Clinical Utility Of Next-generation Sequencing Research Articles

High Diagnostic Yield and Clinical Utility of Next-Generation Sequencing in Children with Epilepsy and Neurodevelopmental Delays: A Retrospective Study.

Advances in genetics led to the identification of hundreds of epilepsy-related genes, some of which are treatable with etiology-specific interventions. However, the diagnostic yield of next-generation sequencing (NGS) in unexplained epilepsy is highly variable (10-50%). We sought to determine the diagnostic yield and clinical utility of NGS in children with unexplained epilepsy that is accompanied by neurodevelopmental delays and/or is medically intractable. A 5-year retrospective review was conducted at the American University of Beirut Medical Center to identify children who underwent whole exome sequencing (WES) or whole genome sequencing (WGS). Data on patient demographics, neurodevelopment, seizures, and treatments were collected. Forty-nine children underwent NGS with an overall diagnostic rate of 68.9% (27/38 for WES, and 4/7 for WGS). Most children (42) had neurodevelopmental delays with (18) or without (24) refractory epilepsy, and only three had refractory epilepsy without delays. The diagnostic yield was 77.8% in consanguineous families (18), and 61.5% in non-consanguineous families (26); consanguinity information was not available for one family. Genetic test results led to anti-seizure medication optimization or dietary therapies in six children, with subsequent improvements in seizure control and neurodevelopmental trajectories. Not only is the diagnostic rate of NGS high in children with unexplained epilepsy and neurodevelopmental delays, but also genetic testing in this population may often lead to potentially life-altering interventions.

Mutated KRASas a promising target in pancreas cancer: PURPLE registry data to inform real-world incidence and prognostic significance and to aid trial recruitment.

11150 Background: With around 90% KRASmutation (mt) frequency, pancreatic ductal adenocarcinoma (PDAC) is considered the most RAS-addicted cancer. Despite guideline recommendations, genomic testing is not routine in all PDAC patients (pts) in Australia, due to unproven clinical impact. As more novel KRAS-direct therapeutics enter clinical trials, understanding the real-world frequency of individual mutations and prognostic significance, and facilitating trial recruitment are important goals, all of which can be supported by registry data. Methods: Data extracted from the PURPLE pancreatic cancer registry from 9 participating Australian cancer centres, between 2016-2022, was analysed to compare clinicopathological features, survival based on KRASmt status, and assess feasibility of the platform to identify and molecularly stratify patients for future clinical trials. Survival estimates were calculated using Kaplan-Meier curves and log-rank testing on SPSS (Macintosh v.29). Results: Of 721 PDAC routine care pts identified, next generation sequencing (NGS) was undertaken in 378/721 (52%). Median patient age was 68 years (range 52-83); 152 (40%) had resectable, 111 (29%) locally advanced and 115 (30%) metastatic disease. 57/378 (15%) were KRAS wildtype. Of 321 pts with a KRAS mt, codons 12,13, and 61 were the most common sites of mt, including G12D (45%), and G12V (30%), with lower frequencies of G12R (13%), Q61H (6%), G12A (3%), G12C (1%), and G13D (1%). Comparing KRAS wildtype to KRASmt pts, there was no difference in median age (68 vs 67, p=0.63), gender (male: 49% vs 60%, p=0.23), Charlson comorbidity index (p=0.30), or stage at first presentation (p=0.99). Overall KRAS wildtype pts were more likely to be ECOG PS 0 at diagnosis (p=0.01) and to receive at least 1 modality of treatment (p=0.004). For all pts, median overall survival (OS) in KRASwildtype versus KRAS mt pts was 29.0 months versus 19.7 months (p=0.007), and for the 115 metastatic pts 15.1 versus 10.4 months (p=0.28). Further analysis of the impact by disease stage and by individual RAS mt is underway. Conclusions: Registry based analysis informs understanding of KRAS mt status of PDAC in a community setting. Here, KRAS mt status was associated with worse OS outcomes, likely in part due to the association with ECOG PS and receiving less active treatment. With newer promising KRAS-targeted therapies becoming available in clinical trials, known RAS status will aid identification of trial candidates. The clinical utility of NGS and rationale for reflex testing in PDAC is increasing. Clinical Registry information (ACTRN12617001474347).

HER2/ERBB2 copy number analysis by targeted next-generation sequencing in breast cancer.

A combination of immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH) is the current standard of care for HER2 evaluation in breast cancer. Here, we investigate the potential clinical utility of next-generation sequencing (NGS)-derived HER2/ERBB2 copy number (CN) data for predicting HER2 status as defined by American Society of Clinical Oncology (ASCO)/College of American Pathologists (CAP) guidelines. In total, 294 locally recurrent and metastatic breast cancers previously tested by targeted hybrid capture-based NGS and by HER2 IHC/FISH were included. Analyses focused on the ERBB2 median log2 ratios and start-end genomic coordinates from NGS, average HER2 CN and HER2/CEP17 ratios from FISH, and the HER2 IHC scores. We also determined a more stringent log2 ratio cutoff to predict HER2-positive status with 100% specificity. Sixty-four (22%) cases were HER2 positive and 230 (78%) were HER2 negative by ASCO/CAP guidelines. The ERBB2 median log2 ratios from NGS strongly correlated with HER2 status by IHC/FISH (area under receiver operator characteristic curve = 0.951). ERBB2 log2 ratio more than 1.7 was 100% specific for HER2-positive results by IHC/FISH. Start and end genomic coordinates for regions of gain near ERBB2 by NGS also predicted HER2 status. Copy number data from our NGS panel strongly correlate with HER2 status. Using a stringent cutoff, ERBB2 log2 ratio accurately predicts HER2 positivity with high specificity. The NGS CN assessment may have utility in determining HER2 status in certain clinical settings.

The diagnostic value of next-generation sequencing technology in sepsis.

ObjectiveThis study aims to assess the clinical utility of next-generation sequencing (NGS) in sepsis diagnosis.MethodsA prospective study was conducted on patients with a high suspicion of sepsis by unknown pathogens from January 2017 to December 2021. Blood samples were taken from patients to perform NGS, blood culture (BC), leucocyte (WBC), procalcitonin (PCT), creatinine (CREA), Albumin (ALB) and C-reactive protein (CRP) tests.ResultsThe feedback time for BC was 3~5 days for bacteria and 5~7 days for fungi, while the turnover time for NGS was only 24 h. The clinical diagnosis was considered the “gold standard”. 83 patients passed our inclusion criteria and were separated into two groups by clinical diagnosis. 62 met the clinical diagnosis criteria for sepsis and 21 were non-sepsis. The data from the two groups were retrospectively compared and analyzed. Of 62 sepsis in 83 patients, 8(9.64%) were diagnosed by both BC and NGS, 51 (61.45%) by NGS only, 1(1.20%) by BC and 2 (2.41%) by conventional testing only; PCT, CREA, CRP levels and the detection rate of NGS and BC were higher in the sepsis group than in the non-sepsis group, while ALB levels were lower (p<0.05). The logistic regression results in our study revealed that NGS and ALB were independent prediction factors for sepsis (p<0.05), the area under the receiver operating characteristic curve (AUC), sensitivity and specificity of NGS for diagnosing sepsis was 0.857, 95.16% and 76.19%, while ALB was 0.728, 58.06%, 80.95%, respectively. The combination’s sensitivity, specificity and AUC of NGS and ALB were 93.55%, 85.71% and 0.935, greater than that of Albumin or NGS only (both p<0.05).ConclusionNGS can effectively and quickly identify pathogens, thereby emerges as a promising technology for sepsis diagnosis. Combination of NGS and ALB can be used for early screening and is more powerful than NGS or ALB only.

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.

Clinical utility of next-generation sequencing for prostate cancer in the context of a changing treatment landscape.

112 Background: Next-generation sequencing (NGS) is increasingly common in clinical practice, but its clinical utility may depend on the availability of sequencing-directed therapies (SDT). There were no FDA-approved SDTs in prostate cancer (PCa) until 2020, when PARP inhibitors olaparib and rucaparib were approved for tumors bearing alterations in certain homologous recombination repair (HRR) genes. We assessed the clinical utility of NGS in PCa before and after the approval of these agents in a single academic medical center. Methods: This was a retrospective single-center study including all PCa patients seen at Mount Sinai Hospital (New York, NY) between 2018–2021 who received NGS via the 161-gene Sema4 Signal Solid Tumor Panel. Clinical data were extracted from the Mount Sinai electronic medical record using a proprietary automated pipeline with limited manual curation (Sema4 PRODB). The primary outcome was clinical utility in metastatic PCa, defined as the proportion of metastatic PCa patients who received SDT. Secondary outcomes included time-to-next-treatment (TTNT, defined as time from SDT start to the start of next systemic therapy) and the proportion of patients with clinically actionable (as of 9/2021) alterations, defined as either Tier 1 (associated with FDA-approved treatments in prostate cancer) or Tier 2 (associated with either off-label or investigational agents). Results: The cohort consisted of 332 PCa patients; 51% (N = 170) were sequenced in 2020 or later. The median age at diagnosis was 65 (IQR 12). The most advanced stage documented was localized for 39% (N = 129) and metastatic for 61% (N = 203). Overall, 167 actionable alterations were identified in 125 patients (38% of cohort). Of the actionable alterations, 31% (N = 51) were Tier 1 and 69% (N = 116) were Tier 2. Of the 44 patients with Tier 1 alterations, 8 (18%) received SDT (all received olaparib). The proportion of metastatic patients receiving olaparib increased from 1% (2/145) before 2020 to 10% (6/58) during or after 2020 (p = 0.008). Of the 36 patients not receiving olaparib: 20 were sequenced before FDA approval and were treated with an alternative systemic therapy; 8 had localized disease and were not eligible; 8 had limited follow-up or unknown treatment status. For those who received olaparib, median TTNT was 5 months. Conclusions: In this single-center retrospective cohort, clinical utility of NGS was linked to treatment landscape. Increases in NGS test volume and olaparib use coincided with the approval of PARP inhibitors for patients with HRR-mutated prostate cancers. Notably, NGS was used to match patients to off-label/ investigational olaparib before its FDA approval.[Table: see text]

Clinical Utility of Next-generation Sequencing in Real-world Cases: A Single-institution Study of Nine Cases.

Targeted next-generation sequencing (NGS) is a well-established technique to detect pathogenic alterations in tumors. Indeed, it is the cornerstone of targeted therapy in precision medicine. We investigated the clinical utility of next-generation sequencing in real-world cases. We retrospectively selected six representative cancer cases, wherein targeted NGS played a pivotal role in the diagnosis and treatment of patients. Additionally, we analyzed three cases with rare, unusual pathogenic alterations. Our NGS analysis revealed that four patients had TPR-ROS1, EGFR-RAD51, and NCOA4-RET fusions and MET exon 14 skipping mutation, respectively, which can be treated with targeted therapy. Furthermore, we used NGS as a diagnostic tool to confirm the origin of unknown primary malignant tumors in two cases. Interestingly, NGS also helped us identify the following cases: patients exhibiting BRCA1 and TP53 mutations that exhibited histological and immunohistochemical characteristics consistent with endometrioid carcinoma, patients with high-grade serous carcinoma not possessing a TP53 mutation, and patients with small cell lung cancer with a ERBB2 mutation and displaying no loss of RB1. We recommend targeted NGS for the diagnoses and targeted therapy of cancer patients.

47. Tumour-only NGS profiling: Application of data filters to identify candidate germline variants

Identifying potential germline pathogenic variants can inform eligibility for certain therapies, management of future cancer risk, and cascade familial variant testing. The OCTANE study is an ongoing Ontario-wide initiative to assess the clinical utility of next-generation sequencing (NGS) for patients with advanced solid tumours (NCT02906943). As the study performs tumour-only profiling rather than testing of paired tumour-normal samples, we sought to devise a variant filtration approach to identify potentially pathogenic germline variants in tumour tissue for confirmatory testing and clinical follow-up.

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.

What's Next in CML - a Prospective Study Evaluating Sanger Sequencing and Next Generation Sequencing (NGS) for BCR-ABL1 Kinase Domain (KD) Mutation Screening

What's Next in CML - a Prospective Study Evaluating Sanger Sequencing and Next Generation Sequencing (NGS) for BCR-ABL1 Kinase Domain (KD) Mutation Screening

Myeloid Sarcoma Type of Acute Promyelocytic Leukemia With a Cryptic Insertion of RARA Into FIP1L1: The Clinical Utility of NGS and Bioinformatic Analyses

BackgroundAcute promyelocytic leukemia (APL) is characterized by the presence of coagulopathy at onset and translocation t (15; 17) (q22; 21), meanwhile, other translocation variants of APL have also been reported. The FIP1L1–RARA fusion gene has recently been reported as a novel RARA-associated fusion gene.ObjectivesWe report a case of de novo myeloid sarcoma (MS) type of APL with FIP1L1–RARA found by next-generation sequencing (NGS) that was not detected by conventional analyze analysis for RARA translocations.MethodsWe performed typical morphological, magnetic resonance imaging (MRI), conventional tests for PML–RARA dual-fusion translocation probe, high-through sequencing and NGS. Meanwhile, bioinformatics analyses were done by using public repositories, including ONCOMINE, COSMIC, and GeneMANIA analysis.ResultsA 28-month-old girl with a complex karyotype that includes 46,XX,t(4;17)(q12;q22)[9]/46,idem,del(16)(q22)[3]/45,idem,-x,-4,-9,-15,del(16)(q22),+marl,+mar2,+mar3[7]/46,xx[3], c.38G>A (p.Gly13Asp) in the KRAS gene, and a cryptic insertion of RARA gene into the FIP1L1 gene was diagnosed with APL complicated by the de novo MS.ConclusionWe report a FIP1L1–RARA fusion in a child with APL who presented with an extramedullary tumor in the skull without the classic karyotype using NGS, whom we treated with good results. NGS analysis should be considered for APL variant cases. Further experimental studies to the association between the mutation in KRAS gene and FIP1L1–RARA fusion on the clinical phenotype and progression of APL are needed to identify more effective therapeutic targets for APL.

Next generation sequencing in children with unexplained epilepsy: A retrospective cohort study

ObjectiveTo evaluate the clinical utility of next-generation sequencing (NGS) in unexplained pediatric epilepsy, and to identify the potential predictors associated with Mendelian genetic causes. MethodsTwo hundred and ten children with unexplained epilepsy, who underwent NGS test were included. We analyzed the demographic, clinical and genetic characteristics, and executed a Logistic regression analysis for identifying predictors for Mendelian genetic causes. Patients were classified as either with isolated epilepsy or syndromic epilepsy with concurrent neurodevelopmental phenotypes. ResultsThe overall diagnostic yield was 29.0% (61/210). A total of 68 variants spanning 39 genes were identified in 58 patients (27.6%, 58/210) from exome sequencing based testing. Of the 68 variants, 33 were novel ones. Besides, STAR and CNTN2 were identified to be a candidate gene for epilepsy. Patients with syndromic epilepsy had a much higher diagnostic yield than that of isolated epilepsy (53/135, 39.3% vs. 8/75, 10.7%, p = 0.000). The odds ratio of detecting genetic cause was 3.939 (95% CI 1.369–11.332) for syndromic epilepsy without epileptic encephalopathy (EE), 5.814 (95% CI 2.208–15.306) for EE, 2.958 (95% CI 1.093–8.000) for patients with seizure onset <12 months, and 2.932 (95%CI 1.414–6.080) for female. Of the 210 patients, 78.4% of patients (145/185) had at least a 50% reduction in seizure frequency and 58.9% (109/185) reached seizure freedom. There was no difference between seizure prognosis and diagnostic outcomes. SignificanceNGS is effective for Mendelian genetic etiological diagnosis for unexplained pediatric epilepsy. Female patients with syndromic epilepsy with onset within the first year of life are most likely to yield a positive test result.

Real-world utility of next-generation sequencing for targeted gene analysis and its application to treatment in lung adenocarcinoma.

PurposeThis study investigated the clinical utility of next‐generation sequencing (NGS) for detection of genetic alterations and its implications on treatment of lung adenocarcinoma in real‐world practice.Patients and MethodsData were reviewed for 391 patients with lung adenocarcinoma who underwent NGS between March 2017 and October 2018. Formalin‐fixed, paraffin‐embedded archival samples were used for performing NGS targeting 382 genes, including all exons of 199 genes, 184 hotspots, and the partial introns of 8 genes often rearranged in cancer. Survival analysis was performed for stage IV disease.ResultsAmong the 391 patients, at least one actionable mutation was identified in 294 patients (75.2%). The most commonly mutated gene was EGFR (n = 130, 33.2%), involving EGFR exon 19 deletion (n = 48, 12.3%), L858R (n = 47, 12%), and others (n = 35, 9%), followed by KRAS (n = 48, 12.3%), ALK (n = 40, 10.2%), RET (6%), MET (3%), ROS‐1 (3%), and BRAF (2%) mutations. TP53 (46.9%) and CDKN2A (12.6%) mutations were common co‐mutations in patients with AMs. With a median follow‐up duration of 16.8 months, median overall survival was 36.8 months in patients with stage IV disease. Patients treated with the corresponding targeted therapy for AMs based on NGS reports lived significantly longer than those not treated with such therapy (p < 0.001). After multivariate analysis, targeted therapy for AM was a significantly favorable factor for survival (AM without targeted therapy vs. AM with targeted therapy, hazard ratio 2.58, 95% confidence interval 1.57–4.25; p < 0.001).ConclusionThis study revealed that AMs could be comparably detected using NGS. Based on these NGS results, a suitable targeted therapy can be selected, which may improve survival in patients with lung adenocarcinoma. This NGS‐based approach is useful in real‐world practice to provide guidance when selecting targeted therapy.

Cover, Volume 42, Issue 4

The cover image is based on the Original Article Diagnostic and clinical utility of next-generation sequencing in children born with multiple congenital anomalies in the China neonatal genomes project (CNGP) by Wenhao Zhou et al., https://doi.org/10.1002/humu.24170.

Next-generation sequencing in children of West India with suspected inherited tubulopathies

Background: The renal tubules maintain homeostasis through an array of proteins coded by multiple genes, which directly or indirectly transport water and solutes in the tubules. Malfunction of these transport proteins leads to inherited tubulopathies. Objective: The objective of the study was to analyze the clinical utility of next-generation sequencing (NGS) in the diagnosis and management of children with tubulopathies. Materials and Methods: This retrospective study was conducted in a tertiary care nephrology center in West India between September 2016 and September 2020. All children ?18 years with suspected inherited tubulopathy, where an NGS was sent, were included. Clinical exome sequencing (CES) covering 6670 genes was done in 14 children. CES included 73 tubulopathy genes. The test result was interpreted as per American College of Medical Genetics classification: No pathogenic variant, variant of unknown significance (VUS), and likely pathogenic and pathogenic variant. Results: The median age (IQR) of the cohort at the onset of disease was 18 months (4–65). History of consanguinity was present in 2 children (14.2%). Five children (35.7%) had Fanconi syndrome, two had distal renal tubular acidosis (d-RTA), two had Bartter/Gitelman syndrome, two had rickets, two had nephrocalcinosis, and one had low-molecular-weight proteinuria. Nine children (64%) had pathogenic variants detected in eight genes: ATP6V0A4, CTNS1, FAH1, PHEX, SLC12A1, SLC4A1, SLCA2, and CLDN 16. Five (35.7%) were novel variants. Two children had three VUS in FAT1, EYA1, and KCNJ1 gene. Three children (21.4%) had no genetic variant. Bartter syndrome type 1 and d-RTA were the most common genetic diagnoses with two patients each. Other diagnoses were tyrosinemia type 1, nephropathic cystinosis, Fanconi Bickel syndrome, X-linked hypophosphatemic rickets, and familial hypomagnesemia, hypercalciuria, and nephrocalcinosis in one each. Conclusion: The yield of NGS in children with tubulopathy was remarkably high. NGS was useful in the diagnosis and management in these children.

Diagnostic and clinical utility of next-generation sequencing in children born with multiple congenital anomalies in the China neonatal genomes project.

Multiple congenital anomalies (MCAs) at birth have emerged as an important cause of neonatal morbidity and mortality. This study aimed to investigate the genetic causes and characteristics of clinical outcomes in a large cohort of neonates with MCAs. Clinical exome sequencing/exome sequencing/genome sequencing were undertaken from December 1, 2016 to December 1, 2019 to detect single nucleotide variations (SNVs) and copy number variations (CNVs) simultaneously in individuals who met the inclusion criteria. A total of 588 neonates with MCAs were enrolled. One hundred sixty-one patients received diagnosis, with 71 CNVs and 90 SNVs detected, the overall diagnostic rate being 27.38%. Cardiovascular malformation was the most common anomaly (60%) and accounted for the top symptomatic proportion in both CNVs and SNVs. As the number of involved system increased from 2 to 3-4, and then to ≥5, the overall diagnostic rate increased gradually from 23.1% to 30.5%, and then to 52.2%, respectively. Patients who received genetic diagnoses were offered better clinical management or were referred to the specific disease clinic. In conclusion, this large cohort study demonstrates that both CNVs and SNVs contribute to the genetic causes of MCAs, and earlier genetic assertion may lead to better clinical management for patients.

First familial cases of type 2 congenital erythrocytosis (ECYT2) with a Chuvash pathogenic variant in VHL gene in Poland: example of the clinical utility of next-generation sequencing in diagnostics of orphan diseases

AbstractIn this article, we report familial cases of type 2 congenital erythrocytosis (ECYT2) in two siblings, a 2-year-old boy and his younger sister. Both patients were diagnosed based on laboratory findings including erythrocytosis, elevated hemoglobin levels, and hematocrit. Acquired erythrocytosis was excluded based on the clinical features and genetic analysis of JAK2/CALR/MPL genes. Next-generation sequencing was employed for older brother revealing NM_000551.4: c.598C&gt;T, p.Arg200Trp homozygous variant in the VHL gene, the similar variant was detected in the younger sibling. Sequencing analysis confirmed the VHL c.598C&gt;T heterozygous variant in both parents. To the best of our knowledge, these are the first confirmed cases of familial erythrocytosis type 2, also known as Chuvash type, in Poland.

Analysis of Chromosomal Copy Number in First-Trimester Pregnancy Loss Using Next-Generation Sequencing.

Embryonic chromosomal abnormality is one of the significant causative factors of early pregnancy loss. Our goal was to evaluate the clinical utility of next-generation sequencing (NGS) technology in identifying chromosomal anomalies associated with first-trimester pregnancy loss. In addition, we attempted to provide fertility guidance to couples anticipating a successful pregnancy. A total of 1,010 miscarriage specimens were collected between March 2016 and January 2019 from women who suffered first-trimester pregnancy loss. Total DNA was isolated from products of conception, and NGS analysis was carried out. We detected a total of 634 cases of chromosomal variants. Among the 634 cases, 462 (72.9%) displayed numerical variants including 383 (60.4%) aneuploidies, 44 (6.9%) polyploidies, and 34 (5.5%) mosaicisms. The other 172 (27.1%) cases showed structural variants including 19 (3.0%) benign copy number variations (CNVs), 52 (8.2%) pathogenic CNVs, and 101 (16%) variants of unknown significance (VOUS) CNVs. When maternal age was ≥ 35 years, the sporadic abortion (SA) group showed an increased frequency of chromosomal variants in comparison with the recurrent miscarriage (RM) group (90/121 vs. 64/104). It was evident that the groups with advanced maternal age had a sharply increased frequency of aneuploidy, whatever the frequency of pregnancy loss (71/121 vs. 155/432, 49/104 vs. 108/349). Our data suggest that NGS could be used for the successful detection of genetic anomalies in pregnancy loss. We recommend that fetal chromosome analysis be offered routinely for all pregnancy losses, regardless of their frequency.

Abstract 3101: Utility of a new targeted next generation sequencing test for liquid biopsy samples from patients with NSCLC

Abstract Blood-based methods for profiling tumor-sourced nucleic acids have become increasingly important in the diagnostic workup for cancer patients, particularly in non-small cell lung cancer (NSCLC). Blood-based assays address several limitations of tissue-based testing approaches such as inaccessible lesions, limited tissue, extended time to results, and invasive specimen collection procedures. Broad-profiling next-generation sequencing (NGS) methods are used to detect rare variants in blood samples. Here we describe the utility of a newly developed 52 gene NGS test, which uses the GeneStudio S5 Prime platform. We report on performance verification of the assay using contrived specimens as well as studies which evaluated clinical specimens at diagnosis and during monitoring. Thirty-eight cell-free nucleic acid (cfNA) specimens were evaluated from donors previously diagnosed with NSCLC. Specimens were collected in blood collection tubes containing preservatives (Streck cfDNA BCT) and shipped at ambient temperature to our central testing Laboratory. cfNA was isolated from plasma and reverse transcribed. The sequencing metrics for these studies included an average of 15 million mapped reads, 94.4% of on target reads, and a mean sequencing depth of 52,000. Additionally, the average molecular coverage was 2400 reads per amplicon with 99.9% uniformity at 500x coverage. Detected variants and their allelic frequencies were compared with reference results from a validated ddPCR method. Eleven variants (6 EGFR and 5 KRAS) were detected in the cohort with allele frequencies ranging from 0.90 - 96.16%. Results were highly concordant between the two methods (R2=0.992) with 90.3% sensitivity and 100% specificity. In addition, we present a case study to further demonstrate clinical utility of NGS for longitudinal monitoring over 540 days for clearance followed by re-emergence of EGFR positive cfNA harboring the EGFR C797S resistance mutation. These studies add to the growing body of knowledge supporting the utility of molecular detection for actionable and emerging biomarkers in liquid biopsy. Citation Format: Jordan Reese, Victoria Maxwell, Leisa Jackson, Gary Pestano, Hestia S. Mellert. Utility of a new targeted next generation sequencing test for liquid biopsy samples from patients with NSCLC [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 3101.