Utility Of Next-generation Sequencing Research Articles

Utility of next generation sequencing in paediatric neurological disorders: experience from South Africa.

Next generation sequencing (NGS)-based tests have become routine first-line investigative modalities in paediatric neurology clinics in many high-income countries (HICs). Studies from these countries show that these tests are both cost-effective and reliable in diagnosing many complex childhood neurological diseases. However, NGS-based testing in low-and middle-income countries (LMICs) is limited due to affordability constraints. The primary objective of this study was to evaluate the diagnostic yield and impact of targeted gene panel sequencing in a selected paediatric cohort attending a tertiary paediatric neurology clinic in the Western Cape Province of South Africa. This retrospective study included 124 consecutive paediatric patients with neurological disease, aged 6 weeks to 17 years, referred for NGS-based multi-gene panel testing over a 41-month period. Twenty-four different disease group-specific panels were utilized. A caregiver experience questionnaire was administered when a pathogenic variant was identified. The overall study diagnostic yield (DY) was 45% (56/124 patients). The diagnostic yield in this study is similar to previously reported paediatric cohorts in HICs. The high yields for neuromuscular disorders (52%) and early epileptic encephalopathies (41%) suggest that NGS-based panels may be more cost-effective as first-line testing in well-defined phenotypes. The latter finding argues for early inclusion of all children with developmental epileptic encephalopathies (DEE), as early diagnosis leads to better treatment and avoidance of unnecessary investigations.

Use of next-generation sequencing in daily routine practice

Developments in molecular diagnosis and implementation of mutation-driven targeted therapy marked a milestone in cancer treatment. Next-generation sequencing allows sequencing of a high number of nucleotides in a short time and from a limited quantity of pathology or cytology specimens. This is a review of actual indications, utility of next-generation sequencing, and availability of targeted therapies in different neoplasms. We present the European Society for Medical Oncology Precision Medicine Working Group recommendations for tumor multigene sequencing use with the Scale for Clinical Actionability of molecular Targets ranking determined for each alteration.

Abstract B017: Clinical utility of next generation sequencing in advanced colorectal cancer: The earlier the better

Abstract Background. Since next generation sequencing (NGS) was approved in solid tumor in Korea, it has been usually performed when patients were progressed on standard chemotherapy in advanced colorectal cancer (CRC). Recently, new targeted agents have been developed rapidly, so the NGS is recommended as early as possible. In this study, we analyzed NGS results and investigated clinical benefit in advanced CRC. Patient and method. Between October 2018 to December 2022, NGS data in 85 advanced CRC patients in St. Vincent’s Hospital, Suwon, Korea were retrospectively analyzed. Results. Seven patients had metachronous, and others had synchronous metastasis. The median age was 58 (34-84), and 59 (69.4%) were male. Fourteen (16.5%) were right-side, 40 (47.1%) were left-side colon, and 30 (35.3%) were rectum. The median time from diagnosis of advanced CRC to NGS was 13 (0-81) months, and median time to death after NGS was 8 (0.3-43.0) months. NGS platform were ‘Oncomine comprehensive assay Plus’ in 71 (83.5%), and the others were various platforms in K-master project. NGS analyses revealed new therapeutic target in 17 (20%): high tumor mutational burden (n = 2), KRAS G12C (n = 4), KRAS (n = 1) or BRAF (n = 2) mutation not routinely screened at initial, BRAF class II mutation (n = 2), Her2/3 mutation (n = 2), and NTRK fusion (n = 1). Among those, two patients with KRAS G12C mutation enrolled in clinical trial of KRAS G12 inhibitor, and one patient with NTRK fusion received NTRK inhibitor (3.5% of total). Among RAS wild type population (n=30), we identified hyperselected patients (n = 18, [60%], absence of either KRAS, NRAS, PTEN, and extracellular domain EGFR mutations, HER2 and MET amplifications, and ALK, RET or NTRK1 fusions by PARADIGM trial), which showed longer survival in cetuximab-treated population (p<0.001). Specific mutation including KRAS, NRAS, BRAF, TP53, or APC did not impact on prognosis. High TMB (≥10Mut/Mb) were associated with longer survival (p = 0.020). For patients with oligometastasis, hyperselected patients (p = 0.062) showed good prognosis. Conclusion. The NGS results provided additional predictive or prognostic information in advanced CRC patients. Application of NGS as early as possible might help to make a clinical decision. Citation Format: Ho Jung An, Hyunho Kim, Hyung Soon Park, Hyeon-Min Cho, Bong-Hyeon Kye, Ri Na Yoo, Ji-Yeon Moon, Sung Hwan Kim, Jonghoon Lee, Hyo Chun Lee. Clinical utility of next generation sequencing in advanced colorectal cancer: The earlier the better [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr B017.

Utility of Next-Generation Sequencing in the Detection of RNA Fusion Genes in Myeloid Malignancies in Singapore

Background Oncogenic fusion genes play an instrumental role in the pathogenesis of haematological malignancies and the identification of these genes is crucial for diagnosis, risk stratification and treatment decisions. Conventional diagnostic methods such as karyotyping and fluorescence in situ hybridization (FISH) are the current standard of care but are limited in the sensitivity and availability of different FISH probes for detection of fusion genes. In our institution, we have routinely integrated a targeted next-generation sequencing (NGS) panel in our diagnostic workflow for patients with suspected myeloid malignancies. Here we aim to review the cases with RNA fusion genes detected via NGS and compare these results with that of conventional diagnostic methods. Methods We analysed bone marrow samples from patients with suspected myeloid malignancies at the time of diagnosis in Singapore General Hospital (SGH). Extracted genomic DNA and RNA were amplified, and library preparation was performed with the Oncomine TM Myeloid Research assay (Thermo Fisher Scientific) to interrogate 40 DNA genes and 29 fusion driver genes. NGS libraries were sequenced on the Ion GeneStudio S5 System (Thermo Fisher Scientific) and analyzed using in-house bioinformatics pipelines and the Ion Reporter software. Limit of detection was determined as 3% allele frequency for DNA variants and 10 -2 for RNA fusions. RNA fusions detected by the NGS panel were consolidated and compared with cytogenetics (karyotyping and FISH) results. Results A total of 436 samples from adult patients with suspected myeloid malignancies were received for NGS testing from October 2021 to March 2023 in SGH, the largest tertiary hospital in Singapore. 306 had paired cytogenetics and NGS testing performed. 27 of the 306 cases (9%) had RNA fusions detected by NGS as summarized in Table 1 and Figure 1; their disease subgroups comprised of 16 acute myeloid leukaemia (AML), 5 chronic myeloid leukaemia (CML), 3 BCR-ABL1-negative myeloproliferative neoplasm (MPN), 2 acute promyelocytic leukaemia (APL) and 1 myelodysplastic syndrome (MDS). Eight AML cases had RUNX1-RUNX1T1.R3R3 fusions, with 6/8 (75%) harbouring additional DNA single nucleotide variants or indel mutations. Two AML cases had CBFB-MYH11 fusions, with 1 associated with KIT D419 deletion, and the other with U2AF1, NF1, and ASXL1 mutations. We also identified 2 adult AML cases (aged 49 and 65 years respectively) with DEK-NUP214.D9N18 fusions, both of which had associated FLT3-ITD mutations. There were 5 cases with KMT2A fusions, with a variety of partners including MLLT3.K9M6 (n=2), ELL.K9E2 (n=1), MLLT1.K8M4 (n=1), and MLLT6.K8M9 (n=1). Two patients with APL had PML-RARA detected for different variants: P3R3 and P6R3. Amongst the 8 MPN cases, 5 had BCR-ABL1 fusions consistent with diagnosis of CML. There was 1 case of MPN-Eo with FLP1L1-PDGFRA fusion, and the remaining 2 with ETV6-ABL1 and KMT2A-ELL.K9E2 rare fusions. The detected RNA fusions had clinical impact in establishing diagnosis (93%), prognosis (96%), and guidance of treatment decisions (89%). Out of the 27 cases with RNA fusions detected by NGS, 23 (85%) had concordant karyotype and FISH results. Four RNA fusions, namely KMT2A-ELL, CBFB-MYH11, FIP1L1-PDGFRA and ETV6-ABL1, were not detected by karyotyping or conventional FISH probes performed in our institution. It likely reflects the sensitivity of the NGS panel in detecting RNA fusions in cases of cryptic translocations. Conclusion We have demonstrated that NGS has a high sensitivity for identification of RNA fusion genes, is complementary to conventional cytogenetics testing, and has vast clinical impact in terms of diagnosis, prognostication and clinical management. We advocate for the integration of NGS with DNA and RNA sequencing into routine investigation of suspected myeloid malignancies for a more precise and comprehensive diagnostic approach.

591P The utility of next generation sequencing for KRAS gene variants prevalence in cytological and tissue samples in real-world NSCLC patients: A large single institution real-world study

591P The utility of next generation sequencing for KRAS gene variants prevalence in cytological and tissue samples in real-world NSCLC patients: A large single institution real-world study

Access to prenatal exome sequencing for fetal malformations: A qualitative landscape analysis in the US.

There is increasing evidence supporting the clinical utility of next generation sequencing for identifying fetal genetic disorders. However, there are limited data on the demand for and accessibility of these tests, as well as payer coverage in the prenatal context. We sought to identify clinician perspectives on the utility of prenatal exome sequencing (ES) and on equitable access to genomic technologies for the care of pregnancies complicated by fetal structural anomalies. We conducted two focus group discussions and six interviews with a total of 13 clinicians (11 genetic counselors; 2 Maternal Fetal Medicine/Geneticists) from U.S. academic centers and community clinics. Participants strongly supported ES for prenatal diagnostic testing in pregnancies with fetal structural anomalies. Participants emphasized the value of prenatal ES as an opportunity for a continuum of care before, during, and after a pregnancy, not solely as informing decisions about abortions. Cost and coverage of the test was the main access barrier, and research was the main pathway to access ES in academic centers. Further integrating the perspectives of additional key stakeholders are important for understanding clinical utility, developing policies and practices to address access barriers, and assuring equitable provision of prenatal diagnostic testing.

Clinical utility of next generation sequencing (NGS) on circulating tumor DNA (ctDNA) in patients (pts) with pancreatic cancer (PC).

4154 Background: ctDNA provides opportunities for identifying targeted therapies, a source to conduct NGS when tissue acquisition is infeasible, detection of minimal residual disease (MRD), and identification of resistance mutations. There are limited large data sets to inform clinical utility and limited correlation of NGS of ctDNA and tumor in PC. Herein, we evaluate the ctDNA detection rate in multiple cohorts of PC and report the ctDNA-tissue genotype concordance in PC at Memorial Sloan Kettering (MSK). Methods: Pts with PC at MSK who had ctDNA prospectively collected using the MSK-ACCESS 129 gene ctDNA NGS assay, were identified. ctDNA detection was defined as the identification of a mutation, copy number (no.) alteration or structural variant. Tissue-based NGS using MSK-IMPACT gene assay was performed for pts with adequate tissue, and matched with ctDNA by date. Clinical, pathologic and outcome data were abstracted. Data are summarized with descriptive statistics. Overall survival (OS) estimated by Kaplan-Meier method from date of ctDNA draw to death/last follow up. Results: From 08/2019 to 07/2022, N= 414 pts with PC and ≥ 1 ctDNA sample included. Median age 69 years (range 29-92), female N=206 (50%). Stage at ctDNA collection: Stage I-III N= 203 (49%); Stage IV N= 211 (51%). ctDNA detection rate by no. of involved organs; 0 organs (MRD), N=7/30 (23%); 1-2 organs, N=158/270 (58.5%); 3-4 organs, N=82/102 (80.4%); >5 organs, 12/12 (100%). CtDNA detection rate by site of metastasis; Liver only, 75/99 (76%); peritoneum only, 15/25 (60%); lung only, 10/22 (45%). In pts with detected ctDNA median CA 19-9 was 494 U/mL (range 0, 247674) vs 100 U/mL (range 1, 22360) in pts with undetected ctDNA. In pts with detected ctDNA median CEA was 7 ng/mL (range 1, 2020) vs 4 ng/mL (range 1, 99) in pts with undetected ctDNA. In stage IV untreated pts, median OS 13 months(m) (95% CI; 7.3, 16) and 10 m (95% CI; 5.6, -) for ctDNA detected vs undetected, respectively. ctDNA detection rates by stage, and concordance between ctDNA and tissue-based NGS for common driver mutations KRAS, TP53, CDKN2A and SMAD4 in N=131 matched pairs are summarized in the table. Conclusions: ctDNA detection rates are high (89%) in pts with untreated stage IV PC, with high concordance between ctDNA and tissue-based NGS (87% - 95%). In untreated stage I-III PC, detection and concordance rates are lower (<50%). Detection rates are associated with disease burden, site of metastasis, CA 19-9/CEA levels. ctDNA is a promising tool in detection of somatic alterations in PC, particularly in stage IV disease, and is a complementary adjunct to tumor-based NGS. [Table: see text]

Utility of next generation sequencing (NGS) in advanced malignancies in a community hospital in an underserved area: A game changer?

e15037 Background: Next Generation Sequencing (NGS) is a revolutionary test used in advanced malignancies to study specific mutations, define tumor burdens, & design personalized treatment plans based on the target mutations. With this study, our aim is to highlight the role of NGS, especially in a community hospital & emphasize on its implementation as a standard of care in advanced metastatic cancers. Methods: We retrospectively reviewed the charts of 51 patients after IRB approval, aged > 18 years with no specific demographic restrictions, with advanced malignancies who had NGS at Mercy Catholic Medical Center, PA. Results: Of 51 patients, 25 were males & 26 were females, 44 patients had positive results on NGS. 100 types of mutations were identified & 338 mutations in total were found. Lung cancer had highest mutations (41.17%-21/51), followed by colon (25.49% - 13/51), breast, ovarian & endometrium (13.72% 7/51), & the rest comprised of GI malignancies & prostate cancers. Of 100 mutations, 32% (32/100) had actionable targets & received NGS directed chemoimmunotherapy, 22% (22/100) mutations had experimental chemo-immunotherapeutic drugs, & 49% (49/100) had no specific NGS directed therapy actionable/target mutation. The most common (1,2,3) & some rare mutations (4,5 and 6) with directed immunotherapy noted on NGS are listed in Table 1. Conclusions: As per our study, 86.27% of the sample size showed positive results on NGS. 32% of these mutations had NGS directed chemoimmunotherapy against these target mutations. In contrary to standard treatment regimens, a personalized treatment plan was developed for these patients, thereby curbing further progression & improving survival in patients with advanced cancer. For example, a patient with stage IV colon cancer who was progressing on the standard treatment with FOLFOX (Folinic acid, flurouracil and oxaliplatin), was started on Ivosedenib, after undergoing NGS and showed marked improvement clinically and radiologically. 29 out of 43 guidelines for various malignancies established by National Comprehensive Cancer Network highlight the significance of NGS. With this study, our goal is to emphasize on the utility of NGS in patients with advanced cancer & the need for it to be incorporated as the standard of care, in small & mid-tier community hospitals, which would need a devised plan for its implementation. [Table: see text]

Diagnostic utility of next generation sequencing for somatic variants in cancer: A seven year cumulative cohort.

e15060 Background: Next generation sequencing (NGS) makes it possible to identify the specific molecular biomarkers to define patient eligibility for targeted therapies via multigene panels. Since then, even though the interpretation of data is complex and not all tumors have genomic alterations within actionable genes, using somatic sequencing for cancer patients becomes a daily routine in clinical oncology. Thus, here we present the data derived from our experience in NGS testing of different cancer patients and biological samples over the course of 7 years in our genetic diagnosis center. Methods: The cohort was consisted of total 5511 cases. While 2590 (46.99%) of them were formalin fixed paraffin embedded (FFPE) tissues, 2906 (52.73%) were liquid biopsies and 15 (0.27%) were other types of biological samples. Total of 15 different cancers were classified (Table 1) and were sequenced using 3 different multigene NGS panels (additional Table 1, BRCAs’ testing for breast/ovarian cancer is not included). NGS was performed via GeneReader NGS System (Qiagen, Germany). Bioinformatics analyzes and variant classification were performed using QCI-Analyze and QCI-Interpret interfaces. Results: As a result, the cases of 93.36% (n = 5145) among all samples were successfully sequenced. The lung cancer group was the most frequent with 47.96% (n = 2643 patients), followed by 491 (%8.9) colorectal 288 (5.23%) and breast cancer patients respectively, while 781 cases (14.17%) grouped as other including many rare cancers. Overall positivity rate was 42.57% (n = 2346) and 366 samples (6.64%) were not sufficient enough to be sequenced. The distribution of positivity rates in relation to cancer type and biological samples used were listed in Table 1. Classification and diagnosis of all tested cases, specimens and Tier 1-2 variants included in this cohort are available on the Additional Table 2. Conclusions: This report is a comprehensive review of the aggregate data across the entire cohort of cancer patients referred to a genetics diagnosis center. While our findings highlight the importance of NGS testing in the somatic setting for cancer patients, also these data stress the importance of diagnosis, biological sample types tested and test results. [Table: see text]

PD46-03 UTILITY OF URINARY TUMOR DNA DETECTION IN TREATMENT-NAÏVE UTUC

You have accessJournal of UrologyCME1 Apr 2023PD46-03 UTILITY OF URINARY TUMOR DNA DETECTION IN TREATMENT-NA&IUML;VE UTUC Heather Huelster, Facundo Davaro, Kyle Rose, Billie Gould, Tiantian Zheng, Prithvi Murthy, Shreyas Naidu, Lucia Camperlengo, Pan Du, Shidong Jia, Aram Vosoughi, and Roger Li Heather HuelsterHeather Huelster More articles by this author , Facundo DavaroFacundo Davaro More articles by this author , Kyle RoseKyle Rose More articles by this author , Billie GouldBillie Gould More articles by this author , Tiantian ZhengTiantian Zheng More articles by this author , Prithvi MurthyPrithvi Murthy More articles by this author , Shreyas NaiduShreyas Naidu More articles by this author , Lucia CamperlengoLucia Camperlengo More articles by this author , Pan DuPan Du More articles by this author , Shidong JiaShidong Jia More articles by this author , Aram VosoughiAram Vosoughi More articles by this author , and Roger LiRoger Li More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000003359.03AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Urine-based molecular biomarkers correlate with tumor variants in urothelial carcinoma of the bladder, showing promise for noninvasive detection of newly diagnosed tumors. However, the utility of next-generation sequencing (NGS) to detect urinary tumor DNA (utDNA) in upper tract urothelial carcinoma (UTUC) remains to be explored. Herein, we prospectively sequenced tumors and utDNA at the time of and serially after radical nephroureterectomy (RNU) to examine the feasibility of using utDNA in detection and surveillance of UTUC. METHODS: Fourteen systemic therapy naïve patients with high-grade, clinically-localized UTUC undergoing RNU were prospectively accrued. Urine was collected preoperatively on the day of surgery and urinary supernatant and cell pellets (UCP) processed for extraction of utDNA. NGS used shallow whole-genome sequencing (WGS) to detect genome-wide copy number changes and a 152-pancancer panel (PredicineCARE) to profile single nucleotide variants, small insertions/deletions, and gene-level copy number changes. Variants with mutation allele frequency ≥0.25% in supernatant, ≥0.3% in UCP, and cancer hotspot variants >0.1% were reported. RESULTS: Prior to RNU, utDNA variants were detected in 11/11 (100%) of urinary supernatants and 9/14 (64%) of UCPs using NGS. The most common alterations detected in the urine were in TP53 (81%), TERT promoter (55%), FGFR3 (55%), and ERBB2 (45%). More tumor variants were detected in urine supernatant than UCP (22.9 vs 2.4, p=0.004). Use of a bespoke tumor-informed approach to adjust MAF thresholds for calling variants increased utDNA detection by a mean 0.5 (range 0-3) variants in urinary supernatant and mean 2.3 (range 0-7) variants in UCP. There was no significant difference in the number of variants detected in urine of patients with UTUC staged as <pT2 compared to ≥pT2 or pN+. CONCLUSIONS: UTUC tumor variants are detectable in the urine of treatment-naïve patients undergoing RNU for UTUC using both a panel-based approach and WGS. utDNA detection and dynamics may present an opportunity for personalized monitoring of UTUC. Source of Funding: None © 2023 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 209Issue Supplement 4April 2023Page: e1170 Advertisement Copyright & Permissions© 2023 by American Urological Association Education and Research, Inc.MetricsAuthor Information Heather Huelster More articles by this author Facundo Davaro More articles by this author Kyle Rose More articles by this author Billie Gould More articles by this author Tiantian Zheng More articles by this author Prithvi Murthy More articles by this author Shreyas Naidu More articles by this author Lucia Camperlengo More articles by this author Pan Du More articles by this author Shidong Jia More articles by this author Aram Vosoughi More articles by this author Roger Li More articles by this author Expand All Advertisement PDF downloadLoading ...

Utility of next-generation sequencing in the diagnosis of neurological disorders

Utility of next-generation sequencing in the diagnosis of neurological disorders

Bioinformatically Expanded Next-Generation Sequencing Analysis Optimizes Identification of Therapeutically Relevant MET Copy Number Alterations in >50,000 Tumors.

Clinical relevance thresholds and laboratory methods are poorly defined for MET amplification, a targetable biomarker across malignancies. The utility of next-generation sequencing (NGS) in assessing MET copy number alterations was determined in >50,000 solid tumors. Using fluorescence in situ hybridization as reference, we validated and optimized NGS analysis. Incorporating read-depth and focality analyses achieved 91% concordance, 97% sensitivity, and 89% specificity. Tumor heterogeneity, neoplastic cell proportions, and genomic focality affected MET amplification assessment. NGS methodology showed superiority in capturing overall amplification status in heterogeneous tumors and defining amplification focality among other genomic alterations. MET copy gains and amplifications were found in 408 samples across 23 malignancies. Total MET copy number inversely correlated with amplified segment size. High-level/focal amplification was enriched in certain genomic subgroups and associated with targeted therapy response. Leveraging our integrated bioinformatic approach, targeted therapy benefit was observed across diverse MET amplification contexts.

Abstract 5872: Investigation on the prevalence of germline and somatic mutations in gynecological malignancies: Implications for risk assessment and therapeutic decision-making

Abstract Introduction: Heritable mutations in cancer predisposition genes increase the relative risk of developing malignant gynecological tumors, whereas actionable somatic mutations could serve as biomarker for therapeutic decision-making. Our study explored the prevalence of germline pathogenic/likely-pathogenic (P/LP) mutations and actionable somatic mutations to understand the clinical utility of next-generation sequencing (NGS) in gynecological malignancies. Methods: Genomic data was retrospectively analyzed for 1,610 women with various stages of gynecological malignancies including ovarian (OC, n=945), cervical (CC, n=358), and endometrial cancers (EC, n=307). Genomic data were derived from targeted NGS of paired white blood cell and other biological samples using a 520-gene panel that included 62 cancer predisposition genes. Genomic signatures including tissue-based tumor mutation burden (tTMB) and microsatellite instability (MSI) were also estimated from somatic mutation profiles. Results: The prevalence of germline P/LP mutations in cancer predisposition genes was 20.6% for OC, 13.4% for EC, and 6.4% for CC. OC had the highest mutation rate in genes involved in the homologous recombination repair pathway (18.8%, 178/945), while EC had the highest mutation rate in genes involved in mismatch repair (MMR) pathway (5.5%, 17/307). The frequency of actionable germline P/LP mutations was 19.8% for OC, 11.7% for EC, and 5.0% for CC. The detection rate for actionable somatic mutations were comparable between carrier and non-carrier of germline mutations (OC, 80.5% vs 76.3%, p=0.2; EC, 73.2% vs 80.5%, p=0.4; and CC, 56.5% vs 63.0%, p=0.7). tTMB was also comparable and generally low among carrier and non-carrier of germline mutations (OC, 3.0 [range: 2.0-5.0] vs 3.0 [2.0-5.0], p=0.9; EC, 6.2 [4.8-45.1] vs. 4.8 [2.8-13.2], p=0.1; and CC, 4.8 [2.4-6.0] vs 4.0 [2.4-7.9], p=0.8). The proportion of MSI-H was comparable for OC patients with or without germline mutations (2.6% vs 1.2%, p=0.2); however, the proportion of MSI-H was significantly higher for EC (29.3% vs 11.7%, p=0.019) and CC (13.0% vs 1.2%, p&amp;lt;0.001) who were carriers of germline mutations than non-carriers. The proportion of MSI-H was comparable between EC patients with germline (n=5) and somatic (n=29) MMR gene mutations (40.0%, 2/5 vs 48.3%, 14/29; p=1). EC patients with MSI-H were more likely to have high tTMB (cut-off ≥7 mutations/Mb) (p&amp;lt;0.001). Conclusions: The comparable frequency of actionable somatic mutations in women with or without germline mutations indicates that both populations could benefit from NGS testing. Our findings raise the importance of NGS-based genetic testing with a large gene panel as part of gynecological cancer screening and diagnosis to explore heritable and acquired mutation status for risk assessment and therapeutic decision-making. Citation Format: Hao Wen, Qin Xu, Xiujie Sheng, Huawen Li, Xipeng Wang, Xiaohua Wu. Investigation on the prevalence of germline and somatic mutations in gynecological malignancies: Implications for risk assessment and therapeutic decision-making [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 5872.

Analysis of genomic alterations and treatment landscape in patients with advanced tumors using real-world data for precision oncology.

6584 Background: Analysis of real-world data is critical for clear understanding of treatment effectiveness. Here, we explore the clinical utility of next-generation sequencing (NGS) and the impact of mutational landscapes on targeted therapy and outcomes in almost 10,000 patients in real-world setting. Methods: We constructed a data analysis platform that integrated NGS results with machine and manually curated electronic medical records data from a single large healthcare system. We assessed eligibility for a targeted therapy based on the constellation of mutations detected by NGS, investigated whether a matched therapy was given and how this affected patient outcome. Results: The study comprised of 9848 patients with NGS results across 90 cancer types including lung (2208;22%), colorectal (1495;15%), multiple myeloma (1153;12%), breast (783;8%), and prostate (480;5%) and other remaining cancers (3,729; 38%) from 2011 to 2021. Overall, 95% of patients had a positive NGS result, of whom 6364 (65%) had a targetable mutation in 34 genes per any NCCN guidelines in any cancer type. Of these, 2930 (46%) were late-stage (3-4). 820 patients across 31 cancer types received any of 54 targeted therapies based on an NGS result; 605/820 (74%) were stage 3-4; overall 605/2930 (21%) of all tested stage 3-4 patients received targeted therapy. Overall stage 4 patients with a targetable mutation who were treated with a targeted therapy had better 5-year overall survival (OS) than patients who did not (HR = 0.71, 95% CI 0.57-0.89, p = 0.003). We further analyzed important biomarkers in specific cancers. Stage 4 lung cancer patients carrying EGFR, ALK, ROS1 or MET alteration and receiving targeted therapy had better 5-year OS (HR 0.56, 95% CI 0.40-0.80, p = 0.001). We observed that multiple myeloma patients carrying KRAS and NRAS mutation received off-label Trametinib, however it did not improve their OS (HR 1.28, 95% CI 0.76-2.15, p = 0.4). Moreover, there are 665 patients with tumor mutational burden (TMB) across 64 cancer types. TMB has been categorized into high (55, 8%), intermediate (177, 27%) and low (433, 65%). OS was significantly longer in TMB-low patients compared to TMB-high and TMB-intermediate patients across all cancers (HR 0.63, 95% CI 0.43-0.92, p = 0.016). There are 5051 patients with microsatellite instability status across 72 cancers categorized into high (828, 16%), and low (4223, 84%) with no significant survival difference between groups. Conclusions: We show that real-world data offers important insights into clinical practice and patient outcome, with respect to genomic alterations and associated targeted therapies. Moreover, we have built an analytics platform which allows users to ask relevant clinical questions around outcome of patients treated with targeted therapy by genomics biomarker and potential to identify off-label use and clinical response in any cancer type.

Utility of Next-Generation Sequencing in the Reconstruction of Clonal Architecture in a Patient with an EGFR Mutated Advanced Non-Small Cell Lung Cancer: A Case Report

EGFR tyrosine kinase inhibitors (EGFR-TKIs) have revolutionized the treatment of non-small cell lung cancer (NSCLC) patients with activating EGFR mutations. However, targeted therapies impose a strong selective pressure against the coexisting tumor populations that lead to the emergence of resistant clones. Molecular characterization of the disease is essential for the clinical management of the patient, both at diagnosis and after progression. Next-generation sequencing (NGS) has been established as a technique capable of providing clinically useful molecular profiling of the disease in tissue samples and in non-invasive liquid biopsy samples (LB). Here, we describe a case report of a patient with metastatic NSCLC harboring EGFR mutation who developed two independent resistance mechanisms (EGFR-T790M and TP53 + RB1 mutations) to dacomitinib. Osimertinib given as a second-line treatment eliminated the EGFR-T790M population and simultaneously consolidated the proliferation of the TP53 + RB1 clone that eventually led to the histologic transformation to small-cell lung cancer (SCLC). Comprehensive NGS profiling revealed the presence of the TP53 + RB1 clone in the pretreatment biopsy, while EGFR-T790M was only detected after progression on dacomitinib. Implementation of NGS studies in routine molecular diagnosis of tissue and LB samples provides a more comprehensive view of the clonal architecture of the disease in order to guide therapeutic decision-making.

Diagnostic Impact of Next-Generation Sequencing Panels for Lymphoproliferative Neoplasms on Small-Volume Biopsies

We investigated the feasibility and utility of next-generation sequencing (NGS)-based targeted somatic mutation panels and IG/TR gene rearrangement assays in the diagnosis of lymphoproliferative disorders (LPDs) in small-volume biopsies. We performed a retrospective, single-institution review of all NGS assays requested over a 3-year period by hematopathologists for diagnostic purposes on small-volume biopsies. We identified 59 small-volume biopsies. The TR assay was most commonly requested (42 [71%]), followed by the somatic mutation panel (32 [54%]) and IG assay (26 [44%]). NGS studies were associated with a change in the diagnostic line in about half of cases (28 [47%]) and in a change in the likelihood of a diagnosis in a further 16 cases (27%); there was no diagnostic impact of NGS testing in 15 cases (25%). Implementation of NGS panel somatic mutation or IG/TR gene rearrangement assays on small-volume biopsies contributes to the diagnosis of LPDs in the majority of select cases for diagnostic purposes. The molecular diagnosis is considered in the context of the clinical, histologic, and immunophenotypic findings and does not by itself lead to a definitive diagnosis in small-volume biopsies.

Utility of next-generation sequencing in genetic testing and counseling of disorders involving the musculoskeletal system\u2014trends observed from a single genetic unit

BackgroundDisorders involving the musculoskeletal system are often identified with short stature and a range of orthopedic problems. The clinical and genetic heterogeneity of these diseases along with several characteristic overlaps makes definitive diagnosis difficult for clinicians. Hence, using molecular testing in addition to conventional tests becomes essential for appropriate diagnosis and management.MethodsComprehensive clinical examination, detailed pretest and posttest counseling, molecular diagnosis with next-generation sequencing (NGS), genotype–phenotype correlation and Sanger sequencing for targeted variant analysis.ResultsThis manuscript reports a molecular spectrum of variants in 34 orthopedic cases referred to a single genetic unit attached to a tertiary care hospital. The diagnostic yield of NGS-based tests coupled with genetic counseling and segregation analysis was 79% which included 7 novel variants. In about 53% (i.e. 18/34 cases), molecular testing outcome was actionable since 8 of the 18 underwent prenatal diagnosis, as they were either in their early gestation or had planned a pregnancy subsequent to molecular testing, while ten cases were premaritally/prenatally counseled for the families to take informed decisions as they were in the reproductive age.ConclusionsThe report highlights the importance of NGS-based tests even in a low resource setting as it helps patients, families and healthcare providers in reducing the economic, social and emotional burden of these disorders.

Multiple viruses detected in human DNA are associated with Alzheimer disease risk.

Multiple infectious agents, including viruses, bacteria, fungi, and protozoa, have been linked to Alzheimer disease (AD) by independent lines of evidence. We quantified DNA sequence reads that align to viral and bacterial reference sequences present in whole exome (WES, N= 15,125) and whole genome (WGS, N= 4,443) sequences measured in blood (N=17,817) and brain (N=1,751) tissue from non-Hispanic white, African American and Caribbean Hispanic participants of the AD Sequencing Project including 9,380 AD cases and 10,188 controls. We tested the association of individual and cumulative normalized viral read counts with AD using logistic and LASSO regression models adjusting for tissue source, sequencing site, sex, APOE genotype, and population. Additional models were tested in subsets stratified by WES/WGS and tissue source. We used PathSeq, a software tool that performs subtraction on high-throughput sequencing data, to identify inserted microbial DNA and tested those species for association with AD. We detected sequences from 175 unique species of human viruses and 17,219 bacteria. Many individual and the cumulative viral read counts varied significantly by sequence center, tissue source, sex, and ancestry. Cumulative normalized human viral read count was associated with AD in every subset of the data (ORmeta = 1.05 per 100,000 units, Pmeta < 7.81x10-12 ). In the WES dataset, the presence of inserted herpes virus 6A sequence measured by PathSeq was protective against AD (OR=0.41, P=0.0007). We also found significant associations using LASSO. In the WES dataset, Hepatitis C was associated with increased AD risk. Herpes 6B was associated with decreased AD risk in both the WES and WGS datasets. In brain samples, herpes 4 and 6B were negatively associated with AD in both WES and WGS datasets, whereas herpes 8 was positively associated with AD and human herpes 3 was negatively associated in the WES dataset. No bacterial species were significantly associated with AD. These results are consistent with the hypothesis that viral infections contribute to AD pathogenesis and highlight the utility of next generation sequencing to detect microbial agents in multiple tissues. Contrary to previous results, except for human herpes 8, herpes viral DNA species are protective against AD.

The clinical utility of next-generation sequencing for bone and soft tissue sarcoma

Background: Sarcomas are a rare and heterogeneous tumor group composed of a variety of histologic subtypes. Targeted next-generation sequencing (NGS) of bone and soft tissue sarcomas is a nascent field with limited evidence for its use within clinical practice. Therefore, further research is needed to validate NGS in sarcoma and assess the clinical utility of these techniques with the hope of improving treatment options. Methods: Comprehensive molecular profiling with NGS was performed on 136 tumors (116 soft tissue, 20 bone) using two commercial vendors. Patient records were retrospectively reviewed, and the clinical impact of NGS-related findings were qualitatively analyzed to determine actionable mutations and number of changes in treatment. Results: The median age was 55.0 years (IQR 42–67 years), and most patients were non-metastatic at presentation (80.9%, n = 110). Prior to performing NGS, 72.1% (n = 98) were treated with a mean 1.1 ± 1.2 lines of systemic chemotherapy. NGS identified 341 putative alterations with at least one mutation present in 89.7% (n = 122) of samples. In a subset of 111 patients with available TMB data, 78.7% (n = 107) had a low (<6 m/Mb) mutational burden. Among all 136 cases, 47.1% (n = 64) contained clinically actionable alterations, and 12 patients had a change in medical treatment based on NGS. Those who underwent a treatment change all had metastatic or recurrent disease; three of these patients experienced a clinical benefit. Conclusion: Most bone and soft tissue sarcomas harbor at least one genetic alteration, and it appears a sizeable number of tumors contain mutations that are clinically actionable. While a change in treatment based off NGS-related findings occurred in 12 cases, three patients experienced a clinical benefit. Our data provide further proof-of-concept for NGS in sarcoma and suggest a clinical benefit may be observed in select patients.

Identification and Classification of Novel Genetic Variants: En Route to the Diagnosis of Primary Ciliary Dyskinesia.

Primary ciliary dyskinesia (PCD) is a disease caused by impaired function of motile cilia. PCD mainly affects the lungs and reproductive organs. Inheritance is autosomal recessive and X-linked. PCD patients have diverse clinical manifestations, thus making the establishment of proper diagnosis challenging. The utility of next-generation sequencing (NGS) technology for diagnostic purposes allows for better understanding of the PCD genetic background. However, identification of specific disease-causing variants is difficult. The main aim of this study was to create a unique guideline that will enable the standardization of the assessment of novel genetic variants within PCD-associated genes. The designed pipeline consists of three main steps: (1) sequencing, detection, and identification of genes/variants; (2) classification of variants according to their effect; and (3) variant characterization using in silico structural and functional analysis. The pipeline was validated through the analysis of the variants detected in a well-known PCD disease-causing gene (DNAI1) and the novel candidate gene (SPAG16). The application of this pipeline resulted in identification of potential disease-causing variants, as well as validation of the variants pathogenicity, through their analysis on transcriptional, translational, and posttranslational levels. The application of this pipeline leads to the confirmation of PCD diagnosis and enables a shift from candidate to PCD disease-causing gene.