Identification Of Somatic Mutations Research Articles

Identification of somatic mutations in cancer through Bayesian-based analysis of sequenced genome pairs

BackgroundThe field of cancer genomics has rapidly adopted next-generation sequencing (NGS) in order to study and characterize malignant tumors with unprecedented resolution. In particular for cancer, one is often trying to identify somatic mutations – changes specific to a tumor and not within an individual’s germline. However, false positive and false negative detections often result from lack of sufficient variant evidence, contamination of the biopsy by stromal tissue, sequencing errors, and the erroneous classification of germline variation as tumor-specific.ResultsWe have developed a generalized Bayesian analysis framework for matched tumor/normal samples with the purpose of identifying tumor-specific alterations such as single nucleotide mutations, small insertions/deletions, and structural variation. We describe our methodology, and discuss its application to other types of paired-tissue analysis such as the detection of loss of heterozygosity as well as allelic imbalance. We also demonstrate the high level of sensitivity and specificity in discovering simulated somatic mutations, for various combinations of a) genomic coverage and b) emulated heterogeneity.ConclusionWe present a Java-based implementation of our methods named Seurat, which is made available for free academic use. We have demonstrated and reported on the discovery of different types of somatic change by applying Seurat to an experimentally-derived cancer dataset using our methods; and have discussed considerations and practices regarding the accurate detection of somatic events in cancer genomes. Seurat is available at https://sites.google.com/site/seuratsomatic.

Diagnose und Behandlung des Retinoblastoms: aktuelle Konzepte zur sicheren Tumorkontrolle bei Erhalt des Sehvermögens

There are approximately 40 new cases of retinoblastoma in Germany per year. Children in whom the tumour is detected when still intraocular have an excellent overall survival rate (> 95%). However, the prognosis of metastasised retinoblastoma remains poor. About 40% of retinoblastoma patients have tumours in both eyes. For these children in particular it is important to save the eye and visual function as much as possible. There are several options for conservative treatment of localised retinoblastoma including laser coagulation, thermotherapy, cryotherapy, brachytherapy and chemotherapy. In recent years, systemic chemotherapy has become the established standard for primary treatment of intraocular retinoblastoma. In case series, intra-arterial, intravitreal and periocular applications of chemotherapy were also shown to be effective in treating intraocular retinoblastoma. Genetic testing is an integral part of the routine diagnostics of all patients. Mutation analysis of tumour material is invaluable for identification of somatic mutations including mutational mosaicism. Genetic testing also identifies children with heritable retinoblastoma, which represent 50% of cases. These children also have a predisposition for the development of tumours outside of the eye (second primary neoplasm). To adequately address these and other late effects in survivors of retinoblastoma, a multidisciplinary approach is needed that optimises therapy and long-term follow-up. Upcoming multicentre clinical trials will evaluate treatment concepts for localised and metastasised retinoblastoma to improve survival rates and quality of life of children with retinoblastoma. This article was translated and modified and was primarily published in Klin Padiatr 2012; 224: 339-347.

Identification of somatic mutations in human prostate cancer by RNA-Seq

RNA-Seq is a recently developed tool to characterize transcriptomes using a massively parallel sequencing technique. In spite of its broad usage in gene expression profiling, RNA-Seq can also be used to discover single nucleotide variants in transcribed regions. Here we report the result of an analysis of transcriptome sequencing data of 5 human prostate cancer tissues. A total of 116 disruptive mutations (frameshift indels or nonsynonymous nucleotide substitutions) in 92 genes are revealed with high confidence. Among these genes, several candidates are of particular interest. For example, a frameshift insertion/deletion (indel) is found in the coding region of TNFSF10, which disrupts the intact open reading frame and undermines the ability of TNFSF10 to induce apoptosis, in consequence promoting abnormal tumor progression. In summary, our findings demonstrate the use of RNA-Seq in somatic mutation screening, and provide a list of candidate genes which can be used in prostate cancer diagnosis and treatment.

Transgenic Mice Overexpressing Neuregulin-1 Model Neurofibroma-Malignant Peripheral Nerve Sheath Tumor Progression and Implicate Specific Chromosomal Copy Number Variations in Tumorigenesis

Patients with neurofibromatosis type 1 (NF1) develop benign plexiform neurofibromas that frequently progress to become malignant peripheral nerve sheath tumors (MPNSTs). A genetically engineered mouse model that accurately models plexiform neurofibroma-MPNST progression in humans would facilitate identification of somatic mutations driving this process. We previously reported that transgenic mice overexpressing the growth factor neuregulin-1 in Schwann cells (P(0)-GGFβ3 mice) develop MPNSTs. To determine whether P(0)-GGFβ3 mice accurately model human neurofibroma-MPNST progression, cohorts of these animals were monitored through death and were necropsied; 94% developed multiple neurofibromas, with 70% carrying smaller numbers of MPNSTs. Nascent MPNSTs were identified within neurofibromas, suggesting that these sarcomas arise from neurofibromas. Although neurofibromin expression was maintained, P(0)-GGFβ3 MPNSTs exhibited Ras hyperactivation, as in human NF1-associated MPNSTs. P(0)-GGFβ3 MPNSTs also exhibited abnormalities in the p16(INK4A)-cyclin D/CDK4-Rb and p19(ARF)-Mdm-p53 pathways, analogous to their human counterparts. Array comparative genomic hybridization (CGH) demonstrated reproducible chromosomal alterations in P(0)-GGFβ3 MPNST cells (including universal chromosome 11 gains) and focal gains and losses affecting 39 neoplasia-associated genes (including Pten, Tpd52, Myc, Gli1, Xiap, and Bbc3/PUMA). Array comparative genomic hybridization also identified recurrent focal copy number variations affecting genes not previously linked to neurofibroma or MPNST pathogenesis. We conclude that P(0)-GGFβ3 mice represent a robust model of neurofibroma-MPNST progression useful for identifying novel genes driving neurofibroma and MPNST pathogenesis.

SAVI: a statistical algorithm for variant frequency identification

BackgroundMany problems in biomedical research can be posed as a comparison between related samples (healthy vs. disease, subtypes of the same disease, longitudinal data representing the progression of a disease, etc). In the cases in which the distinction has a genetic or epigenetic basis, next-generation sequencing technologies have become a major tool for obtaining the difference between the samples. A commonly occurring application is the identification of somatic mutations occurring in tumor tissue samples driving a single cell to expand clonally. In this case, the progression of the disease can be traced through the trajectory of the frequency of the oncogenic alleles. Thus obtaining precise estimates of the frequency of abnormal alleles at various stages of the disease is paramount to understanding the processes driving it. Although the procedure is conceptually simple, technical difficulties arise due to inhomogeneous samples, existence of competing subclonal populations, and systematic and non-systematic errors introduced by the sequencing technologies.ResultsWe present a method, Statistical Algorithm for Variant Frequency Identification (SAVI), to estimate the frequency of alleles in a set of samples. The method employs Bayesian analysis and uses an iterative procedure to derive empirical priors. The approach allows for the comparison of allele frequencies across several samples, e.g. normal/tumor pairs and more complex experimental designs comparing multiple samples in tumor progression, as well as analyzing sequencing data from RNA sequencing experiments.ConclusionsAnalyzing sequencing data through estimating allele frequencies using empirical Bayes methods is a powerful complement to the ever-increasing throughput of the sequencing technologies.

Abstract P2-06-07: Exome sequencing identifies somatic mutations in basal-like breast cancer before and after neoadjuvant chemotherapy

Abstract Purpose: Among the five breast cancer subtypes, basal-like breast cancers (BLBCs) have drawn much attention, because they are associated with poor clinicopathological features, lack of effective treatment targets, and have poor prognosis. We designed the experiment to test the hypothesis that significant mutational evolution of BLBCs might occur during chemotherapy. Methods: We performed exome sequencing (100-fold coverage) to examine the exomes of 11 paired BLBC samples (pretreatment tumor biopsies and posttreatment tumors). The molecular subtype of BLBC was proven by gene-expression profile as well as by immunohistochemistry assay. Firstly, we sequenced the tumor genomic DNA obtained before and after neoadjuvant chemotherapy and their matched normal breast tissues. After analyzing and comparing potential somatic single nucleotide variations (SNVs) and insertions & deletions (indels), we focused on the functional mutations in certain genes or pathways. Furthermore, after validation of the mutations by Sanger sequencing, we investigated their biological functions (e.g. drug-resistance, cell proliferation, etc.). Results: Compared with pretreatment biopsies, analysis of whole-exome sequence data identified and validated TEKT4 (tektin 4) — a constitutive protein of microtubules in cilia, flagella, basal bodies and centrioles — as a significantly mutated gene in posttreatment tumors with a mutation frequency of approximately 17.6% in an independent extension cohort of 84 paired BLBC samples. TEKT4 mutations are biologically relevant, as ectopic expression of mutant TEKT4 increased paclitaxel resistance of BLBC cell lines both in vitro and in vivo. Additionally, compared with the normal breast tissues, 16 significantly mutated genes were identified and validated in the paired cancer samples, including TP53, ARID1A, BEND5, LCT, COL4A4, GAP43, COL6A6, LOC728369, STAP1, KCNK17, LZTS2, TST, IL9R, HDLBP, RASL11A and FAM22D. These genes have functions that could be broadly grouped into five biological classes: (i) MAPK signaling, (ii) PI3K/AKT signaling, (iii) cell cycle, (iv) metabolism and (v) loss of genome stability. Conclusion: Our data reveal the novel concept that significant evolution might occur during chemotherapy and mutational heterogeneity could be a property of BLBCs. In addition, the identification of somatic mutations as biomarkers for pCR (pathologic complete remission) prediction and prognosis might help us optimize choice for sequential therapy and improve patients' survival. Prospective clinical trials based on these findings might illustrate the drug-resistant mechanism and identify resistance pathways that can be exploited for therapeutic selection. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P2-06-07.

Abstract PD05-08: Genomic characterisation of invasive breast cancers with heterogeneous HER2 gene amplification

Abstract Aims: HER2 gene amplification is observed in up to 15% of breast carcinomas. In a rare subset of breast cancers classified as HER2-positive by immunohistochemistry and in situ hybridisation, HER2 overexpression and gene amplification are restricted to a subset of >30% but not all cancer cells. Here we sought to characterise the repertoire of gene copy number aberrations and somatic mutations in the HER2-positive and HER2-negative components of cases with heterogeneous HER2 overexpression and gene amplification. Material and methods: Cases diagnosed as HER2 positive but with >30% but <100% of cells displaying HER2 overexpression were retrieved from the authors' institutions. HER2 heterogeneity status was re-assessed using immunohistochemistry and chromogenic and/ or fluorescence in situ hybridisation. For cases with confirmed HER2 gene amplification heterogeneity, HER2-positive and HER2-negative components were microdissected from tissue sections stained with the Herceptest antibody. DNA samples extracted from both components of each case were subjected to microarray-based comparative genomic hybridisation (aCGH), using a 32K BAC array platform with 50Kb resolution. The HER2-positive and HER2-negative components of cases with frozen material were also subjected to massively parallel targeted exome sequencing. Results: Twelve cases yielded sufficient DNA for aCGH analysis. Tumours were preferentially ER positive (83%) and of histological grade 3 (67%). The HER2-positive and HER2-negative components of all cases shared most of the copy number aberrations. A pairwise comparison of the genomic profiles of the two components from each case revealed that in ten of the twelve cases, copy number aberrations in addition to 17q12 amplification encompassing the HER2 gene locus were restricted to one of the two components. Exome sequencing of two cases suggested that the HER2-positive and HER2-negative components from each case harboured >30 somatic mutations in common, including identical TP53 somatic mutations in both components of each case. The HER2-negative component of one of the cases displayed a somatic mutation in NRG2, an ERBB receptor ligand, and the HER2-negative component of the other case harboured a mutation in PTTG1IP, a proto-oncogene with putative oestrogen receptor elements. Conclusions: Our results demonstrate that in HER2-positive breast cancers with heterogeneous HER2 gene amplification, the HER2-positive and HER2-negative components are clonally related. The distinct genomic profiles of HER2-positive and HER2-negative components, however, suggest that, at least in some of these cases, HER2 amplification may constitute a relatively late event in tumour evolution. Exome sequencing revealed mutations restricted to the HER2-negative components of HER2-positive tumours with heterogeneous HER2 overexpression/gene amplification, which may constitute potential drivers in the absence of HER2 overexpression/gene amplification. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr PD05-08.

The Molecular and Cytokine Profile of Triple-Negative (JAK2 V617F, JAK2 exon 12, MPL negative) Myelofibrosis, a Myeloproliferative Neoplasm with Distinct Clinico-Pathologic Characteristics

AbstractAbstract 3805Myelofibrosis (MF) is a clonal Philadelphia chromosome negative myeloid neoplasm characterized by cytopenias with or without proliferative hematologic changes, extramedullary hematopoiesis and an increased propensity for transformation to acute myeloid leukemia. Identification of somatic mutations in Janus kinase 2 (JAK2) has revolutionized our understanding of the pathogenesis of some MF patients. However, there is a wide range of clinical heterogeneity in MF patients and somatic mutations of JAK2 (exon 14 or exon12) and MPL are only found in 50–60%/1–2% and 5–10% of MF patients respectively. Therefore, a large proportion of MF patients remain molecularly unaccounted after sequencing for these commonly analyzed mutations. We hypothesized that MF patients who are lack of JAK2 and MPL mutations, collectively called “triple-negative MF,” have a distinct clinicopathologic profile compared to their mutant counterparts.Our study included 76 MF patients (primary MF=48, post-essential thrombocythemic-MF=21, post-polycythemic-MF=7), with amedian age of 65 years (25–80) and predominantly intermediate 1 or 2 risk by the D-IPSS grouping (low=8, Int-1=20, Int-2=42, high=6). We compared hematologic and clinical variables between JAK2 mutant (N=40) and triple-negative MF (N=36) and found that triple-negative MF patients had a higher WBC count (14.4±23.6. vs 11±14.9) and higher platelet counts (365±434.1 vs 252±220.5) but similar hemoglobin levels, LDH levels and D-IPSS plus risk profiles. No difference in overall survival was noted (25.5 months in triple-negative vs 24 months in JAK2 positive).Newly discovered molecular mutations in myeloid malignancies have provided insight into the biology of MDS and some MPN cases. We performed direct sequencing on 9 genes including TET2 (Exon 3), DNMT3A (Exon 18/19, 20, 21, 22 and 23), IDH1/2 (Exon4), LNK (exon2–8), EZH2 (Exon 18/19, N/KRAS (Exon 1–2), CBL (Exon 8–9) and did not find any mutation in the triple-negative cohort. Recently, L3MBTL1 has been reported to be important in the pathogenesis of polycythemia vera, but sequencing of this gene in MF patients did not yield any mutations.Next generation sequencing technologies have been instrumental in the discovery of new genes important in disease pathogenesis in myeloid neoplasias. We performed whole exome sequencing in the triple-negative cases and found somatic mutations in the following genes, ODZ1 (cellular signal transducer), ZNFE391 (transcriptional regulator), DENND3 (protein-coding), ARHGEF11 (modulator of cellular process through G protein) and PIK3CD (Phosphorylate Inositol Lipids regulator involving in Immune response) in one patient and subsequently confirmed as somatic by direct sequencing. This is the first time, which these genetic mutations were reported in hematologic malignancies specifically in MF. However, these mutations did not recur when screening, a limited cohort of patients (N=20).For better understanding of this disease subtype, we compared the cytokine profile in these two groups based on the fact that, cytokines have been crucial in the pathogenesis of MF, and we found marked elevation in the levels of IL-1B (1.63 vs 0.42), IL-8 (2.89 vs 0.56), IL-17A (2.56 vs 1.12) and IFN-δ (2.07 vs 0.37) in the JAK2 positive patients compared to the triple-negative cases. Comparison of both groups showed similar responses to JAK inhibitors by spleen size shrinkage and constitutional symptoms.In conclusion, despite the discovery of JAK2 and MPL mutations, a subset of MF patients remains molecularly uncharacterized. We found that triple negative MF patients had higher leukocyte and platelet counts, but a similar prognosis. Moreover, we identified novel somatic mutations in 1 triple-negative MF patient, which has not been previously described interestingly, despite lower cytokine levels; responses to JAK-inhibitors were still noted. Whole exome sequencing and other immunogenetic studies may reveal novel genes which may elucidate the pathogenesis of these MF cases. Disclosures:No relevant conflicts of interest to declare.

Whole Exome Sequencing Analysis in AML with Normal Karyotype Not Harboring FLT3/ITD Mutation Reveals Novel Genetic Alterations.

AbstractAbstract 2593 Background:NK-AML represents genetically heterogeneous group of disease. However genetic lesions affecting treatment outcome in patients with NK-AML are relatively unknown. Methods:The discovery cohort consists of 67 NK-AML patients in complete remission (median age: 49.2, ranges: 19–70) without FLT-3 mutations. Genomic DNA was extracted from enriched AML cells at diagnosis or control specimens obtained after complete remission. Whole exomes were captured using Agilent SureSelect and sequencing were performed by HiSeq2000 with 41∼89× coverage. Bioinformatics analysis and identification of somatic mutation has been done by series of software such as BWA, Picard, GATK, VarScan 2, and custom-made scripts. All the data has been re-checked by manual inspection. Validation has been done independent set of cohort (358 NK-AML patients, median age: 51, ranges: 15–85) with Sanger sequencing on highly mutated target sites. Results:Filtering against dbSNP and COSMIC database generated 485 genes with somatic and structural variations. Among them, 41 genes were detected in more than two patients. In addition to well-known 28 mutations, 13 novel mutations with different frequencies were identified including genes responsible for structural maintenance of chromosome (SMC1A, 6.0%) and tumor suppressor function (FAT1, 6.0%). Most common type of mutation was missense mutation (70.8%), and substantial fraction of mutation was splicing site mutations (3.8%). The hematological system development and hematologic function were most highly enriched by the Ingenuity Pathway Analysis (IPA) as expected. CIRCOS plot analysis showed similar co-occurring pattern of recurrent mutations with previous reports. Hierarchical clustering analysis divided into four different groups according to the number of harboring mutations. In network analysis four distinct subgroups were observed ranging 21 to 3 gene network. Conclusion:Using whole exome sequencing approach, a catalog of recurrent mutations was successfully defined in the patients with NK-AML without FLT3/ITD mutation. This candidate list of novel mutations should be tested further for therapeutic target and prognostic marker in the patients with NK-AML. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Whole Genome Sequencing of Four CD34+-Derived iPSC Polycythemia Vera Clones From a Single Female

AbstractAbstract 1755Polycythemia vera (PV) is an acquired clonal hematopoietic disorder characterized by the JAK2V617F somatic mutation, which, however, constitutes only a variable part of the PV clone. Even within the JAK2V617F subclone, heterozygous and homozygous JAK2V617F progenitors coexist. Further, family clustering of PV suggests the existence of predisposing germline mutations. The nature of these pre-JAK2V617Fsomatic and germline mutations has not yet been identified, and the search for these predisposing genetic lesions using marrow and blood cells is hampered by their genetic heterogeneity. In order to overcome this obstacle, we generated 5 inducible pluripotent stem cell lines (iPSC) from the same PV female with different JAK2 genomic configuration (4 from CD34+ blood cells, one from marrow mesenchymal cells having germ-line DNA configuration). Genomic DNA was isolated from the cell lines, followed by whole genome sequencing (WGS) at >20X depth, and whole exome sequencing (WES) at >130X depth.We have developed a comprehensive WGS/WES pipeline for sequence alignment and variant calling. The major components are: a) Pre-processing and quality control of raw reads outputted from sequencing instruments; b) Align the read against the Human Genome Project reference using BWA (Burrows-Wheeler Aligner); c) Local realignment at indels using GATK (Genome Analysis Toolkit); d) Identify germline/somatic single nucleotide variants (SNVs), copy number variants (CNV) and structural variants (SV) using GATK, Varscan2, Breakdancer, CNVnator, and Pindel. The variant calls are intersected and merged. Calls detected by two or more algorithms are regarded as high confidence.Two somatic mutations, rs1047840, and rs3795677 were identified by WGS and WES in the CD34+ cell derived iPSCs. rs1047840 (E589K) is a SNP in EXO1 gene that encodes a protein with 5' to 3' exonuclease activity as well as an RNase H activity. EXO1 is involved in DNA mismatch repair and homologous recombination. rs1047840 conferred increased risk of breast, lung, and gastric cancer. rs3795677 (R64H) is a SNP in FMN2 gene, which is required for microtubule-independent chromatin positioning during metaphase. Interestingly, these two mutations are all common SNVs, and they are located in a duplicated region on 1q43. Whole genome analysis is ongoing to characterize and validate the complex mixture of SVs in this region, identify potential translocations and determine the exact genomic breakpoints. The variants will be validated in non-iPSC cells from the same individual to rule out iPSC artifacts.This work identifies somatic mutations in EXO1 and FMN2, and CNVs in 1q43 as the potential genetic defects involved in the early pathogenesis of PV. It also demonstrates the value of combining WGS and WES for the identification of somatic mutations. Disclosures:No relevant conflicts of interest to declare.

SF3B1 haploinsufficiency leads to formation of ring sideroblasts in myelodysplastic syndromes

AbstractWhole exome/genome sequencing has been fundamental in the identification of somatic mutations in the spliceosome machinery in myelodysplastic syndromes (MDSs) and other hematologic disorders. SF3B1, splicing factor 3b subunit 1 is mutated in 60%-80% of refractory anemia with ring sideroblasts (RARS) and RARS associated with thrombocytosis (RARS-T), 2 distinct subtypes of MDS and MDS/myeloproliferative neoplasms (MDSs/MPNs). An idiosyncratic feature of RARS/RARS-T is the presence of abnormal sideroblasts characterized by iron overload in the mitochondria, called RS. Based on the high frequency of mutations of SF3B1 in RARS/RARS-T, we investigated the consequences of SF3B1 alterations. Ultrastructurally, SF3B1 mutants showed altered iron distribution characterized by coarse iron deposits compared with wild-type RARS patients by transmission electron microscopy. SF3B1 knockdown experiments in K562 cells resulted in down-regulation of U2-type intron-splicing by RT-PCR. RNA-sequencing analysis of SF3B1 mutants showed differentially used genes relevant in MDS pathogenesis, such as ASXL1, CBL, EZH, and RUNX families. A SF3B pharmacologic inhibitor, meayamycin, induced the formation of RS in healthy BM cells. Further, BM aspirates of Sf3b1 heterozygous knockout mice showed RS by Prussian blue. In conclusion, we report the first experimental evidence of the association between SF3B1 and RS phenotype. Our data suggest that SF3B1 haploinsufficiency leads to RS formation.

21IN - Is there a Role for New Cytotoxic Agents in TNBC?

ABSTRACT Triple-negative breast cancer (TNBC), which accounts for 15% to 20% of all cases of breast cancer, is characterized by having estrogen-receptor (ER)-negative and progesterone-receptor (PR)-negative and has no overexpression of human epidermal growth factor receptor 2 (HER2). It is an aggressive subtype of breast cancer marked by higher rates of visceral and central nervous system metastases and poor disease-free survival compared with hormone receptor-positive sybtypes. In recent years, an increasing appreciation and identification of somatic mutations and other genetic aberrations that drive human malignancies have led us within reach of personalized cancer medicine. However, after a high expectation with iniparib, a non optimal PARP inhibitor, final data from a randomized phase III trial did not demonstrate this drug to improve outcomes in combination with chemotherapy in patients with advanced TNBC and bevacizumab is the only “targeted” agent which has shown to improve outcomes in combination with chemotherapy in patients with metastatic TNBC. Thus, chemotherapy continues to be the standard of care in these patients. Although taxanes and anthracyclines are still the most important compounds for patients with metastatic TNBC, the majority of them will progress and patients will need new chemotherapeutic drugs. Thus, new cytotoxic drugs are being studied in patients with TNBC in late-line. Among them, new antimicrotubule agents such as eribulin mesylate have shown to increase survival versus the treatment of physicians choice and seems to be active also in this tumor type. Other agents which seem to play a role are vinflunine and KNTR-102, a new polymeric conjugate of irinotecan, both in phase III trials. NKTR-102 has shown an impressive response rate in patients with very heavily pretreated TNBC, including taxanes, anthacyclines and capecitabine. Disclosure J. Cortes: Consultant: Roche, Novartis, Celgene Honoraria: Roche, Celgene, Novartis, Cephalon/Teva

Identification of Somatic Mutations in Parathyroid Tumors Using Whole-Exome Sequencing

The underlying molecular alterations causing sporadic parathyroid adenomas that drive primary hyperparathyroidism have not been thoroughly defined. The aim of the study was to investigate the occurrence of somatic mutations driving tumor formation and progression in sporadic parathyroid adenoma using whole-exome sequencing. Eight matched tumor-constitutional DNA pairs from patients with sporadic parathyroid adenomas underwent whole-exome capture and high-throughput sequencing. Selected genes were analyzed for mutations in an additional 185 parathyroid adenomas. Four of eight tumors displayed a frame shift deletion or nonsense mutation in MEN1, which was accompanied by loss of heterozygosity of the remaining wild-type allele. No other mutated genes were shared among the eight tumors. One tumor harbored a Y641N mutation of the histone methyltransferase EZH2 gene, previously linked to myeloid and lymphoid malignancy formation. Targeted sequencing in the additional 185 parathyroid adenomas revealed a high rate of MEN1 mutations (35%). Furthermore, this targeted sequencing identified an additional parathyroid adenoma that contained the identical, somatic EZH2 mutation that was found by exome sequencing. This study confirms the frequent role of the loss of heterozygosity of chromosome 11 and MEN1 gene alterations in sporadic parathyroid adenomas and implicates a previously unassociated methyltransferase gene, EZH2, in endocrine tumorigenesis.

La génétique des paragangliomes et des phéochromocytomes

Pheochromocytomas and paragangliomas are rare tumors that arise from chromaffin tissues of the adrenal medulla and from sympathetic and parasympathetic ganglia. Ten susceptibility genes (VHL, RET, NF1, SDHA, B, C and D, SDHAF2, TMEM127 and finally MAX) are responsible for at least 30% of the cases. These patients can either have a familial history of the disease, a syndromic presentation, but can also emerge as apparently sporadic forms. Moreover, recent transcriptomic studies have led to the identification of somatic mutations in VHL and RET genes in 15% of sporadic cases, thus bringing a molecular explanation for nearly half of all cases. These findings have had a major impact on the understanding of the molecular mechanisms of tumorigenesis in pheochromocytomas and paragangliomas. It was shown that the genetic status of tumors is associated with particular transcription signatures, such as the activation of the pseudohypoxic response or the activation of MAPK or mTOR signalling. From nowadays to a close future, these advances will have important consequences on the monitoring of patients, from genetic counseling to a personalized clinical management.

Identification of somatic mutations in non-small cell lung carcinomas using whole-exome sequencing

Lung cancer is the leading cause of cancer-related death, with non-small cell lung cancer (NSCLC) being the predominant form of the disease. Most lung cancer is caused by the accumulation of genomic alterations due to tobacco exposure. To uncover its mutational landscape, we performed whole-exome sequencing in 31 NSCLCs and their matched normal tissue samples. We identified both common and unique mutation spectra and pathway activation in lung adenocarcinomas and squamous cell carcinomas, two major histologies in NSCLC. In addition to identifying previously known lung cancer genes (TP53, KRAS, EGFR, CDKN2A and RB1), the analysis revealed many genes not previously implicated in this malignancy. Notably, a novel gene CSMD3 was identified as the second most frequently mutated gene (next to TP53) in lung cancer. We further demonstrated that loss of CSMD3 results in increased proliferation of airway epithelial cells. The study provides unprecedented insights into mutational processes, cellular pathways and gene networks associated with lung cancer. Of potential immediate clinical relevance, several highly mutated genes identified in our study are promising druggable targets in cancer therapy including ALK, CTNNA3, DCC, MLL3, PCDHIIX, PIK3C2B, PIK3CG and ROCK2.

Abstract 2351: Identification of somatic mutations in esophageal squamous cell carcinoma and corresponding xenograft by next-generation sequencing

Abstract The genomes of all cancer cells carry somatic mutations, including both passenger and driver mutations. Driver mutations, which are implicated in oncogenesis, are most likely preserved in xenografted tumor cells in immunedeficient mice. Esophageal squamous cell carcinoma (ESCC) is one of the most aggressive carcinomas in the gastrointenstinal tract. It is prevalent in the developing world and associated with substantial morbidity and mortality. Molecular mechanisms contributing to initiation and progression of ESCC are still poorly understood. Systematic identification of genetic variations and putative driver mutations may lead to understanding of pathogenesis of the disease. In this study, using next-generation sequencing we performed exome sequencing of eight paired Chinese esophageal squamous cell carcinoma and xenograft. Comparing with control genomes obtained from case-matched peripheral blood cells, we identified a wide variety of somatic variations in both primary and xenograft tumors, including TP53, RGMA, MUC4, TCEAL6, PRSS3, etc. Moreover, we found that mouse sequences accounted for a significant fraction of sequence reads from xenograft samples. Excluding these mouse sequences significantly improved the accuracy of mutation detection in xenograft samples. Overall, our findings shed a light on important somatic mutations involved in ESCC. In addition, comparison between primary and xenograft tumors may lead to identification of driver mutations that confer growth advantage. These mutations represent therapeutic opportunities for development of targeted therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2351. doi:1538-7445.AM2012-2351

Germ-line GATA2 p.THR354MET mutation in familial myelodysplastic syndrome with acquired monosomy 7 and ASXL1 mutation demonstrating rapid onset and poor survival

While most myelodysplastic syndrome/acute myeloid leukemia cases are sporadic, rare familial cases occur and provide some insight into leukemogenesis. The most clearly defined familial cases result from inherited mutations in RUNX1 or CEBPA. Recently, novel germline mutations in GATA2 have been reported. We, therefore, investigated individuals from families with one or more first-degree relatives with myelodysplastic syndrome/acute myeloid leukemia with wild-type RUNX1 and CEBPA, for GATA2 mutations. Screening for other recurrent mutations was also performed. A GATA2 p.Thr354Met mutation was observed in a pedigree in which 2 first-degree cousins developed high-risk myelodys-plastic syndrome with monosomy 7. They were also observed to have acquired identical somatic ASXL1 mutations and both died despite stem cell transplantation. These findings confirm that germline GATA2 mutations predispose to familial myelodysplastic syndrome/acute myeloid leukemia, and that monosomy 7 and ASXL1 mutations may be recurrent secondary genetic abnormalities triggering overt malignancy in these families.

The human urothelium consists of multiple clonal units, each maintained by a stem cell

Little is known about the clonal architecture of human urothelium. It is likely that urothelial stem cells reside within the basal epithelial layer, yet lineage tracing from a single stem cell as a means to show the presence of a urothelial stem cell has never been performed. Here, we identify clonally related cell areas within human bladder mucosa in order to visualize epithelial fields maintained by a single founder/stem cell. Sixteen frozen cystectomy specimens were serially sectioned. Patches of cells deficient for the mitochondrially encoded enzyme cytochrome c oxidase (CCO) were identified using dual-colour enzyme histochemistry. To show that these patches represent clonal proliferations, small CCO-proficient and -deficient areas were individually laser-capture microdissected and the entire mitochondrial genome (mtDNA) in each area was PCR amplified and sequenced to identify mtDNA mutations. Immunohistochemistry was performed for the different cell layers of the urothelium and adjacent mesenchyme. CCO-deficient patches could be observed in normal urothelium of all cystectomy specimens. The two-dimensional length of these negative patches varied from 2-3 cells (about 30 µm) to 4.7 mm. Each cell area within a CCO-deficient patch contained an identical somatic mtDNA mutation, indicating that the patch was a clonal unit. Patches contained all the mature cell differentiation stages present in the urothelium, suggesting the presence of a stem cell. Our results demonstrate that the normal mucosa of human bladder contains stem cell-derived clonal units that actively replenish the urothelium during ageing. The size of the clonal unit attributable to each stem cell was broadly distributed, suggesting replacement of one stem cell clone by another.

Comprehensive Identification of Somatic Mutations in Chronic Lymphocytic Leukemia

Massively parallel sequencing enables the sequencing of whole genomes, exomes, and transcriptomes from many tumor samples. Thus, it now is possible to comprehensively identify somatic mutations, including single base changes, deletions, insertions, and genomic rearrangements. Early results for hematopoietic tumors show great promise, but many questions remain to be answered.

Abstract 924: Identification of somatic mutations of the PPP2R1A gene in subtypes of endometrial and ovarian carcinomas

Abstract Somatic mutations in PPP2R1A have been found at low frequencies in lung, breast cancer and melanoma and most recently through whole-exome sequencing in ovarian clear cell carcinoma. We examined whole-transcriptome sequencing data from ovarian clear cell carcinomas (OCCC) and identified a cluster of mutations in exon 5 of PPP2R1A. The protein phosphatase 2, regulatory subunit A, alpha (PPP2R1A, or PR65a) gene is the scaffolding subunit of the serine-threonine protein phosphatase 2A (PP2A) holoenzyme. PP2A is involved in growth and survival cellular pathways, and has been suggested to play a role as a tumor suppressor gene in some human cancers. The PP2A holoenzyme is made up of the A scaffolding subunit (isoforms α and β), the C catalytic subunit (isoforms α and β and multiple regulatory B subunits also with many isoforms. PPP2R1A is made up of 15 HEAT domains forming a horseshoe-like multi-helical structure that interacts with both the C and B subunits of PP2A. We have examined the frequency of PPP2R1A mutations across the spectrum of both ovarian and endometrial carcinomas. DNA from endometrial and ovarian subtypes was sequenced across exons 5 and 6 of PPP2R1A using PCR based Sanger sequencing, and somatic mutation status was verified by sequencing matched normal DNA. Targeted sequencing of PPP2R1A revealed somatic missense mutations in 36.7% (18 of 49) of endometrial high-grade serous primary tumors. This high frequency of mutations has not previously been reported and suggests a strong link to the pathology of this disease subtype. Mutations were also identified at a lower frequency of 4.9% (3 of 61) in endometrial endometrioid carcinomas, and ovarian primary tumors at 11.6% (5 of 43) in endometrioid, 4% (2 of 50) in clear cell, 0% (0 of 50) in high-grade serous and 0% (0 of 12) of low-grade serous carcinomas. These nucleotide missense mutations result in the modification of amino acid residues that are highly conserved within the intrarepeat helix of the HEAT domains of PPP2R1A. Previous mutation based biochemical studies have identified these amino acid residues as important in the interaction between the regulatory B subunit and PPP2R1A. This indicates a strong functional role of PPP2R1A in stabilization of the PP2A complex, and a probable role in cellular tumorgenesis. A closer survey of mutations found within PPP2R1A and the PP2A complex, as well as functional characterization of this pathway, may pave the way to defining biologically relevant pathways and targets within endometrial carcinomas, ultimately leading to improved patient-targeted therapies and survival. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 924. doi:10.1158/1538-7445.AM2011-924