Frequency Of Genetic Alterations Research Articles

Comprehensive analysis of genomic alterations detected by next-generation sequencing-based tissue and circulating tumor DNA assays in Chinese patients with non-small cell lung cancer.

e20534 Background: While tumor genotyping is the standard treatment for patients with non-small cell lung cancer (NSCLC), spatial and temporal tumor heterogeneity and insufficient specimens can lead to limitations in the use of tissue-based sequencing. Circulating tumor DNA (ctDNA) fully encompasses tumor-specific sequence alterations and offers an alternative to tissue sample biopsies. The aim of the study was to evaluate whether the frequency of multiple genomic alterations observed following ctDNA sequencing was similar to that observed following tissue sequencing in NSCLC. Methods: A total of 99 NSCLC patients were enrolled in this study, including 40 tissue and 59 plasma samples. All kinds of variants of oncogenic drivers in NSCLC were identified by next-generation sequencing (NGS) with Acornmed panel. Results: The frequencies of genetic alterations detected in ctDNA were significantly correlated to those detected via tissue profiling (Spearman’s r = 0.812, P = 0.022). Genomic data revealed significant mutual exclusivity between alterations in epidermal growth factor receptor ( EGFR) and tumor protein 53 ( TP53; P = 0.020) and between those in EGFR and Kirsten rat sarcoma viral oncogene homolog ( KRAS; P = 0.008), as well as potential mutual exclusivity between alterations in EGFR and Erb-B2 receptor tyrosine kinase 2 ( ERBB2; P = 0.059). Furthermore, the EGFR mutant allele frequency (MAF) was strongly correlated with the TP53 MAF in individual tumors (Spearman’s r = 0.773, P = 0.005), and there was a marked difference in the EGFR MAF between patients with and without the TP53 mutation (P = 0.001). Levels of the tumor serum marker CA242 in patients with ctDNA-detectable mutations were higher than those in patients without ctDNA-detectable mutations. Conclusions: This study provides a better understanding of the spectra of genomic alterations detected by tissue and plasma ctDNA assays in Chinese patients with NSCLC. The present data also highlights the importance of tissue and plasma ctDNA screening by NGS to guide personalized therapy and promote the clinical management of NSCLC patients.

Genetic alteration of histone lysine methyltransferases and their significance in renal cell carcinoma.

BackgroundHistone lysine methyltransferases (HMTs), a category of enzymes, play essential roles in regulating transcription, cellular differentiation, and chromatin construction. The genomic landscape and clinical significance of HMTs in renal cell carcinoma (RCC) remain uncovered.MethodsWe conducted an integrative analysis of 50 HMTs in RCC and discovered the internal relations among copy number alterations (CNAs), expressive abundance, mutations, and clinical outcome.ResultsWe confirmed 12 HMTs with the highest frequency of genetic alterations, including seven HMTs with high-level amplification, two HMTs with somatic mutation, and three HMTs with putative homozygous deletion. Patterns of copy number and expression varied among different subtypes of RCC, including clear cell renal cell carcinoma, papillary cell carcinoma, and chromophobe renal carcinoma. Kaplan–Meier survival analysis and multivariate analysis identified that CNA or mRNA expression in some HMTs were significantly associated with shorter overall patient survival. Systematic analysis identified six HMTs (ASH1L, PRDM6, NSD1, EZH2, WHSC1L1, SETD2) which were dysregulated by genetic alterations as candidate therapeutic targets.DiscussionIn summary, our findings strongly evidenced that genetic alteration of HMTs may play an important role in generation and development of RCC, which lays a solid foundation for the mechanism for further research in the future.

Clinical and biological relevance of genetic alterations in pediatric T-cell acute lymphoblastic leukemia in Taiwan.

The leukemogenesis of T-cell acute lymphoblastic leukemia (T-ALL) involves multistep processes of genetic alterations. We aimed to determine the genetic alterations including common fusion transcripts, overexpression of T-cell transcription factor oncogenes, and deletion or mutation of targeted genes in pediatric T-ALL in Taiwan as well as their impact on outcomes in those treated with the Taiwan Pediatric Oncology Group-ALL-2002 protocol. Between 1995 and 2015, bone marrow samples obtained from 102 children aged <18 years consecutively diagnosed with T-ALL were examined. Thirty-two genetic alterations were examined by reverse transcription polymerase chain reaction (PCR) assays-PCR-based assays-followed by direct sequencing, real time quantitative PCR with TaqMan assays, or multiplex ligase probe amplification. TAL1 overexpression, CDKN2A/2B deletions, and NOTCH1 mutation were the most frequent aberrations while none had NF1, SUZ12 deletion, JAK1 or JAK2 mutations, or NUP214-ABL1 fusion in our cohort. The most frequent cooperating occurrence of genetic alterations included CDKN2A/2B and MTAP, MTAP and CDKN2B, LEF1 and PTPN2, and HOX11L2 and PHF6 mutation/deletion. NOTCH1 mutations conferred a favorable overall survival, whereas SIL-TAL1 fusion, TAL overexpression, LEF1 deletion, and PHF6 deletion/mutation were associated with an inferior outcome. By multivariate analysis, PHF6 mutation/deletion was the only independent predictor for inferior overall survival. The present study showed that the frequencies of genetic alterations in Taiwanese children with T-ALL differed considerably from those reported in Western countries. PHF6 mutation/deletion was an independently adverse predictor.

ARID1A-deficiency in urothelial bladder cancer: No predictive biomarker for EZH2-inhibitor treatment response?

Bladder cancer therapy relies on aggressive treatments highlighting the need for new, targeted therapies with reduced side effects. SWI/SNF complexes are mutated in ~20% across human cancers and dependency of SWI/SNF-deficient tumors on EZH2 has been uncovered recently. To systematically dissect the frequency of genetic alterations in SWI/SNF complexes potentially contributing to their inactivation, mutations and copy number variations in 25 SWI/SNF subunit genes were analyzed making use of publicly available sequencing data for 408 muscle-invasive bladder carcinoma samples. ARID1A truncating mutations were identified as the by far most common alterations of SWI/SNF complexes in urothelial bladder cancer. As current ARID1A protein expression data in bladder cancer are inconsistent and incomplete we examined if the frequency of truncating ARID1A mutations translates into a similar frequency of cases showing ARID1A protein loss. We applied a validated ARID1A antibody conducting a comprehensive immunohistochemistry-based expression analysis in urothelial bladder cancer (n = 362) including carcinoma in situ (CIS) cases. While observing increased median ARID1A protein levels in all carcinoma subgroups compared to normal urothelial controls (n = 21), the percentage of cases showing ARID1A protein loss was positively correlated with increasing stage and grade culminating in a rate of 14.1% in muscle-invasive disease. ARID1A-depletion did neither increase EZH2 protein or trimethylated H3K27 levels in vitro nor did ARID1A expression correlate with EZH2 or H3K27me3 amounts in human bladder carcinomas. Importantly, ARID1A-deficiency was neither associated with enhanced sensitivity towards inhibition of EZH2 enzymatic activity nor depletion of EZH2 protein. In summary, ARID1A truncating mutations, potentially translating into ARID1A protein loss in a subset of high-grade bladder cancers, are the most common SWI/SNF genetic alterations in bladder cancer. Our data do not support ARID1A-deficiency as predictive biomarker for EZH2-inhibitor treatment response in bladder cancer underlining the need for future bladder cancer-specific, drug screens for successfull discovery of ARID1A-deficiency-based targeted drugs.

Association of Broad-Based Genomic Sequencing With Survival Among Patients With Advanced Non–Small Cell Lung Cancer in the Community Oncology Setting

Broad-based genomic sequencing is being used more frequently for patients with advanced non-small cell lung cancer (NSCLC). However, little is known about the association between broad-based genomic sequencing and treatment selection or survival among patients with advanced NSCLC in a community oncology setting. To compare clinical outcomes between patients with advanced NSCLC who received broad-based genomic sequencing vs a control group of patients who received routine testing for EGFR mutations and/or ALK rearrangements alone. Retrospective cohort study of patients with chart-confirmed advanced NSCLC between January 1, 2011, and July 31, 2016, and who received care at 1 of 191 oncology practices across the United States using the Flatiron Health Database. Patients were diagnosed with stage IIIB/IV or unresectable nonsquamous NSCLC who received at least 1 line of antineoplastic treatment. Receipt of either broad-based genomic sequencing or routine testing (EGFR and/or ALK only). Broad-based genomic sequencing included any multigene panel sequencing assay examining more than 30 genes prior to third-line treatment. Primary outcomes were 12-month mortality and overall survival from the start of first-line treatment. Secondary outcomes included frequency of genetic alterations and treatments received. Among 5688 individuals with advanced NSCLC (median age, 67 years [interquartile range, 41-85], 63.6% white, 80% with a history of smoking); 875 (15.4%) received broad-based genomic sequencing and 4813 (84.6%) received routine testing. Among patients who received broad-based genomic sequencing, 4.5% received targeted treatment based on testing results, 9.8% received routine EGFR/ALK targeted treatment, and 85.1% received no targeted treatment. Unadjusted mortality rates at 12 months were 49.2% for patients undergoing broad-based genomic sequencing and 35.9% for patients undergoing routine testing. Using an instrumental variable analysis, there was no significant association between broad-based genomic sequencing and 12-month mortality (predicted probability of death at 12 months, 41.1% for broad-based genomic sequencing vs 44.4% for routine testing; difference -3.6% [95% CI, -18.4% to 11.1%]; P = .63). The results were consistent in the propensity score-matched survival analysis (42.0% vs 45.1%; hazard ratio, 0.92 [95% CI, 0.73 to 1.11]; P = .40) vs unmatched cohort (hazard ratio, 0.69 [95% CI, 0.62 to 0.77]; log-rank P < .001). Among patients with advanced non-small cell lung cancer receiving care in the community oncology setting, broad-based genomic sequencing directly informed treatment in a minority of patients and was not independently associated with better survival.

Molecular heterogeneity and CXorf67 alterations in posterior fossa group A (PFA) ependymomas.

Of nine ependymoma molecular groups detected by DNA methylation profiling, the posterior fossa type A (PFA) is most prevalent. We used DNA methylation profiling to look for further molecular heterogeneity among 675 PFA ependymomas. Two major subgroups, PFA-1 and PFA-2, and nine minor subtypes were discovered. Transcriptome profiling suggested a distinct histogenesis for PFA-1 and PFA-2, but their clinical parameters were similar. In contrast, PFA subtypes differed with respect to age at diagnosis, gender ratio, outcome, and frequencies of genetic alterations. One subtype, PFA-1c, was enriched for 1q gain and had a relatively poor outcome, while patients with PFA-2c ependymomas showed an overall survival at 5years of > 90%. Unlike other ependymomas, PFA-2c tumors express high levels of OTX2, a potential biomarker for this ependymoma subtype with a good prognosis. We also discovered recurrent mutations among PFA ependymomas. H3 K27M mutations were present in 4.2%, occurring only in PFA-1 tumors, and missense mutations in an uncharacterized gene, CXorf67, were found in 9.4% of PFA ependymomas, but not in other groups. We detected high levels of wildtype or mutant CXorf67 expression in all PFA subtypes except PFA-1f, which is enriched for H3 K27M mutations. PFA ependymomas are characterized by lack of H3 K27 trimethylation (H3 K27-me3), and we tested the hypothesis that CXorf67 binds to PRC2 and can modulate levels of H3 K27-me3. Immunoprecipitation/mass spectrometry detected EZH2, SUZ12, and EED, core components of the PRC2 complex, bound to CXorf67 in the Daoy cell line, which shows high levels of CXorf67 and no expression of H3 K27-me3. Enforced reduction of CXorf67 in Daoy cells restored H3 K27-me3 levels, while enforced expression of CXorf67 in HEK293T and neural stem cells reduced H3 K27-me3 levels. Our data suggest that heterogeneity among PFA ependymomas could have clinicopathologic utility and that CXorf67 may have a functional role in these tumors.

Multiomic molecular comparison of primary versus metastatic pancreatic tumors.

213 Background: Pancreatic cancer metastasizes very early, as evidenced by the fact that &gt; 70% of patients with operable disease ultimately develop metastases. Thus, it is likely that the molecular characteristics of primary pancreatic tumors are similar to metastatic lesions. We compared the frequency of genetic alterations and protein expression from primary vs. metastatic pancreatic tumors, and from metastases from different sites. By focusing on actionable genetic and proteomic information, we sought to explore whether targeted therapies could be tailored to patients at metastatic progression based on primary surgical material. Methods: Next generation DNA sequencing (NGS) data of 208 genes and a limited set of protein markers were analyzed from pancreatic tumors of 431 patients enrolled in the Know Your Tumor initiative. Of the 208 genes tested, mutations in 70 were considered potentially actionable based on preclinical and clinical evidence. We compared 146 primary pancreatic tumors against 285 metastatic lesions, and examined subgroups for liver vs. lung vs. other metastatic lesions. Molecular alterations were compared between independent groups for each gene/protein using Fisher’s exact test. Significance was assessed using a false discovery rate adjusted q-value threshold of 0.05. Results: No differences in the specific mutation or expression pattern were observed between primary vs. metastatic lesions, nor across the site of metastasis after correcting for multiple hypotheses. Even the proportion of actionable alterations (including mutations in the homologous recombination DNA repair pathway) was similar across subgroups. Conclusions: Comparison of the muli-omic profile of primary vs. metastatic pancreatic adenocarcinoma reveals that the molecular architecture is very similar, and that actionable alterations are identified at the same frequency. This is unlike the data observed from other solid tumors, (e.g. colon and breast cancer), in which substantial molecular discordance and heterogeneity exists between primary tumors and metastatic sites, but is consistent with the belief that primary pancreatic cancers metastasize early and thus are molecularly indistinguishable from metastatic lesions.

Unraveling the heterogeneity of IgM monoclonal gammopathies: a gene mutational and gene expression study.

Immunoglobulin M (IgM) monoclonal gammopathies show considerable variability, involving three different stages of presentation: IgM monoclonal gammopathy of undetermined significance (IgM-MGUS), asymptomatic Waldenström's macroglobulinemia (AWM), and symptomatic WM (SWM). Despite recent findings about the genomic and transcriptomic characteristics of such disorders, we know little about the causes of this clinical heterogeneity or the mechanisms involved in the progression from indolent to symptomatic forms. To clarify these matters, we have performed a gene expression and mutational study in a well-characterized cohort of 69 patients, distinguishing between the three disease presentations in an attempt to establish the relationship with the clinical and biological features of the patients. Results showed that the frequency of genetic alterations progressively increased from IgM-MGUS to AWM and SWM. This means that, in contrast to MYD88 p.L265P and CXCR4 WHIM mutations, present from the beginning of the pathogenesis, most of them would be acquired during the course of the disease. Moreover, the expression study revealed a higher level of expression of genes belonging to the Toll-like receptor (TLR) signaling pathway in symptomatic versus indolent forms, which was also reflected in the disease presentation and prognosis. In conclusion, our findings showed that IgM monoclonal gammopathies present higher mutational burden as the disease progresses, in parallel to the upregulation of relevant pathogenic pathways. This study provides a translational view of the genomic basis of WM pathogenesis.

Genomic landscape of ovarian clear cell carcinoma via whole exome sequencing

ObjectiveTo analyze whole exome sequencing (WES) data on ovarian clear cell carcinoma (OCCC) in Korean patients via the technique of next generation sequencing (NGS). Genomic profiles were compared between endometriosis-associated OCCC (EMS-OCCC) and Non-EMS-OCCC. MethodsWe used serum samples and cancer tissues, stored at the Seoul National University Hospital Human Biobank, that were initially collected from women diagnosed with OCCC between 2012 and 2016. In total, 15 patients were enrolled: 5 with pathologically confirmed EMS-OCCC and 10 with Non-EMS-OCCC. We performed NGS WES on 15 fresh frozen OCCC tissues and matched serum samples, enabling comprehensive genomic characterization of OCCC. ResultsOCCC was characterized by complex genomic alterations, with a median of 178 exonic mutations (range, 111-25,798) and a median of 343 somatic copy number variations (range, 43-1,820) per tumor sample. In all, 54 somatic mutations were discovered across 14 genes, including PIK3CA (40%), ARID1A (40%), and KRAS (20%) in the 15 Korean OCCCs. Copy number gains in NTRK1 (33%), MYC (40%), and GNAS (47%) and copy number losses in TET2 (73%), TSC1 (67%), BRCA2 (60%), and SMAD4 (47%) were frequent. The significantly altered pathways were associated with proliferation and survival (including the PI3K/AKT, TP53, and ERBB2 pathways) in 87% of OCCCs and with chromatin remodeling in 47% of OCCCs. No significant differences in frequencies of genetic alterations were detected between EMS-OCCC and Non-EMS-OCCC groups. ConclusionWe successfully characterized the genomic landscape of 15 Korean patients with OCCC. We identified potential therapeutic targets for the treatment of this malignancy.

Genomic comparison of matched primary and metastatic germ cell tumors (GCT).

4556 Background: Tumor genomic analysis may be useful in patients with GCT as a means of identifying potentially actionable genomic alterations or mutations such as TP53 that confer resistance to chemotherapy. As GCTs often exhibit significant morphologic heterogeneity, we evaluated the level of concordance between genomic alterations in matched primary and metastatic GCT samples. Methods: GCT patients enrolled on an institutional prospective sequencing protocol with available primary and metastatic tumor tissue were eligible. Each tumor was subjected to MSK-IMPACT, an exon capture sequencing assay, which detects copy number alterations (CNAs) and mutations in 410 cancer-related genes. For each primary-metastasis pair, concordance and clonality was assessed using the FACETS algorithm. Results: Matched primary-metastasis tumor pairs were available for 36 patients (78% nonseminoma, 22% seminoma, median age 33.5 years). All patients received chemotherapy, with 25 (69%) receiving treatment prior to analysis of the metastatic samples. The frequency of genetic alterations was low with a median of 3 mutations (1-7), 7 amplifications (1-26) and 1 deletion (1-9) detected per sample, with no significant difference in mutational/CNA burden between primaries and metastases. Of 109 unique mutations across patients, only 44 (40%) were concordant between the primary and matched metastasis, including 5 of 9 hotspot mutations. For CNAs, 184 (81%) of 226 were concordant. Only 24 of 109 (22%) mutations were clonal (defined as predicted to be present in all cancer cells) in either the primary or metastatic matched samples; of these, only 4 were clonal in both the primary and metastatic samples, including 2 hotspots. However, 4 of 5 alterations in TP53/MDM2 were shared by both the primary and metastasis pairs. In a separate exploratory cohort, 4 TP53 mutations were identified in 3 primary tumors and 1 metastasis, and all 4 mutations were also detected by cell-free DNA profiling. Conclusions: Genomic concordance, particularly for mutations, is poor between primary and metastatic GCT samples. Cell-free DNA analysis may help overcome this limitation by identifying alterations in progressive tumors without need for a new biopsy.

Screening of BRCA 1 - 185delAG mutation in Ovarian Cancer patients in a Tertiary care centre from Telangana

Aim: Ovarian cancer (OC) occurs due to genetic alterations and mutations in BRCA gene. The aim of the study is to assess the frequency of genetic alterations that persist in Ashkenazi founder mutation BRCA1, 185delAG in patients with ovarian cancer from South Indian origin. Materials and Methods: A total of 100 ovarian cancer patients and an equal number of control subjects were included in the present study. Screening of 185delAG mutation BRCA1 gene was carried out by ARMS PCR followed by agarose gel electrophoresis. Statistical analysis was applied to test for the significance of the results obtained. Results: The genotype distribution of WW, WM, MM showed a significant difference between the two subjects, 95%, 4% and 1% in controls and 52%, 36% and 12% in cases respectively. An increased frequency of homozygotic mutant genotypes (MM) were found in patients compared to controls. Similarly, a significant difference in the distribution of M allele in cases and control subjects (W v/s M: χ2 P<0.0001, OR 18.06, 95% CI 6.31-51.65) was observed. Conclusion: The demographic details of the patients and controls revealed that females of age greater than 40 years are associated with high risk of ovarian cancer. The postmenopausal women have a very high susceptibility to OC (6.5 times riskier). Therefore, 185delAG mutation BRCA1 has a possible association in the etiology of ovarian cancer.

Unexpected frequency of genomic alterations in histologically normal colonic tissue from colon cancer patients.

As shown by genomic studies, colorectal cancer (CRC) is a highly heterogeneous disease, where copy number alterations (CNAs) may greatly vary among different patients. To explore whether CNAs may be present also in histologically normal tissues from patients affected by CRC, we performed CGH + SNP Microarray on 15 paired tumoral and normal samples. Here, we report for the first time the occurrence of CNAs as a common feature of the histologically normal tissue from CRC patients, particularly CNAs affecting different oncogenes and tumor-suppressor genes, including some not previously reported in CRC and others known as being involved in tumor progression. Moreover, from the comparison of normal vs paired tumoral tissue, we were able to identify three groups: samples with an increased number of CNAs in tumoral vs normal tissue, samples with a similar number of CNAs in both tissues, and samples with a decrease of CNAs in tumoral vs normal tissue, which may be likely due to a selection of the cell population within the tumor. In conclusion, our approach allowed us to uncover for the first time an unexpected frequency of genetic alteration in normal tissue, suggesting that tumorigenic genetic lesions are already present in histologically normal colonic tissue and that the use in array comparative genomic hybridization (CGH) studies of normal samples as reference for the paired tumors can lead to misrepresented genomic data, which may be incomplete or limited, especially if used for the research of target molecules for personalized therapy and for the possible correlation with clinical outcome.Electronic supplementary materialThe online version of this article (doi:10.1007/s13277-016-5181-0) contains supplementary material, which is available to authorized users.

Non-myogenic tumors display altered expression of dystrophin (DMD) and a high frequency of genetic alterations.

DMD gene mutations have been associated with the development of Dystrophinopathies. Interestingly, it has been recently reported that DMD is involved in the development and progression of myogenic tumors, assigning DMD a tumor suppressor activity in these types of cancer. However, there are only few reports that analyze DMD in non-myogenic tumors. Our study was designed to examine DMD expression and genetic alterations in non-myogenic tumors using public repositories. We also evaluated the overall survival of patients with and without DMD mutations. We studied 59 gene expression microarrays (GEO database) and RNAseq (cBioPortal) datasets that included 9817 human samples. We found reduced DMD expression in 15/27 (56%) pairwise comparisons performed (Fold-Change (FC) ≤ 0.70; p-value range = 0.04-1.5×10−20). The analysis of RNAseq studies revealed a median frequency of DMD genetic alterations of 3.4%, higher or similar to other well-known tumor suppressor genes. In addition, we observed significant poorer overall survival for patients with DMD mutations. The analyses of paired tumor/normal tissues showed that the majority of tumor specimens had lower DMD expression compared to their normal adjacent counterpart. Interestingly, statistical significant over-expression of DMD was found in 6/27 studies (FC ≥ 1.4; p-value range = 0.03-3.4×10−15). These results support that DMD expression and genetic alterations are frequent and relevant in non-myogenic tumors. The study and validation of DMD as a new player in tumor development and as a new prognostic factor for tumor progression and survival are warranted.

Epigenetic mechanisms regulating the development of hepatocellular carcinoma and their promise for therapeutics.

Hepatocellular carcinoma (HCC) is one of the most common cancers around the globe and third most fatal malignancy. Chronic liver disorders such as chronic hepatitis and liver cirrhosis often lead to the development of HCC. Accumulation of genetic and epigenetic alterations are involved in the development of HCC. Genetic research sparked by recent developments in next generation sequencing has identified the frequency of genetic alterations that occur in HCC and has led to the identification of genetic hotspots. Emerging evidence suggests that epigenetic aberrations are strongly associated with the initiation and development of HCC. Various important genes encoding tumor suppressors including P16, RASSF1A, DLC-1, RUNX3 and SOCS-1 are targets of epigenetic dysregulation during the development of HCC. The present review discusses the importance of epigenetic regulations including DNA methylation, histone modification and microRNA mediated regulation of gene expression during tumorigenesis and their use as disease biomarkers. Furthermore, these epigenetic alterations have been discussed in relationship with promising therapeutic perspectives for HCC and related cancers.

Assessment of the frequency of genetic alterations (LOH/MSI) in patients with intraepithelial cervical lesions with HPV infection: a pilot study.

In the present study, we analyzed (1) the type of HPV infection and (2) the frequency of loss of heterozygosity and microsatellite imbalance (LOH/MSI) in normal cytology and cervical intraepithelial neoplasia (CIN1-3). The cytological material included: low-grade squamous intraepithelial lesions (CIN1, n=11), high-grade lesions (CIN2 and CIN3, n=13), and cytologically normal cells from non-neoplastic cervical samples (n=8). HPV genotyping was done using RealLine HPV 16/18 kit. We used 20 microsatellite markers from: 1p31.2, 3p14.3, 3p21.3, 3p22.2, 3p24.2, 3p25.3, 7q32.2, 9p21.3, 11p15.5, 12q23.2, and 16q22.1. LOH/MSI was correlated with clinicopathological parameters. The presence of HPV DNA was revealed in 78.13% samples, including normal cytology. LOH/MSI was the most frequent for: 3p25.3 (39%), 3p22.2 (20.83%), 3p24.2 (20%), and 3p14.3 (16.67%). It was demonstrated that D3S1234 (FHIT; 3p14.3), D3S1611 (MLH1; 3p22.2), D3S1583 (RARB; 3p24.2), D3S1317 and D3S3611 (VHL; 3p25.3) could differentiate patients with CIN2/CIN3 versus CIN1, showing significantly higher frequency in CIN2/CIN3. LOH/MSI frequency for other than 3p markers was lower, 10-22.2%. The simultaneous occurrence of LOH/MSI for several markers (OFAL) was higher in CIN2/CIN3. Significant differences in OFAL were found between samples with versus without HPV infection. In HPV-positive patients, significant differences in OFAL were found between normal cytology, CIN1 and CIN2/CIN3. HPV infection influences the increase in LOH/MSI frequency, especially in tumor suppressor gene loci. Several studied microsatellite markers seem to be useful for CIN grading. Hopefully, the obtained results, if confirmed on larger patient cohort, would allow creating a panel of markers supporting clinical diagnosis in patients with HPV infection.

Routine molecular profiling of patients with advanced non-small-cell lung cancer: results of a 1-year nationwide programme of the French Cooperative Thoracic Intergroup (IFCT)

The molecular profiling of patients with advanced non-small-cell lung cancer (NSCLC) for known oncogenic drivers is recommended during routine care. Nationally, however, the feasibility and effects on outcomes of this policy are unknown. We aimed to assess the characteristics, molecular profiles, and clinical outcomes of patients who were screened during a 1-year period by a nationwide programme funded by the French National Cancer Institute. This study included patients with advanced NSCLC, who were routinely screened for EGFR mutations, ALK rearrangements, as well as HER2 (ERBB2), KRAS, BRAF, and PIK3CA mutations by 28 certified regional genetics centres in France. Patients were assessed consecutively during a 1-year period from April, 2012, to April, 2013. We measured the frequency of molecular alterations in the six routinely screened genes, the turnaround time in obtaining molecular results, and patients' clinical outcomes. This study is registered with ClinicalTrials.gov, number NCT01700582. 18,679 molecular analyses of 17,664 patients with NSCLC were done (of patients with known data, median age was 64·5 years [range 18-98], 65% were men, 81% were smokers or former smokers, and 76% had adenocarcinoma). The median interval between the initiation of analysis and provision of the written report was 11 days (IQR 7-16). A genetic alteration was recorded in about 50% of the analyses; EGFR mutations were reported in 1947 (11%) of 17,706 analyses for which data were available, HER2 mutations in 98 (1%) of 11,723, KRAS mutations in 4894 (29%) of 17,001, BRAF mutations in 262 (2%) of 13,906, and PIK3CA mutations in 252 (2%) of 10,678; ALK rearrangements were reported in 388 (5%) of 8134 analyses. The median duration of follow-up at the time of analysis was 24·9 months (95% CI 24·8-25·0). The presence of a genetic alteration affected first-line treatment for 4176 (51%) of 8147 patients and was associated with a significant improvement in the proportion of patients achieving an overall response in first-line treatment (37% [95% CI 34·7-38·2] for presence of a genetic alteration vs 33% [29·5-35·6] for absence of a genetic alteration; p=0·03) and in second-line treatment (17% [15·0-18·8] vs 9% [6·7-11·9]; p<0·0001). Presence of a genetic alteration was also associated with improved first-line progression-free survival (10·0 months [95% CI 9·2-10·7] vs 7·1 months [6·1-7·9]; p<0·0001) and overall survival (16·5 months [15·0-18·3] vs 11·8 months [10·1-13·5]; p<0·0001) compared with absence of a genetic alteration. Routine nationwide molecular profiling of patients with advanced NSCLC is feasible. The frequency of genetic alterations, acceptable turnaround times in obtaining analysis results, and the clinical advantage provided by detection of a genetic alteration suggest that this policy provides a clinical benefit. French National Cancer Institute (INCa).

Medical treatment involving investigational drugs and genetic profile of thymic carcinoma

BackgroundThymic carcinoma is a rare neoplasm of the thymus, and information regarding its genetic profile and optimal medical treatment is limited. We sought to characterize the genetic profile of thymic carcinoma and to evaluate the efficacy of various medical treatments, including treatment with tyrosine kinase inhibitors (TKIs), cytotoxic agents, and immune checkpoint inhibitors. MethodsWe retrospectively reviewed medical records of 64 consecutive patients with thymic carcinoma at the National Cancer Center Hospital between April 1973 and March 2014. We analyzed treatment course of patients who underwent medical treatment involving investigational drugs. For patients with available tissue samples, targeted sequencing of 50 cancer-related genes using next-generation sequencing was performed. ResultsThirty-six patients had received chemotherapy. Median progression-free survival in patients receiving first-line chemotherapy was 7.07 months (95% confidence interval, 5.67–8.93). Median survival time was 32.6 months (95% confidence interval, 23.2–43.4). As second- or later-line chemotherapy, a total of 13 patients were treated with 24 investigational drugs, including 8 multi-targeted TKIs, 5 cytotoxic agents, and 2 immune checkpoint inhibitors. Six (24%) of the patients treated with investigational drugs maintained disease control for at least 6 months. Tissue samples of 52 patients (81.3%) were available for targeted sequencing, consisting of 52 formalin-fixed, paraffin-embedded (FFPE) and 16 fresh frozen tissue samples. The genetic alterations of TP53, KRAS, FBXW7, and NRAS were detected in 7 patients (13.5%), and no KIT mutations were noted. ConclusionsMulti-targeted TKIs exhibited potential clinical efficacy for previously-treated thymic carcinoma. The frequency of genetic alterations in this study was low, with no apparent relationship with the efficacy of chemotherapy.

PARKIN Inactivation Links Parkinson's Disease to Melanoma.

Melanoma incidence is higher in patients affected by Parkinson's disease (PD) and vice versa, but the genetic link shared by both diseases is unknown. As PARK2 is both a tumor suppressor gene and frequently mutated in young onset PD, we evaluated the role of PARK2 in melanoma predisposition and progression. An in-depth PARK2 gene dosage analysis and sequencing was performed on 512 French case patients and 562 healthy control patients, as well as sporadic tumors and melanoma cell lines. The frequency of genetic alterations was compared between case patients and control patients using two-sided Fisher's exact tests and odds ratio (OR) calculations. We used western blotting to determine PARKIN expression in melanocytes and melanoma cell lines and transfection followed by clonogenic assays to evaluate the effect of PARKIN expression on cellular proliferation. All statistical tests were two-sided. Germline PARK2 mutations (including copy number variations, splicing, and putative deleterious missense mutations) were present in 25 case patients but only four control patients (OR = 3.95, 95% confidence interval = 1.34 to 15.75). Copy number variations (CNVs) and loss of heterozygosity were present in 60% and 74%, respectively, of primary tumors. PARKIN protein was expressed in melanocytes but not in most melanoma cell lines, and its expression decreased following melanocyte transformation by oncogenic NRAS. Re-expression of PARKIN in melanoma cell lines resulted in a drastic reduction of cell proliferation and inhibition of PARKIN in melanocytes stimulated their proliferation. Our results show an important role for PARK2 as a tumor suppressor both in melanoma predisposition and progression, which could explain the epidemiological association of these diseases.

The Genomic and Epigenomic Landscapes of Blast Crisis Transformation in Chronic Myeloid Leukemia

The transition from chronic phase (CP) to blast crisis (BC) chronic myeloid leukemia (CML) is characterized by reprogramming of the CML transcriptome (Radich et al. PNAS 2006), and shortened survival. Current models propose genomic instability as causal in BC transformation with enhanced DNA damage and impaired DNA repair inducing genetic mutations (ranging from large chromosomal aberrations to point mutations), altered gene function, and eventually BC transformation (Perrotti et al. JCI 2010). Consistent with this model are the phenomena of BC clonal evolution, and the increased frequency of ABL kinase domain mutations found in BC. Because different mutational processes are associated with distinct cancer-specific mutation signatures (Alexandrov et al. Nature 2013), this model also predicts the existence of a CML-specific mutation signature. In addition, recent work has highlighted the importance of epigenetic alterations in hematologic malignancies (Shih et al., Nat. Rev. Cancer, 2012). However, we lack a complete understanding of the type or frequency of genetic alterations in BC, and the relative contribution of genetic vs. epigenetic events in reprogramming the BC transcriptome.To address these knowledge gaps, we analyzed the CML progression genome, epigenome, and transcriptome in 12 CP/BC sample pairs. Whole-genome sequencing revealed the CML genome to be relatively stable with respect to structural variations, indels, and somatic single nucleotide variants. The average number of nonsynonymous coding mutations per BC genome was 5, placing the BC coding genome in the same mutation frequency range as AML and ALL genomes (Alexandrov et al. Nature 2013). In addition, we identified a novel mutation signature in all CML samples suggesting a CML-specific mutational process. 1175 genes were 'hit' by genomic, mostly copy number, alterations in >1 sample, and included TCR genes and Ikaros (IKZF1) among lymphoid BC pairs. Only 21 recurrently altered genes were affected by somatic SNVs or indels, with resistance-associated ABL1 mutations being commonest.We next used DNA methylation arrays to assess the BC epigenome, and found 20,651 CpG sites (out of 455,187) to be hyper-methylated, and 3225 to be hypo-methylated in BC compared to CP. Combined methylome and transcriptome analysis demonstrated an inverse relationship between methylation and expression changes at a subset of CpG sites enriched at promoters. Genes with increased methylation/decreased expression or decreased methylation/increased expression included those involved in cell cycle control/heme biosynthesis, and molecular mechanisms of cancer/G-protein coupled receptor signaling/MAPK signaling respectively. Unsupervised methylation-based clustering segregated samples into CP, lymphoid BC and myeloid BC groups, recapitulating expression-based clustering, and further supporting a functional role for DNA methylation in BC transcriptional reprogramming.We next performed an integrative analysis by combining the genome, methylome, and transcriptome datasets, and included data from 34 additional CML samples. Top ranking candidate genes included epigenetic modifiers, and hematopoetic differentiation- and stem cell-related genes. Functional analysis of candidate genes and epigenetic processes using genetic and epigenetic drug-based approaches are ongoing.In summary, we conclude that: 1. The genomic and epigenomic landscapes in BC are characterized by a modest number of recurring events in the former, but consistent and striking differences in the latter, 2. The BC methylome is functionally associated with the robust gene expression changes found in BC, and 3. Epigenetic modifier drugs may be of use in reversing the gene expression changes characteristic of BC. DisclosuresChuah:Children International: Honoraria; Novartis: Honoraria; Bristol Meyers Squibb: Honoraria. Takahashi:Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Sysmex: Research Funding, Speakers Bureau; Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Celgene: Speakers Bureau; Masis: Consultancy; Otsuka: Membership on an entity's Board of Directors or advisory committees; Astellas: Speakers Bureau; BMS: Honoraria, Research Funding, Speakers Bureau.

Abstract 4804: High throughput whole exome DNA and transcriptome RNA sequencing to identify genetic drivers and alterations in HPV-negative and HPV-positive HNSCC cell lines

Abstract Head and neck squamous cell carcinoma (HNSCC) is among the top cancer types with high frequencies of genetic alterations, including mutation and copy number variation (CNV). The Cancer Genome Atlas (TCGA) has profiled over 279 HNSCC tumors and generated a comprehensive genomic characterization of HNSCC. This has led to an urgent need for a panel of head and neck cell line models with genomic alterations representative of those found by TCGA. We performed whole exome DNA sequencing (exome DNA-seq) and transcriptome RNA sequencing (RNA-seq) on 15 HPV negative and 11 HPV positive HNSCC lines, which were compared with three normal human oral mucosa lines and 8 matched blood samples. Exome DNA- and RNA-seq were performed on the ABI SOLiD platform with an average depth of 87X and 44X respectively. Using an in-house analysis pipeline, we determined the CNVs and single nucleotide variants (SNV) obtained from DNA-seq to be able to compare with the genomic alterations found in TCGA, and also to cross-validate these with the SNVs identified in our RNA-seq. We identified chromosome losses in 3p, 5q, 8p, 9p and 18q and gains in 3q, 7p and 11q in a significant portion of cell lines with software CONTRA (COpy Number Targeted Resequencing Analysis), which are consistent with previous karyotype and TCGA CNV studies. Integrative analysis between CNV by exome-seq and gene expression by RNA-seq of these cell lines revealed a significant positive correlation in multiple oncogenes including PIK3CA, TP63, CCND1, FADD, BIRC2 and YAP1, which is in concordance with TCGA results. We established a workflow to determine deleterious mutations and somatic mutations using software ANNOVAR, in combination with functional prediction tool Mutation Assessor, and Sanger Institute's somatic mutation database, COSMIC, in order to characterize legacy and newer HNSCC lines without and with matched samples. We identified a median of 1588 potentially deleterious and/or somatic mutations for each cell line. The most recurrently mutated genes in TCGA with a functional impact are also frequently mutated in cell lines, including TP53, FAT1 and NOTCH1, etc. Many of the genomic alterations identified converge on the networks we previously defined in HNSCC, including the PI3K/AKT/mTOR, NFκB, and RAS/MAPK pathways. Our findings suggest that these cell lines can serve as HNSCC models for mechanistic and therapeutic studies, and thereby provide a valuable resource for the wider biomedical research community. Supported by NIDCD intramural projects ZIA-DC-000016, 73 and 74. Citation Format: Hui Cheng, Xinping Yang, Han Si, Anthony Saleh, Jamie Coupar, Robert L. Ferris, Wendell G. Yarbrough, Mark E. Prince, Thomas E. Carey, Carter Van Waes, Zhong Chen. High throughput whole exome DNA and transcriptome RNA sequencing to identify genetic drivers and alterations in HPV-negative and HPV-positive HNSCC cell lines. [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 4804. doi:10.1158/1538-7445.AM2015-4804