Prevalence Of Somatic Mutations Research Articles

Abstract 283: Identification of a high-risk subgroup in primary prostate cancers presenting with targetable immune biology

Abstract Background Recent studies have demonstrated limited success of immune checkpoint therapies in unselected prostate cancer. We therefore assessed an immune based DNA Damage Repair Deficiency (DDRD) assay, that we have previously reported represents activation of the cGAS STING pathway in the TCGA dataset of primary prostate cancers, to investigate the presence of targetable immune biology in prostate cancer. In addition we applied a second assay (the prostate cancer metastatic signature-PCM) that predicts risk of metastatic recurrence for early prostate cancer to assess if immune therapy could have a role in treating high risk disease. Methods 498 samples in the TCGA dataset with RNA sequencing data were scored with the PCM and DDRD assays. Integrative analysis was performed on 488 of those samples with matched RNA sequencing, promoter site methylation, somatic mutation and somatic copy number variation. Gene expression of n=6 immune checkpoint targets was investigated with the subgroups identified using T-tests. The prevalence of immune infiltration in each subgroup was tested by applying a cut off to the leukocyte fraction. The viability of reproducing those subgroups with RNA sequencing alone was tested in the TCGA dataset and an independent validation dataset of 321 resected primary prostate cancers. Cox proportional hazards regression analysis was performed for biochemical recurrence and metastatic events in both datasets. Results Integrative analysis of the TCGA dataset identified four patient subgroups characterised primarily by variances in copy number and genomic mutation. One of these subgroups ‘Metastatic-like DDRD' had significantly higher PCM scores and DDRD immune scores compared to the other subgroups (p < 2E-12). This subgroup of patients showed elevated leukocyte fraction and expression of immune checkpoint genes: CD274 (PDL1), CTLA4, ICOS, IDO1, HAVCR2 (TIM3) & LAG3 (p < 2E-6). Genomic instability with amplification of 8q and a larger prevalence of somatic mutations including that of TP53 was also detected in this subgroup. The ‘Metastatic-like DDRD' subgroup was found to have a significant association with poor survival outcome in TCGA (multivariable: p < 0.008), a result that has also been replicated in an independent dataset in both univariate (p < 0.001) and multivariable analysis (p < 0.01). Conclusions We have identified and validated a poor prognostic subgroup, representing 10-20% of early prostate cancer patients that are at increased risk of developing metastatic disease and present with targetable immune biology. These patients may represent a viable target population for immune checkpoint and DNA damaging therapies in prostate cancer. Citation Format: Emma Reilly, Andrena McCavigan, Steven M. Walker, Nuala McCabe, Eileen Parkes, Denis P. Harkin, Richard D. Kennedy, Laura A. Knight. Identification of a high-risk subgroup in primary prostate cancers presenting with targetable immune biology [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 283.

Exploration of the cGAS-STING pathway in prostate cancer.

103 Background: Recent studies have demonstrated limited success of immune checkpoint therapies in unselected prostate cancer. We therefore assessed an immune based DNA Damage Repair Deficiency (DDRD) assay, that we have previously reported represents activation of the cGAS STING pathway, in the TCGA prostate cancer dataset to investigate the presence of targetable immune biology in prostate cancer. In addition we applied a second assay (the prostate cancer metastatic signature-PCM) that predicts the risk of metastatic recurrence for early prostate cancer, in order to assess if immune therapy could have a role in treating high risk disease. Methods: 498 samples with RNA sequencing data were scored with the PCM and DDRD assays. Integrative analysis was performed on 488 samples with RNA sequencing, promoter site methylation, somatic mutation and somatic copy number variation. Gene expression of n = 6 immune checkpoint targets was investigated with the subgroups identified using T-tests. The prevalence of immune infiltration in each subgroup was tested by applying a cut off to the leukocyte fraction. Cox proportional hazards regression analysis of 441 patients was assessed for biochemical recurrence. Results: Integrative analysis identified four patient subgroups characterised primarily by variances in copy number and genomic mutation. One of these subgroups ‘Metastatic-like DDRD’ had significantly higher PCM scores and DDRD immune scores compared to the other subgroups (p < 2E-12). This subgroup of patients showed elevated leukocyte fraction and expression of immune checkpoint genes: CD274 (PDL1), CTLA4, ICOS, IDO1, HAVCR2 (TIM3) & LAG3 (p < 2E-6). Genomic instability with amplification of 8q and a larger prevalence of somatic mutations including that of TP53 was also detected in this subgroup. These patients had an increased risk of biochemical relapse in both univariate (p < 2E-5) and multivariate (p < 0.008) analysis. Conclusions: We identified a poor prognostic subgroup, representing 17% of early prostate cancer patients that are at increased risk of developing metastatic disease and present with targetable immune biology. These patients may represent a viable target population for immune checkpoint and DNA damaging therapies in prostate cancer.

The landscape of somatic mutations in Indonesian cervical cancer is predominated by the PI3K pathway

ObjectiveTo investigate the prevalence of somatic mutations in Indonesian cervical carcinoma patients in the context of histology and human papillomavirus (HPV) type. MethodsIn total 174 somatic hot-spot mutations in 13 genes were analyzed by mass spectrometry in 137 Indonesian cervical carcinomas. ResultsIn 66/137 tumors (48%) 95 mutations were identified. PIK3CA was most frequently mutated (24%), followed by FBXW7 (7%), CTNNB1 (6%), and PTEN (6%). In squamous cell carcinomas more often multiple mutations per sample (p=0.040), and more PIK3CA (p=0.039) and CTNNB1 (p=0.038) mutations were detected compared to adenocarcinomas. PIK3CA mutations were associated with HPV 16 positivity, CDKN2A mutations with HPV 52 positivity, and, interestingly, PTEN mutations with HPV negativity. Balinese tumor samples more often carried multiple mutations (p=0.019), and more CTNNB1, CDKN2A, and NRAS mutations compared to Javanese tumor samples. ConclusionsPotentially targetable somatic mutations occurred in 48% of Indonesian cervical carcinomas. The landscape of mutations is predominated by mutations concerning the PI3K pathway, and we prompt for more research on developing therapies targeting this pathway, explicitly for the more advanced stage cervical carcinoma patients.

Genetic aspects of primary hyperaldosteronism and pheochromocytoma

Pheochromocytoma and primary hyperaldosteronism are the most common causes of secondary hypertension. In a group of patients with primary hyperaldosteronism the prevalence of somatic mutation has been established in patients with aldosterone-producing adenomas (APA), genetic mutations have been identified in patients with familytypes of the disease. The authors declare that aldosterone-producing cellular clusters, which derived from zona glomerulosa, appear as a result of somatic mutations and might be a precursor of APA. Development of bilateral adrenal hyperplasia and APA might be explained by an existence of autoantibodies and their chronic stimulation of zona glomerulosa. The assessment of somatic and germline mutations in patients with pheochromocytoma and paraganglioma facilitates early diagnostics other tumors within syndromic neoplasia. Implementation of new genetic test in practice would improve early diagnosis of adrenal pathology in hypertensive patients.

Association between the Prevalence of Somatic Mutations in PIK3CA Gene in Tumors and Clinical and Morphological Characteristics of Breast Cancer Patients.

The presence of activating somatic mutations in codons 542 and 545 of exon 9 (p.E542K c.1624G>A and p.E545K c.1633G>A) and in codon 1047 of exon 20 (p.H1047R c.3140A>G and p.H1047L c.3140A>T) of PIK3CA gene encoding catalytic p110α-subunit of phosphatidylinositol-3-kinase was studied in tumors of 473 breast cancer patients by multiplex allele-specific real-time PCR. Fifty-eight (12.3%) different mutations were found. An increase in the frequency of PIK3CA gene mutations with disease progression (from 2.4 to 28.7% with tumor progression from I-IIa to III-IV stage; p=0.0001) and a trend towards its increase in the tumors with unfavorable prognostic characteristics (high histological grade, triple negative phenotype) were demonstrated. The presence of the studied PIK3CA gene mutations in tumors significantly reduces relapse-free survival in the total group and in stage III cancer patients.

Somatic mutations in children with GATA2-associated myelodysplastic syndrome who lack other features of GATA2 deficiency

AbstractApproximately 10% of children with primary myelodysplastic syndrome (MDS) have germ line GATA2 mutations, leading to the proposal that all children with primary MDS and certain cytogenetic findings, including monosomy 7, be tested for germ line GATA2 mutations regardless of family history or other clinical features associated with GATA2 deficiency. In adults with familial GATA2-MDS, those with somatic mutations in ASXL1 experience rapid disease progression to acute myeloid leukemia (AML) and poor prognosis after stem cell transplantation; however, the prevalence of somatic mutations in primary pediatric GATA2-MDS is unclear. Here, we studied a cohort of 8 pediatric patients with MDS and lacking additional GATA2-associated clinical features or significant family history and identified heterozygous germ line GATA2 mutations in 5 patients, including 1 with a normal karyotype. For those with GATA2-MDS, we screened for somatic mutations in genes with prognostic relevance in AML/MDS, using a targeted next-generation sequencing panel. Although no somatic mutations in ASXL1 were observed, somatic mutations were found in RUNX1, SETBP1, IKZF1, and CRLF2. One subject with deleterious mutations in RUNX1, SETBP1, and IKZF1 rapidly progressed to AML with disease that was refractory to treatment. Our findings confirm the importance of GATA2 testing in primary pediatric MDS, even in the absence of other clinical features of GATA2 deficiency. Further, similar to what has been observed in adults with GATA2-MDS, somatic mutations with potential prognostic effect occur in children with MDS associated with mutations in GATA2.

Abstract P1-05-15: Multi-omics and immuno-oncology profiling reveal distinct molecular signatures of young Asian breast cancers

Abstract Breast cancers (BC) in younger, premenopausal patients (YBC) tend to be more aggressive with worse prognosis, higher chance of relapse and poorer response to endocrine therapies compared to breast cancers in older patients. The proportion of YBC (age ≤ 40) among BC in East Asia is estimated to be 16-32%, significantly higher than the 7% reported in Western countries. To characterize the molecular bases of Asian YBC, we have performed whole-exome sequencing (WES) and whole-transcriptome sequencing (WTS) on tumor and matched normal samples from 134 Korean BC patients consisting of 74 YBC cases (age ≤ 40) and 60 OBC cases (age > 40). We then performed comparison analyses and integrative analyses with the TCGA BC cohort consisting of 1,116 tumors from primarily Caucasian patients, also grouped by age into YBC (age ≤ 40), IBC (40 < age ≤ 60) and OBC (age > 60). Somatic mutation prevalence analysis identified 7 significantly mutated genes and the same top three genes – TP53, GATA3 and PIK3CA – were reported by the TCGA BC study. To identify differentially expressed (DE) genes and pathways in YBCs vs. OBCs, we performed logistic regression analyses while controlling for the confounding effects of tumor purity and stage. We were surprised to see a significant overlap in DE pathways between a comparison of adjacent normal tissues in younger vs. older TCGA cohorts and a comparison of YBC vs. OBC tumors, indicating that normal tissue compartment could contribute to observed differences between bulk tumors. To separately examine molecular signatures from tumor, stroma and normal compartments, we used non-negative matrix factorization (NMF) analyses to virtually dissect bulk tumor expression data and identified 14 factors including 3 factors associated with normal tissues, 1 factor associated with stroma and 1 factor associated with tumor infiltrating lymphocytes (TIL). Integrative analyses of tumor associated factors and DE pathways revealed that estrogen response, endocrine therapy resistance, and oxidative phosphorylation pathways are up-regulated in YBCs compared to OBCs while cell cycle and proliferation pathways are up-regulated in Asian OBCs. Interestingly, many immune and inflammation pathways correlated with the TIL factor were significantly upregulated in OBCs vs. YBCs. Using gene expression signatures representing distinct immune cell types, we classified our cohort into four subtypes of varying TIL activities and observed significant enrichment of the TIL-high subtype in OBCs compared to YBCs. These observations were confirmed by IHC analyses of four TIL markers (CD45, CD4, CD8 and CD163) in 120 tumors. To our knowledge, this is the first large-scale multi-omics study of Asian breast cancer and would significantly contribute to the compendium of molecular data available for studying young breast cancers. The major landmarks in the molecular landscape looked similar across BCs of different ethnicities and ages, however, we have identified a number of distinguishing molecular characteristics associated with Asian YBC. The sources for some signatures were further traced to non-tumor intrinsic compartments, indicating that tumor microenvironment may play potentially important roles in driving the carcinogenesis of young breast cancers. Citation Format: Kan Z, Ding Y, Cho S, Lee S-H, Powell E, Jung HH, Chung W, Deng S, Choi Y-l, Kim J, Park W-Y, Vizcarra P, Fernandez-Banet J, Nichols T, Ram S, Lee SK, Kim SW, Lee JE, Ching KA, Kim J-Y, Ahn JS, Im Y-H, Nam SJ, Park YH. Multi-omics and immuno-oncology profiling reveal distinct molecular signatures of young Asian breast cancers [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P1-05-15.

Genetic and Functional Analysis of GRIN2A in Tumor Samples.

Ionotropic glutamate receptors (iGluRs) are large integral membrane multi-protein complexes that create ion channels in plasma membranes. Upon binding of receptor specific ligands (e.g., glutamate), increased efflux or influx of mono- or divalent cations (e.g., Ca2+) promotes synaptic transmission, cellular migration, and survival. Three classes of iGluRs were originally defined after their respective agonists: AMPA, kainate, and NMDA receptors (NMDARs). Recently, we examined iGluR families at the genetic level using Next-Generation Sequencing (NGS) (whole-exome sequencing (WES)) and discovered a high prevalence of somatic mutations within the gene for one of the NMDAR subunits, GRIN2A, specifically in malignant melanoma. Following confirmation of the somatic mutations, we focused on functional characterization of a subset of the GRIN2A mutants that demonstrated a loss of NMDAR functionality. We used gene expression and protein biochemistry to examine complex formation between GluN1 subunit (encoded by GRIN1) and GluN2A subunit (encoded by GRIN2A), anchorage-independent growth in soft agar and cellular migration. Furthermore, we used shRNA depletion of endogenous GRIN2A in melanoma cells expressing either wild-type GRIN2A or mutant GRIN2A and measured cellular proliferation compared to negative controls. Our data show that somatic mutation of certain residues in GluN2A results in increased survival and is the first such report to demonstrate the functional importance of GRIN2A mutations in melanoma and the significance ionotropic glutamate receptor signaling plays in malignant melanoma.

Chemoradiation Increases PD-L1 Expression in Certain Melanoma and Glioblastoma Cells

Immunotherapy approaches currently make their way into the clinics to improve the outcome of standard radiochemotherapy (RCT). The programed cell death receptor ligand 1 (PD-L1) is one possible target that, upon blockade, allows T cell-dependent antitumor immune responses to be executed. To date, it is unclear which RCT protocol and which fractionation scheme leads to increased PD-L1 expression and thereby renders blockade of this immune suppressive pathway reasonable. We therefore investigated the impact of radiotherapy (RT), chemotherapy (CT), and RCT on PD-L1 surface expression on tumor cells of tumor entities with differing somatic mutation prevalence. Murine melanoma (B16-F10), glioblastoma (GL261-luc2), and colorectal (CT26) tumor cells were treated with dacarbazine, temozolomide, and a combination of irinotecan, oxaliplatin, and fluorouracil, respectively. Additionally, they were irradiated with a single dose [10 Gray (Gy)] or hypo-fractionated (2 × 5 Gy), respectively, norm-fractionated (5 × 2 Gy) radiation protocols were used. PD-L1 surface and intracellular interferon (IFN)-gamma expression was measured by flow cytometry, and IL-6 release was determined by ELISA. Furthermore, tumor cell death was monitored by AnnexinV-FITC/7-AAD staining. For first in vivo analyses, the B16-F10 mouse melanoma model was chosen. In B16-F10 and GL261-luc2 cells, particularly norm-fractionated and hypo-fractionated radiation led to a significant increase of surface PD-L1, which could not be observed in CT26 cells. Furthermore, PD-L1 expression is more pronounced on vital tumor cells and goes along with increased levels of IFN-gamma in the tumor cells. In melanoma cells CT was the main trigger for IL-6 release, while in glioblastoma cells it was norm-fractionated RT. In vivo, fractionated RT only in combination with dacarbazine induced PD-L1 expression on melanoma cells. Our results suggest a tumor cell-mediated upregulation of PD-L1 expression following in particular chemoradiation that is not only dependent on the somatic mutation prevalence of the tumor entity.

Targeted Next Generation Sequencing Identifies Novel Genetic Mutations in Patients with Waldenstrom's Macroglobulinemia/Lymphoplasmacytic Lymphoma or IgM-Monoclonal Gammopathies of Undetermined Significance

Background. TheMYD88 (L265P) somatic mutation is present in more than 90% of patients (pts) with Waldenström’s Macroglobulinemia (WM)/lymphoplasmacitic lymphoma (LPL). The second most common mutations are nonsense (NS) or frameshift (FS) mutations in the CXCR4 gene, detectable in approximately 25-30% of WM pts by Sanger sequencing. Limited data are available about other genetic mutations in WM/LPL and its precursor condition IgM-monoclonal gammopathy of undetermined significance (IgM-MGUS).Pts and methods. Using targeted next generation sequencing (NGS), we evaluated the prevalence of somatic mutations of 11 genes selected on the basis of evidences available from the literature (MYD88, CXCR4, ARID1A, KMT2D, TP53, NOTCH2, PRDM1, CD79b, TRAF3,TNFAIP3, MYDBBP1A) in 119 pts, classified as WM/LPL (n=63) or IgM-MGUS (n=56) according to International Consensus Criteria. Median age of pts (67 males, 52 females) was 65 years (range: 38-82). Samples were collected at diagnosis (n=101), after diagnosis but before any treatment (n=9) or at progression after therapy (n=9). Paired tumor and germline DNA extracted respectively from CD19-selected and CD19-depleted bone marrow (BM) mononuclear cells was available in all pts. Mean resequencing depth across gene panel was 1009x. Only mutations tagged as oncogenic or possibly oncogenic based on information derived from the literature and on in silico prediction effect were considered in the analysis. For MYD88 (L265P) and CXCR4 mutations, results obtained with NGS were compared with those obtained respectively with allele-specific PCR (AS-PCR) and Sanger sequencing.Results. Overall, we found 151 mutations in 88 pts (74%). The median number of mutations was significantly higher in WM/LPL as compared with IgM-MGUS and in pts previously treated as compared with untreated ones (median 2 versus 1, P < 0.001 for both comparisons). MYD88 mutations were found in 80/119 pts (67%), with a median allele burden of 34.2% (range: 2.5-93.3%). The prevalence of MYD88 mutations was significantly higher in WM/LPL as compared with IgM-MGUS (86% versus 46%, P <0.001). MYD88 mutations other than classical L265P (n=76) were found in 4 pts and were represented by V217F (n=2), S219C (n=1), M232T (n=1). Fifteen pts who were MYD88 (L265P) wild-type by NGS were found to be mutated by AS-PCR (K coefficient of concordance between NGS and AS-PCR: 70%, P < 0.001). CXCR4 mutations were found in 19/119 pts (16%), with a median allele burden of 34% (range: 4.2-84%). The prevalence of CXCR4 mutations was significantly higher in WM/LPL as compared with IgM-MGUS (24% versus 7%, P < 0.02). The K coefficient of concordance between NGS and Sanger was 83% (P < 0.001), with 2 pts mutated only by NGS and 2 pts mutated only by Sanger. Somatic mutations were also found in KMT2D (formerly known as MLL2) (16% of pts), TP53 (8%), NOTCH2 (7%), PRDM1 (4%), ARID1A (3%), CD79b (2%), and TRAF3 (1%). No mutations were found in MYBBP1A and TNFAI3. Overall, the prevalence of these mutations was significantly lower in pts wild-type either for MYD88 or CXCR4 as compared with those with MYD88 and/or CXCR4 mutations (15% versus 41%, P = 0.04). The prevalence of KMT2D mutations was significantly higher in WM/LPL as compared with MGUS (25% versus 5%), while for the other genes the distribution was not statistically different according to diagnosis. With a median follow-up of 20 months (range: 0-264), we did not find a statistically significant correlation between genetic mutations and pts’ outcome in terms of overall survival or time to first treatment.Conclusions. In this cohort of pts with WM/LPL and IgM-MGUS studied with NGS we could demonstrate that: i) NGS identifies MYD88 mutations other than L265P in a small proportion of pts; ii) the prevalence of CXCR4 mutations by Sanger is confirmed by NGS, despite the higher sensitivity of the latter method; iii) the subgroup of pts wild type either for MYD88 or CXCR4 shows a low incidence of other genetic mutations; iv) 25% of pts with WM/LPL were found to carry KMT2D mutations, a prevalence similar to that reported in marginal zone lymphoma; v) genetic mutations are more common in WM/LPL than in IgM-MGUS in agreement with the hypothesis that multiple genetic hits are required for progression from a pre-benign condition to a neoplastic disease; vi) due to the indolent nature of these disorders, longer follow-up is probably needed to see the prognostic impact of these mutations, if any. DisclosuresNo relevant conflicts of interest to declare.

Prevalence and clinical significance of BRCA1/2 germline and somatic mutations in Taiwanese patients with ovarian cancer.

Germline and somatic BRCA1/2 mutations define a subset of patients with ovarian cancer who may benefit from treatment with poly (ADP-ribose) polymerase inhibitors. Unfortunately, data on the frequency of BRCA1/2 germline mutations in Taiwanese patients with ovarian cancer are scarce, with the prevalence of somatic mutations being unknown. We aim to investigate the occurrence of BRCA1/2 mutations in 99 Taiwanese patients with ovarian cancer which included serous (n = 46), endometrioid (n = 24), and clear cell (n = 29) carcinomas. BRCA1/2 mutations were identified using next-generation sequencing of formalin-fixed paraffin-embedded tumor samples. Pathogenic variants (BRCA1: n = 7; BRCA2: n = 6) were detected in 12.1% (12/99) of the study patients. Somatic and germline BRCA1/2 mutation rates in serous ovarian cancer are 4/46 (8.7%) and 8/46 (17%), respectively. All of the pathogenic BRCA1/2 mutations were identified in serous carcinoma samples (12/46; 26.1%). One-third (4/12) of the deleterious BRCA1/2 mutations occurred in tumor tissues only (somatic mutations). All of them coexisted with loss of heterozygosity, resulting in biallelic BRCA inactivation. Five novel pathogenic mutations were identified, including four somatic variants (BRCA1 p.S242fs, BRCA1 p.F989fs, BRCA1 p.G1738fs, and BRCA2 p.D1451fs) and a germline variant (BRCA2 p.E260fs). We also detected additional six novel mutations (three in BRCA1 and three in BRCA2) with pathogenic potentials. We conclude that BRCA1/2 mutations are common in Taiwanese patients with serous ovarian carcinoma and similar to mutation rates in other ethnic groups. The analysis of BRCA1/2 somatic mutations is crucial for guiding therapeutic decisions in ovarian cancer.

SY 03-3 OVERVIEW OF SOMATIC MUTATIONS AND EPIGENETIC REGULATION OF ALDOSTERONE PRODUCING ADENOMA (APA)

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Abstract LB-340: A novel method for estimating somatic mutations cellular prevalence in cancer using whole genome haplotype phasing

Abstract The process of malignant transformation results in the acquisition of complex chromosomal copy number alterations and somatic mutations. Moreover, cancer exhibits extensive intra-tumor heterogeneity with multiple sub-populations of tumor cells containing both common and private genomic alterations. Within- and between-patient tumor heterogeneity is now seen as one of the major obstacles for precision medicine and the development of effective treatment strategies. Revealing the proportion of cells affected by particular somatic changes is emerging as a key factor for designing targeted therapies, characterizing tumor evolution and understanding chemotherapy-resistance mechanisms. Determining the proportion of cells harboring a particular mutation (the so called cellular prevalence) is a difficult task as it is confounded by several variables. Normal cell contamination, local copy number profile and the temporal relationship between the mutation and the copy number alteration represent some of the key variables. In order to more effectively quantify the cellular prevalence of somatic mutations we developed a novel method, which uses a combination of side, and haplotype phase information obtained from long fragment read sequencing to more accurately compute mutational prevalence. The method utilizes three sources of information: the phasing information, the copy number variation and the allele counts for prevalence estimation using a simple equation in both a linear and a non-linear form. We present theoretical examples of known genomics alterations and show using simulation that our method was able to accurately estimate the cellular prevalence compared to standard methods. We also apply our method to a real data set obtained from dense long fragment read sequencing of a single ovarian cancer and demonstrate the highly accurate estimation of cellular prevalence. Finally we provide an implementation of the method in the statistical software. Citation Format: Donatien Chedom-Fotso, Ahmed Ahmed, Christopher Yau. A novel method for estimating somatic mutations cellular prevalence in cancer using whole genome haplotype phasing. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-340.

A promising road in colorectal cancer treatment: personalized immunotherapy based on molecular and immune classification system

Immunotherapy has increasingly proven to be a key treatment modality that can make a significant impact on the lives of many cancer patients. In particular, immune checkpoint inhibitors targeting the programmed cell death protein 1 (PD-1) pathway have led to remarkable clinical benefits in various cancers, including melanoma and lung cancer, and active investigations are ongoing to expand their therapeutic utility in other cancer types (1). In fact, several clinical trials have revealed a link between high somatic mutation prevalence and the clinical success in PD-1 axis blockade. Considering that colorectal cancers (CRC) are known to have very high rates of somatic mutations in comparison to other solid cancers (2), the potential of immunotherapeutic agents in treating patients with CRC is encouraging (3,4).

Prevalence of somatic mutations in risk genes including BRCA1/2 in consecutive ovarian cancer patients (AGO-TR-1 study).

5544Background: The mutation status of BRCA1/2 and other ovarian cancer (OC) predisposition genes is a valuable prognostic and predictive factor and widely used for clinical decision making. Howeve...

Genomic Profiling of Pediatric Acute Myeloid Leukemia Reveals a Changing Mutational Landscape from Disease Diagnosis to Relapse.

The genomic and clinical information used to develop and implement therapeutic approaches for acute myelogenous leukemia (AML) originated primarily from adult patients and has been generalized to patients with pediatric AML. However, age-specific molecular alterations are becoming more evident and may signify the need to age-stratify treatment regimens. The NCI/COG TARGET-AML initiative used whole exome capture sequencing (WXS) to interrogate the genomic landscape of matched trios representing specimens collected upon diagnosis, remission, and relapse from 20 cases of de novo childhood AML. One hundred forty-five somatic variants at diagnosis (median 6 mutations/patient) and 149 variants at relapse (median 6.5 mutations) were identified and verified by orthogonal methodologies. Recurrent somatic variants [in (greater than or equal to) 2 patients] were identified for 10 genes (FLT3, NRAS, PTPN11, WT1, TET2, DHX15, DHX30, KIT, ETV6, KRAS), with variable persistence at relapse. The variant allele fraction (VAF), used to measure the prevalence of somatic mutations, varied widely at diagnosis. Mutations that persisted from diagnosis to relapse had a significantly higher diagnostic VAF compared with those that resolved at relapse (median VAF 0.43 vs. 0.24, P < 0.001). Further analysis revealed that 90% of the diagnostic variants with VAF >0.4 persisted to relapse compared with 28% with VAF <0.2 (P < 0.001). This study demonstrates significant variability in the mutational profile and clonal evolution of pediatric AML from diagnosis to relapse. Furthermore, mutations with high VAF at diagnosis, representing variants shared across a leukemic clonal structure, may constrain the genomic landscape at relapse and help to define key pathways for therapeutic targeting. Cancer Res; 76(8); 2197-205. ©2016 AACR.

Genetics of Aldosterone-Producing Adenoma in Korean Patients.

ObjectivesRecently, somatic mutations in KCNJ5, ATP1A1, ATP2B3, and CACNA1D genes were found to be associated with the pathogenesis of aldosterone-producing adenoma (APA). This study aimed to investigate the prevalence of somatic mutations in KCNJ5, ATP1A1, ATP2B3, and CACNA1D and examine the correlations between these mutations and the clinical and biochemical characteristics in Korean patients with APA.MethodsWe performed targeted gene sequencing in 66 patients with APA to detect somatic mutations in these genes.ResultsSomatic KCNJ5 mutations were found in 47 (71.2%) of the 66 patients with APA (31 cases of p.G151R and 16 cases of p.L168R); these two mutations were mutually exclusive. Somatic mutations in the ATP1A1, ATP2B3, and CACNA1D genes were not observed. Somatic KCNJ5 mutations were more prevalent in female patients (66% versus 36.8%, respectively; P = 0.030). Moreover, patients with KCNJ5 mutations comprised a significantly higher proportion of patients younger than 35 years of age (19.1% versus 0%, respectively; P = 0.040). There were no significant differences in pre-operative blood pressure, plasma aldosterone, serum potassium, lateralization index, and adenoma size according to mutational status. Patients with KCNJ5 mutations were less likely to need antihypertensive medications after adrenalectomy compared with those without mutation (36.2% versus 63.2%; P = 0.045).ConclusionsThe present study demonstrated the high prevalence of somatic KCNJ5 mutations in Korean patients with APA. Carriers of somatic KCNJ5 mutations were more likely to be female. Early diagnosis and better therapeutic outcomes were associated with somatic KCNJ5 mutations in APA.

Age-Associated Acquired Mutations in Hematopoietic Cells Predominantly Affect Epigenetic Regulators TET2 and DNMT3A and Are Associated with Distinct Biological and Hematological Profiles

Abstract BACKGROUND. Recent studies using whole genome approaches have shown that acquired mutations in genes such as DNMT3A, TET2, ASXL1, JAK2 and TP53 are observed in a subset of elderly subjects without hematologic malignancies, and are associated with an increased relative risk of developing myeloid cancers, including acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS). The goal of this study is to precisely determine, using a more sensitive approach, the prevalence of somatic mutations and their allelic frequencies in elderly subjects, and assess the impact of somatic mutations on biological characteristics. METHODS. Cohort: 1101 hematologically healthy women aged 70 to 101 years old were recruited from the general population. All patients in the cohort were characterized with a i) medical questionnaire; ii) complete blood counts (CBC), and iii) X-chromosome inactivation (XCI) patterns in polymorphonuclear (PMN), using HUMARA to assess the clonality of myeloid-derived cells. Sequencing: All individuals were sequenced using a targeted approach at high coverage (95% &gt;500x) on an Ion Proton NGS instrument. Libraries were generated using an amplicon-based panel covering 19 recurrently mutated genes in myeloid malignancies (ASXL1, BRAF, CBL, CEBPA, DNMT3A, FLT3, GATA2, IDH1, IDH2, JAK2, KIT, KRAS, NPM1, NRAS, PTPN11, RUNX1, TET2, TP53 and WT1) with 237 amplicons over 22kb. Statistical analyses: Mutational status was correlated with the biological characteristics of subjects using linear regression analysis (GLM). All age-dependent variables were corrected for age (XCI skewing PMN↑, monocytes↑, hemoglobin↓, lymphocytes↓ and platelets↓). RESULTS. Mutations. We identified 249 somatic mutations in 210 of the 1101 individuals (19%) with variant allele frequencies (VAF) ranging from 3.6% to 74.8%. Mutations were found in DNMT3A (n=131), TET2 (n=100), ASXL1 (n=5), JAK2 (N=4), TP53 (n=4), CBL (n=2), IDH1 (n=1), RUNX1 (n=1) and CEBPA (n=1). TET2 and DNMT3A mutations accounted for 93% of all somatic mutations. More than one somatic mutation was observed in 32 individuals (3%), with one subject harboring 5 mutations. Correlation between mutational status and biological characteristics. The presence of mutations significantly increased with age (all genes: P =0.00024, TET2: P =0.00016, TET2 and DNMT3A (double mutations): P =0.03268), although no age effect was documented for DNMT3A mutation alone. The presence of a mutation in TET2, but not in other disease alleles correlated with increased myeloid skewing (P= 0.0008). The presence of a mutation (all genes) or a mutation in DNMT3A correlated with a decreased mean corpuscular volume (MCV) (P= 0.046). TET2 mutations were associated with decreased total white blood cell counts (P =0.0262) and PMN counts (P =0.0137). DNMT3A correlated with increased total white blood cell counts (P =0.035) and increased lymphocyte counts (P =0.0256). No mutational phenotype correlated with alterations in monocyte counts. Despite these global mutation-associated hematological phenotypes, we observed several individuals with a high VAF (&gt;30%) in DNMT3A or TET2 with completely normal CBC. CONCLUSION: Acquired mutations in the normal aging population occur at very high frequency, most commonly TET2 and DNMT3A. This suggests that mutation-driven epigenetic dysregulation is key in the development of age-associated hematological cancers. TET2 and DNMT3A mutations are associated with distinctive characteristics such as increased clonal dominance, aging and lower PMN counts for TET2; increased lymphocyte counts and decreased MCV for DNMT3A. This suggests that these two genes may have a different impact on transformation to malignant phenotype. Prospective evaluation of this cohort and sequential analysis of blood specimens will provide informative insight on the sequence of events leading to age-associated hematological cancers. Disclosures Mollica: Pfizer: Consultancy; BMS: Consultancy; Novartis: Consultancy. Busque:Novartis: Consultancy; BMS: Consultancy; PFIZER: Consultancy.

Abstract A1-30: Integrated genomic analysis identified sequential molecular aberrations in stepwise progression from early to classical hepatocellular carcinomas

Abstract Early hepatocellular carcinoma (HCC) is defined as a very well differentiated liver cancer with the presence of stromal invasion to the remaining Glisson's triad within the tumor (carcinoma in situ), which consists of small hepatocytes with little cellular atypia but with structural atypia. Different from classical HCC, early HCC is macroscopically characterized by indistinct margins and clinically detected as hypovascular lesion by imaging modalities. When such small-sized precursor lesions reach 1.5 to 2.0 cm in diameter, less differentiated cancerous tissues arise within the tumor and develop, finally replacing the very well differentiated HCC. Thus, early HCC originates from chronic liver disease and develops into classical HCC in stepwise manner. To identify sequential genetic alterations in stepwise hepatocarcinogenesis, transcriptome and whole-exome sequences were performed on 47 early and 105 classical HCCs, and molecular aberrations containing somatic mutations, gene expression, fusion events, and copy number aberrations were compared between two groups. In addition, integrating the molecular alterations from different methods, we distinguished genetic driver aberrations from by-chance changes that are not relevant to cancer genome and elucidated recurrent pathways responsible for initiation and progression in stepwise hepatocarcinogenesis. Expression profile of early HCC was different from that of classical HCC, and chromosomal aberrations, somatic mutations, and gene fusion events were significantly frequent in classical HCC. By exome sequencing, mutation of CTNNB1, CSMD3, AXIN1, COL11A1, TSC2, and ATM were often observed in both groups, while those of TP53, ARID2 and ALPK2 were significantly frequent in classical HCC. Integrated analysis showed that distinct expression profile in early HCC would define it as another molecular entity from classical HCC due to its different mutation pattern. Prevalence of somatic mutations of CTNNB1 and p53/RB pathway genes were common, while those of ARID2 and AKT/PI3K pathway genes were significantly frequent in classical HCC. Unexpectedly, WNT target genes were up-regulated only in classical HCC with CTNNB1 mutation, and even down-regulated in those with AXIN1 mutation. Similarly, cell cycle genes related with p53/RB pathway were up-regulated only in classical HCC. Taken together, despite of frequent mutations in CTNNB1 and TP53 in early HCC, additional mutational events such as SWI/SNF complexes and AKT/PI3K pathway will be required for transcriptional activation of downstream targets in HCC progression. Furthermore, TERT is the most frequently up-regulated in early HCC by various mechanisms: recurrent gene fusion including 3 prime portion of TERT such as SLC12A7-TERT (8.5%), focal gain of 5p15.33 (19.1% vs 12.3%), hepatitis B virus integration (2.1% vs 3.8%), and TERT promoter mutation (63.8% vs 59.0%). Despite of driver genes mutation, their pathways are not aberrantly activated and therefore, early HCC is a precursor in accordance with clinical characteristics different from classical HCC. Citation Format: Yutaka Midorikawa, Shogo Yamamoto, Kenji Tatsuno, Hiroki Ueda, Shingo Tsuji, Genta Nagae, Tadatoshi Takayama, Hiroyuki Aburatani. Integrated genomic analysis identified sequential molecular aberrations in stepwise progression from early to classical hepatocellular carcinomas. [abstract]. In: Proceedings of the AACR Special Conference on Translation of the Cancer Genome; Feb 7-9, 2015; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(22 Suppl 1):Abstract nr A1-30.

Abstract 4810: Exome sequencing of refractory diffuse large B-cell lymphomas highlights candidate genes for targeted resequencing

Abstract As the most common lymphoid malignancy, Diffuse Large B-Cell Lymphoma (DLBCL) has largely benefited from immunochemotherapy combinations developed in the past decade. However 30% to 40% of patients still do not respond to treatment, or relapse in a few months. The mechanisms involved in short term treatment failure are poorly understood, and biomarkers to predict refractoriness in newly diagnosed patients are still lacking. To address this issue, 14 normal / tumoral pairs of exomes from patients who progressed or relapsed within 12 months were sequenced on a HiSeq2500 platform. Somatic events were called using VarScan2, and significantly mutated pathways were highlighted by PathScan (False Discovery Rate, FDR &amp;lt; 0.001). Genes from 175 pathways were found to be more mutated than expected according to the background mutation rate observed throughout the exomes (2.68 non synonymous mutations per megabase, ranging from 0.69 to 4.66), including genes encoding histone monomers (HIST1H1/2) and transcription regulators (CREBBP, EP300, TP53), members and relatives of the NFKB signaling pathway (MYD88, TNFAIP3, NFKBIA/E), or other signaling cascades (IRF4, CIITA, SOCS1). To pinpoint genes and pathways unusually enriched in such refractory patients, a published series of 39 DLBCL genome pairs was used (Morin et al, Blood 2013). Variant annotation and filtering was reapplied to enforce target region consistency, and Fisher tests were conducted independently in the two series. 22 of the 175 pathways selected by PathScan were found to be enriched in refractory patients (FDR &amp;lt; 0.01) but not in the comparative series (FDR &amp;gt; 0.5). One of the most relevant was “IL4 mediated signaling events”, which includes genes such as STAT6, IRF4 or SOCS1. These 3 genes, along with 31 other genes frequently mutated in DLBCL, were sequenced in a series of 216 DLBCLs with an orthogonal method (PGM sequencer). Although the survival impact of IL4 related gene mutations could not be confirmed, their prevalence (17.5%) was far above what was previously described in DLBCL. Molecular profiling by Affymetrix U133+2 arrays revealed a strong association with the Primary Mediastinal B-Cell lymphoma (PMBL) subtype (Fisher p = 5e-10), a molecular subtype observed in 4/14 and 22/216 patients of the refractory and extended series respectively. We propose here an exhaustive description of mutations found in 14 exomes from refractory DLBCL patients, compared to 39 previously published genomes. The extension to 216 patients enrolled in the LNH03 LYSA (LYmphoma Study Association) clinical trial program using targeted resequencing and transcriptomic arrays allowed us to refine these results, and highlight the high prevalence of somatic mutations in IL4 signaling related genes in PMBLs. Citation Format: Sylvain Mareschal, Pierre-Julien Viailly, Philippe Bertrand, Elodie Bohers, Jean-Philippe Jais, Martin Figeac, Thierry J. Molina, Fabienne Desmots, Thierry Fest, Gilles Salles, Corinne Haioun, Hervé Tilly, Karen Leroy, Fabrice Jardin. Exome sequencing of refractory diffuse large B-cell lymphomas highlights candidate genes for targeted resequencing. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4810. doi:10.1158/1538-7445.AM2015-4810