malignant and non-malignant samples from primary tumor and non-malignant sites. Using the CRC method, four institutions independently cultured tumor tissues from 48 non-small cell lung cancers (NSCLC, mostly from primary resected tumors) and 22 non-malignant lungs. We found that epithelial cells could be cultured from tumor and non-malignant lung. However, epithelial cells cultured from tumors had features of non-malignant respiratory epithelial cells which include: 1) among 22 mutations found in the original tumors only two mutations were found in the CRC cultures with reduced frequency (31% to 13% and 92% to 15% from original tumor and CRC culture respectively); 2) copy number variation was analyzed in 9 tumor and their CRC cultures and only diploid patterns were found in CRC cultures; 3) mRNA expression profiles were similar to those of normal respiratory epithelial cells; and 4) co-culture of tumor and non-malignant lung epithelial cells resulted in mostly non-malignant cells. We conclude that CRC method is a highly selective and useful method for the growth of non-malignant respiratory epithelial cells from tumor specimens and only occasionally do such CRC cultures contain a small subpopulation of cancer cells marked by oncogenic mutations. While our findings are restricted to resected primary NSCLC, they indicated the necessity to fully characterize all CRC cultures and the need to develop culture technology that facilitates the growth of primary lung cancers.

Pancreatic cancer (PC) is characterized by racial/ethnic disparities and the debilitating muscle-wasting condition, cancer cachexia. Florida ranks second in the number of PC deaths and has a large and understudied minority population. We examined the primary hypothesis that PC incidence and mortality rates may be highest among Black Floridians and the secondary hypothesis that biological correlates of cancer cachexia may underlie disparities. PC incidence and mortality rates were estimated by race/ethnicity, gender, and county using publicly available state-wide cancer registry data that included approximately 2700 Black, 25 200 Non-Hispanic White (NHW), and 3300 Hispanic/Latino (H/L) Floridians diagnosed between 2004 and 2014. Blacks within Florida experienced a significantly (P < 0.05) higher incidence (12.5/100 000) and mortality (10.97/100 000) compared to NHW (incidence = 11.2/100 000; mortality = 10.3/100 000) and H/L (incidence = 9.6/100 000; mortality = 8.7/100 000), especially in rural counties. To investigate radiologic and blood-based correlates of cachexia, we leveraged data from a subset of patients evaluated at two geographically distinct Florida Cancer Centers. In Blacks compared to NHW matched on stage, markers of PC-induced cachexia were more frequent and included greater decreases in core musculature compared to corresponding healthy control patients (25.0% vs 10.1% lower), greater decreases in psoas musculature over time (10.5% vs 4.8% loss), lower baseline serum albumin levels (3.8 vs 4.0 gm/dL), and higher platelet counts (332.8 vs 268.7 k/UL). Together, these findings suggest for the first time that PC and cachexia may affect Blacks disproportionately. Given its nearly universal contribution to illness and PC-related deaths, the early diagnosis and treatment of cachexia may represent an avenue to improve health equity, quality of life, and survival.

Acral skin may develop nevi, but their mutational status and association with acral melanoma is unclear.To perform targeted next-generation sequencing on a cohort of acral nevi to determine their mutational spectrum.Acral nevi specimens (n = 50) that had been obtained for diagnostic purposes were identified from the pathology archives of a tertiary care academic cancer center and a university dermatology clinic. Next-generation sequencing was performed on DNA extracted from the specimens, and mutations called. A subset of samples was stained immunohistochemically for the BRAF V600E mutation.A total of 50 nevi from 49 patients (19 males and 30 females; median [range] age, 48 [13-85] years) were examined. Analysis of the sequencing data revealed a high prevalence of BRAF mutations (n = 43), with a lower frequency of NRAS mutations (n = 5). Mutations in BRAF and NRAS were mutually exclusive.In this cohort study, nevi arising on mostly sun-protected acral skin showed a rate of BRAF mutation similar to that of acquired nevi on sun-exposed skin but far higher than that of acral melanoma. These findings are in contrast to the well-characterized mutational landscape of acral melanoma.

Replication initiation must be a carefully regulated process to avoid genomic instability caused by aberrant replication. In eukaryotic cells, distinct steps of protein loading (origin licensing) and replication activation are choreographed such that a cell can replicate only once per cell cycle. The first proteins recruited to the origins form the pre-replication complex. Of these proteins, Cdt1 is of interest, as it is the focus of several pathways to control replication initiation. It is degraded by two different pathways, mediated by the interaction of Cdt1 with proliferating cell nuclear antigen (PCNA) or with cyclin-Cdk2 and inhibited by geminin once cells are in S-phase, presumably to prevent reloading of pre-replication complexes once S-phase has begun. Although the requirement of Cdt1 in loading MCM2–7 is known, the mechanism by which overexpressed Cdt1 stimulates re-replication is unclear. In this study we have designed various mutations in Cdt1 to determine which portion of Cdt1 is important for re-replication, providing insight into possible mechanisms. Surprisingly, we found that mutants of Cdt1 that do not interact with MCM2–7 are able to induce re-replication when overexpressed. The re-replication is not due to titration of geminin from endogenous Cdt1 and is not accompanied by stabilization of endogenous Cdt1. Additionally, the N-terminal one-third of Cdt1 is sufficient to induce re-replication. The N terminus contains the PCNA- and cyclin-interacting motifs, and deletion of both motifs simultaneously in the overexpressed Cdt1 prevents re-replication. These findings suggest that exogenous Cdt1 induces re-replication by de-repressing endogenous Cdt1 through the titration of PCNA and cyclin. Replication initiation must be a carefully regulated process to avoid genomic instability caused by aberrant replication. In eukaryotic cells, distinct steps of protein loading (origin licensing) and replication activation are choreographed such that a cell can replicate only once per cell cycle. The first proteins recruited to the origins form the pre-replication complex. Of these proteins, Cdt1 is of interest, as it is the focus of several pathways to control replication initiation. It is degraded by two different pathways, mediated by the interaction of Cdt1 with proliferating cell nuclear antigen (PCNA) or with cyclin-Cdk2 and inhibited by geminin once cells are in S-phase, presumably to prevent reloading of pre-replication complexes once S-phase has begun. Although the requirement of Cdt1 in loading MCM2–7 is known, the mechanism by which overexpressed Cdt1 stimulates re-replication is unclear. In this study we have designed various mutations in Cdt1 to determine which portion of Cdt1 is important for re-replication, providing insight into possible mechanisms. Surprisingly, we found that mutants of Cdt1 that do not interact with MCM2–7 are able to induce re-replication when overexpressed. The re-replication is not due to titration of geminin from endogenous Cdt1 and is not accompanied by stabilization of endogenous Cdt1. Additionally, the N-terminal one-third of Cdt1 is sufficient to induce re-replication. The N terminus contains the PCNA- and cyclin-interacting motifs, and deletion of both motifs simultaneously in the overexpressed Cdt1 prevents re-replication. These findings suggest that exogenous Cdt1 induces re-replication by de-repressing endogenous Cdt1 through the titration of PCNA and cyclin. DNA replication is a complex process that a cell must perform accurately to ensure the genome is copied correctly with minimal damage. Additionally, replication must be temporally regulated so that the genome is copied exactly once before division. This ensures that daughter cells will not get undesirable amplifications or deletions that may cause abnormal functions, such as the unregulated growth observed in cancer. Work in the last decade has revealed an elaborate system to ensure that DNA replication occurs once, and only once, per cell cycle. This system of replication licensing relies on the sequential recruitment of replication initiation factors to chromatin at the appropriate time and prevents association at all other times. By controlling the chromatin association of these initiation factors, cells are able to limit precisely when replication can begin and prevent it otherwise. In higher eukaryotes, it is generally thought that the origin recognition complex recognizes origins of replication. The origin recognition complex serves as an origin marker and recruits Cdc6 and Cdt1 to chromatin. These proteins are then required to load the MCM2–7 complex. Once the MCM2–7 complex is loaded, downstream members of the preinitiation complex, including Cdc45/Sld3, GINS, MCM10, and Dpb11/Sld2, are loaded onto chromatin, eventually culminating in recruitment of the replication machinery. The recruitment of these factors is controlled by the presence or absence of cyclin-dependent kinase (CDK) 2The abbreviations used are:CDKcyclin-dependent kinasePCNAproliferating cell nuclear antigensiRNAsmall interfering RNAFACSfluorescence-activated cell sorterGFPgreen fluorescent proteinEGFPenhanced GFPCycyclinMNasemicrococcal nucleasePipes1,4-piperazinediethanesulfonic acid. activity. CDK activity is low in G1, when the pre-replication complex (consisting of the origin recognition complex, Cdc6, Cdt1, and MCM2–7) is loaded. Increasing CDK activity is then required for further loading of the preinitiation complex members and the replication machinery (for review see Refs. 1Bell S.P. Dutta A. Annu. Rev. Biochem. 2002; 71: 333-374Crossref PubMed Scopus (1394) Google Scholar and 2Teer J.K. Dutta A. Results Probl. Cell Differ. 2006; 42: 31-63Crossref PubMed Scopus (8) Google Scholar). cyclin-dependent kinase proliferating cell nuclear antigen small interfering RNA fluorescence-activated cell sorter green fluorescent protein enhanced GFP cyclin micrococcal nuclease 1,4-piperazinediethanesulfonic acid. MCM2–7 is thought to be the replicative helicase responsible for unwinding DNA ahead of the replication machinery (3Ishimi Y. J. Biol. Chem. 1997; 272: 24508-24513Abstract Full Text Full Text PDF PubMed Scopus (460) Google Scholar, 4You Z. Komamura Y. Ishimi Y. Mol. Cell. Biol. 1999; 19: 8003-8015Crossref PubMed Scopus (171) Google Scholar, 5Lee J.K. Hurwitz J. J. Biol. Chem. 2000; 275: 18871-18878Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar, 6Moyer S.E. Lewis P.W. Botchan M.R. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 10236-10241Crossref PubMed Scopus (544) Google Scholar). It is loaded in a Cdc6- and Cdt1-dependent manner, and this loading can only occur during low levels of CDK activity, in late M phase and early G1 phase. Cdt1 has been shown to associate physically with MCM2–7 in many organisms (7Tanaka S. Diffley J.F. Nat. Cell Biol. 2002; 4: 198-207Crossref PubMed Scopus (217) Google Scholar, 8Cook J.G. Park C. Burke T.W. Leone G. Degregori J. Engel A. Nevins J.R. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 1347-1352Crossref PubMed Scopus (101) Google Scholar, 9Yanagi K. Mizuno T. You Z. Hanaoka F. J. Biol. Chem. 2002; 277: 40871-40880Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar, 10Ferenbach A. Li A. Brito-Martins M. Blow J.J. Nucleic Acids Res. 2005; 33: 316-324Crossref PubMed Scopus (55) Google Scholar), suggesting that it directly recruits MCM2–7 to chromatin. However, Cdc6 ATPase mutants increase association of Cdt1 and decrease association of MCM2–7 with chromatin, which might suggest mutual exclusion of Cdt1 and MCM2–7 on chromatin (7Tanaka S. Diffley J.F. Nat. Cell Biol. 2002; 4: 198-207Crossref PubMed Scopus (217) Google Scholar, 11Randell J.C. Bowers J.L. Rodriguez H.K. Bell S.P. Mol. Cell. 2006; 21: 29-39Abstract Full Text Full Text PDF PubMed Scopus (206) Google Scholar). Either way, it has been suggested that the interaction between Cdt1 and MCM2–7 is required to load the helicase on chromatin. Recent experiments in Xenopus egg extracts show that the C-terminal region of Cdt1 is required for MCM2–7 loading and that this region is also required for the interaction with MCMs (mini-chromosome maintenance) (10Ferenbach A. Li A. Brito-Martins M. Blow J.J. Nucleic Acids Res. 2005; 33: 316-324Crossref PubMed Scopus (55) Google Scholar). The importance of the C-terminal one-third of Cdt1 is highlighted by the fact that this is the only area that shows sequence identity from Saccharomyces cerevisiae to humans (7Tanaka S. Diffley J.F. Nat. Cell Biol. 2002; 4: 198-207Crossref PubMed Scopus (217) Google Scholar, 12Devault A. Vallen E.A. Yuan T. Green S. Bensimon A. Schwob E. Curr. Biol. 2002; 12: 689-694Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). Once MCM2–7 is loaded, its subsequent activation (and eventual origin firing) requires CDK and CDK-like activity (for review see Ref. 2Teer J.K. Dutta A. Results Probl. Cell Differ. 2006; 42: 31-63Crossref PubMed Scopus (8) Google Scholar). Therefore, MCM2–7 bridges the two CDK activity states that function to license replication: its chromatin loading must occur in low CDK activity, and its activation and origin firing must occur in high CDK activity. One of the key players in the replication licensing system is Cdt1. Cdt1 is required for MCM2–7 loading (7Tanaka S. Diffley J.F. Nat. Cell Biol. 2002; 4: 198-207Crossref PubMed Scopus (217) Google Scholar, 13Maiorano D. Moreau J. Mechali M. Nature. 2000; 404: 622-625Crossref PubMed Scopus (295) Google Scholar, 14Nishitani H. Lygerou Z. Nishimoto T. Nurse P. Nature. 2000; 404: 625-628Crossref PubMed Scopus (370) Google Scholar). MCM2–7 loading is limited to late M and early G1 phases in part due to inhibition of Cdt1 by several mechanisms in S-phase. Cdt1 is degraded in S-phase by two different mechanisms: CDK-dependent degradation by the SCF (Skp1/Cullin/F-box containing) complex and PCNA-dependent degradation by the Cul4-DDB1 complex (for review see Ref. 15Fujita M. Cell Div. 2006; 1: 22Crossref PubMed Scopus (81) Google Scholar). It is also inhibited by geminin (16Wohlschlegel J.A. Dwyer B.T. Dhar S.K. Cvetic C. Walter J.C. Dutta A. Science. 2000; 290: 2309-2312Crossref PubMed Scopus (584) Google Scholar, 17Tada S. Li A. Maiorano D. Mechali M. Blow J.J. Nat. Cell Biol. 2001; 3: 107-113Crossref PubMed Scopus (390) Google Scholar), a small protein that occurs at high levels during S-phase. Disrupting these mechanisms of Cdt1 inhibition can cause re-replication in a variety of systems (18Vaziri C. Saxena S. Jeon Y. Lee C. Murata K. Machida Y. Wagle N. Hwang D.S. Dutta A. Mol. Cell. 2003; 11: 997-1008Abstract Full Text Full Text PDF PubMed Scopus (338) Google Scholar, 19Zhong W. Feng H. Santiago F.E. Kipreos E.T. Nature. 2003; 423: 885-889Crossref PubMed Scopus (259) Google Scholar, 20Zhu W. Chen Y. Dutta A. Mol. Cell. Biol. 2004; 24: 7140-7150Crossref PubMed Scopus (204) Google Scholar, 21Arias E.E. Walter J.C. Genes Dev. 2005; 19: 114-126Crossref PubMed Scopus (166) Google Scholar, 22Li A. Blow J.J. EMBO J. 2004; 24: 395-404Crossref PubMed Scopus (115) Google Scholar). This suggests that when Cdt1 is not inhibited it can stimulate re-replication. Despite the expectation that inappropriate Cdt1 activity in S-phase may cause re-replication by excessive MCM2–7 reloading, the actual role of Cdt1 in re-replication induction is unclear. Recent work has shown that Cdt1 levels are increased in certain cancers (23Karakaidos P. Taraviras S. Vassiliou L.V. Zacharatos P. Kastrinakis N.G. Kougiou D. Kouloukoussa M. Nishitani H. Papavassiliou A.G. Lygerou Z. Gorgoulis V.G. Am. J. Pathol. 2004; 165: 1351-1365Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar). Interestingly, in 73% of cases in which p53 was also mutated, aneuploidy was observed. Our laboratory has reported similar observations; in tumor cell lines lacking p53 and overexpressing Cdt1 and Cdc6, re-replication is observed (18Vaziri C. Saxena S. Jeon Y. Lee C. Murata K. Machida Y. Wagle N. Hwang D.S. Dutta A. Mol. Cell. 2003; 11: 997-1008Abstract Full Text Full Text PDF PubMed Scopus (338) Google Scholar). Furthermore, high levels of Cdt1 appear to have a causative role in cancer. It has been shown that retrovirally activated Cdt1 can be oncogenic in mice (24Arentson E. Faloon P. Seo J. Moon E. Studts J.M. Fremont D.H. Choi K. Oncogene. 2002; 21: 1150-1158Crossref PubMed Google Scholar) and that Cdt1 overexpression in thymocytes can cause lymphomas when p53 is also mutated. As Cdt1 overexpression causes re-replication in a variety of systems, it is possible that high levels of Cdt1 induce genomic instability and tumorigenesis by inducing re-replication. Therefore, an understanding of the mechanism of re-replication initiation is also informative for tumorigenesis and disease progression. Cell Culture—293T cells, human embryonic kidney cells transformed with adenovirus oncogenes E1a and E1b and with simian virus 40 oncogene T antigen, were maintained in Dulbecco's modified Eagle's medium supplemented with 10% iron-supplemented donor calf serum and 1% penicillin/streptomycin. Standard tissue culture growth conditions and methods were used. Lipofectamine 2000 (Invitrogen) was used to transfect cells. Cdt1 Mutant Construction—Cdt1 mutants were subcloned using PCR-based techniques in a vector with an N-terminal FLAG tag. Primer sequences used are available upon request. Because the natural nuclear localization sequence of Cdt1 is in the first 93 residues, the constructs lacking these N-terminal residues (94–X, 163–X) have an artificial nuclear localization sequence fused to the N terminus to ensure normal localization. Western Blotting, Immunoprecipitation, and siRNA—Western blotting was performed as described. In this case, cells were lysed in 50 mm Tris, pH 7.4, 0.2% Nonidet P-40, 150 or 300 mm NaCl, 1 mm EDTA, 10 mm NaF, 0.2 mm Na3VO3, 1 mm phenylmethylsulfonyl fluoride, 2 mm dithiothreitol, and a 1:100 protease inhibitor mixture (Sigma). Equal amounts of protein were loaded on SDS-PAGE followed by transfer to nitrocellulose for standard Western blotting. FLAG and β-actin antibodies were purchased from Sigma, and MCM6 and cyclin A antibodies were purchased from Santa Cruz Biotechnology. Anti-FLAG-agarose beads (Sigma) were used for Cdt1 co-immunoprecipitation. Geminin antibody was described previously (16Wohlschlegel J.A. Dwyer B.T. Dhar S.K. Cvetic C. Walter J.C. Dutta A. Science. 2000; 290: 2309-2312Crossref PubMed Scopus (584) Google Scholar). hsCdt1 antibody was described previously (25Senga T. Sivaprasad U. Zhu W. Park J.H. Arias E.E. Walter J.C. Dutta A. J. Biol. Chem. 2006; 281: 6246-6252Abstract Full Text Full Text PDF PubMed Scopus (210) Google Scholar). siRNA was performed according to common methods using RNAi MAX (Invitrogen). siRNA oligonucleotides against human geminin have been described previously (20Zhu W. Chen Y. Dutta A. Mol. Cell. Biol. 2004; 24: 7140-7150Crossref PubMed Scopus (204) Google Scholar). The Cdt1 siRNA sequence is 5′-GCAAUGUUGGCCAGAUCAA-3′. FACS—Cells were prepared as described previously (18Vaziri C. Saxena S. Jeon Y. Lee C. Murata K. Machida Y. Wagle N. Hwang D.S. Dutta A. Mol. Cell. 2003; 11: 997-1008Abstract Full Text Full Text PDF PubMed Scopus (338) Google Scholar). Cells were cotransfected with farnesylated EGFP and gated for GFP-positive cells to ensure analysis was limited to the Cdt1-transfected cells. Alternately, cells were transiently transfected with a puromycin resistance marker, and 48 h of puromycin treatment was used to enrich transfected cells. The analysis was carried out on a BD Biosciences FACS Calibur using Cellquest and FloJo software. Chromatin Fractionation—Chromatin fractionation protocols used to observe MCM7 and Cdt1 chromatin loading were described previously (Refs. 26Todorov I.T. Attaran A. Kearsey S.E. J. Cell Biol. 1995; 129: 1433-1445Crossref PubMed Scopus (204) Google Scholar and 27Mendez J. Stillman B. Mol. Cell. Biol. 2000; 20: 8602-8612Crossref PubMed Scopus (754) Google Scholar, respectively). Briefly, the MCM7 chromatin loading was observed as follows. Cells (2 × 106) were lysed in 100 μl of CSK buffer (10 mm Pipes, pH 7.0, 100 mm NaCl, 300 mm sucrose, 3 mm MgCl2) containing 0.5% Triton X-100, 1 mm ATP, and 1 mm Na3VO4. Lysates were incubated on ice for 20 min and then centrifuged at 1500 rpm for 5 min at 4 °C. Supernatant (S1) was removed, and pellets were washed with 1 ml of lysis buffer and centrifuged again. Pellets were incubated in 100 μl of lysis buffer containing 1 mm CaCl2 and 120 units of micrococcal nuclease (Worthington) for 10 min at 37 °C and centrifuged. Supernatant (S2, chromatin-bound fraction) was removed, and pellets were washed with 1 ml of lysis buffer and centrifuged again. Pellets were boiled in 100 μl of 1× sample buffer (P2) (26Todorov I.T. Attaran A. Kearsey S.E. J. Cell Biol. 1995; 129: 1433-1445Crossref PubMed Scopus (204) Google Scholar). The Cdt1 chromatin loading was determined as follows. Cells were resuspended (4 × 107 cells/ml) in Buffer A (10 mm HEPES, pH 7.9, 10 mm KCl, 1.5 mm MgCl2, 0.34 m sucrose, 10% glycerol, 1 mm dithiothreitol, a 1:100 protease inhibitor mix (Sigma), 0.1 mm phenylmethylsulfonyl fluoride). 0.1% Triton X-100 was added, and the cells were incubated for 5 min on ice. Nuclei were collected in pellet 1 (P1) by low-speed centrifugation (4 min, 1,300 × g, 4 °C). The supernatant (S1) was further clarified by high-speed centrifugation (15 min, 20,000 × g, 4 °C) to remove cell debris and insoluble aggregates. Nuclei were washed once in Buffer A and then lysed in Buffer B (3 mm EDTA, 0.2 mm EGTA, 1 mm dithiothreitol, protease inhibitors as described above). Insoluble chromatin was collected by centrifugation (4 min, 1,700 × g, 4 °C), washed once in Buffer B, and centrifuged again under the same conditions. The final chromatin pellet (P3) was resuspended in Laemmli buffer and sheared by passing it through a syringe. To release chromatin-bound proteins by nuclease treatment, cell nuclei (P1) were resuspended in Buffer A plus 1 mm CaCl2 and 0.2 unit of micrococcal nuclease (Sigma). After incubation at 37 °C for 2 min, the nuclease reaction was stopped by the addition of 1 mm EGTA. Nuclei were collected by low-speed centrifugation and lysed as above (27Mendez J. Stillman B. Mol. Cell. Biol. 2000; 20: 8602-8612Crossref PubMed Scopus (754) Google Scholar). Recent evidence has suggested that the C terminus of Xenopus and mouse Cdt1 interacts with the MCM2–7 complex. We first sought to confirm this result in human cells. A variety of truncations and internal deletions were made in the regions defined in mouse (9Yanagi K. Mizuno T. You Z. Hanaoka F. J. Biol. Chem. 2002; 277: 40871-40880Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar) and Xenopus (10Ferenbach A. Li A. Brito-Martins M. Blow J.J. Nucleic Acids Res. 2005; 33: 316-324Crossref PubMed Scopus (55) Google Scholar) systems to be important for the interaction with MCM2–7. To determine the analogous amino acids in hsCdt1 (Homo sapiens) (546 amino acids), a multiple alignment was performed using ClustalW (28Thompson J.D. Higgins D.G. Gibson T.J. Nucleic Acids Res. 1994; 22: 4673-4680Crossref PubMed Scopus (55761) Google Scholar). In Xenopus egg extracts, xlCdt1Δ573–620 (X. laevis) did not bind MCM2–7. The analogous hsCdt1 residues (499–546) were therefore deleted. Yeast two-hybrid and in vitro pulldown experiments with mouse Cdt1 suggested that mmCdt1-(407–477) (Mus musculus) was important for direct interaction with mmMCM6. Interestingly, mmCdt1Δ520–577 (a deletion of the extreme C terminus) seemed to interact more strongly with mmMCM6, suggesting that this region may be inhibitory for MCM binding. We synthesized the analogous deletions of the internal required region and C-terminal inhibitory region in human Cdt1: hsCdt1Δ395–465 and hsCdt1Δ508–546. Additional internal deletions were also made based on a secondary structure prediction using the GOR4 algorithm (29Garnier J. Gibrat J.F. Robson B. Methods Enzymol. 1996; 266: 540-553Crossref PubMed Google Scholar). The fourth-from-last helix (hsCdt1Δ444–465), the third-from-last helix (hsCdt1Δ495–505), and the final helix (hsCdt1Δ518–546) were each deleted. These mutations are summarized in Fig. 1. To test the interaction of these hsCdt1 deletions with MCM2–7, we overexpressed FLAG-tagged versions of the proteins in human 293T cells. hsCdt1 was then immunoprecipitated using an anti-FLAG antibody, and hsMCM6 levels were examined by Western blotting. Wild type hsCdt1 was able to pull down endogenous hsMCM6, reproducing earlier results in different systems from other laboratories (Fig. 2A). However, none of the deletion mutants pulled down detectable hsMCM6, indicating that a large portion of the C terminus of hsCdt1 is important for the interaction with hsMCM6. Both the wild type and deletion mutants of hsCdt1 were able to pull down geminin, indicating the proteins are likely to be folded correctly. Interestingly, both Δ499–546 and Δ508–546 did not interact with hsMCM6. In previous studies, the Δ499–546 Xenopus equivalent did not bind to MCM2–7 (10Ferenbach A. Li A. Brito-Martins M. Blow J.J. Nucleic Acids Res. 2005; 33: 316-324Crossref PubMed Scopus (55) Google Scholar), but the mouse Δ508–546 equivalent bound more strongly (9Yanagi K. Mizuno T. You Z. Hanaoka F. J. Biol. Chem. 2002; 277: 40871-40880Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar), suggesting that the extreme C terminus of Cdt1 was inhibitory for MCM binding. Our data agrees with the Xenopus data, as the extreme C terminus appears to be required for MCM interaction. It is possible that the Cdt1-MCM6 interaction in mice is slightly different from that in other organisms. To eliminate the possibility that these deleted regions are also important for a non-MCM binding function of hsCdt1, we made three different sets of point mutants in full-length hsCdt1. These mutants are termed PEHL (P492T, E496A, H498A, L502A), RELAR (R453A, E456A, L457A, A458S, R459A), and RPLVF (R356K, P455H, L461E, V464A, F465A). Each set of point mutants was made in highly conserved residues in the regions deleted previously (see Fig. 1). FLAG-tagged versions of these point mutants were expressed in 293T cells and pulled down with anti-FLAG. Although wild type hsCdt1 interacted with hsMCM6, we did not observe an interaction between any of our point mutants and hsMCM6 (Fig. 2B), suggesting that these mutations interrupt the binding between the two proteins. Again, the hsCdt1 point mutants all bind to geminin. As only 4–5 amino acids have been changed in each mutant, the chance of disrupting another activity of Cdt1 in all three cases is decreased. We tested the re-replication initiation activity of these point mutants by overexpressing them in human 293T cells. Our laboratory has shown previously that by either overexpressing hsCdt1 (18Vaziri C. Saxena S. Jeon Y. Lee C. Murata K. Machida Y. Wagle N. Hwang D.S. Dutta A. Mol. Cell. 2003; 11: 997-1008Abstract Full Text Full Text PDF PubMed Scopus (338) Google Scholar) or relieving its inhibition via knockdown of geminin (20Zhu W. Chen Y. Dutta A. Mol. Cell. Biol. 2004; 24: 7140-7150Crossref PubMed Scopus (204) Google Scholar, 30Machida Y.J. Dutta A. Genes Dev. 2007; 21: 184-194Crossref PubMed Scopus (151) Google Scholar), re-replication can be induced in human cells. As expected, transient overexpression of wild type full-length hsCdt1 induced >4N DNA content as determined by FACS (Fig. 3A). In addition, overexpression of each of the MCM non-interacting point mutants also induces re-replication (Fig. 3A). The percentage of re-replicating cells after hsCdt1 point mutant overexpression is similar or higher than wild type hsCdt1 (quantitated in Fig. 3B). This result is very surprising, as these point mutants of hsCdt1 do not appear to interact with hsMCM6. This suggests that although Cdt1 is required for MCM2–7 loading onto chromatin, the interaction between these proteins is not required for re-replication induction. Because the MCM non-interacting point mutants of Cdt1 were able to induce re-replication, we examined the ability of C-terminal deletion mutants to do the same. In previous Xenopus experiments, a C-terminal truncation neither interacted with MCM2–7 nor functioned in replication licensing. The analogous human mutant, residues 1–498, as well as two mutants with larger C-terminal deletions, residues 1–389 and 1–370, was tested for its ability to induce re-replication. Again, surprisingly, these MCM non-interacting mutants caused re-replication (Fig. 3, C and D). This further supports the notion that direct interaction between the overexpressed Cdt1 and MCM2–7 is not required for re-replication. Additionally, these results indicate that the entire C-terminal region, although the only part conserved from S. cerevisiae to humans, is not required to induce re-replication, suggesting that re-replication may not depend on the normal licensing role of Cdt1. One possible explanation for the induction of re-replication by MCM non-interacting point mutants of Cdt1 is that the overexpressed Cdt1 is simply titrating geminin away from endogenous Cdt1, thereby activating it. We addressed this possibility by overexpressing the central domain of Cdt1 that contains a geminin-interacting region: hsCdt1-(163–370). This FLAG-tagged fragment is able to pull down equal amounts of geminin compared with wild type and point mutant hsCdt1, indicating it is able to titrate geminin just as efficiently (Fig. 4A). However, this fragment is not able to induce re-replication compared with wild type hsCdt1 (Fig. 4B); to ensure proper localization, this fragment and others with large N-terminal deletions have an artificial nuclear localization sequence added to the N terminus. This leads us to believe that the re-replication stimulation observed after overexpression of hsCdt1 is not due to geminin titration. To further demonstrate that Cdt1 overexpression does not simply titrate geminin away from endogenous Cdt1, siRNA was used to knock down geminin followed by Cdt1 overexpression. Although re-replication is induced after geminin siRNA in some cell lines (20Zhu W. Chen Y. Dutta A. Mol. Cell. Biol. 2004; 24: 7140-7150Crossref PubMed Scopus (204) Google Scholar, 31Zhu W. Dutta A. Mol. Cell. Biol. 2006; 26: 4601-4611Crossref PubMed Scopus (73) Google Scholar), we did not observe significant re-replication after geminin siRNA in 293T cells (Fig. 4, C and D, FLAG lanes). Taken on its own, this observation suggests that geminin depletion from endogenous Cdt1 is not sufficient to induce re-replication in this cell line. Wild type Cdt1 was also overexpressed in control or geminin siRNA-treated cells. Geminin depletion in Cdt1-overexpressing cells had very little effect on the percentage of re-replicating cells. (Fig. 4, C and D) Thus, Cdt1 overexpression induces re-replication by a mechanism other than titration of geminin. It is possible that overexpressed Cdt1 may titrate another inhibitor of re-replication. CDK activity is also important for inhibition of re-replication as has been demonstrated recently (30Machida Y.J. Dutta A. Genes Dev. 2007; 21: 184-194Crossref PubMed Scopus (151) Google Scholar). As Cdt1 contains a cyclin (Cy) binding motif that is required for interaction with cyclins, titration of cyclins from endogenous targets (including endogenous Cdt1) could cause re-replication. To test this, we deleted the Cy motif in both full-length Cdt1 and Cdt1-(1–370) (Fig. 4, E and F). Although the ΔCy mutants do not interact with cyclin A (Fig. 4G) (32Sugimoto N. Tatsumi Y. Tsurumi T. Matsukage A. Kiyono T. Nishitani H. Fujita M. J. Biol. Chem. 2004; 279: 19691-19697Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar), they are still able to induce re-replication, suggesting that titration of cyclins is not required for the observed re-replication following Cdt1 overexpression. It is also interesting to note that although the 163–370 fragment of Cdt1 did not induce re-replication, the 1–370 fragment did. This indicates that a critical function of the N terminus of Cdt1 is required for re-replication. To further examine this phenomenon, we overexpressed the 163–546 fragment, in which only the N terminus is deleted. Surprisingly, we found that this fragment induced an S-phase arrest (Fig. 5, A and B). A slightly shorter N-terminal truncation, fragment 94–546, also induced an S-phase arrest. Interestingly, if we disrupted MCM binding by overexpressing a Cdt1 mutant lacking the N terminus and lacking the C terminus or having C-terminal point mutations, the S-phase arrest was lost, and cells behaved like the control FLAG transfection (Fig. 5, A and B). This indicates not only that the N terminus has functions important for normal activity of Cdt1 but also that the cell cycle is inhibited when this fragment is absent. The cell cycle inhibition can be rescued when MCM interaction is disrupted, suggesting that N-terminally deleted Cdt1 inhibits the cell cycle by stably associating with and perturbing the normal function of MCM2–7. As the Cdt1 N terminus seemed to be required for induction of re-replication, we wondered whether the N terminus was sufficient for re-replication. Cdt1-(1–163) was overexpressed and was able to induce re-replication nearly as well as full-length Cdt1 (Fig. 5, C and D). This result suggests that the N terminus is sufficient for re-replication induction and strongly suggests that re-replication stimulation does not occur via the normal function of Cdt1. However, the N terminus does contain both motifs required for Cdt1 degradation. We have shown that removal of the Cy motif (required for CDK/SCF-mediated destruction of Cdt1) did not affect re-replication. It is possible that the N terminus is titrating away factors required for the PCNA/CUL4/DDB1-mediated degradation of Cdt1.

To the editor: The genetic determinants that govern the phenotype associated with chronic myelomonocytic leukemia (CMML) and myelodysplastic syndromes (MDS) are unknown. Many recurrent mutations occur in both diseases, suggesting that the presence or absence of a solitary mutation is insufficient

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.

The role of transforming growth factor beta-type-1 (TGFβ1) in pancreatic ductal adenocarcinoma (PDAC) progression is stage-dependent. We hypothesized that TGFβ1 expression is associated with survival and proliferation markers in patients with early-stage PDAC. We acquired clinicopathologic, treatment, and mRNA expression data from The Cancer Genome Atlas data set for 106 patients identified with stage I/II PDAC who underwent pancreaticoduodenectomy. Patients were categorized as high expression when mRNA expression was ≥75th percentile for each gene. Average log2 mRNA expression of TGFβ1 in patients with high expression was 11.6 ± 0.2 and 10.5 ± 0.6 in patients with low expression (P<0.001). Low TGFβ1 expression is associated with shorter median survival compared with high TGFβ1 expression (17 versus at least 60 months; P=0.005). Patients with tumors demonstrating high MKI67 (the gene encoding Ki-67) expression have shorter median survival versus those with lowerMKI67 expression (16 versus 20 months; P=0.026). TGFβ1 and MKI67 are inversely associated (P=0.009). On multivariate analysis, improved survival is associated with TGFβ1 overexpression (P=0.017), adjuvant chemotherapy (P=0.001), and adjuvant radiotherapy (P=0.017), whereas positive surgical margins are associated with worse survival (P=0.002). In patients who undergo pancreaticoduodenectomy for PDAC, high TGFβ1 expression may counteract the worse survival associated with high proliferation.

IntroductionTo address the lack of genomic data from Hispanic/Latino (H/L) patients with lung cancer, the Latino Lung Cancer Registry was established to collect patient data and biospecimens from H/L patients.MethodsThis retrospective observational study examined lung cancer tumor samples from 163 H/L patients, and tumor-derived DNA was subjected to targeted-exome sequencing (>1000 genes, including EGFR, KRAS, serine/threonine kinase 11 gene [STK11], and tumor protein p53 gene [TP53]) and ancestry analysis. Mutation frequencies in this H/L cohort were compared with those in a similar cohort of non-Hispanic white (NHW) patients and correlated with ancestry, sex, smoking status, and tumor histologic type.ResultsOf the adenocarcinomas in the H/L cohort (n = 120), 31% had EGFR mutations, versus 17% in the NHW control group (p < 0.001). KRAS (20% versus 38% [p = 0.002]) and STK11 (8% versus 16% [p = 0.065]) mutations occurred at lower frequency, and mutations in TP53 occurred at similar frequency (46% versus 40% [p = 0.355]) in H/L and NHW patients, respectively. Within the Hispanic cohort, ancestry influenced the rate of TP53 mutations (p = 0.009) and may have influenced the rate of EGFR, KRAS, and STK11 mutations.ConclusionsDriver mutations in H/L patients with lung adenocarcinoma differ in frequency from those in NHW patients associated with their indigenous American ancestry. The spectrum of driver mutations needs to be further assessed in the H/L population.

Malignant peripheral nerve sheath tumor (MPNST) is a type of soft-tissue sarcoma strongly associated with dysfunction in neurofibromin; an inhibitor of the RAS pathway.We performed high-throughput screening of an array of FDA approved and promising agents in clinical development both alone and in combination at physiologically achievable concentrations against a panel of established MPNST cell line models.We found that drugs targeting a variety of factors in the RAS pathway can effectively lead to cell death in vitro with considerable drug combination synergy in regimens that target MEK or mTOR.We observed that the degree of relative sensitivity to chemotherapeutic agents was associated with the status of neurofibromin in these cell line models.Using a combination of agents that target MEK and mTORC1/2, we effectively silenced RAS/PI3K/MEK/mTOR signaling in vitro.Moreover, we employed RNAi against NF1 to establish that MPNST drug sensitivity is directly proportional to relative level of intracellular neurofibromin.Thus, two-drug combinations that target MEK and mTORC1/2 are most effective in halting the RAS signaling cascade, and the relative success of this and related small molecule interventions in MPNSTs may be predicated upon the molecular status of neurofibromin.www.oncotarget.com

Leukoencephalopathies are a group of white matter disorders related to abnormal formation, maintenance, and turnover of myelin in the central nervous system. These disorders of the brain are categorized according to neuroradiological and pathophysiological criteria. Herein, we have identified a unique form of leukoencephalopathy in seven patients presenting at ages 2 to 4 months with progressive microcephaly, spastic quadriparesis, and global developmental delay. Clinical, metabolic, and imaging characterization of seven patients followed by homozygosity mapping and linkage analysis were performed. Next generation sequencing, bioinformatics, and segregation analyses followed, to determine a loss of function sequence variation in the phospholipase A2-activating protein encoding gene (PLAA). Expression and functional studies of the encoded protein were performed and included measurement of prostaglandin E2 and cytosolic phospholipase A2 activity in membrane fractions of fibroblasts derived from patients and healthy controls. Plaa-null mice were generated and prostaglandin E2 levels were measured in different tissues. The novel phenotype of our patients segregated with a homozygous loss-of-function sequence variant, causing the substitution of leucine at position 752 to phenylalanine, in PLAA, which causes disruption of the protein's ability to induce prostaglandin E2 and cytosolic phospholipase A2 synthesis in patients' fibroblasts. Plaa-null mice were perinatal lethal with reduced brain levels of prostaglandin E2 The non-functional phospholipase A2-activating protein and the associated neurological phenotype, reported herein for the first time, join other complex phospholipid defects that cause leukoencephalopathies in humans, emphasizing the importance of this axis in white matter development and maintenance.

The Origin Recognition Complex (ORC) is a critical component of replication initiation. We have previously reported generation of an Orc2 hypomorph cell line (Δ/–) that expresses very low levels of Orc2 but is viable. We have shown here that Chk2 is phosphorylated, suggesting that DNA damage checkpoint pathways are activated. p53 was inactivated during the derivation of the Orc2 hypomorphic cell lines, accounting for their survival despite active Chk2. These cells also show a defect in the G₁ to S-phase transition. Cdk2 kinase activation in G₁ is decreased due to decreased Cyclin E levels, preventing progression into S-phase. Molecular combing of bromodeoxyuridine-labeled DNA revealed that once the Orc2 hypomorphic cells enter S-phase, fork density and fork progression are approximately comparable with wild type cells. Therefore, the low level of Orc2 hinders normal cell cycle progression by delaying the activation of G₁ cyclin-dependent kinases. The results suggest that hypomorphic mutations in initiation factor genes may be particularly deleterious in cancers with mutant p53 or increased activity of Cyclin E/Cdk2.

Patients with advanced metastatic melanoma have poor prognosis and the genetics underlying its pathogenesis are poorly understood. High-throughput sequencing has allowed comprehensive discovery of somatic mutations in cancer samples. Here, on analysis of our whole-genome and whole-exome sequencing data of 29 melanoma samples, we identified several genes that harbor recurrent nonsynonymous mutations. These included MAP3K5 (mitogen-activated protein kinase kinase kinase-5), which in a prevalence screen of 288 melanomas was found to harbor a R256C substitution in 5 cases. All MAP3K5-mutated samples were wild type for BRAF, suggesting a mutual exclusivity for these mutations. Functional analysis of the MAP3K5 R256C mutation revealed attenuation of MKK4 (mitogen-activated protein kinase kinase 4) activation through increased binding of the inhibitory protein thioredoxin (TXN/TRX-1/Trx), resulting in increased proliferation and anchorage-independent growth of melanoma cells. This mutation represents a potential target for the design of new therapies to treat melanoma.

Abstract Background: EGFR is a major therapeutic target for colorectal cancer. Currently, extended RAS/RAF testing identifies only nonresponders to EGFR inhibitors (EGFRi). We aimed to develop a mutation signature that further refines drug-sensitive subpopulations to improve EGFRi outcomes. Methods: A prespecified, 203-gene expression signature score measuring cetuximab sensitivity (CTX-S) was validated with two independent clinical trial datasets of cetuximab-treated patients with colorectal cancer (n = 44 and n = 80) as well as an in vitro dataset of 147 cell lines. The CTX-S score was then used to decipher mutated genes that predict EGFRi sensitivity. The predictive value of the identified mutation signature was further validated by additional independent datasets. Results: Here, we report the discovery of a 2-gene (APC+TP53) mutation signature that was useful in identifying EGFRi-sensitive colorectal cancer subpopulations. Mutant APC+TP53 tumors were more predominant in left- versus right-sided colorectal cancers (52% vs. 21%, P = 0.0004), in microsatellite stable (MSS) versus microsatellite instable (MSI) cases (47% vs. 2%, P &amp;lt; 0.0001), and in the consensus molecular subtype 2 versus others (75% vs. 37%, P &amp;lt; 0.0001). Moreover, mutant APC+TP53 tumors had favorable outcomes in two cetuximab-treated patient-derived tumor xenograft (PDX) datasets (P = 0.0277, n = 52; P = 0.0008, n = 98). Conclusions: Our findings suggest that the APC and TP53 combination mutation may account for the laterality of EGFRi sensitivity and provide a rationale for refining treated populations. The results also suggest addition of APC+TP53 sequencing to extended RAS/RAF testing that may directly increase the response rates of EGFRi therapy in selected patients. Impact: These findings, if further validated through clinical trials, could also expand the utility of EGFRi therapies that are currently underutilized.

Sézary syndrome (SS) is a genetically and clinically distinct entity among cutaneous T-cell lymphomas (CTCL). SS is characterized by more aggressive disease compared to the most common indolent type of CTCL, mycosis fungoides. However, there are limited available genomic data regarding SS. To characterize and expand current mappings of the genomic landscape of cutaneous T-cell lymphomas, whole exome sequencing was performed on peripheral blood samples from 7 patients with SS. We detected 21 784 variants, of which 21 140 were novel and 644 were previously described. Filtering revealed 551 nonsynonymous variants among 525 mutated genes—25 recurrent mutations and 1 recurrent variant. Several recurrently mutated genes crucial to pathogenesis pathways, including JAK/STAT, PPAR, PI3K-AKT, and FGFR, were identified. Furthermore, genetic mutations spanned both known and novel genes, supporting the idea of a long-tail distribution of mutations in lymphoma. Acknowledging these genetic variants and their affected pathways may inspire future targeted therapies. Whole exome sequencing of a limited number of SS patients revealed both novel findings and corroborated complexities of the ‘long-tail’ distribution of previously reported mutations.

MUC16 is a mucin marker that is frequently mutated in melanoma, but whether MUC16 mutations could be useful as a surrogate biomarker for tumor mutation burden (TMB) remains unclear.This study rigorously evaluates the MUC16 mutation as a clinical biomarker in cutaneous melanoma by utilizing genomic and clinical data from patient samples from The Cancer Genome Atlas (TCGA) and two independent validation cohorts. We further extended the analysis to studies with patients treated with immunotherapies.Analysis results showed that samples with MUC16 mutations had a higher TMB than the samples of wild-type, with strong statistical significance (P < 0.001) in all melanoma cohorts tested. Associations between MUC16 mutations and TMB remained statistically significant after adjusting for potential confounding factors in the TCGA cohort [OR, 9.28 (95% confidence interval (CI), 5.18-17.39); P < 0.001], Moffitt cohort [OR, 31.95 (95% CI, 8.71-163.90); P < 0.001], and Yale cohort [OR, 8.09 (95% CI, 3.12-23.79); P < 0.01]. MUC16 mutations were also found to be associated with overall survival in the TCGA [HR, 0.62; (95% CI, 0.45-0.85); P < 0.01] and Moffitt cohorts [HR, 0.49 (95% CI, 0.28-0.87); P = 0.014]. Strikingly, MUC16 is the only top frequently mutated gene for which prognostic significance was observed. MUC16 mutations were also found valuable in predicting anti-CTLA-4 and anti-PD-1 therapy responses.MUC16 mutation appears to be a useful predictive marker of global TMB and patient survival in melanoma.This is, to the best of our knowledge, the first systematic evaluation of MUC16 mutation as a clinical biomarker and a predictive biomarker for immunotherapy in melanoma.

Abstract Study Objectives: A hallmark of tumor infiltrating lymphocytes (TIL) in melanoma is the potential for a complete response (CR) which can last for decades. This long-lived effect is attributed to persistence of memory T cells. However, clinical efficacy in metastatic non-small cell lung cancer (mNSCLC) has not previously been reported. We launched a phase I trial with the objective to evaluate the safety and efficacy in mNSCLC after evidence of progression on nivolumab. Methodology: Full eligibility criteria is described in trial registry (NCT03215810). Patients were required to have mNSCLC with at least 1 RECIST measurable lesion and also 1 lesion safely accessible for excisional biopsy, usually a supraclavicular lymph node or pleural nodule. This histologically confirmed metastasis was resected and TIL cultured. Autologously reactive cultures were pooled and cryopreserved. TIL was successfully expanded in 95% of patients, to a median dose of 103 billion CD3+ cells. Patients received nivolumab for 4 cycles with two serial CT scans. Patients with tumor enlargement or new lesions proceeded to lymphodepletion cyclophosphamide/fludarabine (Cy/Flu), TIL, and attenuated IL-2. After first CT scan, patients resumed nivolumab for up to 11 doses to augment TIL persistence. Results: Of 32 patients screened, 20 were eligible and enrolled. Median age 54 yrs (range 38 - 75), median PD-L1 proportion score 6%, median estimated TMB 5 mut/MB, 20% EGFR-mutant. The majority had bulky disease with mean sum of target lesion diameters of 7.0 cm. Of 20 enrolled, 13 had evidence of progression on nivolumab and received subsequent Cy/Flu/TIL/IL-2. Two more are progressing on nivolumab and set to receive TIL within next 2 months. Common non-hematologic adverse events (AEs) of the Cy/Flu/TIL/IL2 regimen included hypoalbuminemia (92%), hypophosphatemia (85%), nausea (77%), hyponatremia (69%), diarrhea (62%), the majority of these AEs resolving by Day +10 post-TIL. Initial tumor regression occurred in most patients at their first post-TIL CT scan, with the median best overall change in sum of target lesion diameters of -38% (range +20 to -100). Median time-on-trial post-TIL is 1.4 years. Two patients have achieved durable CRs which are still ongoing almost 1 year post-TIL. One is a never-smoker with EGFREx19 mutation and PD-L1 1%. Three additional patients were able to maintain a clinical remission by local ablative therapy of an isolated new lesion performed 6 to 17 months post-TIL. Persistence of the infused TCR-Vβ clonotypes at post-infusion timepoints was associated with clinical benefit. Neoantigen-specific T cells were detected in the TIL and post-TIL peripheral blood. Conclusions: TIL has manageable toxicity and capacity to achieve durable remission in mNSCLC after nivolumab treatment. TIL may be a promising option for fit mNSCLC patients. Citation Format: Ben Creelan, Chao Wang, Jamie Teer, Eric Toloza, John Mullinax, Jiqiang Yao, John Koomen, Sungjune Kim, Alberto Chiappori, James Saller, Leighann Montoya, Ana Marie Landin, Tawee Tanvetyanon, Bin Fang, Zachary Thompson, Xiaoping Yu, Andreas Saltos, Dung-Tsa Chen, Jose Conejo-Garcia, Eric Haura, Scott Antonia. Durable complete responses to adoptive cell transfer using tumor infiltrating lymphocytes (TIL) in non-small cell lung cancer (NSCLC): A phase I trial [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr CT056.

Aim: Successful treatment with tacrolimus to prevent graft versus host disease (GVHD) and minimize tacrolimus-related toxicities among allogeneic hematopoietic cell transplantation (alloHCT) recipients is contingent upon quickly achieving and maintaining concentrations within a narrow therapeutic range. The primary objective was to investigate associations between CYP3A4, CYP3A5 or ABCB1 genotype and the proportion of patients that attained an initial tacrolimus goal concentration following initiation of intravenous (iv.) and conversion to oral administration. Materials &amp; methods: We retrospectively evaluated 86 patients who underwent HLA-matched (8/8) related donor alloHCT and were prescribed a tacrolimus-based regimen for GVHD prophylaxis. Results &amp; conclusion: The findings of the present study suggests that CYP3A5 genotype may impact attainment of initial therapeutic tacrolimus concentrations with oral administration in alloHCT recipients.

Abstract Intraductal papillary mucinous neoplasms (IPMNs) often precede invasive pancreatic ductal adenocarcinoma, with each IPMN tumor carrying a 10-25% risk of progressing to cancer within 10 years of detection. The objective of this work is to employ AI-augmented analyses of single mutations occurring at specific tri-nucleotide sequences or codons and identify markers associated with the progression of cysts to pancreatic cancer. Despite being precursors of pancreatic cancer there is a lack of validated intervention targets for precision prevention for IPMNs. Currently cancer prevention for patients with IPMN is centered on surgery using a risk-tailored approach; the clinical ability to stratify risk of cancer progression of individual IPMN tumors is poor and essentially no effective non-surgical interventions exist. To advance the precision cancer prevention for IPMNs we develop an intelligent, machine learning (ML) derived framework addressing the challenge of IPMN stratification from samples with limited data space, e.g., somatic mutations. Our study incorporates the text-based statistical and ML analyses of the mutational profiles of patients’ genomes followed by an integration of codon sequence derived mutational features for biomarkers validation. Given the potential of genome spatial organization to understand mutagenesis and genomic instability, we also map the DNA sequence information onto the local spatial genome organization to elucidate intrinsic features and molecular mechanisms that potentially lead to mutations. Results of this study show that information extracted from mutational profiles with the proposed methods significantly improves performance of the ML models in stratification of IPMNs and biomarker identification. We also present how utilization of these ML tools for mutational data, spatial genome organization, and functional analyses from tumor samples, can aid understanding of the underlying mechanisms of mutations in IPMNs and pancreatic cancer. Citation Format: Nam Nguyen, Jamie K. Teer, Margaret A. Park, Patricia McDonald, Jason B. Fleming, Jennifer B. Permuth, Kwang-Cheng Chen, Aleksandra Karolak. Reinforcing risk prediction for intraductal papillary mucinous neoplasms of the pancreas with AI-optimized nucleotide-to-amino acid analyses [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr B109.

Abstract Introduction: Identifying dysregulated gene expression in ovarian cancers has been limited by a deficit of available normal tissues. Here, we generated the largest set of high-grade serous ovarian cancer (HGSOC) tumors with normal precursor tissues for transcriptome analyses to identify lncRNA expression patterns, provide insight into their cellular contexts and functions, and assess their relevance to HGSOC pathogenesis and genetic susceptibility. Methods: We performed RNA sequencing on 220 primary HGSOCs and 116 benign epithelia (micro-dissected fallopian tube, ovarian surface, and inclusion cyst epithelia), and combined samples with 428 HGSOCs from TCGA, 60 HGSOCs from a prior study, and 180 bulk ovary tissues from GTEx. Reads were processed with a uniform bioinformatic and quality control pipeline, combined and batch corrected. Normalized TPM expression values were compared using the Wilcoxon rank sum test with &amp;gt;2-fold change and FDR &amp;lt;1% considered statistically significant. We computed weighted gene co-expression networks for each tissue type and tested for enrichment of differentially expressed genes (DEGs) and gene ontologies within co-expression modules. Results: Transcriptomes included 27,700 expressed genes (8,202 lncRNAs) across 706 HGSOCs, 180 bulk ovary, and 88 ovarian epithelia tissues. Most genes were expressed in all tissues, though 5% each showed HGSOC- or normal tissue-specific expression which comprised ≥50% lncRNA. Comparing HGSOCs to normal tissues identified 11,804 DEGs with 4,522 lncRNAs (DElncRNA) of which ~50% were tissue-specific. DEGs included MUC16 and multiple GWAS/TWAS implicated susceptibility genes including RAD51, BRIP1, BNC2, TIPARP-AS1, PRC1, KANSL1, ANKLE1, CHMP4C, ESRP2, and CCNE1. The most highly expressed DElncRNA in HGSOC were upregulated RMRP (P=1.4 × 10−39), SNHG1 (P=3.0 × 10−27), and HAGLR (P=2.0 × 10−24) at the HOXD risk locus. Lower proportions of lncRNA were seen in the HGSOC co-expression modules compared to those in normal tissues and less (3% vs. 15%) HGSOC modules were considered strongly lncRNA regulated (&amp;gt;50% lncRNA in module). Tissue-specific DElncRNA tended toward upregulation in HGSOC compared to ovarian epithelia with enriched modules associated with cell cycle regulation (P=1.5 × 10−38); and toward downregulation in HGSOC compared to bulk ovary with enriched modules associated with chemokine signaling/response (P=1.7 × 10−113). Conclusions: The lower proportion of lncRNA involved in HGSOC co-expression networks indicates a marked loss of regulation by lncRNA. Preliminary interrogation of HGSOC-dysregulated lncRNA expression revealed tissue-specific differences that highlight unique biological pathways in precursor epithelia and the ovarian microenvironment that contribute to HGSOC pathogenesis. Our results provide additional support for previously nominated risk genes. eQTL analysis has recently been completed and integration with GWAS are underway to elucidate lncRNA and lncRNA-regulated susceptibility genes and will be presented at the conference. Citation Format: Brett M. Reid, Ann Chen, Zhihua Chen, Florian A. Karreth, Peter Kanetsky, Jennifer B. Permuth, Ozlen Saglam, Jamie K. Teer, Xiaoqing Yu, Simon Gayther, Ellen Goode, Paul Pharoah, Thomas A. Sellers, Kate Lawrenson, Jonathan Tyrer. Patterns of lncRNA-regulated gene expression in high-grade serous ovarian carcinomas [abstract]. In: Proceedings of the AACR Special Conference on Ovarian Cancer; 2023 Oct 5-7; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(5 Suppl_2):Abstract nr A004.

Ancestrally diverse and admixed populations, including the Hispanic/Latino/a/x/e community, are underrepresented in cancer genetic and genomic studies. Leveraging the Latino Colorectal Cancer Consortium, we analyzed whole exome sequencing data on tumor/normal pairs from 718 individuals with colorectal cancer (128 Latino, 469 non-Latino) to map somatic mutational features by ethnicity and genetic ancestry. Global proportions of African, East Asian, European, and Native American ancestries were estimated using ADMIXTURE. Associations between global genetic ancestry and somatic mutational features across genes were examined using logistic regression. TP53 , APC , and KRAS were the most recurrently mutated genes. Compared to non-Latino individuals, tumors from Latino individuals had fewer KRAS (OR=0.64, 95%CI=0.41-0.97, p=0.037) and PIK3CA mutations (OR=0.55, 95%CI=0.31-0.98, p=0.043). Genetic ancestry was associated with presence of somatic mutations in 39 genes (FDR-adjusted LRT p<0.05). Among these genes, a 10% increase in African ancestry was associated with significantly higher odds of mutation in KNCN (OR=1.34, 95%CI=1.09-1.66, p=5.74×10 -3 ) and TMEM184B (OR=1.53, 95%CI=1.10-2.12, p=0.011). Among RMGs, we found evidence of association between genetic ancestry and mutation status in CDC27 (LRT p=0.0084) and between SMAD2 mutation status and AFR ancestry (OR=1.14, 95%CI=1.00-1.30, p=0.046). Ancestry was not associated with tumor mutational burden. Individuals with above-average Native American ancestry had a lower frequency of microsatellite instable (MSI-H) vs microsatellite stable tumors (OR=0.45, 95%CI=0.21-0.99, p=0.048). Our findings provide new knowledge about the relationship between ancestral haplotypes and somatic mutational profiles that may be useful in developing precision medicine approaches and provide additional insight into genomic contributions to cancer disparities.Our data in ancestrally diverse populations adds essential information to characterize mutational features in the colorectal cancer genome. These results will help enhance equity in the development of precision medicine strategies.

Abstract Background: Neoantigen-based personalized cancer vaccines carry significant promise in treating solid malignancies. However, there are uncertainties regarding the choice between the primary or the metastatic tumor for neoantigen prediction in individual patients. Here, we conducted a thorough examination of somatic variations in 676 patients who had paired primary and metastatic solid tumors. Methods: Patients were enrolled in the Total Cancer Care protocol (NCT03977402) to which patients provided an IRB-approved written informed consent within the Oncology Research Information Exchange Network (ORIEN). Whole-exome sequencing of 756 primary and metastatic tumor pairs was performed (N = 676 patients). These included Genitourinary (n=83), Gynecological (n=97), Gastrointestinal (n=213), Thoracic (n=33), Cutaneous (n=24), Breast (n=108), Endometrial (n=49), Sarcoma (n=35), Head-and-Neck (n=106) and others (n=8). The data was analyzed through the ORIEN AVATAR Molecular Analysis Pipeline for somatic mutation variant detection and variant annotation. In this analysis, we focused on somatic events that result in an in-frame alteration (such as missense, in-frame deletion and in-frame insertion) and out-of-frame protein-altering mutations (such as frameshifts, de novo start, out-of-frame, and nonstop gain). Clonal population structure was determined based on pyclone-vi. Results: For in-frame events, bladder cancer, melanoma, and gynecological cancers shared close to 75% of the mutations between paired primary and metastatic cases. In contrast, sarcoma and thyroid cancer had a low overlap (~ 25%) of variants. For out-of-frame events, these events tend to have a lower proportion of shared somatic variants between primary and metastasis than in-frame variants. Oncogenic drivers (e.g., BRAF V600E, KRAS G12A, and TP53 loss-of-function) were highly likely to be present in both paired primary and metastatic tumors. Next, we performed additional analysis on evolutionary selection of protein-coding variants via dN/dS calculation. We found no significant global shift in dN/dS ratio between paired primary and metastatic tumors across malignancies. However, we found increased selection of protein-coding variants in brain and liver metastatic sites, which correlated with increased homologous recombination deficiency within these sites. Conclusions: Our analysis demonstrates genetic variations that exist when comparing paired primary and metastatic tumors that appear to vary by histology. Variants are potentially undergoing negative selection supported by the preferential loss of out-of-frame events in metastatic tumors and positive selection in specific metastatic sites. Overall, understanding the clonal structure will be key to neoantigen prediction for effective neoantigen-based vaccines. Citation Format: Alyssa Obermayer, Timothy Shaw, Darwin Chang, Joshua Davis, Jamie K. Teer, Xiaoqing Yu, Xuefeng Wang, Dale Hedges, Aik Choon Tan, Robert Rounbehler, Abdul Rafeh Naqash, Margaret Gatti-Mays, Aakrosh Ratan, Martin McCarter, Howard Colman, Igor Puzanov, Susanne Arnold, Michelle Churchman, Patrick Hwu, William Dalton, George Weiner, Jose Conejo-Garcia, Paulo C. Rodriguez, Bodour Salhia, Ahmad A. Tarhini. Analysis of clonal heterogeneity within paired primary and metastatic tumor samples of patients with solid tumors and implications for neoantigen-based personalized cancer vaccines [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 3886.

Abstract Sarcomas encompass a group of malignant diseases arising from mesenchymal origins. Given their rarity and diversity, a fundamental understanding of the genomic underpinnings for many sarcoma subtypes is still lacking. Studies are often limited to one or several of the more common subtypes or a narrow evaluation of a broader sampling. We therefore report on one of the largest comprehensive omics evaluation in sarcomas to date, including whole exome sequencing (WES; n = 1170) and RNA-sequencing (n = 983) of tissues from 29 different sarcoma histologic subtypes collected at 13 institutions in the US as part of the Oncology Research Information Exchange Network (ORIEN). We identified recurrent somatic mutations previously identified in sarcomas (e.g. TP53, KIT) as well as other cancer types (e.g. BRCA1). The burden of putatively pathogenic driver point mutations was higher in metastatic samples (median = 3) as compared to primary tumor samples (median = 2; p &amp;lt; 0.001). We observed frequent copy number alterations including whole genome doubling more commonly in metastatic compared to primary tumors (23.4% vs. 16.9%; p = 0.0.25). Inspection of gene expression dimensionality reduction (UMAP) showed separation of gastrointestinal stromal tumors (GISTs), leiomyosarcomas, myxoid liposarcomas, and well/de-differentiated liposarcomas from the other histologies. Differential expression analysis for these four histologies with gene set enrichment analysis highlights the diversity of disease-specific pathways and need for sarcoma subtype-specific translational focus. Estimation of immune cell abundances based on RNA-seq followed by hierarchical clustering identified five immune subtypes. The subtypes ranged from low (clusters A, B) to high (clusters D, E) immune infiltration with higher abundance of T, B, Natural Killer (NK), and myeloid cells (FDR &amp;lt; 0.01). Intermediate immune group C was predominantly composed by GISTs and marked by an enrichment for NK cells (FDR &amp;lt; 0.01) compared to all groups except the immune “hot” group E; however, this immune group exhibited modest infiltration by other immune cell types. Notably, we observed significant differences in the overall survival of patients with sarcomas in immune enriched (C, D, E) compared to immune depleted clusters (A, B; p = 0.002). In summary, we report the genomic and expressional landscape of over 1000 sarcomas, representing one of the largest comprehensive profiling efforts in this disease. We identify the mutational and copy number variation landscape and observe differences between primary and metastatic samples. We highlight expression pathways that are enriched in histologic subtypes that cluster most distinctly from others, providing a subtype-specific roadmap for further translational efforts. Finally, we define immune enriched or depleted sarcoma subgroupings that carry a prognostic impact. Citation Format: Alex C. Soupir, Oscar E. Ospina, Dale Hedges, Jamie K. Teer, Michael D. Radmacher, David M. McKean, Nathan Seligson, Martin McCarter, Breelyn Wilkey, Greg Riedlinger, John Groundland, Benjamin J. Miller, Bryan Schneider, Reema Patel, Abdul Rafeh-Naqash, Stephen Edge, Bodour Salhia, Chris Moskaluk, Maggy Johns, Michelle L. Churchman, Oliver Hampton, David Liebner, Brooke L. Fridley, Andrew S. Brohl. Genomic landscape and estimation of immune infiltration of soft tissue sarcoma histology subtypes from the ORIEN network [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 3928.

Abstract Purpose: The Latino Colorectal Cancer Consortium (LC3) was established to fill a gap in representation of the diverse Hispanic/Latino/a/x community in cancer genomic research and to enhance understanding of determinants of disparities in the burden of colorectal cancer (CRC). Methods: The LC3 joins together three established studies with deeply-annotated clinical, epidemiologic, and biologic data available for a heterogenous group of Hispanic/Latino/a/x men and women diagnosed with primary colorectal adenocarcinoma: the Hispanic Colorectal Cancer Study (California), the Total Cancer Care Protocol at Moffitt Cancer Center (Florida), and the Puerto Rico Biobank (southern Puerto Rico). Each contributing study collected data on demographics, medical history, family history, and lifestyle factors. Vital status, cause of death, treatment, and clinical characteristics were obtained through medical chart abstraction and/or linkage to cancer registries. Blood, saliva, or normal colonic tissues were used to extract and genotype germline DNA for a subset of participants. Tumor tissues (snap frozen or formalin-fixed paraffin-embedded) were evaluated by pathologists for diagnosis, tissue content, tumor cellularity, necrosis, immune infiltration, and additional histopathologic characteristics. Results: The LC3 has assembled 2,041 participants across the three studies to date, with recruitment ongoing. Median age at diagnosis is 57 (range: 19-93), 54% of participants are male, and 63% are colon cancer cases (vs. rectal cancer). Participants were diagnosed between 1999 and 2022. The target is to generate comprehensive multi-omic data on 600 LC3 participants. To date, 243 have genome-wide germline genotyping, 323 have paired tumor/normal whole exome sequencing, and 257 have T cell receptor immunosequencing (immunoSEQ, Adaptive Biotechnologies). Further, 206 cases are represented on a set of tissue microarray blocks (target N=281). Bulk RNA-seq data are to be generated on 250 tumors. Conclusions: The LC3 fills an important gap in research infrastructure for the scientific community. Key strengths of this unique consortium are its ability to capture a diversity of Latino CRC patients with respect to nativity and cultural heritage and to represent the spectrum of stages from early to metastatic. Ultimately, research from the LC3 will inform development of equitable precision medicine approaches and predictive models to improve the health of the rapidly-growing, heterogeneous Hispanic/Latino/a population. Citation Format: Nicole C. Loroña, Marco Matejcic, Daniel Sobieski, Nathalie T. Nguyen, Hannah J. Hoehn, Diana B. Diaz, Kritika Shankar, Esther Jean-Baptiste, Domenico Coppola, Clifton Fulmer, Ozlen Saglam, Kun Jiang, Teresita Muñoz-Antonia, Idhaliz Flores, Edna Gordian, José A. Oliveras Torres, Seth I. Felder, Julian A. Sanchez, Jason Fleming, Erin M. Siegel, Douglas Cress, Mariana C. Stern, Jamie K. Teer, Stephanie L. Schmit, Jane C. Figueiredo. The Latino Colorectal Cancer Consortium: A resource for colorectal cancer disparities research [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 6134.

Abstract Somatic evolution selects cancer cell phenotypes that maximize survival and proliferation in dynamic environments. Although cancer cells are molecularly heterogeneous, we hypothesized convergent adaptive strategies to common host selection forces can be inferred from patterns of epigenetic and genetic evolutionary selection in similar tumors. We systematically investigated gene mutations and expression changes in lung adenocarcinomas with no common driver genes ( n = 313). Although 13,461 genes were mutated in at least one sample, only 376 non-synonymous mutations evidenced positive evolutionary selection with conservation of 224 genes, while 1736 and 2430 genes exhibited ≥ two-fold increased and ≥ 50% decreased expression, respectively. Mutations under positive selection are more frequent in genes with significantly altered expression suggesting they often “hardwire” pre-existing epigenetically driven adaptations. Conserved genes averaged 16-fold higher expression in normal lung tissue compared to those with selected mutations demonstrating pathways necessary for both normal cell function and optimal cancer cell fitness. The convergent LUAD phenotype exhibits loss of differentiated functions and cell–cell interactions governing tissue organization. Conservation with increased expression is found in genes associated with cell cycle, DNA repair, p53 pathway, epigenetic modifiers, and glucose metabolism. No canonical driver gene pathways exhibit strong positive selection, but extensive down-regulation of membrane ion channels suggests decreased transmembrane potential may generate persistent proliferative signals. NCD LUADs perform niche construction generating a stiff, immunosuppressive microenvironment through selection of specific collagens and proteases. NCD LUADs evolve to a convergent phenotype through a network of interconnected genetic, epigenetic, and ecological pathways.

While genomic sequencing methods are powerful tools in the discovery of the genetic underpinnings of human disease, incidentally-revealed novel genomic risk factors may be equally important, both scientifically, and as relates to direct patient care. We performed whole-exome sequencing on a child with VACTERL association who suffered severe post-surgical neonatal pulmonary hypertension, and identified a potential novel genetic risk factor for this complication: a heterozygous mutation in CPSI. Newborn screening results from this patient's monozygotic twin provided evidence that this mutation, in combination with an environmental trigger (in this case, surgery), may have resulted in pulmonary artery hypertension due to inadequate nitric oxide production. Identification of this genetic risk factor allows for targeted medical preventative measures in this patient as well as relatives with the same mutation, and illustrates the power of incidental medical information unearthed by whole-exome sequencing.

Discovery proteomics experiments include many options for sample preparation and MS data acquisition, which are capable of creating datasets for quantifying thousands of proteins. To define a strategy that would produce a dataset with sufficient content while optimizing required resources, we compared (1) single-sample LC-MS/MS with data-dependent acquisition to single-sample LC-MS/MS with data-independent acquisition and (2) peptide fractionation with label-free (LF) quantification to peptide fractionation with relative quantification of chemically labeled peptides (sixplex tandem mass tags (TMT)). These strategies were applied to the same set of four frozen lung squamous cell carcinomas and four adjacent tissues, and the overall outcomes of each experiment were assessed. We identified 6656 unique protein groups with LF, 5535 using TMT, 3409 proteins from single-sample analysis with data-independent acquisition, and 2219 proteins from single-sample analysis with data-dependent acquisition. Pathway analysis indicated the number of proteins per pathway was proportional to the total protein identifications from each method, suggesting limited biological bias between experiments. The results suggest the use of single-sample experiments as a rapid tissue assessment tool and digestion quality control or as a technique to maximize output from limited samples and use of TMT or LF quantification as methods for larger amounts of tumor tissue with the selection being driven mainly by instrument time limitations. Data are available via ProteomeXchange with identifiers PXD004682, PXD004683, PXD004684, and PXD005733.

Abstract Inherited pathogenic variants in genes that confer moderate to high risk of breast cancer may explain up to 50% of familial breast cancer. This study aimed at identifying inherited pathogenic variants in breast cancer cases from Puerto Rico that were not linked to BRCA1 or BRCA 2. Forty-eight breast cancer patients that met the clinical criteria for BRCA testing but had received a negative BRCA1 /2 result were recruited. Fifty-three genes previously implicated in hereditary cancer predisposition were captured using the BROCA Agilent cancer risk panel followed by massively parallel sequencing. Missense variants of uncertain clinical significance in CHEK2 were evaluated using an in vitro kinase assays to determine their impact on function. Pathogenic variants were identified in CHEK2 , MUTYH , and RAD51B in four breast cancer patients, which represented 8.3% of the cohort. We identified three rare missense variants of uncertain significance in CHEK 2 and two variants (p.Pro484Leu and p.Glu239Lys) showed markedly decreased kinase activity in vitro comparable to a known pathogenic variant. Interestingly, the local ancestry at the RAD51B locus in the carrier of p.Arg47* was predicted to be of African origin. In this cohort, 12.5% of the BRCA -negative breast cancer patients were found to carry a known pathogenic variant or a variant affecting protein activity. This study reveals an unmet clinical need of genetic testing that could benefit a significant proportion of at-risk Latinas. It also highlights the complexity of Hispanic populations as pathogenic factors may originate from any of the ancestral populations that make up their genetic backgrounds.

Background Uterine carcinosarcoma, a rare gynecological malignancy, often presents at the advanced stage with a poor prognosis because current therapies have not improved rates of survival. Genetic characterization of this tumor may lead to novel, specifically targeted drug targets to provide better treatment options for patients with this malignancy. Methods We present a case of a woman aged 61 years with uterine carcinosarcoma and retrospectively analyzed 100 study patients with uterine carcinosarcoma. From this group, 9 study patients underwent targeted sequencing of 1,321 genes. Results All 9 study patients had at least 1 mutation in JAK2, KRAS, PIK3CA, CTNNB1, PTEN, FBXW7, TP53, and ERBB2; of these, TP53 was the most frequently mutated gene (6/9). In addition, ARID1A and KMT2C, which have been described and identified as part of a set of chromatin-remodeling genes, were also found in our analyses. From our 100-person cohort clinical analyses, study patients with stage 1 cancer had a median survival rate of 33 months (95% confidence interval, 19–109) compared with a median survival rate of 6 months (95% confidence interval, 3–12) in those with stage 4 disease. Conclusions Disease stage alone predicted the rate of clinical survival. Up to 50% in the study group were identified at having early stage disease (stage 1/2), indicating improved rates of overall detection compared with previously reported data. Our mutational analysis findings add to the number of tumors in which these mutations have been found and suggest that chromatin-remodeling dysregulation may play a role in the tumorigenesis of carcinosarcoma.

The clinical utility of histone/protein deacetylase (HDAC) inhibitors in combinatorial regimens with proteasome inhibitors for patients with relapsed and refractory multiple myeloma (MM) is often limited by excessive toxicity due to HDAC inhibitor promiscuity with multiple HDACs. Therefore, more selective inhibition minimizing off-target toxicity may increase the clinical effectiveness of HDAC inhibitors. We demonstrated that plasma cell development and survival are dependent upon HDAC11, suggesting this enzyme is a promising therapeutic target in MM. Mice lacking HDAC11 exhibited markedly decreased plasma cell numbers. Accordingly, in vitro plasma cell differentiation was arrested in B cells lacking functional HDAC11. Mechanistically, we showed that HDAC11 is involved in the deacetylation of IRF4 at lysine103. Further, targeting HDAC11 led to IRF4 hyperacetylation, resulting in impaired IRF4 nuclear localization and target promoter binding. Importantly, transient HDAC11 knockdown or treatment with elevenostat, an HDAC11-selective inhibitor, induced cell death in MM cell lines. Elevenostat produced similar anti-MM activity in vivo, improving survival among mice inoculated with 5TGM1 MM cells. Elevenostat demonstrated nanomolar ex vivo activity in 34 MM patient specimens and synergistic activity when combined with bortezomib. Collectively, our data indicated that HDAC11 regulates an essential pathway in plasma cell biology establishing its potential as an emerging theraputic vulnerability in MM.

Abstract Purpose: Metastatic melanoma is a tumor amenable to immunotherapy in part due to the presence of antigen-specific tumor-infiltrating lymphocytes (TIL). These T cells can be activated and expanded for adoptive cell transfer (ACT), which has resulted in relatively high rates of clinical responses. Similarly, immune checkpoint inhibitors, specifically programmed cell death protein 1 (PD-1) blocking antibodies, augment antitumor immunity and increase the influx of T cells into tumors. Thus, we hypothesized that addition of PD-1 inhibition may improve the outcomes for patients undergoing ACT with TILs. Patients and Methods: Patients with stage III/IV metastatic melanoma with unresectable disease who were anti–PD-1 treatment-naïve were enrolled. TILs were generated in the presence of anti–4-1BB antibody in vitro and expanded for ACT. Patients in cohort 1 received TIL infusion followed by nivolumab. Patients in cohort 2 also received nivolumab prior to surgical harvest and during TIL production. Results: A total of 11 patients were enrolled, all of whom were evaluated for response, and nine completed ACT. Predominantly CD8+ TILs were successfully expanded from all ACT-treated patients and were tumor reactive in vitro. The trial met its safety endpoint, as there were no protocol-defined dose-limiting toxicity events. The objective response rate was 36%, and median progression-free survival was 5 months. Two nonresponders who developed new metastatic lesions were analyzed to determine potential mechanisms of therapeutic resistance, which included clonal divergence and intrinsic TIL dysfunction. Conclusions: Combination therapy with TILs and nivolumab was safe and feasible for patients with metastatic melanoma and provides important insights for future therapeutic developments in ACT with TILs.

Abstract Evidence from human and animal studies suggests that chronic behavioral stress and resulting activation of the sympathetic nervous system may influence initiation and progression of tumors. However, the underlying mechanisms for these observations are poorly understood. The purpose of this study is to explore the effects of adrenergic signaling on cell line models derived from normal cells presumed to originate epithelial ovarian cancers. Here we explored the effects of the stress-related hormone, norepinephrine, on the transcriptional program of normal immortalized ovarian (iOSE) and fallopian tube (iFTSEC) surface epithelial cells. Analysis of RNA-Seq data of treated and untreated cells revealed a significant overlap between the responses in iOSE and iFTSEC cells. Most genes modulated by norepinephrine in ovarian and fallopian tube epithelial cells are already expressed in normal ovarian and fallopian tissue and cells. For several genes, expression changes were reflected at the protein level. Genes in immune-related and developmental pathways were enriched in the set of genes modulated by norepinephrine. We identified HOXA5, SPIB, REL, SRF, SP1, NFKB1, MEF2A, E2F1, and EGR1 transcription factor binding sites to be highly enriched in our dataset. These data represent the early transcriptional response to norepinephrine in cells postulated to originate epithelial ovarian cancer.

Wilms’ tumor is the most common renal malignancy in children. In addition to staging, molecular risk stratification, such as loss of heterozygosity (LOH) in Chromosomes 1 and 16, is being increasingly used. Although genetic predisposition syndromes have been well-characterized in some Wilms’ tumors, recent sequencing and biology efforts are expanding the classification of this malignancy. Here we present a case of siblings with remarkably similar presentations of bilateral Wilms’ tumor at ∼12 mo of age. Thorough exam after the younger sibling was diagnosed did not reveal any signs to suggest one of the known Wilms’ predisposition syndromes. Both were treated with standard therapies with good response and long-term sustained complete remission of 53 and 97 mo, respectively. Whole-exome sequencing was performed on a tumor sample from each patient and matched blood from one, revealing a shared truncation mutation of TRIM28 in all three samples with heterozygosity in the germline sample. TRIM28 loss has been recently implicated in early-stage Wilms’ tumors with epithelioid morphology. These siblings expand the phenotype for presentation with multifocal disease with retained excellent response to standard therapy.