Somatic Single Nucleotide Polymorphisms Research Articles

Abstract 2704: Resolving clone-and haplotype-specific copy number variation and DNA methylation in heterogeneous tumors with nanopore sequencing

Abstract Large-scale somatic genomic copy number variations (CNV) accumulate during cancer progression, resulting in a tumor comprised of collections of cells, or clones, with distinct CNV profiles. Untangling intra-tumor heterogeneity and inferring clone- and haplotype-specific CNV profiles is important for cancer research and can help inform treatment. We present a computational workflow to infer clone- and haplotype-specific cancer CNV profiles by processing long nanopore reads obtained with high-throughput bulk sequencing of a tumor and matching normal sample. The workflow first identifies and phases heterozygous germline single nucleotide polymorphisms (SNPs) in the normal sample. Nanopore reads from the tumor sample are then haplotagged with presence/absence of the phased germline SNPs. Both the overall and the haplotype-specific read counts from the tumor are then tallied over fixed-size bins tiled across the reference genome. Finally, we use the state-of-the art HATCHet matrix factorization algorithm to process the total- and allele-specific read counts and get integer clone- and allele-specific copy number profiles, as well as the clonal cellular fractions of the tumor sample. To test the proposed approach, we have nanopore sequenced to 100x average read-depth coverage a COLO829 tumor and a COLO829BL matching normal cell line. Previous NGS-based bulk and single-cell analyses of the COLO829 cell-line have revealed it to be highly aneuploid and heterogeneous. We show that the proposed nanopore-based workflow identifies clone- and haplotype-specific cancer CNV profiles in concordance with previously published results, with regions of heterogeneity in full agreement with earlier bulk and single-cell studies. Inferred CNV profiles and their clonal fractions are further supported by observed allele-frequencies of somatic SNPs. We demonstrate the stability of the obtained results across lower tumor sample sequencing coverage levels, with CNVs remaining consistent down to 40x tumor coverage, putting the proposed approach on par with the industry standard NGS-based experiments. Notably, because of the unique ability of long nanopore reads to retain single-molecule methylation signals, we were further able to identify haplotype-specific differentially methylated regions both within the tumor sample, as well as in a tumor vs normal comparison, thus shedding light on acquisition/loss of DNA modifications during tumor growth. These results show how nanopore sequencing can be used to resolve some of the complexity that characterizes structurally aberrant heterogeneous cancer samples, while also revealing the previously inaccessible dimension of haplotype-specific tumor methylation. Oxford Nanopore Technologies products are not intended for use for health assessment or to diagnose, treat, mitigate, cure, or prevent any disease or condition. Citation Format: Sergey Aganezov, John Beaulaurier, Eoghan Harrington, Sissel Juul. Resolving clone-and haplotype-specific copy number variation and DNA methylation in heterogeneous tumors with nanopore sequencing [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2704.

Identification of the Immune Signatures for Ovarian Cancer Based on the Tumor Immune Microenvironment Genes.

Ovarian cancer (OV) is a deadly gynecological cancer. The tumor immune microenvironment (TIME) plays a pivotal role in OV development. However, the TIME of OV is not fully known. Therefore, we aimed to provide a comprehensive network of the TIME in OV. Gene expression data and clinical information from OV patients were obtained from the Cancer Genome Atlas Program (TCGA) database. Non-negative Matrix Factorization, NMFConsensus, and nearest template prediction algorithms were used to perform molecular clustering. The biological functions of differentially expressed genes (DEGs) were identified using Metascape, gene set enrichment analysis (GSEA), gene ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis. The copy number variations (CNVs), single nucleotide polymorphisms (SNPs) and tumor mutation burden were analyzed using Gistic 2.0, R package maftools, and TCGA mutations, respectively. Estimation of STromal and Immune cells in MAlignant Tumor tissues using Expression data and CIBERSORT were utilized to elucidate the TIME. Moreover, external data from the International Cancer Genome Consortium (ICGC) and ArrayExpress databases were used to validate the signature. All 361 samples from the TCGA OV dataset were classified into Immune Class and non-Immune Class with immune signatures. By comparing the two classes, we identified 740 DEGs that accumulated in immune-related, cancer-related, inflammation-related biological functions and pathways. There were significant differences in the CNVs between the Immune and non-Immune Classes. The Immune Class was further divided into immune-activated and immune-suppressed subtypes. There was no significant difference in the top 20 genes in somatic SNPs among the three groups. In addition, the immune-activated subtype had significantly increased proportions of CD4 memory resting T cells, T cells, M1 macrophages, and M2 macrophages than the other two groups. The qRT-PCR results indicated that the mRNA expression levels of RYR2, FAT3, MDN1 and RYR1 were significantly down-regulated in OV compared with normal tissues. Moreover, the signatures of the TIME were validated using ICGC cohort and the ArrayExpress cohort. Our study clustered the OV patients into an immune-activated subtype, immune-suppressed subtype, and non-Immune Class and provided potential clues for further research on the molecular mechanisms and immunotherapy strategies of OV.

Multi-omics analyses reveal MdMYB10 hypermethylation being responsible for a bud sport of apple fruit color.

Apple bud sports offer a rich resource for clonal selection of numerous elite cultivars. The accumulation of somatic mutations as plants develop may potentially impact the emergence of bud sports. Previous studies focused on somatic mutation in the essential genes associated with bud sports. However, the rate and function of genome-wide somatic mutations that accumulate when a bud sport arises remain unclear. In this study, we identified a branch from a 10-year-old tree of the apple cultivar 'Oregon SpurII' as a bud sport. The mutant branch showed reduced red coloration on fruit skin. Using this plant material, we assembled a high-quality haplotype reference genome consisting of 649.61Mb sequences with a contig N50 value of 2.04Mb. We then estimated the somatic mutation rate of the apple tree to be 4.56 × 10 -8 per base per year, and further identified 253 somatic single-nucleotide polymorphisms (SNPs), including five non-synonymous SNPs, between the original type and mutant samples. Transcriptome analyses showed that 69 differentially expressed genes between the original type and mutant fruit skin were highly correlated with anthocyanin content. DNA methylation in the promoter of five anthocyanin-associated genes was increased in the mutant compared with the original type as determined using DNA methylation profiling. Among the genetic and epigenetic factors that directly and indirectly influence anthocyanin content in the mutant apple fruit skin, the hypermethylated promoter of MdMYB10 is important. This study indicated that numerous somatic mutations accumulated at the emergence of a bud sport from a genome-wide perspective, some of which contribute to the low coloration of the bud sport.

Ultrasensitive analysis of genetic instability related to chemical exposure

Our concerns have been raised about whether prolonged exposure to ammunition-related chemicals could correlate with genomic instability predisposing to lung carcinogenesis. The group of professional soldiers engaged in routine ammunition analysis and its explosive properties testing. To assess the presence of an innate genetic profile, DNA isolated from swabs was analyzed with LungCarta and HS Lung Panels and MassARRAY Analyzer 4 mass spectrometry. The presence of new somatic single nucleotide polymorphisms (SNPs) was detected with liquid biopsy technique and circulating cell-free DNA (ccfDNA) isolation, which was further analyzed with UltraSeek Lung Panel. Both genetic profiles obtained for each individual were compared. Genetic analysis revealed that in professional soldiers with long-term exposure to ammunition-related toxic chemicals, new SNPs in genes related to lung carcinogenesis are present. The preliminary data indicate that using modern molecular techniques could be a valuable tool for monitoring the genome instability in context of occupational risk of harmful volatile toxic compounds and improving personnel safety. The analyzed group will be further enlarged, and follow-up studies will be continued.

Clonal origin in normal adults of all blood lineages and circulating hematopoietic stem cells

Characterization of human cells that sustain blood cell production lifelong has historically been inferred from phenotypically defined subsets of cells assayed in vitro, in transplanted immunodeficient mice, or in patients transplanted with genetically marked cells. These approaches have led to the concept of a persistent complex hierarchical process of differentiation divisions originating from a rare population of CD34+CD38-CD45RA-CD90+CD49f+ cells with an average self-renewal potential of >0.5 and an ability to produce some or all blood cell types for >1 year. However, the role of these "49f" cells in the unperturbed adult has remained poorly understood. To address this gap, somatic single-nucleotide polymorphisms (SNVs) have recently been exploited as lineage tracing markers to enumerate and characterize active hematopoietic clones in normal adults using a capture and recapture approach. We show here that the use of somatic transversions to identify somatically acquired variant alleles enabled their detection in bulk populations at frequencies of approximately 1 in 80,000 cells. We then applied this method to blood cells isolated from two normal adults (aged 31 and 53 years) over a 1- to 3-year period. The results revealed in both donors a continued clonal output of both T- and B-lymphoid cells as well as myeloid cells identified by the same unique transversions found to distinguish single 49f cells isolated from the same donors' initial blood samples. These findings provide the first evidence of a continuing hematopoietic stem cell-derived source of all mature blood cell types in normal (unperturbed) adult humans.

Sequence conservation of mitochondrial (mt)DNA during expansion of clonal mammary epithelial populations suggests a common mtDNA template in CzechII mice.

One major foundation of cancer etiology is the process of clonal expansion. The mechanisms underlying the complex process of a single cell leading to a clonal dominant tumor, are poorly understood. Our study aims to analyze mitochondrial DNA (mtDNA) for somatic single nucleotide polymorphisms (SNPs) variants, to determine if they are conserved throughout clonal expansion in mammary tissues and tumors. To test this hypothesis, we took advantage of a mouse mammary tumor virus (MMTV)-infected mouse model (CzechII). CzechII mouse mtDNA was extracted, from snap-frozen normal, hyperplastic, and tumor mammary epithelial outgrowth fragments. Next generation deep sequencing was used to determine if mtDNA “de novo” SNP variants are conserved during serial transplantation of both normal and neoplastic mammary clones. Our results support the conclusion that mtDNA “de novo” SNP variants are selected for and maintained during serial passaging of clonal phenotypically heterogeneous normal cellular populations; neoplastic cellular populations; metastatic clonal cellular populations and in individual tumor transplants, grown from the original metastatic tumor. In one case, a mammary tumor arising from a single cell, within a clonal hyperplastic outgrowth, contained only mtDNA copies, harboring a deleterious “de novo” SNP variant, suggesting that only one mtDNA template may act as a template for all mtDNA copies regardless of cell phenotype. This process has been attributed to “heteroplasmic-shifting”. A process that is thought to result from selective pressure and may be responsible for pathogenic mutated mtDNA copies becoming homogeneous in clonal dominant oncogenic tissues.

Diverging Clonal Evolution during Sequential Therapy with Chemoimmunotherapy Followed By BTK Inhibitors

Background: Treatment confers an evolutionary bottleneck for cancer. Little data exists on the genomic evolution of CLL under different treatment pressure and disease compartments. Methods: We identified 13 patients uniformly treated with chemoimmunotherapy (CIT) as first-line therapy and a BTK inhibitor (BTKi) as second-line. We collected peripheral blood (PB) samples before CIT, at relapse after CIT (which was also before starting a BTKi), and 6 months on a BTKi. Five patients on a long-term BTKi were additionally tested at two years on BTKi. Two patients had samples from bone marrow (BM) and/or lymph node (LN) compartments at 6 months on a BTKi. In total, 48 tumor samples paired with saliva serving as a germline control were tested with whole exome sequencing (WES). We used high confidence variants identified by at least two variant callers for somatic single nucleotide polymorphisms and insertion-deletion variants, and TitanCNA for copy number variations. Clonal composition was reconstructed using PhyloWGS. Results: The median age of the cohort was 60 years at the start of CIT. The median duration of follow-up was 7 years. Median time between the start of CIT and a BTKi was 43 months (range 10-135). To date, eleven patients are alive and remain in response to a BTKi. Two patients died, one due to hepatitis B virus reactivation, and one with Richter's transformation. The median tumor mutational burden (TMB), defined as the number of nonsynonymous coding mutations per Mb, was 1.9 before CIT, 2.1 after CIT, and 1.9 after 6 months on a BTKi, consistent with previous reports in CLL. TMB was loosely associated with time since CIT exposure (R2=0.12, P=0.014). All patients had at least one mutation in known driver genes, most commonly NOTCH1 and SF3B1 (31% each; Figure A). Most of these mutations were subclonal and remained subclonal throughout the treatment course. The number of known driver gene mutations per patient remained relatively stable during the treatment course (median: 2 before CIT, 3 after CIT, 3 at 6 months on a BTKi; all P>0.05). We recomposed clonal structures and estimated the cancer cell fraction (CCF) of each clone. Phylogenetic analysis showed branching evolution in most patients with a median of three child clones originating from a parent clone. In these patients, multiple clonal branches and its descendants were simultaneously being selected during therapy. In addition, we observed linear evolution in three (23%) patients, characterized by a repetitive selection of one parent clone and its leading progeny per generation. Treatment with CIT and a BTKi selected for distinct sets of clones. BTKi therapy effectively downsized clones which grew to dominance after CIT, except in one patient whose subclone with concurrent NRAS and TP53 mutations remained dominant throughout CIT and BTKi therapy. The median CCF decrease of CIT-selected clones was 18% during BTKi therapy (range 1-52). In all patients with SF3B1 mutation, clonal fractions of SF3B1 mutated clones were higher at relapse after CIT and lower during BTKi therapy. There was no consistent pattern of clonal selection for NOTCH1 mutation. We compared patterns of clonal evolution in different compartments in two patients who had PB, LN and/or BM samples available. Each compartment had a distinct clonal composition. Notably, one patient who progressed with Richter's transformation after 6 months on a BTKi showed three main clones shared among all compartments and a set of compartment-restricted clones (Figure B). The LN was preferentially enriched with clones carrying HIST1H1E mutation and complex copy number changes, reflecting transformed clones. The NOTCH1 mutated clone and its progeny, reflecting CLL clones, were dominant in PB and BM, but not in LN. Conclusion: The number and clonality of somatic mutations affecting known driver genes, remained relatively stable over the course of treatment with CIT followed by a BTKi at relapse. Distinct sets of clones evolved under each line of therapy. Clonal composition is shaped by the growth potential of individual clones, the selective pressure of the therapy used, and the tumor microenvironment. Figure Disclosures Wiestner: Pharmayclics: Research Funding; Acerta: Research Funding; Nurix: Research Funding; Merck: Research Funding.

Clonal Origin in Normal Adults of All Blood Lineages and Circulating Hematopoietic Stem Cells

Characterization of human cells that sustain blood cell production lifelong has historically been inferred from phenotypically defined subsets of cells assayed in vitro, in transplanted immunodeficient mice, or in patients transplanted with genetically marked cells. These approaches have led to the concept of a persistent complex hierarchical process of differentiation divisions originating from a rare population of CD34+CD38-CD45RA-CD90+CD49f+ cells with an average self-renewal potential of >0.5, and an ability to produce some or all blood cell types for >1 year. However, the role of these “49f” cells in the unperturbed adult has remained poorly understood. To address this gap, somatic single nucleotide polymorphisms (SNPs) have recently been exploited as lineage tracing markers to enumerate and characterize active hematopoietic clones in normal adults using a capture and recapture approach. We show here that the use of somatic transversions to identify somatically acquired variant alleles enabled their detection in bulk populations at frequencies of approximately 1 in 80,000 cells. We then applied this method to blood cells isolated from 2 normal adults over a 1 to 3 year period. The results revealed in both donors a continued clonal output of both T- and B-lymphoid cells as well as myeloid cells identified by the same unique transversions found to distinguish single 49f cells isolated from the same donors’ initial blood samples. These findings provide the first evidence of a continuing hematopoietic stem cell-derived source of all mature blood cell types in unperturbed normal adult humans.

Tumor Mutational Burden and Other Immunotherapy Markers in Histiocytic Neoplasms Using Next Generation Sequencing

IntroductionHistiocytic neoplasms are rare hematological malignancies that have protean clinical manifestations and can pose significant management challenges. Recently, vemurafenib was approved by the US-FDA for treatment of BRAF-V600-mutant Erdheim-Chester disease (ECD). However, there is a lack of FDA-approved therapies for other histiocytic neoplasms such as Langerhans cell histiocytosis (LCH) and Rosai-Dorfman disease (RDD). Over the last 5 years, immune checkpoint inhibitors such as programmed death-1 (PD-1) and programmed death-ligand 1 (PD-L1) inhibitors have shown significant improvement in outcomes among patients with several hematological and solid organ malignancies. In order to identify appropriate treatment candidates for these therapies, predictive biomarkers have been developed in various cancers. Evidence from solid tumors has suggested a favorable response to checkpoint inhibitor therapy with higher tumor mutational burden (TMB), defined as the number of mutations within a tumor genome. Next generation sequencing (NGS) of various tumors has shown an association between TMB of < 5 mutations/megabase (mut/Mb) to be associated with an absence of benefit from checkpoint inhibitors. In addition, high levels of PD-1/PD-L1 expression and microsatellite instability (MSI) are also correlated with response to therapy. The latter may be a result of somatic or germline alterations in DNA mismatch repair genes. In this study, we report the results for these biomarkers using NGS in patients with histiocytic neoplasms.MethodsWe utilized TempusTM NGS platform to analyze the tissue specimen of patients with histiocytic neoplasm. The Tempus xO Assay (Tempus Labs; Chicago, IL) combines a 1,711 gene targeted somatic and germline DNA sequencing panel with RNA-sequencing to detect both germline and somatic single nucleotide polymorphisms, indels, copy number variants, and gene rearrangements causing chimeric mRNA transcript expression in a wide array of solid tumor types. The assay utilizes formalin-fixed paraffin-embedded tumor samples and matched blood samples. TMB was calculated and reported as somatic mutations in tumor tissue per million base-pairs or mut/Mb. RNA sequencing was utilized to assess for PD-L1 and PD-1 gene expressions as compared to matched tumor and normal reference sets. Both PD-L1 and PD-1 gene expressions were reported as percentiles. DNA mismatch repair status was predicted by analysis of alterations in five common mismatch repair genes in somatic and germline DNA (MSH2, MSH6, MLH1, PMS2, and EPCAM). If there were no alterations identified in these genes, the mismatch repair status was predicted as microsatellite stable (MSS).ResultsA total of 13 patients with histiocytic neoplasms were included in the study. The distribution of individual histiocytic neoplasms was as follows: RDD (n=9), ECD (n=3), and LCH (n=1). The median TMB for RDD and ECD patients was 0.17 mut/Mb. For the one patient with LCH, the TMB level was 0.51 mut/Mb. Individual TMB levels are shown in figure 1. PD-L1 and PD-1 expression levels are depicted in table 1. Compared to normal reference sets, the PD-L1 expression was elevated in one patient each with RDD and ECD, and PD-1 expression was elevated in two patients each with RDD and ECD. For both ECD patients with higher PD-1 expression, NGS also showed presence of BRAF-V600E in the tumor tissue. The LCH patient had a low level of PD-L1 and PD-1 expression. For patients where evaluation of DNA mismatch repair was feasible on the tissue specimen (n=4), none showed related somatic or germline alterations.ConclusionsIn our series, the histiocytic neoplasms RDD, ECD, and LCH demonstrated low levels of TMB. None of the RDD and ECD patients were found to have alterations in DNA mismatch repair genes. Other markers of immunotherapy such as PD-L1/PD-1 expression appeared to be higher in ECD patients with BRAF-V600E, but in only a small subset of RDD patients. The low TMB seen in our study suggests that these histiocytic neoplasms may be less likely to respond to immune checkpoint inhibitors such as anti-PD-1 and anti-PD-L1 agents as compared to the tumors with high TMB. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Abstract 3104: Establishment of an oral tongue squamous cell carcinoma cell line from a never-smoking patient

Abstract (Objective) The rising incidence of oral tongue squamous cell carcinoma (OTSCC) in patients who have never smoked and the paucity of knowledge of its biological behavior prompted us to develop a new cell line originating from a never-smoker. (Materials and methods) Fresh tumor tissue of keratinizing OTSCC was collected from a 44-year-old woman who had never smoked. Serum-free media with a low calcium concentration were used in cell culture, and a multifaceted approach was taken to verify and characterize the cell line, designated UCSF-OT-1109.(Results) UCSF-OT-1109 was authenticated by STR DNA fingerprint analysis, presence of an epithelial marker EpCAM, absence of human papilloma virus (HPV) DNA, and SCC-specific microscopic appearance. Sphere-forming assays and mouse xenografts supported its tumorigenic potential. Spectral karyotype (SKY) analysis revealed numerical and structural chromosomal abnormalities. Whole-exome sequencing (WES) identified 46 non-synonymous and 13 synonymous somatic single-nucleotide polymorphisms (SNPs) and one frameshift deletion in the coding regions. Specifically, mutations of CDKN2A, TP53, SPTBN5, NOTCH2, and FAM136A were found in the databases. Copy number aberration (CNA) analysis revealed that the cell line loses chromosome 3p and 9p, but lacks amplification of 3q and 11q (as does HPV-negative, smoking-unrelated OTSCC). It also exhibits four distinctive focal amplifications in chromosome 19p, containing 131 genes without SNPs. Particularly, 52 genes showed &amp;gt;3- to 4-fold amplification and could be potential oncogenic drivers.(Conclusion) We have successfully established a novel OTSCC cell line from a never-smoking patient. UCSF-OT-1109 is potentially a robust experimental model of OTSCC in never-smokers. Citation Format: Steven J. Wang, Saurabh Asthana, Annemieke van Zante, Adam B. Olshen, Frank McCormick, Osamu Tetsu. Establishment of an oral tongue squamous cell carcinoma cell line from a never-smoking patient [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 3104.

A novel germline ARMC5 mutation in a patient with bilateral macronodular adrenal hyperplasia: a case report

BackgroundBilateral macronodular adrenal hyperplasia (BMAH) is a rare cause of Cushing’s syndrome (CS). BMAH is predominantly believed to be caused by two mutations, a germline and somatic one, respectively, as described in the two-hit hypothesis. In many familial cases of BMAH, mutations in armadillo repeat containing 5 (ARMC5), a putative tumor suppressor gene, are thought to induce the disorder. The objective of this study was to report a case in which the patient presented with BMAH induced by a novel heterozygous germline ARMC5 mutation (c. 517C > T, p. Arg173*) alone rather than a two-hit mutation.Case presentationA 51-year-old woman was identified with masses in the bilateral adrenals. Serum cortisol levels were increased significantly both in the morning (08:00 AM) and late at night (24:00 AM), while plasma adrenocorticotropic hormone was normal. The patient underwent a left adrenalectomy and histopathology substantiated the BMAH diagnosis. WES of the germline DNA discovered a novel heterozygous germline ARMC5 mutation (c. 517C > T, p. Arg173*) and in silico analysis predicted that the mutation significantly impaired protein function, resulting in inactivated ARMC5. Subsequently, WES of the tumor specimen identified 79 somatic single nucleotide polymorphisms (SNPs)/insertion-deletion (indel) mutations, including 32 missense/nonsense/splice/stop-loss mutations. None of these mutations were CS-related.ConclusionsA novel germline ARMC5 mutation (c. 517C > T, p. Arg173*) was identified that induced BMAH alone without a second mutation. ARMC5 sequencing may improve the identification of clinical forms of BMAH and allow earlier diagnosis of this disease.

Establishment and characterization of an oral tongue squamous cell carcinoma cell line from a never-smoking patient

ObjectiveThe rising incidence of oral tongue squamous cell carcinoma (OTSCC) in patients who have never smoked and the paucity of knowledge of its biological behavior prompted us to develop a new cell line originating from a never-smoker. Materials and methodsFresh tumor tissue of keratinizing OTSCC was collected from a 44-year-old woman who had never smoked. Serum-free media with a low calcium concentration were used in cell culture, and a multifaceted approach was taken to verify and characterize the cell line, designated UCSF-OT-1109. ResultsUCSF-OT-1109 was authenticated by STR DNA fingerprint analysis, presence of an epithelial marker EpCAM, absence of human papilloma virus (HPV) DNA, and SCC-specific microscopic appearance. Sphere-forming assays supported its tumorigenic potential. Spectral karyotype (SKY) analysis revealed numerical and structural chromosomal abnormalities. Whole-exome sequencing (WES) identified 46 non-synonymous and 13 synonymous somatic single-nucleotide polymorphisms (SNPs) and one frameshift deletion in the coding regions. Specifically, mutations of CDKN2A, TP53, SPTBN5, NOTCH2, and FAM136A were found in the databases. Copy number aberration (CNA) analysis revealed that the cell line loses chromosome 3p and 9p, but lacks amplification of 3q and 11q (as does HPV-negative, smoking-unrelated OTSCC). It also exhibits four distinctive focal amplifications in chromosome 19p, containing 131 genes without SNPs. Particularly, 52 genes showed >3- to 4-fold amplification and could be potential oncogenic drivers. ConclusionWe have successfully established a novel OTSCC cell line from a never-smoking patient. UCSF-OT-1109 is potentially a robust experimental model of OTSCC in never-smokers.

Molecular genetic investigative leads to differentiate monozygotic twins.

Molecular genetic investigative leads to differentiate monozygotic twins.

Abstract 5102: Identifying targets for melanoma therapy: Biological and clinical implications of BRAF L597 mutants

Abstract Recurrent driver mutations in BRAF (V600), NRAS (G12/13, Q61), KIT (W557, V559, L576, K642, D816), GNAQ (Q209) and GNA11 (Q209) have previously been identified in malignant melanomas, but over one-third of cases have no known potentially actionable targets. To identify novel drivers, we performed whole-genome sequencing of a tumor-normal pair from a patient with an aggressive, chemotherapy-naïve metastatic melanoma which was pan-negative for the mutations listed above. Our data analysis pipeline revealed the landscape of somatic single nucleotide polymorphisms (SNPs), insertions and deletions, copy number variations, and structural variants. A subset of the nonsynonymous SNPs was validated by direct sequencing, resulting in the identification of a somatic BRAF L597R mutation. Surrogate kinase assays suggest that this mutant is sensitive to both BRAF and MEK inhibitors. Subsequent mutational analyses showed that BRAF L597 mutations were in 2 of 34 (6%) ‘pan-negative’ tumors. Collectively, these data demonstrate the frequency and potential therapeutic implications of BRAF L597 mutations in melanoma. 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 5102. doi:1538-7445.AM2012-5102

Comprehensive examination of somatic oncogene mutation in normal and pathologic thyroid tissues

It is established that numerous somatic oncogene mutation (BRAF, NRAS, HRAS, KRAS) and gene translocations (RET/PTC, PAX8/PPAR-gamma) are associated with the development of thyroid cancer. In this study 22 intraoperative thyroid tissue samples (11 pathologic and 11 normal) were examined. Somatic single nucleotide polymorphisms were analyzed by LigthCycler melting method, while translocations were identified by real-time polymerase chain reaction technique. In tumorous sample 3 BRAF, 2 NRAS and one HRAS mutations were found, as well as one RET/PTC1 translocation. Results confirm international data showing that these oncogene mutations and translocations are linked to thyroid cancer. Cytological examination completed with genetic data may support the diagnosis of thyroid malignancies. In addition, genetic alterations may indicate malignant transformation and may become prognostic factors in future. Orv. Hetil., 2011, 152, 672–677.