Targeted Next-generation DNA Sequencing Research Articles

Mechanisms of Resistance to Tyrosine Kinase Inhibitors in ROS1 Fusion-Positive Nonsmall Cell Lung Cancer.

ROS1 fusion-positive (ROS1+) nonsmall cell lung cancer (NSCLC) patients are highly sensitive to tyrosine kinase inhibitor (TKI) treatments. However, acquired TKI resistance remains the major hurdle preventing patients from experiencing prolonged benefits. 107 advanced or metastatic ROS1+ NSCLC patients who progressed on crizotinib and lorlatinib were recruited. Tissue and plasma samples were collected at baseline (N = 50), postcrizotinib (N = 91), and postlorlatinib (N = 21), which were all subject to the 139-gene targeted next-generation DNA sequencing. Molecular dynamics modeling was performed to investigate the effects of ROS1 mutations on binding to different TKIs. In patients with postcrizotinib and postlorlatinib samples, an accumulation of on- and off-target resistance alterations after multiple TKI treatments was observed. ROS1 G2032R and MET amplification were the most common on-target and off-target alterations, respectively. Patients with CD74-ROS1 and SLC34A2-ROS1 had longer progression-free survival (PFS) (P < 0.001) and higher rates of resistance mutations (on-target, P = 0.001; off-target, P = 0.077) than other ROS1 fusion variants following crizotinib treatment. Ten distinct on-target resistance mutations were detected after TKI therapies, of which 4 were previously unreported (ROS1 L2010M, G1957A, D1988N, L1982V). Molecular dynamics simulations showed that all 4 mutations were refractory to crizotinib, while G1957A, D1988N, and L1982V were potentially sensitive to lorlatinib and entrectinib. This study provided a comprehensive portrait of TKI-resistance mechanisms in ROS1+ NSCLC patients. Using in silico simulations of TKI activity, novel secondary mutations that may confer TKI resistance were identified and may support clinical therapeutic decision-making.

Epidemiology, Genetics, and DNA Methylation Grouping of Hyperostotic Meningiomas.

Meningiomas are the most common primary intracranial tumors and are among the only tumors that can form lamellar, hyperostotic bone in the tumor microenvironment. Little is known about the epidemiology or molecular features of hyperostotic meningiomas. Using a retrospective database of 342 meningiomas treated with surgery at a single institution, we correlated clinical, tumor-related, targeted next-generation DNA sequencing (n = 39 total, 16 meningioma-induced hyperostosis [MIH]), and surgical variables with the presence of MIH using generalized linear models. Meningioma DNA methylation grouping was analyzed on a separate population of patients from the same institution with preoperative imaging studies sufficient for identification of MIH (n = 200). MIH was significantly correlated with anterior fossa (44.3% of MIH vs 17.5% of non-MIH were in the anterior fossa P < .001, c2) or skull base location (62.5% vs 38.3%, P < .001, c2) and lower MIB-1 labeling index. Gross total resection was accomplished in 27.3% of tumors with MIH and 45.5% of nonhyperostotic meningiomas (P < .05, t test). There was no association between MIH and histological World Health Organization grade (P = .32, c2). MIH was significantly more frequent in meningiomas from the Merlin-intact DNA methylation group (P < .05). Somatic missense mutations in the WD-repeat-containing domain of the TRAF7 gene were the most common genetic alteration associated with MIH (n = 12 of 15, 80%, P < .01, c2). In this article, we show that MIH has a predilection for the anterior skull base and affected tumors are less amenable to gross total resection. We find no association between MIH and histological World Health Organization grade, but show that MIH is more common in the Merlin-intact DNA methylation group and is significantly associated with TRAF7 somatic missense mutations. These data provide a framework for future investigation of biological mechanisms underlying MIH.

Clinicopathologic and genetic analysis of invasive breast carcinomas in women with germline CHEK2 variants

PurposeGermline pathogenic variants in checkpoint kinase 2 (CHEK2) are associated with a moderately increased risk of breast cancer (BC). The spectrum of clinicopathologic features and genetics of these tumors has not been fully established.MethodsWe characterized the histopathologic and clinicopathologic features of 44 CHEK2-associated BCs from 35 women, and assessed responses to neoadjuvant chemotherapy. A subset of cases (n = 23) was additionally analyzed using targeted next-generation DNA sequencing (NGS).ResultsMost (94%, 33/35) patients were heterozygous carriers for germline CHEK2 variants, and 40% had the c.1100delC allele. Two patients were homozygous, and five had additional germline pathogenic variants in ATM (2), PALB2 (1), RAD50 (1), or MUTYH (1). CHEK2-associated BCs occurred in younger women (median age 45 years, range 25–75) and were often multifocal (20%) or bilateral (11%). Most (86%, 38/44) were invasive ductal carcinomas of no special type (IDC-NST). Almost all (95%, 41/43) BCs were ER + (79% ER + HER2-, 16% ER + HER2 + , 5% ER-HER2 +), and most (69%) were luminal B. Nottingham grade, proliferation index, and results of multiparametric molecular testing were heterogeneous. Biallelic CHEK2 alteration with loss of heterozygosity was identified in most BCs (57%, 13/23) by NGS. Additional recurrent alterations included GATA3 (26%), PIK3CA (226%), CCND1 (22%), FGFR1 (22%), ERBB2 (17%), ZNF703 (17%), TP53 (9%), and PPM1D (9%), among others. Responses to neoadjuvant chemotherapy were variable, but few patients (21%, 3/14) achieved pathologic complete response. Most patients (85%) were without evidence of disease at time of study (n = 34). Five patients (15%) developed distant metastasis, and one (3%) died (mean follow-up 50 months).ConclusionAlmost all CHEK2-associated BCs were ER + IDC-NST, with most classified as luminal B with or without HER2 overexpression. NGS supported the luminal-like phenotype and confirmed CHEK2 as an oncogenic driver in the majority of cases. Responses to neoadjuvant chemotherapy were variable but mostly incomplete.

Concurrent PTEN and PDGFRB Alterations Characterize Storiform Collagenoma.

Storiform collagenoma is a rare mesenchymal skin tumor that is composed of thickened collagen bundles arranged in a characteristic storiform pattern with a relatively hypocellular CD34-positive spindle cell component. Storiform collagenoma is most often sporadic, but multiple lesions can occur in Cowden syndrome, which is characterized by germline alterations in PTEN (phosphatase and tensin homolog) on chromosome 10. Here, we investigated the molecular pathogenesis of storiform collagenoma using a targeted next-generation DNA sequencing platform, including 5 sporadic cases and one case associated with Cowden syndrome. Recurrent PTEN alterations were identified in all cases, with biallelic PTEN inactivation observed in the case associated with Cowden syndrome and one sporadic case. Unexpectedly, we also identified recurrent activating mutations in the platelet-derived growth factor receptor beta (PDGFRB) gene. This included a missense substitution in the D5 Ig-like domain of PDGFRB in the Cowden syndrome-associated case. In addition, we report missense alterations in the juxtamembrane domain of PDGFRB in 4 of 5 (80%) sporadic cases, including mutations that have been previously described in sporadic myofibroma and myopericytoma. Therefore, we confirm the neoplastic nature of storiform collagenoma, we expand the spectrum of reported PDGFRB alterations in mesenchymal tumors and we suggest a possible collaborative role for PTEN and PDGFRB in the pathogenesis of storiform collagenoma.

Feasibility of Targeted Next-Generation DNA Sequencing for Expanding Population Newborn Screening.

Newborn screening (NBS) is an effective public health intervention that reduces death and disability from treatable genetic diseases, but many conditions are not screened due to a lack of a suitable assay. Whole genome and whole exome sequencing can potentially expand NBS but there remain many technical challenges preventing their use in population NBS. We investigated if targeted gene sequencing (TGS) is a feasible methodology for expanding NBS. We constructed a TGS panel of 164 genes which screens for a broad range of inherited conditions. We designed a high-volume, low-turnaround laboratory and bioinformatics workflow that avoids the technical and data interpretation challenges associated with whole genome and whole exome sequencing. A methods-based analytical validation of the assay was completed and test performance in 2552 newborns examined. We calculated annual birth estimates for each condition to assess cost-effectiveness. Assay analytical sensitivity was >99% and specificity was 100%. Of the newborns screened, 1.3% tested positive for a condition. On average, each individual had 225 variants to interpret and 1.8% were variants of uncertain significance (VUS). The turnaround time was 7 to 10 days. Maximum batch size was 1536 samples. We demonstrate that a TGS assay could be incorporated into an NBS program soon to increase the number of conditions screened. Additionally, we conclude that NBS using TGS may be cost-effective.

Pediatric-type high-grade neuroepithelial tumors with CIC gene fusion share a common DNA methylation signature

Pediatric neoplasms in the central nervous system (CNS) show extensive clinical and molecular heterogeneity and are fundamentally different from those occurring in adults. Molecular genetic testing contributes to accurate diagnosis and enables an optimal clinical management of affected children. Here, we investigated a rare, molecularly distinct type of pediatric high-grade neuroepithelial tumor (n = 18), that was identified through unsupervised visualization of genome-wide DNA methylation array data, together with copy number profiling, targeted next-generation DNA sequencing, and RNA transcriptome sequencing. DNA and/or RNA sequencing revealed recurrent fusions involving the capicua transcriptional repressor (CIC) gene in 10/10 tumor samples analyzed, with the most common fusion being CIC::LEUTX (n = 9). In addition, a CIC::NUTM1 fusion was detected in one of the tumors. Apart from the detected fusion events, no additional oncogenic alteration was identified in these tumors. The histopathological review demonstrated a morphologically heterogeneous group of high-grade neuroepithelial tumors with positive immunostaining for markers of glial differentiation in combination with weak and focal expression of synaptophysin, CD56 and CD99. All tumors were located in the supratentorial compartment, occurred during childhood (median age 8.5 years) and typically showed early relapses. In summary, we expand the spectrum of pediatric-type tumors of the CNS by reporting a previously uncharacterized group of rare high-grade neuroepithelial tumors that share a common DNA methylation signature and recurrent gene fusions involving the transcriptional repressor CIC. Downstream functional consequences of the fusion protein CIC::LEUTX and potential therapeutic implications need to be further investigated.

Abstract PD7-07: Somatic alterations in primary tumors of patients (pts) with metastatic breast cancer (MBC) may predict likelihood of brain metastasis

Abstract Background: Despite advances in treatment options, outcomes remain poor for many pts with breast cancer brain metastases (BCBMs). Identifying genomic predictors of brain metastasis from primary tumors could lead to better stratification of pts at risk and drive the development of preventative strategies. The objective of this study was to describe the landscape of genomic alterations in primary tumors from pts with MBC who subsequently did or did not develop BCBMs. Methods: We performed a case control study to identify somatic alterations in primary tumors associated with a higher incidence of brain metastases. We reviewed outcomes for 2562 unique MBC patients from a single institution who underwent targeted next-generation DNA sequencing of &amp;gt; 280 cancer-related genes (OncoPanel) from their tumor between July 1, 2013 and December 31, 2020. Pts were included in this analysis if they had at least 2 years of follow-up from date of metastatic diagnosis and OncoPanel testing on a primary breast tumor. We compared single nucleotide variants (oncogenic or likely oncogenic), copy number variation (amplification and deep deletions) and tumor mutation burden in the primary tumors of pts in this cohort. Copy number variation was corrected for Panel version and tumor purity. Wilcoxon rank sum test and Fisher exact test was used to compare genomic differences between groups. False discovery rate was used to correct for multiple hypothesis testing and q &amp;lt; 0.1 was considered significant Results: A total of 369 pts were included in the final analytic cohort. Of these, 115 were diagnosed with brain mets (cases, BM group) and 224 were not (controls, nBM group). The BM group was enriched for patients with HER2-positive breast cancer (33 vs 12.5%), consistent with previous work. In the whole cohort, the most common and clinically significant somatic alterations (oncogenic single nucleotide variants or copy number high amplification or two copy deletion) are shown in Table 1. When adjusting for subtype there were no significantly enriched SNVs in BM vs nBM group. When adjusting for subtype, FGFR1 amplification was significantly enriched in hormone receptor positive HER2 negative (HR+ HER2-) patients with BM (log2 odds ratio 1.22, q &amp;lt; 0.1). Tumor mutation burden was not significantly different in primary tumors between the BM and nBM groups (median TMB 7.3 vs 6.1, Wilcoxon p = 0.08). Pathway analysis combining all subtypes revealed that RTK_RAS pathway (log2 odds ratio 1.64, q value &amp;lt; 0.1) and TP53 pathway (log2 odds ratio 1.15, q value &amp;lt; 0.1) gene sets were significantly enriched in the BM group. When controlling for subtype, pathway analysis revealed that RTK_RAS pathway gene set was significantly enriched in HR+ HER2- BM group (log2 odds ratio 1.36 q &amp;lt; 0.1). Conclusions: In this case control series of patients with metastatic breast cancer with or without brain metastases, we found that primary tumors that are enriched for somatic alterations in the RTK_RAS and TP53 pathway may be associated with higher risk of developing brain metastases. Further validation in larger cohorts is warranted. Table 1. Frequency of somatic alterations in primary tumor by brain metastasis outcome. Citation Format: Sheheryar Kabraji, Yvonne Y. Li, Melissa E. Hughes, Hersh V. Gupta, Lauren Buckley, Janet L. Files, Ayesha Mohammed-Abreu, Anne-Marie Feeney, Greg Kirkner, Ashka Patel, Ana C. Garrido-Castro, Romualdo Barroso-Sousa, Brittany Bychkovsky, Matthew Meyerson, Sara Tolaney, Deborah A. Dillon, Bruce Johnson, Eric Winer, Andrew Cherniack, Nancy U. Lin. Somatic alterations in primary tumors of patients (pts) with metastatic breast cancer (MBC) may predict likelihood of brain metastasis [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr PD7-07.

Genetic analysis of CFH and MCP in Egyptian patients with immune-complex proliferative glomerulonephritis.

Glomerulonephritis (GN) is a complex disease with intricate underlying pathogenic mechanisms. The possible role of underlying complement dysregulation is not fully elucidated in some GN subsets, especially in the setting of autoimmunity or infection. In the current study, diagnosed cases of lupus nephritis (LN) and post-infectious GN (PIGN) were recruited for molecular genetic analysis and targeted next-generation DNA sequencing was performed for two main complement regulating genes: in the fluid phase; CFH, and on tissue surfaces; MCP. Three heterozygous pathogenic variants in CFH (Q172*, W701*, and W1096*) and one likely pathogenic heterozygous variant in MCP (C223R) have been identified in four of the studied LN cases. Additionally, among the several detected variants of uncertain significance, one novel variant (CFH:F614S) was identified in 74% of the studied LN cases and in 65% of the studied PIGN cases. This variant was detected for the first time in the Egyptian population. These findings suggest that subtle mutations may be present in complement regulating genes in patients with immune-complex mediated category of GN that may add to the disease pathogenesis. These findings also call for further studies to delineate the impact of these gene variants on the protein function, the disease course, and outcome.

Genetic Polymorphisms Associated with Vincristine Pharmacokinetics and Vincristine-Induced Peripheral Neuropathy in Pediatric Oncology Patients.

Simple SummaryVincristine is a type of chemotherapy that is often used in the treatment of children with cancer. The main side effect of vincristine is nerve damage. Patients experience symptoms such as tingling, pain or muscle weakness. Some children are more sensitive to vincristine than others, which may depend on variations in genes and in the breakdown of vincristine by the body. In this study, we investigated the effect of variations in genes on nerve damage due to vincristine and breakdown of vincristine by the body. We found that nine variations in seven genes were associated with nerve damage due to vincristine, whereas three variations in three genes were associated with the breakdown of vincristine by the body. It is important that future studies try to replicate these findings. Our findings help us towards the goal of tailoring vincristine treatment to each child, with optimal therapeutic effect while limiting nerve damage. Vincristine (VCR) is an important component of curative chemotherapy for many childhood cancers. Its main side effect is VCR-induced peripheral neuropathy (VIPN), a dose limiting toxicity. Some children are more susceptible to VIPN, which is at least partially dependent on genetic factors and pharmacokinetics (PK). In this study, we identify and replicate genetic variants associated with VCR PK and VIPN. Patient samples from a randomized clinical trial studying the effect of administration duration of VCR on VIPN in 90 patients were used. PK sampling was conducted on between one and five occasions at multiple time points. A linear two-compartment model with first-order elimination was used, and targeted next-generation DNA sequencing was performed. Genotype–trait associations were analyzed using mixed-effect models or logistic regression analysis for repeated measures, or Poisson regression analysis in which the highest VIPN score per patient was included. Nine single-nucleotide polymorphisms (SNPs) in seven genes (NDRG1, GARS, FIG4, FGD4, SEPTIN9, CEP72, and ETAA1) were associated with VIPN. Furthermore, three SNPs in three genes (MTNR1B, RAB7A and SNU13) were associated with PK of VCR. In conclusion, PK of VCR and VIPN are influenced by SNPs; upfront identification of those that lead to an altered susceptibility to VIPN or VCR exposure could help individualize VCR treatment.

Tracking Clonal Evolution of Multiple Myeloma Using Targeted Next-Generation DNA Sequencing.

Clonal evolution drives treatment failure in multiple myeloma (MM). Here, we used a custom 372-gene panel to track genetic changes occurring during MM progression at different stages of the disease. A tumor-only targeted next-generation DNA sequencing was performed on 69 samples sequentially collected from 30 MM patients. The MAPK/ERK pathway was mostly affected with KRAS mutated in 47% of patients. Acquisition and loss of mutations were observed in 63% and 37% of patients, respectively. Four different patterns of mutation evolution were found: branching-, mutation acquisition-, mutation loss- and a stable mutational pathway. Better response to anti-myeloma therapy was more frequently observed in patients who followed the mutation loss—compared to the mutation acquisition pathway. More than two-thirds of patients had druggable genes mutated (including cases of heavily pre-treated disease). Only 7% of patients had a stable copy number variants profile. Consequently, a redistribution in stages according to R-ISS between the first and paired samples (R-ISS″) was seen. The higher the R-ISS″, the higher the risk of MM progression and death. We provided new insights into the genetics of MM evolution, especially in heavily pre-treated patients. Additionally, we confirmed that redefining R-ISS at MM relapse is of high clinical value.

Abstract 3634: Integrative analysis of the contribution of genomic and clinical factors to worse clinical outcomes in non-Hispanic Black patients with colorectal cancer

Abstract Non-Hispanic Black (NHB) patients have higher incidence and higher mortality from colorectal cancer (CRC) than non-Hispanic white (NHW) patients. The extent to which this aggressive behavior in NHB patients is driven by differences in cancer genomics is presently unknown. We analyzed clinical and genomic data from 3,963 CRC patients treated at Memorial Sloan Kettering, including 336 (8%) NHB and 3,627 (92%) NHW patients. Tumors were sequenced using the clinical MSK-IMPACT test, a targeted next-generation DNA sequencing assay that identifies mutations, copy number changes and structural rearrangements in 341-505 genes using solid tissue and matched blood. No significant differences by race were observed for stage at diagnosis or sex. While the difference in median age at diagnosis was small (53.4 for NHB vs. 55 years for NHW patients, p=0.012), significantly fewer NHB patients were diagnosed after 65 years of age (17% vs. 27%, p=0.001). Right-sided colon tumors were more common among NHB patients (37% vs. 25%, p=0.02); the proportion of rectal tumors was similar in both racial groups (31% in NHB vs. 34% in NHW patients, n.s.). Hypermutated tumors, including tumors with microsatellite instability (MSI) and POLE hypermutants, were more frequent among NHW patients (12% vs. 8%, p=0.02). Right-sided tumors were more frequently MSI/hypermutated in the NHW patients (27% vs. 13%, p=0.008), whereas no differences by race were observed for left-sided colon (5% vs. 1%, n.s.) or rectal primaries (4% vs. 5%, n.s.). Genomic analysis of the non-hypermutated tumors revealed no significant differences in tumor mutational burden or fraction of genome altered between racial groups. Tumors in NHB patients were enriched in KRAS mutations (60% vs. 45%, p&amp;lt;0.001), but G12C mutations accounted only for 3% of all driver KRAS mutations in NHB patients vs. 8% of all driver mutations in NHW patients (p=0.01). BRAF mutations were more frequent in non-hypermutated NHW patients (7% vs. 3%, p=0.006), but the overall frequency of RTK/RAS pathway alterations was higher in the NHB group (74% vs. 66%, p=0.004). Even though all patients were treated at the same single institution during the study, NHB patients had shorter overall survival (OS) from the time of sequencing (median 28 months [95% CI 25-38] vs. 50 months [95% CI 47-53], p&amp;lt;0.001). NHB patients also had worse OS [HR 1.64, CI 1.3 - 2.1, p&amp;lt;0.001] in a multivariate analysis using Cox Proportional-Hazards and accounting for age at diagnosis, stage at diagnosis, body mass index, primary tumor location, MSI status and oncogenic alterations in APC, TP53, KRAS, SMAD4, and BRAF. Our data confirms that NHB patients with CRC have worse clinical outcomes than NHW patients. Clinically relevant differences in the frequency of alterations in cancer driver genes are observed based on racial origin, but they do not fully explain the difference in survival. Citation Format: Henry Walch, Anisha Luthra, Walid K. Chatila, Fan Wu, Debyani Chakravarty, Chin-Tung Chen, Rupa Sood, Dana M. Omer, Jesse J. Smith, Nikolaus Schultz, Karuna Ganesh, Julio Garcia-Aguilar, Rona Yaeger, Francisco Sanchez-Vega. Integrative analysis of the contribution of genomic and clinical factors to worse clinical outcomes in non-Hispanic Black patients with colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3634.

Design of a targeted next-generation DNA sequencing panel for pediatric T-cell lymphoblastic lymphoma to unravel biology and optimize treatment.

Low incidence and molecular heterogeneity of pediatric T-cell lymphoblastic lymphoma (T-LBL) require an international, large-scale effort to identify novel clinical biomarkers. The ongoing international clinical trial LBL2018 (NCT04043494) represents an ideal opportunity to implement a common analytic approach. Targeted next-generation sequencing is well-suited for this purpose; however, selection of relevant target genes for T-LBL remains subject of ongoing debates. Our group has recently designed and evaluated a first target panel of 80 candidate genes for T-LBL. The present study aimed at developing a novel optimized gene panel for large-scale application and to promote an international agreement on a common core panel. Small sequence variants obtained from our former study were systematically analyzed and classified with regards to pathogenic relevance, to prioritize candidate genes. Additional genes were curated from literature and online databases for a more comprehensive analysis of relevant functions and signaling pathways. The new target panel TGP-T-LBL entails 84 candidate genes which are key actors in NOTCH, PI3K-AKT, JAK-STAT, RAS signaling, epigenetic regulation, transcription, DNA repair, cell cycle regulation, and ribosomal function. From our former gene panel, 35 out of 80 candidate genes were selected for the novel panel. Forty-six out of 84 genes are currently being analyzed in the ongoing international trial LBL2018. Exploratory analysis of prognostic relevance on mutation-level suggested a potential association of PIK3CA variants c.1624G>A(p.Glu542Lys) and c.1633G>A(p.Glu545Lys) to occurrence of relapse, emphasizing particular relevance of mutation analysis in PI3K-AKT signaling. Our approach promotes comprehensive and clinically relevant mutational profiling of pediatric T-LBL.

DNA double-strand break repair gene mutation and the risk of papillary thyroid microcarcinoma: a case\u2013control study

ObjectiveTo study the relationship between DNA double-strand break (DSB) repair gene mutations and the risk of papillary thyroid microcarcinoma (PTMC).MethodsOne hundred patients with PTMC or benign thyroid nodules (BTNs) at Henan Cancer Hospital were retrospectively analyzed. The DSB repair capacity of peripheral blood T lymphocytes in the two groups was assessed by flow cytometry. Data were compared using Student’s t-test to evaluate the relationship between DSB repair capacity and the risk of PTMC. Factors influencing DSB repair capacity were analyzed by multivariate logistic regression analysis. The relationship between PTMC and DSB repair capacity was analyzed by univariate analysis. Targeted next-generation DNA sequencing was applied to screen and analyze DSB repair genes related to PTMC.ResultsThe DSB repair capacity was 31.30% in the PTMC group and 44.40% in the BTN group, with that of the former being significantly lower (P < 0.05). Multivariate logistic regression analysis of age, sex, obesity status, radiation and other factors showed that radiation exposure was positively correlated with reduced DSB repair capacity(OR = 3.642; 95% CI 1.484–8.935, P = 0.020). Moreover, univariate analysis showed that a reduction in DSB repair capacity was a risk factor for PTMC(OR = 2.333; 95% CI 1.027–5.300, P = 0.043).Targeted next-generation DNA sequencing was performed on the DSB repair genes discovered, and those that were mutated in association with PTMC were Rad50 and FANCA; Rad51 mutations were related to BTN.ConclusionRadiation exposure is positively associated with induced DSB repair gene mutations, which may cause a reduced capacity for DSB repair and eventually lead to PTMC.

EMBR-25. GENOME-WIDE GENETIC AND EPIGENETIC ASSESSMENT OF GROUP 4 MEDULLOBLASTOMA FOR IMPROVED, BIOMARKER DRIVEN, PROGNOSTICATION AND RISK-STRATIFICATION

IntroductionMedulloblastoma (MB) is the most common malignant brain tumour in children. The most frequent molecular subgroup, Group 4 (MBGrp4) accounts for ~35/40% of cases, however it has the least understood underlying biology. Clinical outcomes are heterogeneous in MBGrp4 and are not accounted for by established clinico-pathological risk factors. There is now a requirement for a comprehensive study of MBGrp4, considering established clinico-pathological features and novel molecular biomarkers to enhance risk-stratification and identify novel therapeutic targets. MethodsA clinically-annotated, retrospective MBGrp4 discovery cohort (n = 420) was generated from UK CCLG institutions, collaborating European centres and SIOP-UKCCSG-PNET3 and HIT-SIOP-PNET4 clinical trials. Contemporary, multi-omics profiling was performed. Focal and arm level copy number aberrations (CNAs) were determined from molecular inversion probe (MIP) or DNA methylation array which additionally provided next generation non-WNT/non-SHH (Grp3/Grp4) subtype classifications. Targeted next-generation DNA sequencing was performed to overlay the mutational landscape. Survival modelling was carried out with patients >3 years old who received craniospinal irradiation. ResultsMBGrp4 subtypes were assigned to 88% of tumours with available data. Subtype VIII was strongly associated with i17q (p<0.0001). The favourable-risk cytogenetic signature (2 or 3 of; chromosome 7 gain, chromosome 8 loss and/or chromosome 11 loss) associated with both subtypes VI and VII (p<0.0001). MYCN amplifications were strongly associated with subtype V (p<0.0001) in addition to 16q loss (p<0.0001). The high-risk CNA group was enriched for mutations in genes involved in chromatin remodelling (p<0.0001). Risk factors were identified from multivariate survival modelling. Subtype and CNA groups contributed to improved risk-stratification models that outperformed current clinical schemes. ConclusionComprehensive genetic and epigenetic profiling in this large retrospective cohort has improved our understanding of the molecular and clinical heterogeneity within MBGrp4. Incorporation of molecular biomarkers improved risk-stratification for MBGrp4.

TP53 mutations and CDKN2A mutations/deletions are highly recurrent molecular alterations in the malignant progression of sinonasal papillomas.

Sinonasal papillomas are benign epithelial tumors of the sinonasal tract that are associated with a synchronous or metachronous sinonasal carcinoma in a subset of cases. Our group recently identified mutually exclusive EGFR mutations and human papillomavirus (HPV) infection in inverted sinonasal papillomas and frequent KRAS mutations in oncocytic sinonasal papillomas. We also demonstrated concordant mutational and HPV infection status in sinonasal papilloma-associated sinonasal carcinomas, confirming a clonal relationship between these tumors. Despite our emerging understanding of the oncogenic mechanisms driving formation of sinonasal papillomas, little is currently known about the molecular mechanisms of malignant progression to sinonasal carcinoma. In the present study, we utilized targeted next-generation DNA sequencing to characterize the molecular landscape of a large cohort of sinonasal papilloma-associated sinonasal carcinomas. As expected, EGFR or KRAS mutations were present in the vast majority of tumors. In addition, highly recurrent TP53 mutations, CDKN2A mutations, and/or CDKN2A copy number losses were detected; overall, nearly all tumors (n = 28/29; 96.6%) harbored at least one TP53 or CDKN2A alteration. TERT copy number gains also occurred frequently (27.6%); however, no TERT promoter mutations were identified. Other recurrent molecular alterations included NFE2L2 and PIK3CA mutations and SOX2, CCND1, MYC, FGFR1, and EGFR copy number gains. Importantly, TP53 mutations and CDKN2A alterations were not detected in matched sinonasal papillomas, suggesting that these molecular events are associated with malignant transformation. Compared to aerodigestive tract squamous cell carcinomas from The Cancer Genome Atlas (TCGA) project, sinonasal papilloma-associated sinonasal carcinomas have a distinct molecular phenotype, including more frequent EGFR, KRAS, and CDKN2A mutations, TERT copy number gains, and low-risk human papillomavirus (HPV) infection. These findings shed light on the molecular mechanisms of malignant progression of sinonasal papillomas and may have important diagnostic and therapeutic implications for patients with advanced sinonasal cancer.

Targeted Next-Generation Sequencing Identifies Molecular and Genetic Events in Dedifferentiated Chondrosarcoma.

Dedifferentiated chondrosarcoma is a rare adult bone tumor with a dismal prognosis and is composed of a conventional chondrosarcoma juxtaposed to high-grade nonchondrogenic sarcoma. Dedifferentiated chondrosarcomas may represent tumor progression from a differentiated to a primitive histotype. To determine the genetic and molecular events that drive progression from a conventional chondrosarcoma to high grade nonchondrogenic sarcoma. We analyzed the genomic landscape of paired conventional and dedifferentiated components of 11 dedifferentiated chondrosarcoma using targeted next-generation DNA sequencing with immunohistochemical validation. Clinical, radiographic, and pathologic features of tumors were reviewed. Capture-based DNA sequencing targeting the coding regions of 479 cancer genes and select introns was performed. The tumors arose in the femur (n = 4; 36%), scapula (n = 3; 27%), pelvis (n = 3; 27%), and humerus (n = 1; 9%) of 7 men (64%) and 4 women (36%; median age, 61 years). DNA was adequate for sequencing from all 11 dedifferentiated components (100%) and 9 paired conventional chondrosarcoma components (82%). All tumors (100%) harbored either IDH1 p.R132 or IDH2 p.R172S hotspot mutations. Seven tumors (64%) displayed COL2A1 alterations. TERT promoter mutations were present in 5 of 9 pairs (56%) and 2 (22%) additional unpaired dedifferentiated components. IDH1/2, COL2A1, and TERT mutations were identical in both components of the paired samples. Pathogenic missense or truncating mutations in TP53 and large-scale copy number alterations were more common in dedifferentiated components than in those of matched conventional components. The results support IDH1/2, COL2A1, and TERT promoter mutations being common in dedifferentiated chondrosarcoma and as likely early events in progression, whereas inactivating mutation of TP53 and high-level copy number alterations may be later events in the dedifferentiated phenotype.

Abstract 1273: RASSF1 promoter hypermethylation is associated with activated CTNNB1 pathways in hepatocellular carcinoma

Abstract BACKGROUND: The promoter-region of the Ras association domain family member 1 (RASSF1) gene is frequently hypermethylated in several solid tumors, including hepatocellular carcinoma (HCC). Wnt-pathway deregulation and increased accumulation of nuclear catenin-beta-1 (CTNNB1) has been associated with RASSF1 inactivation, and more recently, with diminished response to immune checkpoint blockade in HCC. There is limited data from clinical tumor and liquid biopsy samples on the relationship between RASSF1 promoter methylation and Wnt beta-catenin pathway activation status in HCC. METHODS: Pyrosequencing was performed to assess RASSF1 promoter methylation in DNA from paired HCC tumor/adjacent-liver tissues as well as cell-free DNA (cfDNA) and buffy coat DNA from blood samples collected in Streck cfDNA tubes from 40 patients with surgically-resectable HCC participating in an IRB-approved diagnostic clinical trial (NCT01395030). All blood samples were collected pre-operatively while all tissue samples were collected upon surgical tumor resection. The DNA methylation analysis results from tumors were compared to a CTNNB1 pathway activation gene expression signature obtained from the same tumor samples. Targeted next-generation DNA sequencing (NGS) was also performed on a limited number of cfDNA samples using a 56 gene Oncology Panel (56G v2, Swift Bioscience) to obtain mutational profiles (including CTNNB1 mutation status) from each HCC case. Allele variant detection was performed using the ERASE-Seq algorithm (Fluxion Biosciences). RESULTS: RASSF1 promoter hypermethylation was detected in 35/40 tumor samples. Levels of RASSF1 promoter methylation were significantly higher in tumor as compared to liver tissue DNA (55.2% vs. 23.5%, p &amp;lt; 0.0001). Methylation levels in the RASSF1 promoter were also significantly higher in cfDNA as compared to buffy-coat DNA (7.5% vs. 0.4%, p &amp;lt; 0.0001). Tumors enriched for genes associated with Wnt beta-catenin pathway activation demonstrated significantly higher RASSF1 promoter methylation as compared to tumors enriched for immune/interferon-related genes (63% vs. 20%, Tukey p = 0.0004). Pilot liquid biopsy studies involving DNA NGS and methylation analysis in a subset of pre-operative blood samples also revealed higher RASSF1 promoter methylation among the cell-free DNA samples carrying activating CTNNB1 exon 3 mutations as compared to p53 mutations (23.4% vs. 1.4%, Tukey p = 0.009) and PIK3 mutations (23.4% vs. 1.5%, Tukey p = 0.031). RASSF1 promoter methylation in tumor tissue DNA was also significantly higher in patients with liquid biopsy detected CTNNB1-mutations as compared to p53-mutations (65.5% versus 37%, p = 0.030). CONCLUSION: CTNNB1 mutations and related gene expression patterns may be associated with RASSF1 promoter hypermethylation in HCC. The clinical implications for immunotherapy and the mechanisms underlying these associations deserve further investigation. Citation Format: Sandi Alexander Kwee, Karolina Peplowska, Christine Farrar, Linda Wong, Min-ae Song, Maarit Tiirikainen. RASSF1 promoter hypermethylation is associated with activated CTNNB1 pathways in hepatocellular carcinoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1273.

Novel Genetic Variations in Acute Myeloid Leukemia in Pakistani Population.

Acute myeloid leukemia (AML) is a hematological malignancy characterized by clonal expansion of blast cells that exhibit great genetic heterogeneity. In this study, we describe the mutational landscape and its clinico-pathological significance in 26 myeloid neoplasm patients from a South Asian population (Pakistan) by using ultra-deep targeted next-generation DNA sequencing of 54 genes (∼5000×) and its subsequent bioinformatics analysis. The data analysis indicated novel non-silent somatic mutational events previously not reported in AML, including nine non-synonymous and one stop-gain mutations. Notably, two recurrent somatic non-synonymous mutations, i.e., STAG2 (causing p.L526F) and BCORL1 (p.A400V), were observed in three unrelated cases each. The BCOR was found to have three independent non-synonymous somatic mutations in three cases. Further, the SRSF2 with a protein truncating somatic mutation (p.Q88X) was observed for the first time in AML in this study. The prioritization of germline mutations with ClinVar, SIFT, Polyphen2, and Combined Annotation Dependent Depletion (CADD) highlighted 18 predicted deleterious/pathogenic mutations, including two recurrent deleterious mutations, i.e., a novel heterozygous non-synonymous SNV in GATA2 (p.T358P) and a frameshift insertion in NPM1 (p.L258fs), found in two unrelated cases each. The WT1 was observed with three independent potential detrimental germline mutations in three different cases. Collectively, non-silent somatic and/or germline mutations were observed in 23 (88.46%) of the cases (0.92 mutation per case). Furthermore, the pharmGKB database exploration showed a missense SNV rs1042522 in TP53, exhibiting decreased response to anti-cancer drugs, in 19 (73%) of the cases. This genomic profiling of AML provides deep insight into the disease pathophysiology. Identification of pharmacogenomics markers will help to adopt personalized approach for the management of AML patients in Pakistan.

Genomic profiling of breast cancer brain metastases reveals targetable alterations.

2525 Background: Genomic characterization of breast cancer brain metastases (BCBMs) has thus far been limited. The objective of this study was to describe the landscape of genomic alterations in patients (pts) with BCBMs. Methods: Targeted next-generation DNA sequencing of &gt; 300 cancer-related genes (OncoPanel) was prospectively performed on primary and metastatic (met) tumors in 321 pts with a diagnosis of BCBM between August 2016 and April 2019 at Dana-Farber Cancer Institute (table). Enrichment analysis of genomic alterations was performed using a two-sided Fisher exact test and differences in tumor mutation burden (TMB) between groups were assessed using two-sided Mann-Whitney U test. Multiple comparison correction was performed using the Benjamini-Hochberg procedure. Results: All subtypes were represented in BCBM (25 HR+/HER2-; 24 HR+/HER2+; 27 HR-/HER2+; 18 TNBC; 5 unknown; n = 99) and extracranial (EC) samples: (96 HR+/HER2-; 32 HR+/HER2+; 22 HR-/HER2+; 41 TNBC; 31 unknown; n = 222). BCBMs were found most commonly to have mutations or copy number alterations in TP53, ERBB2, PIK3CA, GATA3, PTEN, ESR1, CDH1, BRCA2, ARID1A, BRCA1 (&gt;5% frequency, table). Two pts acquired ERBB2 amplification (amp) between the matched primary breast sample and brain met. In pair-wise comparisons of BCBMs to unmatched primaries or EC mets, only ERBB2 amp was significantly enriched (table, † = adjusted p &lt; 0.05). There was no significant difference in TMB between BCBM and EC mets (median 9.12 vs 7.26, p = 0.15). In contrast, TMB was significantly higher in BCBMs compared to unmatched primaries (median 9.12 vs 7.26, p=0.005). Conclusions: BCBMs display similar mutations and copy number alterations compared to primary tumors and EC mets in pts with BCBM. These data suggest that BCBMs contain actionable genomic alterations that are most often also reflected in EC disease. Alterations in ERBB2, PIK3CA/PTEN, and BRCA1/2 represent potentially targetable alterations in pts with BCBM. [Table: see text]

Rare Variants in Specific Lysosomal Genes Are Associated With Parkinson's Disease.

Impaired lysosomal degradation of α-synuclein and other cellular constituents may play an important role in Parkinson's disease (PD). Rare genetic variants in the glucocerebrosidase (GBA) gene were consistently associated with PD. Here we examine the association between rare variants in lysosomal candidate genes and PD. We investigated the association between PD and rare genetic variants in 23 lysosomal candidate genes in 4096 patients with PD and an equal number of controls using pooled targeted next-generation DNA sequencing. Genewise association of rare variants in cases or controls was analyzed using the optimized sequence kernel association test with Bonferroni correction for the 23 tested genes. We confirm the association of rare variants in GBA with PD and report novel associations for rare variants in ATP13A2, LAMP1, TMEM175, and VPS13C. Rare variants in selected lysosomal genes, first and foremost GBA, are associated with PD. Rare variants in ATP13A2 and VPC13C previously linked to monogenic PD and more common variants in TMEM175 and VPS13C previously linked to sporadic PD in genome-wide association studies are associated with PD. © 2020 International Parkinson and Movement Disorder Society.