Targeted Next-generation Sequencing Platform Research Articles

Tumour purity as an underlying key factor in tumour mutation detection in colorectal cancer

Tumour purity as an underlying key factor in tumour mutation detection in colorectal cancer

Emerging Epidemiological Data on Rare Intellectual Disability Syndromes from Analyzing the Data of a Large Iranian Cohort.

Intellectual disability (ID) is a genetically heterogeneous condition, and so far, 1679 human genes have been identified for this phenotype. Countries with a high rate of parental consanguinity, such as Iran, provide an excellent opportunity to identify the remaining novel ID genes, especially those with an autosomal recessive (AR) mode of inheritance. This study aimed to investigate the most prevalent ID genes identified via next-generation sequencing (NGS) in a large ID cohort at the Genetics Research Center (GRC) of the University of Social Welfare and Rehabilitation Sciences. First, we surveyed the epidemiological data of 619 of 1295 families in our ID cohort, who referred to the Genetics Research Center from all over the country between 2004 and 2021 for genetic investigation via the NGS pipeline. We then compared our data with those of several prominent studies conducted in consanguineous countries. Data analysis, including cohort data extraction, categorization, and comparison, was performed using the R program version 4.1.2. We categorized the most common ID genes that were mutated in more than two families into 17 categories. The most common syndromic ID in our cohort was AP4 deficiency syndrome, and the most common non-syndromic autosomal recessive intellectual disability (ARID) gene was ASPM. We identified two unrelated families for the 36 ID genes. We found 14 genes in common between our cohort and the Arab and Pakistani groups, of which three genes (AP4M1, AP4S1, and ADGRG1) were repeated more than once. To date, there has been no comprehensive targeted NGS platform for the detection of ID genes in our country. Due to the large sample size of our study, our data may provide the initial step toward designing an indigenously targeted NGS platform for the diagnosis of ID, especially common ARID in our population.

Circulating Tumor DNA Dynamics and Treatment Outcome of Regorafenib in Metastatic Colorectal Cancer.

Circulating tumor DNA (ctDNA) is emerging as a valuable non-invasive tool to identify tumor heterogeneity and tumor burden. This study investigated ctDNA dynamics in metastatic colorectal cancer patients treated with regorafenib. In this prospective biomarker study, plasma cell-free DNA (cfDNA) samples obtained at baseline, at the first response evaluation after 2 cycles of treatment, and at the time of progressive disease were sequenced using a targeted next-generation sequencing platform which included 106 genes. A total of 285 blood samples from 110 patients were analyzed. Higher baseline cfDNA concentration was associated with worse progression-free survival (PFS) and overall survival (OS). After 2 cycles of treatment, variant allele frequency (VAF) in the majority of ctDNA mutations decreased with a mean relative change of -31.6%. Decreases in the VAF of TP53, APC, TCF7L2, and ROS1 after 2 cycles of regorafenib were associated with longer PFS. We used the sum of VAF at each time point as a surrogate for the overall ctDNA burden. A reduction in sum (VAF) of ≥ 50% after 2 cycles was associated with longer PFS (6.1 vs. 2.7 months, p=0.002), OS (11.3 vs. 5.9 months, p=0.001), and higher disease control rate (86.3% vs. 51.1%, p < 0.001). VAF of the majority of the ctDNA mutations increased at the time of disease progression, and VAF of BRAF increased markedly. Reduction in ctDNA burden as estimated by sum (VAF) could be used to predict treatment outcome of regorafenib.

Clinical and Genomic Characterization of Bladder Carcinomas With Glandular Phenotype.

To compare oncologic outcomes and genomic alteration profiles in patients with bladder and urachal adenocarcinoma, urothelial carcinoma (UC) with glandular differentiation, and UC, not otherwise specified (NOS) undergoing surgical resection, with emphasis on response to systemic therapy. We identified patients with bladder cancer with glandular variants who underwent surgical resection at Memorial Sloan Kettering from 1995 to 2018 (surgical cohort) and/or patients who had tumor sequencing using a targeted next-generation sequencing platform (genomics cohort). Pathologic complete and partial response rates to neoadjuvant chemotherapy (NAC) and recurrence-free and cancer-specific survival were measured. Alteration frequencies between histologic subtypes were compared. Thirty-seven patients with bladder adenocarcinoma, 46 with urachal adenocarcinoma, 84 with UC with glandular differentiation, and 1,049 with UC, NOS comprised the surgical cohort. Despite more advanced disease in patients with bladder and urachal adenocarcinoma, no significant differences in recurrence or cancer-specific survival by histology were observed after adjusting for stage. In patients with UC with glandular differentiation, NAC resulted in partial (≤ pT1N0) and complete (pT0N0) responses in 28% and 17%, respectively. Bladder and urachal adenocarcinoma genomic profiles resembled colorectal adenocarcinoma with frequent TP53, KRAS, and PIK3CA alterations while the genomic profile of UC with glandular differentiation more closely resembled UC, NOS. Limitations include retrospective nature of analysis and small numbers of nonurothelial histology specimens. The genomic profile of bladder adenocarcinomas resembled colorectal adenocarcinomas, whereas UC with glandular differentiation more closely resembled UC, NOS. Differences in outcomes among patients with glandular bladder cancer variants undergoing surgical resection were largely driven by differences in stage. Cisplatin-based NAC demonstrated activity in UC with glandular differentiation, suggesting NAC should be considered for this histologic variant.

Dynamic changes in longitudinal circulating tumour DNA profile during metastatic colorectal cancer treatment.

Circulating tumour DNA (ctDNA) has been spotlighted as an attractive biomarker because of its easy accessibility and real-time representation of tumour genetic profile. However, the clinical utility of longitudinal ctDNA monitoring has not been clearly defined. Serial blood samples were obtained from metastatic colorectal cancer patients undergoing first-line chemotherapy. ctDNA was sequenced using a targeted next-generation sequencing platform which included 106 genes. Changes in ctDNA profile and treatment outcome were comprehensively analysed. A total of 272 samples from 62 patients were analysed. In all, 90.3% of patients had detectable ctDNA mutation before treatment. ctDNA clearance after chemotherapy was associated with longer progression-free survival which was independent of radiological response (adjusted hazard ratio 0.22, 95% confidence interval 0.10-0.46). Longitudinal monitoring was able to detect ctDNA progression which preceded radiological progressive disease (PD) in 58.1% (median 3.3 months). Diverse resistant mutations (34.9%) and gene amplification (7.0%) at the time of PD were discovered. For 16.3% of the PD patients, the newly identified mutations could be potential candidates of targeted therapy or clinical trial. ctDNA profile provided a more accurate landscape of tumour and dynamic changes compared to radiological evaluation. Longitudinal ctDNA monitoring may improve personalised treatment decision-making.

The concordance rates of an initial trophectoderm biopsy with the rest of the embryo using PGTseq, a targeted next-generation sequencing platform for preimplantation genetic testing-aneuploidy

To determine how often the results of a single trophectoderm (TE) biopsy tested by PGTseq, a targeted next-generation sequencing preimplantation genetic testing for aneuploidy technology, reflect the biology of the rest of the embryo. Blinded prospective cohort study. University-affiliated private practice. A total of 300 blastocysts were donated; 113 of these embryos were euploid; 163 embryos possessed at least one whole chromosome aneuploidy; and 24 embryos were negative for whole chromosome aneuploidy but possessed at least one secondary finding on initial TE biopsy. All blastocysts underwent rebiopsy and preimplantation genetic testing for aneuploidy on the PGTseq platform. Partial concordance rate per embryo, total concordance rate per embryo, and total concordance rate per chromosomal event. An initial TE biopsy result of euploidy or whole chromosome aneuploidy was reconfirmed in >99% of rebiopsied samples, affirming that meiotic errors are manifested in almost the entire embryo. In contrast, results of whole chromosome or segmental mosaicism were confirmed in 15%-18% of subsequent rebiopsies, suggesting that mitotic events are only sporadically seen throughout the embryo. Segmental aneuploidy was confirmed in 56.6% of rebiopsied samples, identifying a mixed meiotic and mitotic etiology for such abnormalities. A euploid or aneuploid result on the PGTseq platform is highly concordant with the rest of the embryo's ploidy status. The rarer confirmation of whole chromosome mosaic and segmental mosaic results suggest that these mosaics are suitable for embryo transfer. Segmental aneuploidy, with higher concordance rates throughout the embryo, may represent a different biologic etiology compared to mosaic embryos.

Expanding the spectrum of thyroid carcinoma with somatic DICER1 mutation: a survey of 829 thyroid carcinomas using MSK-IMPACT next-generation sequencing platform.

DICER1 gene encodes an RNaseIII endoribonuclease essential for the cleavage of pre-microRNA to mature microRNA. Germline DICER1 mutation results in DICER syndrome, a cancer predisposition syndrome which manifests in the thyroid gland as early-onset multinodular goiter and increased risk for differentiated thyroid carcinoma. Recently, somatic DICER1 mutations were described in various thyroid neoplasms, including follicular adenoma, papillary thyroid carcinoma, follicular carcinoma, and poorly differentiated thyroid carcinoma. In this study, we identified and described 14 cases (1.7%) with somatic DICER1 mutations from a cohort of 829 patients with thyroid follicular cell-derived thyroid carcinomas which were sequenced using MSK-IMPACT targeted next-generation sequencing platform. We expanded the histologic spectrum of thyroid carcinomas with somatic DICER1 mutations to include Hurthle cell carcinoma, high-grade differentiated thyroid carcinoma, and anaplastic thyroid carcinoma. All patients were adults with a median age of diagnosis of 59years (range: 22-82). Although rare, a subset of thyroid cancers, including the aggressive subtypes, display somatic DICER1 mutations, some of which have oncogenic potential.

Clinical application of next-generation sequencing in cancer patients.

e15090 Background: Next-generation sequencing (NGS) has been moving rapidly from pure cancer genomics research towards clinical oncology to provide a rigorous personalized approach to treating cancer. Still there is a number of limitations to overcome before we are ready to fully introduce NGS into everyday clinical decision-making. Here we are sharing our experience with using targeted NGS platforms in clinical practice. Methods: We retrospectively evaluated the NGS results and the treatments, provided to the patients after NGS testing between Marсh 2019 and January 2021. The patients’ clinical characteristics, response to treatment and genomic mutation profiles were reviewed. Coprimary endpoints were the percentage of patients with NGS revealed targeted therapy options and the percentage of patients who received mutation-driven therapy. Results: Samples from 95 patients were tested, most frequently GI tumors (n=25, 26%), hepatobiliary tumors (n=11, 12%), breast cancer (n=10, 11%) gynecological tumors (n=10, 11%), central nervous system tumors (n=7, 7%) and others (n=32, 33%). An average of two gene alterations were identified per patient (range 0-15). The 95% (n=90) of samples had at least one detected mutation, and in 42% (n=40) of cases gene mutations happened to be targetable with existing drugs, including off-label prescriptions. Mutation-driven therapy was performed in 9,5% cases (n=10) with clinically meaningful response in 5 patients (2 patients with ovarian cancer, 2 patients with colon cancer and 1 melanoma). Conclusion: Mutational profiling using a targeted NGS panel has identified potentially targetable alterations in a vast majority of advanced cancer patients. The assay has revealed additional therapeutic options in 42% of patients. However, due to the short-term follow-up, just a small number of patients have received mutation-driven therapy. Further study will be required to determine if the mutation-driven therapy will prove to be clinically meaningful in these patients.

Minimalist approaches to cancer tissue-of-origin classification by DNA methylation

Classification of cancers by tissue-of-origin is fundamental to diagnostic pathology. While the combination of clinical data, tissue histology, and immunohistochemistry is usually sufficient, there remains a small but not insignificant proportion of difficult-to-classify cases. These challenging cases provide justification for ancillary molecular testing, including high-throughput DNA methylation array profiling, which promises cell-of-origin information and compatibility with formalin-fixed specimens. While diagnostically powerful, methylation profiling platforms are costly and technically challenging to implement, particularly for less well-resourced laboratories. To address this, we simulated the performance of “minimalist” methylation-based tests for cancer classification using publicly-available and internal institutional profiling data. These analyses showed that small and focused sets of the most informative CpG biomarkers from the arrays are sufficient for accurate diagnoses. As an illustrative example, one classifier, using information from just 53 out of about 450,000 available CpG probes, achieved an accuracy of 94.5% on 2575 fresh primary validation cases across 28 cancer types from The Cancer Genome Atlas Network. By training minimalist classifiers on formalin-fixed primary and metastatic cases, generally high accuracies were also achieved on additional datasets. These results support the potential of minimalist methylation testing, possibly via quantitative PCR and targeted next-generation sequencing platforms, in cancer classification.

Adverse histology, homozygous loss of CDKN2A/B, and complex genomic alterations in locally advanced/metastatic renal mucinous tubular and spindle cell carcinoma.

Mucinous tubular and spindle cell carcinoma (MTSCC) is a rare subtype of renal cell carcinoma with characteristic histologic features and chromosomal alterations. Although typically indolent, a small subset of cases has been reported to exhibit aggressive clinical behavior. We retrospectively identified 33 patients with MTSCC, consisting of 10 cases of locally advanced / metastatic-MTSCC (pT3 or N1 or M1) and 23 kidney confined-MTSCC (pT1/T2) without disease recurrence or progression. Utilizing a single nucleotide polymorphism array and a targeted next-generation sequencing platform, we examined genome-wide molecular alterations in 24 cases, including 11 available samples from 8 patients with locally advanced / metastatic-MTSCC. Ten patients with locally advanced / metastatic-MTSCC were 8 females (80%) and 2 males (20%). At nephrectomy, 7 of these 10 cases (70%) were pT3 or pN1 while the remaining 3 (30%) were pT1/T2. Eight patients (80%) developed metastases and common sites included lymph node (4, 40%), bone (4, 40%), and retroperitoneum (3, 30%). Four patients died of disease (40%) during follow-up. Locally advanced / metastatic-MTSCCs shared typical MTSCC genomic profiles with loss of chromosomes 1, 4, 6, 8, 9, 13, 14, 15, and 22, while some exhibited additional complex genomic alterations, most frequently a relative gain of 1q (7/8). Homozygous loss of CDKN2A/B was observed in 3 (38%) locally advanced / metastatic-MTSCCs. Tumor necrosis, solid nested/sheet pattern, irregular trabecular/single-file infiltration in a desmoplastic stroma, lymphovascular space invasion, and increased mitotic activity were associated with locally advanced / metastatic-MTSCCs (all p < 0.05). Our findings reveal that MTSCCs with aggressive clinical behavior have progressed through clonal evolution; CDKN2A/B deletion and additional complex genomic abnormalities may contribute to this process. Recognizing the morphologic presentation of high grade MTSCC and evaluating adverse histologic features seen in these tumors can help establish a definitive diagnosis and stratify patients for treatment and prognostication.

Impact of Somatic Mutations in Non-Small-Cell Lung Cancer: A Retrospective Study of a Chinese Cohort.

BackgroundSomatic mutations are important biomarkers for selecting an optimal targeted therapy and predicting outcomes for non-small-cell lung cancer (NSCLC) patients that are often detected from tissue samples. However, tissue samples are not always readily available from these patients. The exploration of using circulating tumor DNA (ctDNA) to identify somatic mutations offers an alternative source that should be explored.MethodsIn this retrospective study, we included 280 patients diagnosed with adenocarcinoma between 2017 and 2018 in a hospital in eastern China. Tissue or ctDNA was collected, and a wide spectrum of somatic mutations was analyzed by targeted next-generation sequencing platforms. Associations among the mutation status, biomarkers, screening methods, disease stages, and interaction with treatment with overall survival (OS) were investigated.ResultsWe found that the EGFR L858R mutation was the most frequently identified mutation in adenocarcinoma in this population by both methods, followed by KRAS (p=3.7e-09), PIK3CA (p=5e-04), and HER2 mutations (p=6.3e-03). We observed that EGFR mutations were significantly mutually exclusive with KRAS, HER2, and MET. FGFR1 mutations were significantly more abundantly detected in the ctDNA group. We found an interaction effect between EGFR mutation and target therapies. The ability of the targeted therapy to improve OS in patients with a single EGFR mutation (HR=0.069, p=0.07) approached significance, but this was not the case for the patients with more than one EGFR mutation or without an EGFR mutation (HR=0.813, p=0.725). Furthermore, the effect of chemotherapy was more predominant in the EGFR group in comparison to the control group.ConclusionThese findings provide useful information on the distribution of somatic mutations via different screening methods and how this related to the optimal treatment selection in Chinese patients with NSCLC.

Abstract P4-09-12: A prospective evaluation of comprehensive tumor profiling busing a targeted Next-generation sequencing for Japanese breast cancer patients

Abstract Background: Oncogenic genetic alterations leading to identify patients who may benefit from the target therapy are essential biomarkers for them, and to review widely what kind of driver gene alteration that each cancer has is important for effective cancer treatments. Next-generation sequencing (NGS) is a powerful tool to comprehensively analyze driver gene mutations. In Japan, some multiplex cancer genetic testings were covered from national health insurance from June, 2019. Therefore, we reviewed our data using a targeted NGS platform (FoundationOne CDx (F1CDx)) in Japanese breast cancer patients. Methods: This study included 105 local advanced or metastatic breast cancer patients in Kyushu University Hospital between October 2018 and June 2019. We prospectively assessed NGS results, clinical characteristics and therapies received for the patients. Primary endpoint was a frequency at which actionable genetic alterations were detected, and coprimary secondary endpoints were the sequence success rate, the rate at which the corresponding therapeutic agent was administered, the percentage of agreement to results with approved in vitro diagnostic agents, and the overall survival. This study was approved by the Institutional Review Board of our hospital (No. 758-00). Results: Samples from 105 breast cancer patients were tested, all of who were women. Then, 99 samples (94.3 %) were success for sequencing and 6 (5.7 %) were failure. Among the succeeded samples, tested from primary tumor is 71 (71.7 %) and metastasis is 34 (34.3 %), while 62 (62.6 %) were biopsied tumor specimen and 43 (37.4 %) resected. The clinical subtypes of them were 45 HR +/ HER2 - (45.5 %), 22 HER2+ (22.2 %), and 32 TNBC (32.3 %). Based on the biomarker findings from F1CDx, microsatellite statuses in 97 samples were stable and 1 sample high, who was diagnosed with Lynch syndrome. Regarding tumor mutation burden (TMB), of 98 patients, 3 had high TMB (&amp;gt;19 mutations/mb), 31 had intermediate TMB (6-19 mutations/mb), 63 had low TMB (1-5 mutations/mb) and 1 were unable to be determinated. The average was 6.7 mutations/mb. The most frequent alteration is TP53 (54.5 %), PIK3CA (39.4 %) and ERBB2 (29.3 %). 87 patients (87.8 %) detected the alterations leading to some therapeutic options based on genetic profiling. 25 patients with metastases had taken genetic testing for BRCA1/2 germline mutation (gBRCA1/2mut), which isnamed BRACAnalysis. In all of 5 patients who had gBRCA1/2muts the same spot mutations were also detected with F1CDx. In 2 of 20 patients had negative gBRCA1/2mut, somatic BRCA1/2muts were newly detected with F1CDx. In 9 of the other patients had not taken the genetic testing, somatic BRCA1/2muts were founded. 18 tumors with HER2/Immunohistochemical staining (IHC) =3+ were judged as ERBB2 amplification, 3 tumors with HER2/IHC=2+ and HER2/FISH positive as low amplification, and 18 tumors with HER2/IHC=2+ and HER2/FISH negative as no amplification. Moreover, there are were 3 patients who newly confirmed ERBB2 amplification and 4 patients who confirmed ERBB2 mutations in this study. Conclusions: The multiplex cancer genetic testing could help identify actionable alterations for the breast cancer patients. NGS results may add some new therapeutic options to standard therapies. Citation Format: Hitomi Kawaji, Makoto Kubo, Nami Yamashita, Yurina Harada, Akiko Shimazaki, Saori Hayashi, Kanako Kurata, Mai Yamada, Kazuhisa Kaneshiro, Masaya Kai, Eiji Baba, Yoshinao Oda, Masafumi Nakamura. A prospective evaluation of comprehensive tumor profiling busing a targeted Next-generation sequencing for Japanese breast cancer patients [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P4-09-12.

Targeted next-generation sequencing of 565 neuro-oncology patients at UCLA: A single-institution experience.

BackgroundTargeted next-generation sequencing (NGS) is frequently obtained at the University of California, Los Angeles (UCLA) for clinical characterization of CNS tumors. In this study, we describe the diagnostic reliability of the Foundation Medicine (FM) targeted NGS platform and its ability to explore and identify tumor characteristics of prognostic significance in gliomas.MethodsNeuro-oncology patients seen at UCLA who have received FM testing between August 2012 and March 2019 were included in this study, and all mutations from FM test reports were recorded. Initial tumor diagnoses and diagnostic markers found via standard clinical methods were obtained from pathology reports. With overall and progression-free survival data, elastic net regularized Cox regression and Cox proportional hazards models were used to determine whether any mutations of unknown significance detected by FM could predict patient outcome in glioblastoma (GBM).ResultsSix hundred and three samples tested by FM from 565 distinct patients were identified. Concordance of diagnostic markers was high between standard clinical testing methods and FM. Oligodendroglial markers detected via FM were highly correlated with 1p19q codeletion in IDH mutated gliomas. FM testing of multiple tumor samples from the same patient demonstrated temporal and spatial mutational heterogeneity. Mutations in BCORL1, ERBB4, and PALB2, which are mutations of unknown significance in GBM, were shown to be statistically significant in predicting patient outcome.ConclusionsIn our large cohort, we found that targeted NGS can both reliably and efficiently detect important diagnostic markers in CNS tumors.

Tumour mutational burden ring trial: Evaluation of targeted next-generation sequencing platforms for implementation in clinical practice

Abstract Background Tumour mutational burden (TMB) is a measurement of DNA variants in a tumour and is a potential biomarker of response to Immune Checkpoint Inhibitor (ICI) therapy. Patients with non-small cell lung cancer (NSCLC) whose tumours have a high TMB (≥10 mutations/Mb) might benefit from upfront ICI combination therapy. Clinical trials have measured TMB using Whole Exome Sequencing (WES) and/or the FoundationOne CDx (F1CDx) assay. In parallel, several commercial next-generation sequencing (NGS) assays have become available. Before implementation of TMB testing in clinical practice, technical and clinical validation are needed. Hence, a multi-center study was organised to establish concordance between several TMB assays. Methods Fifteen resection NSCLC formalin-fixed paraffin-embedded (FFPE) samples with broad TMB range were selected. Each participant received extracted DNA and was asked to report TMB values using their own protocol. In parallel, FFPE slides were analysed with F1CDx. Eight labs participated using five different methods: Oncomine TML assay (Thermofisher; n = 4), TSO500 assay (Illumina; n = 1), NEOplus assay (NEO New Oncology; n = 1), a 0,4 Mb targeted resequencing lab-developed assay (LDT; n = 1) and WES using three different TMB calculation methods (n = 1). Correlations between each platform and F1CDx were calculated. Also, the reported TMB category (high vs low) of the different platforms in comparison to F1CDx was evaluated for the fifteen samples. Results Assessment of TMB values obtained by the platforms and F1CDx demonstrated a high correlation (R2 between 0.81 and 0.94), except for the smaller LDT (R2 = 0.53). The TMB category (high vs low) reported by each platform showed concordance with the F1CDx category for eleven (73%) to thirteen (93%) of the fifteen samples. From the fifteen samples, the same category was reported by all different platforms for seven (47%) samples. Conclusion Our data show that assays from different providers can be used to predict TMB. However, samples with a TMB value around the cut-off of 10 mut/Mb are challenging and interpretation should occur with caution. Further studies are required before implementing these assays in routine clinical diagnosis. Legal entity responsible for the study The authors. Funding Bristol-Myers Squibb. Disclosure S. Lambin: Research grant / Funding (institution): Bristol-Myers Squibb. All other authors have declared no conflicts of interest.

Distinctive mechanisms underlie the loss of SMARCB1 protein expression in renal medullary carcinoma: morphologic and molecular analysis of 20 cases

Renal medullary carcinoma is a rare but highly aggressive type of renal cancer occurring in patients with sickle cell trait or rarely with other hemoglobinopathies. Loss of SMARCB1 protein expression, a core subunit of the switch/sucrose nonfermentable (SWI/SNF) chromatin remodeling complex, has emerged as a key diagnostic feature of these tumors. However, the molecular mechanism underlying this loss remains unclear. We retrospectively identified 20 patients diagnosed with renal medullary carcinoma at two institutions from 1996 to 2017. All patients were confirmed to have sickle cell trait, and all tumors exhibited a loss of SMARCB1 protein expression by immunohistochemistry. The status of SMARCB1 locus was examined by fluorescence in situ hybridization (FISH) using 3-color probes, and somatic alterations were detected by targeted next-generation sequencing platforms. FISH analysis of all 20 cases revealed 11 (55%) with concurrent hemizygous loss and translocation of SMARCB1, 6 (30%) with homozygous loss of SMARCB1, and 3 (15%) without structural or copy number alterations of SMARCB1 despite protein loss. Targeted sequencing revealed a pathogenic somatic mutation of SMARCB1 in one of these 3 cases that were negative by FISH. Tumors in the 3 subsets with different FISH findings largely exhibited similar clinicopathologic features, however, homozygous SMARCB1 deletion was found to show a significant association with the solid growth pattern, whereas tumors dominated by reticular/cribriform growth were enriched for SMARCB1 translocation. Taken together, we demonstrate that different molecular mechanisms underlie the loss of SMARCB1 expression in renal medullary carcinoma. Biallelic inactivation of SMARCB1 occurs in a large majority of cases either via concurrent hemizygous loss and translocation disrupting SMARCB1 or by homozygous loss.

Targeted NGS Platforms for Genetic Screening and Gene Discovery in Primary Immunodeficiencies.

Background: Primary Immunodeficiencies (PIDs) are a heterogeneous group of genetic immune disorders. While some PIDs can manifest with more than one phenotype, signs, and symptoms of various PIDs overlap considerably. Recently, novel defects in immune-related genes and additional variants in previously reported genes responsible for PIDs have been successfully identified by Next Generation Sequencing (NGS), allowing the recognition of a broad spectrum of disorders.Objective: To evaluate the strength and weakness of targeted NGS sequencing using custom-made Ion Torrent and Haloplex (Agilent) panels for diagnostics and research purposes.Methods: Five different panels including known and candidate genes were used to screen 105 patients with distinct PID features divided in three main PID categories: T cell defects, Humoral defects and Other PIDs. The Ion Torrent sequencing platform was used in 73 patients. Among these, 18 selected patients without a molecular diagnosis and 32 additional patients were analyzed by Haloplex enrichment technology.Results: The complementary use of the two custom-made targeted sequencing approaches allowed the identification of causative variants in 28.6% (n = 30) of patients. Twenty-two out of 73 (34.6%) patients were diagnosed by Ion Torrent. In this group 20 were included in the SCID/CID category. Eight out of 50 (16%) patients were diagnosed by Haloplex workflow. Ion Torrent method was highly successful for those cases with well-defined phenotypes for immunological and clinical presentation. The Haloplex approach was able to diagnose 4 SCID/CID patients and 4 additional patients with complex and extended phenotypes, embracing all three PID categories in which this approach was more efficient. Both technologies showed good gene coverage.Conclusions: NGS technology represents a powerful approach in the complex field of rare disorders but its different application should be weighted. A relatively small NGS target panel can be successfully applied for a robust diagnostic suspicion, while when the spectrum of clinical phenotypes overlaps more than one PID an in-depth NGS analysis is required, including also whole exome/genome sequencing to identify the causative gene.

Somatic Mutations of TSC2 or MTOR Characterize a Morphologically Distinct Subset of Sporadic Renal Cell Carcinoma With Eosinophilic and Vacuolated Cytoplasm.

The differential diagnosis of renal cell neoplasms with solid or nested architecture and eosinophilic cytoplasm has become increasingly complex. Despite recent advances in classifying a number of entities exhibiting this morphology, some tumors remain in the unclassified category. Here we describe a morphologically distinct group of sporadic renal cell carcinoma (RCC) with predominantly nested architecture, eosinophilic, and remarkably vacuolated cytoplasm retrospectively identified from a cohort of previously unclassified tumors. We examined the clinicopathologic and immunohistochemical features of these tumors and investigated their mutational and copy number alterations using a targeted next-generation sequencing platform. The study included 7 patients with a mean age of 54 years (range: 40 to 68 y) and a male to female ratio of 3:4. All patients presented with a solitary renal mass and had no prior medical or family history raising concern for syndromic conditions. Tumors were well-circumscribed, unencapsulated, and comprised of nests of eosinophilic cells in a hypocellular and often edematous stroma. Tumor cells had round nuclei with prominent nucleoli and granular cytoplasm with striking vacuolization. Thick-walled vessels and calcifications were also frequently present, whereas increased mitotic activity, necrosis, foamy histiocytes or lymphocytic infiltrates were not identified. All cases were positive for PAX8, had retained expression of SDHB and FH, and exhibited a CK7-/CK20- phenotype. While cathepsin-K was positive in 5 cases, none exhibited immunoreactivity to HMB45 or Melan A, or TFE3 immunostaining. Next-generation sequencing identified somatic inactivating mutations of TSC2 (3/5 tumors tested) or activating mutations of MTOR (2/5) as the primary molecular alterations, consistent with hyperactive mTOR complex 1 signaling which was further demonstrated by phospho-S6 and phospho-4E-BP1 immunostaining. Copy number analysis revealed a loss of chromosome 1 in both cases with MTOR mutation. These tumors represent a novel subset of sporadic RCC characterized by alterations in TSC1-TSC2 complex or the mTOR complex 1 pathway. Recognition of their characteristic morphologic and immunophenotypic features will allow them to be readily identified and separated from the unclassified RCC category.

AluScanCNV2: An R package for copy number variation calling and cancer risk prediction with next-generation sequencing data

The usage of next-generation sequencing (NGS) to detect copy number variation (CNV) is widely accepted in cancer research. Based on an AluScanCNV software developed by us previously, an AluScanCNV2 software has been developed in the present study as an R package that performs CNV detection from NGS data obtained through AluScan, whole-genome sequencing or other targeted NGS platforms. Its applications would include the expedited usage of somatic CNVs for cancer subtyping, and usage of recurrent germline CNVs to perform machine learning-assisted prediction of a test subject's susceptibility to cancer.

Rapid Targeted Next-Generation Sequencing Platform for Molecular Screening and Clinical Genotyping in Subjects with Hemoglobinopathies

Hemoglobinopathies are among the most common autosomal-recessive disorders worldwide. A comprehensive next-generation sequencing (NGS) test would greatly facilitate screening and diagnosis of these disorders. An NGS panel targeting the coding regions of hemoglobin genes and four modifier genes was designed. We validated the assay by using 2522 subjects affected with hemoglobinopathies and applied it to carrier testing in a cohort of 10,111 couples who were also screened through traditional methods. In the clinical genotyping analysis of 1182 β-thalassemia subjects, we identified a group of additional variants that can be used for accurate diagnosis. In the molecular screening analysis of the 10,111 couples, we detected 4180 individuals in total who carried 4840 mutant alleles, and identified 186 couples at risk of having affected offspring. 12.1% of the pathogenic or likely pathogenic variants identified by our NGS assay, which were undetectable by traditional methods. Compared with the traditional methods, our assay identified an additional at-risk 35 couples. We describe a comprehensive NGS-based test that offers advantages over the traditional screening/molecular testing methods. To our knowledge, this is among the first large-scale population study to systematically evaluate the application of an NGS technique in carrier screening and molecular diagnosis of hemoglobinopathies.

Abstract 4737: A lung cancer risk classifier comprising genome maintenance genes measured in normal bronchial epithelial cells

Abstract Background: Annual low dose CT (LDCT) screening of individuals at high demographic risk reduces lung cancer mortality by more than 20%. However, subjects selected for screening based on demographic criteria typically have less than a 10% lifetime risk for lung cancer. Thus, there is need for a biomarker that better stratifies subjects for LDCT screening. Toward this goal, we previously reported a lung cancer risk test (LCRT) biomarker comprising 14 genome-maintenance (GM) pathway genes measured in normal bronchial epithelial cells (NBEC) that accurately classified cancer (CA) from non-cancer (NC) subjects. The primary goal of the studies reported here was to optimize the LCRT biomarker for high specificity and ease of clinical implementation. Methods: Targeted competitive multiplex PCR amplicon libraries were prepared for next generation sequencing (NGS) analysis of transcript abundance at 68 sites among 33 GM target genes in NBEC specimens collected from a retrospective cohort of 120 subjects, including 60 CA cases and 60 NC controls. Genes were selected for analysis based on contribution to the previously reported LCRT biomarker and/or prior evidence for association with lung cancer risk. Linear discriminant analysis was used to identify the most accurate classifier suitable to stratify subjects for screening. Results: The best classifier comprised 11 features including age and 10 transcript abundance assays representing nine genes; specifically, CDKN1A, E2F1, ERCC5, GPX1, GSTP1, KEAP1, TP63, XPA, and XRCC1. After cross-validation, the Receiver Operator Characteristic area under the curve was 0.94 (95% CI: 0.92-0.96) and overall classification accuracy was 88.3% (95% CI 82.4%-94.1%). Using a threshold set at 95% sensitivity, specificity was &amp;gt; 70%. Conclusions: The LCRT biomarker reported here displayed high specificity and ease of implementation on a high throughput, quality-controlled targeted NGS platform. As such, it is optimized for clinical validation in specimens from the ongoing LCRT blinded prospective cohort study. Following validation, the biomarker is expected to have clinical utility by better stratifying subjects for annual lung cancer screening compared to current demographic criteria alone. Citation Format: Jiyoun Yeo, Erin Crawford, Xiaolu Zhang, Sadik Khuder, Tian Chen, Albert Levin, Thomas Blomquist, James Willey. A lung cancer risk classifier comprising genome maintenance genes measured in normal bronchial epithelial cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4737. doi:10.1158/1538-7445.AM2017-4737