Matched Tumor DNA Research Articles

Multiplex Gene Profiling of Cell-Free DNA in Patients With Metastatic Melanoma for Monitoring Disease.

Hotspot blood cell-free DNA (cfDNA) biomarker assays have limited utility in profiling tumor heterogeneity and burden and in capturing regional metastasis with low disease burden in patients with melanoma. We investigated the utility of a sensitive 54-cancer gene digital next-generation sequencing approach targeting blood cfDNA single nucleotide variants (SNVs) and copy number amplification for monitoring disease in patients with melanoma with regional or distant organ metastasis (DOM). A total of 142 blood samples were evaluated by digital next-generation sequencing across two patient cohorts. Cohort 1 contained 44 patients with stage II, III, or IV disease with matched tumor DNA at the time of surgery or DOM. Cohort 2 consisted of 12 overlapping patients who were longitudinally monitored after complete lymph node dissection to DOM. In cohort 1, cfDNA SNVs were detected in 75% of patients. Tumor-cfDNA somatic SNV concordance was 85% at a variant allele fraction of ≥ 0.5%. An SNV load (number of unique SNVs detected) of greater than two SNVs and an SNV burden (total cumulative SNV VAF) of > 0.5% were significantly associated with worse overall survival (P < .05) in stage IV patients. In cohort 2, 98 longitudinal blood samples along with matched regional and distant metastases from 12 stage III patients were analyzed before complete lymph node dissection and throughout disease progression. cfDNA SNV levels correlated with tumor burden (P = .019), enabled earlier detection of recurrence compared with radiologic imaging (P < .01), captured tumor heterogeneity, and identified increasing SNVs levels before recurrence. This study demonstrates significant utility for cfDNA profiling in patients with melanoma with regional and/or distant metastasis for earlier detection of recurrence and progression and in capturing tumor evolution and heterogeneity, thus impacting how patients with melanoma are monitored.

Systematic pan-cancer analysis of somatic allele frequency

Imbalanced expression of somatic alleles in cancer can suggest functional and selective features, and can therefore indicate possible driving potential of the underlying genetic variants. To explore the correlation between allele frequency of somatic variants and total gene expression of their harboring gene, we used the unique data set of matched tumor and normal RNA and DNA sequencing data of 5523 distinct single nucleotide variants in 381 individuals across 10 cancer types obtained from The Cancer Genome Atlas (TCGA). We analyzed the allele frequency in the context of the variant and gene functional features and linked it with changes in the total gene expression. We documented higher allele frequency of somatic variants in cancer-implicated genes (Cancer Gene Census, CGC). Furthermore, somatic alleles bearing premature terminating variants (PTVs), when positioned in CGC genes, appeared to be less frequently degraded via nonsense-mediated mRNA decay, indicating possible favoring of truncated proteins by the tumor transcriptome. Among the genes with multiple PTVs with high allele frequency, ARID1, TP53 and NSD1 were known key cancer genes. All together, our analyses suggest that high allele frequency of tumor somatic variants can indicate driving functionality and can serve to identify potential cancer-implicated genes.

Targeted next-generation sequencing for differential diagnosis of neurofibromatosis type 2, schwannomatosis, and meningiomatosis.

Clinical overlap between neurofibromatosis type 2 (NF2), schwannomatosis, and meningiomatosis can make clinical diagnosis difficult. Hence, molecular investigation of germline and tumor tissues may improve the diagnosis. We present the targeted next-generation sequencing (NGS) of NF2, SMARCB1, LZTR1, SMARCE1, and SUFU tumor suppressor genes, using an amplicon-based approach. We analyzed blood DNA from a cohort of 196 patients, including patients with NF2 (N = 79), schwannomatosis (N = 40), meningiomatosis (N = 12), and no clearly established diagnosis (N = 65). Matched tumor DNA was analyzed when available. Forty-seven NF2-/SMARCB1-negative schwannomatosis patients and 27 NF2-negative meningiomatosis patients were also evaluated. A NF2 variant was found in 41/79 (52%) NF2 patients. SMARCB1 or LZTR1 variants were identified in 5/40 (12.5%) and 13/40 (∼32%) patients in the schwannomatosis cohort. Potentially pathogenic variants were found in 12/65 (18.5%) patients with no clearly established diagnosis. A LZTR1 variant was identified in 16/47 (34%) NF2/SMARCB1-negative schwannomatosis patients. A SMARCE1 variant was found in 3/39 (∼8%) meningiomatosis patients. No SUFU variant was found in the cohort. NGS was an effective and sensitive method to detect mutant alleles in blood or tumor DNA of mosaic NF2 patients. Interestingly, we identified a 4-hit mechanism resulting in the complete NF2 loss-of-function combined with SMARCB1 and LZTR1 haploinsufficiency in two-thirds of tumors from NF2 patients. Simultaneous investigation of NF2, SMARCB1, LZTR1, and SMARCE1 is a key element in the differential diagnosis of NF2, schwannomatosis, and meningiomatosis. The targeted NGS strategy is suitable for the identification of NF2 mosaicism in blood and for the investigation of tumors from these patients.

Non-invasive detection of somatic mutations using next-generation sequencing in primary central nervous system lymphoma.

PurposePrimary central nervous system lymphomas (PCNSL) have recurrent genomic alterations. The main objective of our study was to demonstrate that targeted sequencing of circulating cell-free DNA (cfDNA) released by PCNSL at the time of diagnosis could identify somatic mutations by next-generation sequencing (NGS).Patients and MethodsPlasmacfDNA and matched tumor DNA (tDNA) from 25 PCNSL patients were sequenced using an Ion Torrent Personal Genome Machine (Life Technologies®). First, patient-specific targeted sequencing of identified somatic mutations in tDNA was performed. Then, a second sequencing targeting MYD88 c.T778C was performed and compared to plasma samples from 25 age-matched control patients suffering from other types of cancer.ResultsAccording to the patient-specific targeted sequencing, eight patients (32% [95% CI 15-54%]) had detectable somatic mutations in cfDNA. Considering MYD88 sequencing, six patients had the specific c.T778C alteration detected in plasma. Using a control group, the sensitivity was 24% [9-45%] and the specificity was 100%. Tumor volume or deep brain structure involvement did not influence the detection of somatic mutations in plasma.ConclusionThis pilot study provided evidence that somatic mutations can be detected by NGS in the cfDNA of a subset of patients suffering from PCNSL.

Prevalence of incidental germline pathogenic (PV) and likely pathogenic (LPV) variants in hereditary cancer-related genes identified in matched tumor/normal sequencing of advanced solid tumors.

1524 Background: Next-generation sequencing (NGS) for tumor molecular profiling can reveal germline incidental mutations in hereditary cancer-related genes. The American College of Medical Genetics and Genomics (ACMG) has recommended that laboratories performing clinical sequencing seek and report PV and LPV in 56 genes. We assessed the prevalence of incidental germline LPV and PV in other cancer-related genes among patients undergoing hybrid capture sequencing of 201 cancer-related genes. Methods: Matched tumor and germline DNA NGS of a targeted panel of 201 genes was performed in 1000 patients (pts) with advanced or metastatic solid tumors enrolled in a molecular testing protocol (NCT01772771) in a research laboratory. We previously reported germline alterations in the putative most actionable genes as designated by ACMG (PMID: 26787237). We assessed the germline LPV and PV in 54 additional cancer-related genes. Results: Among the 1000 patients who underwent tumor and normal DNA sequencing, 37 patients (3.7%) were found to have a germline PV or LPV in the following genes: ATM (4); BAP1 (1); CDH1 (1); CDKN2A (1); CHEK1 (2); CHEK2 (10); EGFR (1); ERCC3 (4); ERCC5 (1); HNF1B (1); HRAS (1); MLL3 (1); NF1 (3); PKHD1 (4); PTCH1 (1) and SMARCA4 (1). Eight pts (22%) had previous genetic counseling and testing for various reasons, but only 3 pts (8%) had previously identified alterations (all with NF1 mutations). After discussion in our return of germline results board, it was decided to return the findings in established hereditary cancer predisposition genes with high penetrance: BAP1 (p.Y401X), CDH1 (p.C688X), CDKN2A (p.G101W), EGFR (p.T790M) and SMARCA4 (p.S332FfsX55) after validation in a CLIA laboratory. Conclusions: Return of the previously unrecognized germline LPV or PV in patients with advanced or metastatic cancers who undergo somatic profiling is of great interest. The exact genes for which the germline results should be returned is controversial. Broader genomic testing is likely to identify additional incidental germline alterations with potential clinical utility to patients and their relatives.

Circulating Tumor DNA Detection in Early-Stage Non-Small Cell Lung Cancer Patients by Targeted Sequencing.

Circulating tumor DNA (ctDNA) isolated from peripheral blood has recently been shown to be an alternative source to detect gene mutations in primary tumors; however, most previous studies have focused on advanced stage cancers, and few have evaluated ctDNA detection in early-stage lung cancer. In the present study, blood and tumor samples were collected prospectively from 58 early-stage non-small lung cancer (NSCLC) patients (stages IA, IB, and IIA) and a targeted sequencing approach was used to detect somatic driver mutations in matched tumor DNA (tDNA) and plasma ctDNA. We identified frequent driver mutations in plasma ctDNA and tDNA in EGFR, KRAS, PIK3CA, and TP53, and less frequent mutations in other genes, with an overall study concordance of 50.4% and sensitivity and specificity of 53.8% and 47.3%, respectively. Cell-free (cfDNA) concentrations were found to be significantly associated with some clinical features, including tumor stage and subtype. Importantly, the presence of cfDNA had a higher positive predictive value than that of currently used protein tumor biomarkers. This study demonstrates the feasibility of identifying plasma ctDNA mutations in the earliest stage lung cancer patients via targeted sequencing, demonstrating a potential utility of targeted sequencing of ctDNA in the clinical management of NSCLC.

Noninvasive detection of tumor-associated mutations from circulating cell-free DNA in hepatocellular carcinoma patients by targeted deep sequencing.

BackgroundDetection of circulating cell-free DNA (cfDNA) has potential clinical value for assessing tumor biology in patients with hepatocellular carcinoma (HCC), yet many traditional assays lack robustness. This study was the first to apply a high-throughput sequencing platform to detect tumor-associated mutations in HCC from circulating tumor-derived DNA (ctDNA) and to evaluate the utility and feasibility of this approach.MethodsUsing the MiSeq™ system, plasma and matched tumor DNA samples were analyzed for hotspot mutations in the TERT, CTNNB1, and TP53 genes that had been verified as the most prevalent mutations in HCC. We compared tumor and plasma data and prospectively investigated the association between significant mutations detected in ctDNA and the patients' clinical outcomes.ResultsIn 41 patients, we detected tumor-associated mutations for HCC in 8 (19.5%) plasma samples. Among them, one showed a tumor-associated mutation in ctDNA but not in the tumor tissue which we used to detect. We also found that ctDNA with mutations could be detected more easily in patients who suffered vascular invasion (P=0.041) and predicted a shorter recurrence-free survival time (P<0.001). There was no relationship between detectable mutations and concentration of cfDNA (P=0.818).ConclusionsThe results of our study suggest that tumor-associated mutations detected in plasma are associated with vascular invasion and might be used to predict a shorter recurrence-free survival time for HCC patients. This kind of biomarker can overcome the limitations of tumor heterogeneity. Moreover, the diagnostic performance is improved if multiple mutations in different genes are combined.

M3P Sequencing Panel Identifies TP53 Mutational Status As a Prognostic Factor in Chemotherapy-Naive Multiple Myeloma

▪IntroductionMultiple myeloma (MM) is characterized by a highly variable disease course, which can be traced to initiating and acquired genomic events. Whole exome analysis of matched tumor and germline DNA from 287 MM patients identified recurrently somatically mutated genes (RSMGs) (Lohr et al. - Cancer Cell 2014, Bolli et al. - Nat Commun 2014). Despite the fact that these RSMGs affect pathways that are biologically important in MM, the clinical relevance of many of these genes in the context of conventional prognostic markers remains to be elucidated.AimsThe aims of this pilot study were: (1) To validate the prevalence of RSMGs in our newly diagnosed MM patient cohort; (2) To assess the correlation between RSMGs, clinical parameters and outcome; (3) To thereby identify the potential clinical usefulness of introducing RSMG mutational profiling in larger MM trial cohorts.Material and MethodsCD138+ enriched MM cells and peripheral blood were obtained with informed consent from chemotherapy-naive patients, participating in 3 clinical trials: HOVON-65/GMMG-HD4, HOVON-87/NMSG-18 and Carthadex (EudraCT number 2004-000944-26, 2007-004007-34 and 2009-014922-40, respectively).Matched tumor and germline DNA were sequenced on an Ion Torrent sequencing platform (PGM, Life Technologies), using the M3 P Mutational Panel v3.0, comprising 1327 customized oligos (Life Technologies), targeted at the coding sequences of 88 MM-relevant genes, including the RSMGs. Somatic mutations were considered positive when present in >=10% of tumor reads and <=10% germline reads, with a minimal coverage of 10x and being non-synonymous, or splice donor variants.All statistical analyses were performed in SPSS version 23, using the log-rank and Mann-Whitney U-test, with the Bonferroni test to correct for multiple comparisons.ResultsA total of 206 DNA samples were sequenced from 103 patients (HOVON-65/GMMG-HD4 (n=16), HOVON-87/NMSG-18 (n=67), Carthadex (n=20)) with an average coverage of 574x in tumor DNA, 451x in germline DNA and an overall coverage of 98%. We collected follow-up data from 102/103 patients, with a median follow-up time of 30 months.168 somatic mutations were detected in 44/88 genes. 82% of patients had at least 1 somatic mutation. Genes most frequently mutated were: (1) NRAS (26%), (2) KRAS (22%), (3) DIS3 (14%), (4) FAM46C (9%), (5) TP53 (7%) and (6) BRAF (6%) (Figure 1). Of note, NRAS and KRAS mutations were mutually exclusive in our cohort. Moreover, all TP53 mutations were located in its DNA binding domain. Three out of 6 BRAF mutations were predicted to cause a V600E amino acid change.We focused on these 6 RSMGs in all further analyses. Correlating mutational status with Progression Free Survival (PFS) and Overall Survival (OS) showed that TP53 mutated patients had a significantly shorter PFS compared to those with wildtype TP53 (adj. p-value=0,018; n=7 versus n=95). Comparing the mutational status of the 6 RSMGs, transplant versus non-transplant protocol, number of mutated genes in the M3 P panel, del17p and t(4;14) status, EMC92 score and ISS stage between patients with a PFS <=1 year and >1 year (n=23 versus n=79), only showed a significant correlation with TP53 mutational status (adj. p-value=0,012). TP53 mutational status remained the only significant prognostic factor when comparing patients with an OS <=1 year and >1 year (adj. p-value=0,003; n=13 versus n=89).When comparing the number of mutated genes, del17p and t(4;14) status, EMC92 score, transplant versus non-transplant protocol and ISS stage between TP53 mutated and wildtype MM, TP53 mutated patients had a significantly higher number of mutated genes in the M3 P panel (adj. p-value=0,001).Conclusions(1) With the M3 P Mutational Panel, we confirm the published prevalence of RSMGs in MM in our cohort of chemotherapy-naive patients. NRAS, KRAS, DIS3, FAM46C, TP53 and BRAF are the most frequently mutated genes.(2) TP53 mutational status is the strongest unfavorable prognostic factor in our cohort and it seems to be associated with greater mutational burden. Validation in a more extensive population is planned.(3) This warrants further investigation of the mutational status of these genes in larger clinical trial cohorts, enabling a more robust comparison with conventional prognostic markers in a multivariate analysis. [Display omitted] DisclosuresBroijl:Celgene: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees. Zweegman:Takeda: Research Funding; Janssen: Research Funding; Celgene: Research Funding. Sonneveld:Janssen: Honoraria, Research Funding; Celgene: Honoraria, Research Funding; Amgen: Honoraria, Research Funding; Karyopharm: Research Funding; SkylineDx: Membership on an entity's Board of Directors or advisory committees.

The Mutational Landscape of CTCL and Sezary Syndrome

Mycosis fungoides and Sézary syndrome (SS) are cutaneous T-cell lymphomas (CTCL) derived from mature CD4-positive skin-homing T cells with Sézary representing the most aggressive leukemic form of the disease. To investigate the pathogenic mechanisms of Sézary syndrome and aggressive CTCLs, we performed whole exome sequencing of matched tumor and normal DNA samples from 42 CTCL cases, including 26 Sézary syndrome. Copy number analysis revealed a distinctive pattern of somatic copy number alterations in Sézary syndrome including highly prevalent recurrent chromosomal deletions involving the TP53, RB1, PTEN, DNMT3A and CDKN1B tumor suppressor genes. Moreover, exome-wide somatic mutation analysis identified a broad spectrum of somatic mutations in key genes involved in epigenetic regulation (TET2, CREBBP, MLL2, MLL3, BRD9, SMARCA4 and CHD3) and signaling, including mutations in MAPK1, BRAF, CARD11 and PRKG1.In this context, we identified three mutations involving position E322 in MAPK1 (p.Glu322Ala and p.Glu322Lys). Functional characterization of these recurrent alleles showed increased ERK1/2 activation upon expression of both MAPK1 E322K and E322A. In addition we identified two mutations (p.Ser615Phe and p.Ser626Lys) in CARD11, an important mediator of NFκB activation. Notably, and in contrast with diffuse B-cell lymphomas where CARD11 mutations are primarily located in the coiled-coil domain, CARD11 alleles present in CTCL cases are located in the linker domain region of the protein. Functional characterization of CTCL associated CARD11 S615F and CARD11 E626K linker mutations revealed markedly increased NFκΒ activity in reporter assays, which was further enhanced upon TCR activation. Finally, we also noted the presence of two closely located mutations (p.Glu17Lys and p.Arg21Gln) in the N-terminal dimerization domain of the cGKIβ protein, in Sézary syndrome samples. cGKIβ, PRKG1 gene, is a cGMP-binding protein implicated in nitric oxide signaling. cGKIβ signaling in T-cells antagonizes TCR activation-induced interleukin 2 release and proliferation. Structural and functional characterization of the two PRKG1 mutations found in Sézary syndrome revealed impaired dimerization and decreased cGKIβ-mediated activation after stimulation with the cGMP analog 8-CPT-cGMP. Moreover, analysis of the effects of cGKIβ E17K and cGKIβ R21Q expression in JURKAT T cells revealed increased NFAT activity after PMA plus ionomycin stimulation, supporting a positive role for these mutations in enhancing the TCR signaling response. In all, these results mechanistically involve increased MAPK-ERK, NFκB and NFAT signaling in the pathogenesis of CTCLs.To analyze the therapeutic potential of targeting these oncogenic signaling pathways in CTCL we performed analysis of MAPK, JNK and NFκB signaling in CTCL cell lines. Therapeutically, inhibition of NFκB signaling with Mi-2 and bortezomib was broadly and highly active across all cell lines tested, while MEK1/2 inhibition with U0126 and inhibition of NFAT with FK506 showed more modest antitumor effects. Collectively, our findings provide new insights into the genetics of Sézary syndrome and CTCL and support the development of personalized therapies targeting key oncogenically activated signaling pathways for the treatment of these diseases. DisclosuresNo relevant conflicts of interest to declare.

Abstract LB-A05: Profiling cell free DNA in breast cancer and non-small cell lung cancer using broad NGS assessment

Abstract Introduction: Cell free DNA (cfDNA) has become a promising approach for non-invasive assessment of the tumor genome. Many cfDNA assays target hotspot alterations in a focused set of genes, but do not provide a broad characterization of the cancer. We have developed and optimized a large next generation sequencing (NGS) panel covering the coding regions of over 500 genes. Using this panel, we sequenced cell free DNA from plasma and matched tumor DNA in patients with early stage breast cancer, late stage breast cancer and late stage lung cancer. Methods: Plasma was collected from patients with cancer using a double spin protocol and, when available, matched archival tumor tissue (representing different time interval with blood collection) was obtained. Next generation sequencing libraries were generated from cell free DNA isolated from 70 plasma samples and genomic DNA from 58 matched tumor samples. The NGS libraries were enriched for the gene panel of interest and were sequenced to a targeted depth of 1,000X for plasma and 300X for matched tumors. We optimized parameters of standard bioinformatics tools to robustly call low allelic fraction events, detecting single nucleotide variants down to 1%, as well as indels and copy number alterations. Results: We identified 8 PIK3CA hotspot alterations in plasma from late stage breast and lung cancers, in addition to many alterations across driver genes such as AKT1, EGFR, IDH2, NRAS, PTEN and TP53. In plasma samples from patients with late stage breast cancer, we found 4 ESR1 mutations exclusive to the plasma samples, of which 3 are known resistance mutations to endocrine therapy. Copy number alterations in EGFR, CCND1 and KRAS were also identified in patient plasma. When comparing the number of alterations across tumor stages, we found that late stage breast (mean = 12.5 variants) and lung cancers (mean = 12.5 variants) had a larger number of alterations present in plasma than early stage breast cancers (mean = 4.5 variants). We compared somatic mutations calls in plasma and matched tumor samples and found a concordance of 53%-67% at the variant level across patients with late stage cancers (N = 37 pairs). Higher variant level concordance was observed among plasma-tumor pairs collected less than a year apart (N = 11 pairs; 76%-84%) versus more than 5 years apart (N = 8 pairs; 41%-50%). Conclusion: We have developed and optimized a 500+ gene panel for direct sequencing of cfDNA, and we demonstrate that this broad assessment of circulating tumor DNA can be used for non-invasive characterization of the cancer genome landscape. The number of alterations identified in patient plasma is consistent with higher levels of ctDNA being present in late stage disease than in early stage disease. The time dependent degree of concordance between plasma and tumor collection suggests that cell free DNA assays may provide a more accurate characterization of the current tumor mutational landscape than an archival tumor sample. The identification of plasma specific ESR1 alterations highlights the importance of cfDNA in the context of identifying mechanisms of resistance, particularly for metastatic disease when tumor tissue collection may not be feasible. In addition, a broad NGS panel provides the opportunity to identify lesions unevaluated by targeted assays and to discover resistance mutations. Citation Format: Nadia Solovieff, Matt Hims, Rebecca Leary, Derek Chiang, Caroline Germa, Cristian Massacesi, Samit Hirawat, Stefan J. Scherer, Michael Morrissey, Wendy Winckler, Emmanuelle di Tomaso. Profiling cell free DNA in breast cancer and non-small cell lung cancer using broad NGS assessment. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr LB-A05.

Abstract 615: Noninvasive diagnosis of actionable mutations by deep sequencing of circulating tumor DNA in multiple myeloma

Abstract Background: Genome sequencing of multiple myeloma (MM) tumors has revealed recurrent mutations that serve as a fertile ground for targeted therapies. Indeed, activating mutations of KRAS, NRAS and BRAF have been reported in approximately 27%, 24% and 4% of MM cases. Based on this observation, we initiated a Phase II NCI-CTEP sponsored clinical trial of trametinib in patients with MM (PHL-9460). Detection of mutations currently require bone marrow aspirates that are invasive and can yield suboptimal samples. Targeted, ultra-deep sequencing of circulating tumor DNA (ctDNA) is a promising tool for accessing the tumor genome that has not been well studied in MM. We set out to determine the feasibility of detecting ctDNA in MM and of identifying actionable mutations and mutational load using liquid biopsy through ctDNA analysis. Methods: MM patients enrolled onto PHL-9460 or those with heavy tumor burden were identified and consented to have their peripheral blood drawn for analysis. Where possible, matched tumor DNA was also obtained. Cell-free DNA was extracted from 7-15 mL of plasma isolated within ¬1 hour of blood draw using the QIAamp Circulating Nucleic Acid Kit and tagged with barcoded sequencing adapters for subsequent pooling. All exons of KRAS, NRAS, BRAF, PIK3CA and EGFR genes were isolated using a custom hybrid capture panel (IDT xGen Lockdown) and sequenced on an Illumina HiSeq 2000. Reads were aligned to the human genome reference (hg19) using bwa and somatic mutations were detected using muTect. Results: We have collected 25 samples from 23 patients, 7 from patients on PHL-9460, 5 newly diagnosed, and 13 from relapsed patients having received 3.3 median prior lines of therapy (range 1-7). To date, 11 samples from 10 patients have been sequenced. The sample with the lowest DNA yield failed due to low library complexity (range 16.6-3872 ng, median yield: 197 ng). From the remaining 10 samples, the mean target coverage ranged from 31,500 to 32,500. Somatic mutations in KRAS, NRAS, or PIK3CA genes were present in 5 of 9 patients with mutant allele frequencies ranging from 1.1% to 32% (3 KRAS and 2 NRAS of which 2 cases also had a low frequency PIK3CA mutation). We did not uncover mutations in BRAF or EGFR. For patients with matched tumor DNA, mutations in ctDNA concurred with those found in tumor DNA sequencing (4 of 4 tumors with known genotypes). Two patients with NRAS mutations enrolled onto PHL-9460 have responded to trametinib (1 partial and 1 minor response) and remain on therapy. Conclusion: ctDNA analysis in this cohort has identified key mutations in MM. The rate of RAS or RAF mutations in ctDNA compared to matched tumors and correlation to response to trametinib is ongoing and will be presented. Preliminary data suggest that ctDNA may be a reliable method of detecting mutations in MM and an alternative to bone marrow biopsy. Citation Format: Rayan Kaedbey, Olena Kis, Arnavaz Danesh, Mark Dowar, Tiantian Li, Zhihua Li, Jessica Liu, Mark Mansour, Mahadeo Sukhai, Tong Zhang, Suzanne Kamel-Reid, Trevor J. Pugh, Suzanne Trudel. Noninvasive diagnosis of actionable mutations by deep sequencing of circulating tumor DNA in multiple myeloma. [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 615. doi:10.1158/1538-7445.AM2015-615

Abstract 3894: The importance of matched tumor and normal DNA for somatic mutation discovery and clinical interpretation

Abstract Massively parallel sequencing approaches are beginning to be used clinically to characterize individual patient tumors and to identify targeted therapies based on mutations identified. A major question in these analyses is the extent to which these methods identify clinically actionable alterations in patients and whether the examination of the tumor tissue alone is sufficient or whether matched normal DNA should also be analyzed to accurately identify tumor-specific (somatic) alterations. To address these issues, we comprehensively evaluated tumor-normal paired samples from 753 cancers from fourteen tumor types. We analyzed somatic alterations using whole exome or targeted next generation sequencing approaches that were validated with high sensitivity and specificity. These analyses revealed an average of 140 and 4.34 somatic changes per exome and targeted analyses, respectively. Approximately 65% of cases had somatic alterations in genes associated with known therapies or current clinical trials. In contrast, a tumor-only sequencing approach followed by bioinformatic removal of common germline variants from existing databases led to a 36% and 58% false discovery rate in alterations identified in targeted and exome analyses, respectively, including in potentially actionable genes. These data suggest that matched tumor-normal sequencing analyses are essential for precise identification and interpretation of somatic alterations and have important implications for the diagnostic and therapeutic management of cancer patients. Citation Format: Siân Jones, Mark Sausen, Valsamo Anagnostou, Samuel V. Angiuoli, Bryan Chesnick, Kevin Galens, Maura Kadan, Lisa Kann, Karli Lytle, Derek Murphy, Monica Nesselbush, Eniko Papp, Sonya Parpart-Li, David Riley, Manish Shukla, Theresa Zhang, Luis A. Diaz, Victor E. Velculescu. The importance of matched tumor and normal DNA for somatic mutation discovery and clinical interpretation. [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 3894. doi:10.1158/1538-7445.AM2015-3894

Abstract 4424: Molecular diagnostics of drug resistant multiple myeloma cases using targeted next generation sequencing

Abstract In multiple myeloma (MM), there are two distinct genetic subtypes based on copy number alterations and translocations. About half of all MM cases are a hyperdiploid type, which is characterized by multiple trisomies of chromosomes 3, 5, 7, 9 11, 15, 19 and 21 and a lower prevalence of primary translocations involving the immunoglobulin heavy chain (IgH) locus at 14q32. The remaining cases are the non-hyperdiploid type, and chromosomes 8, 13, 14, and 16 are frequently lost. Non-hyperdiploid myeloma is strongly association with translocations of IgH alleles with various partner chromosomes, such as t(11;14)(q13;q32), t(4;14)(p16.3; q32), t(14;16)(q32;q23), t(6;14)(p21;q32) or t(14;20) (q32;q11). The copy number alterations in other chromosome regions such as 1q, 6q, 8p, and 16q occur in both subtypes. Overall, non-hyperdiploid MM is associated with worse survival than hyperdiploid MM. In this study, we sequenced all exons of 409 cancer-related genes in matched tumor and normal DNA from 5 non-hyperdiploid MM patients using a next-generation semiconductor sequencing protocol. DNA was extracted from magnetic bead-enriched bone marrow CD138 positive tumor cells from the patients and CD138 negative cells were used as matched normal cells. We detected both point mutations and copy number variations (CNVs). One individual with refractory MM (IgG λ type, ISS stage II) displayed 8 non-synonymous somatic mutations in addition to numbers of CNVs including CCND1 and RB1. We observed 2 point mutations and 3 CNVs, affecting NF-kB pathway genes: IKBKB, CYLD, IKBKE, CD79B and SYK. Although the basis of NF-kB pathway activation is only partially understood, our findings greatly expand the mechanisms by which NF-kB may be activated in this patient. We also detected several gene alterations, which may be associated with poor prognosis and poor response to chemotherapy in patients with refractory MM. Although its value should be further confirmed in larger samples, the targeted next generation sequencing is a valuable tool for high-throughput genetic testing in clinical research. Citation Format: Hiroshi Ikeda, Yasushi Sasaki, Tetsuyuki Igarashi, Yuka Aoki, Toshiaki Hayashi, Tadao Ishida, Takashi Tokino, Yasuhisa Sinomura. Molecular diagnostics of drug resistant multiple myeloma cases using targeted next generation sequencing. [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 4424. doi:10.1158/1538-7445.AM2015-4424

Abstract 4905: Comprehensive genomic analyses of oral squamous cell carcinoma tissues by semiconductor-based next-generation sequencing

Abstract Somatic mutation analysis is standard of practice for human cancers in order to identify therapeutic sensitizing and resistance mutations. We performed comprehensive genomic analyses that use PCR target enrichment and next-generation sequencing on the Ion Torrent Personal Genome Machine (PGM). We first validated a multigene sequencing screen interrogating 2855 mutational hotspots in 50 cancer-related genes using the Ion Torrent AmpliSeq cancer panel v2. Ten nanogram of DNA was used as template to amplify 207 regions in oral squamous cell carcinoma (OSCC). Approximately 1000 average coverage was obtained with more than 90% of target bases having at least 100 sequence reads. We detected mutations of the TP53 gene in 96% (22 of 23) of OSCC cell lines. Of the 62 OSCC specimens, 42 presented at least one somatic mutation among the 50 investigated genes, and 21 of these showed multiple gene somatic mutations. The most frequent mutations were detected on TP53 (26 of 62; 41.9%). CDKN2A mutations were identified in 9 cases; STK11 mutations were observed in 8 cases; and PIK3CA were mutated 5 cases. In addition, we sequenced 409 cancer-related genes in matched tumor and normal DNA from OSCC patients using the Ion Ampliseq Comprehensive Cancer Panel. This panel targets all exons of 409 tumor suppressor genes and oncogenes frequently cited and frequently mutated (covered regions: 95.4% of total), and 15,992 amplicons amplify more than 1.2 megabases of target sequence. We piloted the use of this platform to identify somatic mutations in 37 OSCC specimens including formalin-fixed paraffin-embedded (FFPE) samples. We also detected copy number variations (CNVs) in which segments of the genome can be duplicated or deleted from sequencing data. This targeted next generation sequencing using low amounts of FFPE DNA is a valuable tool for high-throughput genetic testing in research and clinical settings. Citation Format: Takafumi Nakagaki, Yasushi Sasaki, Kenta Kobashi, Kousuke Takeda, Miyuki Tamura, Tomoko Ohashi, Kazuhiro Ogi, Masashi Idogawa, Hiroyoshi Hiratsuka, Takashi Tokino. Comprehensive genomic analyses of oral squamous cell carcinoma tissues by semiconductor-based next-generation sequencing. [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 4905. doi:10.1158/1538-7445.AM2015-4905

Personalized genomic analyses for cancer mutation discovery and interpretation.

Massively parallel sequencing approaches are beginning to be used clinically to characterize individual patient tumors and to select therapies based on the identified mutations. A major question in these analyses is the extent to which these methods identify clinically actionable alterations and whether the examination of the tumor tissue alone is sufficient or whether matched normal DNA should also be analyzed to accurately identify tumor-specific (somatic) alterations. To address these issues, we comprehensively evaluated 815 tumor-normal paired samples from patients of 15 tumor types. We identified genomic alterations using next-generation sequencing of whole exomes or 111 targeted genes that were validated with sensitivities >95% and >99%, respectively, and specificities >99.99%. These analyses revealed an average of 140 and 4.3 somatic mutations per exome and targeted analysis, respectively. More than 75% of cases had somatic alterations in genes associated with known therapies or current clinical trials. Analyses of matched normal DNA identified germline alterations in cancer-predisposing genes in 3% of patients with apparently sporadic cancers. In contrast, a tumor-only sequencing approach could not definitively identify germline changes in cancer-predisposing genes and led to additional false-positive findings comprising 31% and 65% of alterations identified in targeted and exome analyses, respectively, including in potentially actionable genes. These data suggest that matched tumor-normal sequencing analyses are essential for precise identification and interpretation of somatic and germline alterations and have important implications for the diagnostic and therapeutic management of cancer patients.

Analysis of the AHR gene proximal promoter GGGGC-repeat polymorphism in lung, breast, and colon cancer

The aryl hydrocarbon receptor (AhR) regulates expression of numerous genes, including those of the CYP1 gene family. With the goal of determining factors that control AHR gene expression, our studies are focused on the role of the short tandem repeat polymorphism, (GGGGC)n, located in the proximal promoter of the human AHR gene. When luciferase constructs containing varying GGGGC repeats were transfected into cancer cell lines derived from the lung, colon, and breast, the number of GGGGC repeats affected AHR promoter activity. The number of GGGGC repeats was determined in DNA from 327 humans and from 38 samples representing 5 species of non-human primates. In chimpanzees and 3 species of macaques, only (GGGGC)2 alleles were observed; however, in western gorilla, (GGGGC)n alleles with n=2, 4, 5, 6, 7, and 8 were identified. In all human populations examined, the frequency of (GGGGC)n was n=4>5≫2, 6. When frequencies of the (GGGGC)n alleles in DNA from patients with lung, colon, or breast cancer were evaluated, the occurrence of (GGGGC)2 was found to be 8-fold more frequent among lung cancer patients in comparison with its incidence in the general population, as represented by New York State neonates. Analysis of matched tumor and non-tumor DNA samples from the same individuals provided no evidence of microsatellite instability. These studies indicate that the (GGGGC)n short tandem repeats are inherited, and that the (GGGGC)2 allele in the AHR proximal promoter region should be further investigated with regard to its potential association with lung cancer susceptibility.

Noninvasive diagnosis of actionable mutations by deep sequencing of circulating free DNA in lung cancer from never-smokers: a proof-of-concept study from BioCAST/IFCT-1002.

Tumor somatic mutation analysis is part of the standard management of metastatic lung cancer. However, physicians often have to deal with small biopsies and consequently with challenging mutation testing. Circulating free DNA (cfDNA) is a promising tool for accessing the tumor genome as a liquid biopsy. Here, we evaluated next-generation sequencing (NGS) on cfDNA samples obtained from a consecutive series of patients for the screening of a range of clinically relevant mutations. A total of 107 plasma samples were collected from the BioCAST/IFCT-1002 lung cancer study (never-smokers cohort). Matched tumor DNA (tDNA) was obtained for 68 cases. Multiplex PCR-based assays were designed to target specific coding regions in EGFR, KRAS, BRAF, ERBB2, and PI3KCA genes, and amplicon sequencing was performed at deep coverage on the cfDNA/tDNA pairs using the NGS IonTorrent Personal Genome Machine Platform. CfDNA concentration in plasma was significantly associated with both stage and number of metastatic sites. In tDNA, 50 mutations (36 EGFR, 5 ERBB2, 4 KRAS, 3 BRAF, and 2 PIK3CA) were identified, of which 26 were detected in cfDNA. Sensitivity of the test was 58% (95% confidence interval, 43%-71%) and the estimated specificity was 87% (62%-96%). These data demonstrate the feasibility and potential utility of mutation screening in cfDNA using IonTorrent NGS for the detection of a range of tumor biomarkers in patients with metastatic lung cancer.

Recurrent Rhoa Mutations In Peripheral T-Cell Lymphoma

Peripheral T-cell lymphomas (PTCLs) are a heterogeneous and poorly understood group of aggressive non Hodgkin lymphomas with poor prognosis. To gain further insight on the genetics and pathogenic mechanisms of aggressive PTCLs we performed whole exome sequencing of matched tumor and normal DNA samples from 12 PTCL patients including 6 PTCL not otherwise specified (PTCL-NOS) tumors, 3 angioimunoablastic (AITL) T-cell lymphomas, 2 nasal type NK-/T-cell lymphomas and one enteropathy-associated T-cell lymphoma (EATL). This analysis identified 288 candidate coding somatic mutations in 268 genes and a mean mutation load of 24 non synonymous mutations per sample (range 4 – 57). Among these we noted the presence of a recurrent heterozygous mutation in the RHOA small GTPase gene (RHOA G17V) present in two independent AITL samples and one PTCL NOS biopsy. Analysis of a broad and diverse panel of 126 PTCL samples identified the presence of the RHOA G17V allele in 32 samples with a high prevalence in AITL (24/36, 67%, P < 0.001) and PTCL NOS cases (8/44, 18%, P < 0.002). The RHOA protein belongs to the Rho family of small GTPases, a group of Ras-like proteins responsible for linking a variety of cell-surface receptors to different intracellular signaling proteins. As is the case for RAS and most other small GTPases, RHOA activation is mediated by guanine exchange factors (GEFs), which catalyze the switch of RHOA from an inactive GDP-bound to an active GTP-bound state. Thus, and to test the functional significance of the RHOA G17V mutation we analyzed the capacity of this mutant to load GTP. This analysis revealed that RHOA G17V fails to incorporate GTP in response to an activated GEF in vitro. Moreover, and consistent with its inability to bind GTP, RHOA G17V failed to interact with rhotekin, a RHOA effector protein that selectively interacts with the GTP-bound active form of RHOA. However and most notably, the lack of RHOA G17V activation is not the result of a defect in RHOA-GEF interaction as RHOA G17V pull down assays demonstrated effective binding of this mutant protein to activated GEF proteins in T-cells. Based on these results we proposed an inhibitory role for RHOA G17V via sequestration of active GEF proteins. Consistently, while forced activation of RHOA signaling by GFP-RHOA overexpression induced loss of adhesion and round cell morphology in HEK293T cells, transfection of GFP-RHOA-G17V induced increased elongation and cellular protrusions as result of RHOA inactivation. In addition, immunoflourescence analysis of actin stress fiber formation by RHOA demonstrated effective abrogation of RHOA mediated cytoskeleton remodeling in cells expressing RHOA G17V. Overall these results show novel insight on the genetic basis of PTCLs and demonstrate a prominent role for RHOA G17V in the pathogenesis of AITL via disruption of RHOA signaling. Disclosures:No relevant conflicts of interest to declare.

Abstract 4594: Recurrent FGFR1 hotspot mutations represent a novel therapeutic target in childhood astrocytoma.

Abstract Introduction: Pilocytic astrocytoma (PA) is the most common childhood brain tumor. This tumor can occur throughout the central nervous system, with roughly 50% of cases arising outside of the cerebellum. Tumors in these non-cerebellar locations are often difficult to treat surgically, leading to multiple tumor recurrences. PA can therefore become a chronic disease, with patients experiencing substantial morbidities. Alterations in the MAPK pathway, particularly BRAF, have previously been identified in ∼80% of PAs. Interestingly, however, the majority of those cases without a recognized change are non-cerebellar. Methods: To investigate the full range of genetic alterations occurring in PA, we used Illumina HiSeq technologies to perform whole-genome sequencing of matched tumor and germline DNA from 47 patients, with corresponding RNA sequencing data for 35 tumors. Results: The average somatic mutation rate in PA was extremely low, at 0.065/Mb, with an average of only 1.8 non-synonymous coding single nucleotide variants (SNVs) per tumor - almost ten times lower than we have recently reported for medulloblastoma. We found several novel alterations in known PA-related genes, including two new oncogenic BRAF fusions. Most strikingly, however, we identified mutations at two hotspots in the FGFR1 receptor tyrosine kinase in 4/6 centrally located PAs lacking any other MAPK pathway change. Two of these cases also carried a mutation in a downstream adaptor protein, PTPN11 (Shp2). Interestingly, germline mutations of PTPN11 are associated with Noonan syndrome (NS), and there are case reports of NS patients developing PAs. Screening of additional non-cerebellar PAs revealed four further cases with an FGFR1 mutation. All PAs, regardless of MAPK pathway alteration, displayed highly elevated expression of FGF2, indicating a general role for ligand-mediated activation of the FGFR1/MAPK pathway in PA tumorigenesis. Notably, the same FGFR1 mutations were also identified in four midline pediatric glioblastomas (GBM), a highly malignant brain tumor, suggesting a possible common origin for a subset of these two entities, despite their dramatically different clinical course. Conclusion: Altogether, MAPK alterations were identified in 96% of PAs, with very few other changes, confirming the concept of PA as a single-pathway disease. Our results also suggest that a subset of centrally located, FGFR1-driven pediatric PAs and GBMs may share common origins. Most importantly, they reveal a novel therapeutic target in clinically relevant subsets of childhood glioma. Citation Format: David TW Jones, Barbara Hutter, Natalie Jäger, Andrey Korshunov, Marcel Kool, Sally R. Lambert, Dong Anh Khuong Quang, Adam M. Fontebasso, Marina Ryzhova, Hans-Jörg Warnatz, Thomas Zichner, Jan O. Korbel, Stephan Wolf, Marie-Laure Yaspo, Keith L. Ligon, Mark W. Kieran, Benedikt Brors, Jörg Felsberg, Guido Reifenberger, V. Peter Collins, Nada Jabado, Roland Eils, Peter Lichter, Stefan M. Pfister, ICGC PedBrain Tumor Project. Recurrent FGFR1 hotspot mutations represent a novel therapeutic target in childhood astrocytoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4594. doi:10.1158/1538-7445.AM2013-4594

Recurrent Mutations of MYD88 and TBL1XR1 in Primary Central Nervous System Lymphomas

Our objective was to identify the genetic changes involved in primary central nervous system lymphoma (PCNSL) oncogenesis and evaluate their clinical relevance. We investigated a series of 29 newly diagnosed, HIV-negative, PCNSL patients using high-resolution single-nucleotide polymorphism (SNP) arrays (n = 29) and whole-exome sequencing (n = 4) approaches. Recurrent homozygous deletions and somatic gene mutations found were validated by quantitative real-time PCR and Sanger sequencing, respectively. Molecular results were correlated with prognosis. All PCNSLs were diffuse large B-cell lymphomas, and the patients received chemotherapy without radiotherapy as initial treatment. The SNP analysis revealed recurrent large and focal chromosome imbalances that target candidate genes in PCNSL oncogenesis. The most frequent genomic abnormalities were (i) 6p21.32 loss (HLA locus), (ii) 6q loss, (iii) CDKN2A homozygous deletions, (iv) 12q12-q22, and (v) chromosome 7q21 and 7q31 gains. Homozygous deletions of PRMD1, TOX, and DOCK5 and the amplification of HDAC9 were also detected. Sequencing of matched tumor and blood DNA samples identified novel somatic mutations in MYD88 and TBL1XR1 in 38% and 14% of the cases, respectively. The correlation of genetic abnormalities with clinical outcomes using multivariate analysis showed that 6q22 loss (P = 0.006 and P = 0.01) and CDKN2A homozygous deletion (P = 0.02 and P = 0.01) were significantly associated with shorter progression-free survival and overall survival. Our study provides new insights into the molecular tumorigenesis of PCNSL and identifies novel genetic alterations in this disease, especially MYD88 and TBL1XR1 mutations activating the NF-κB signaling pathway, which may be promising targets for future therapeutic strategies.