Next-generation Sequencing Assay Research Articles

Prevalence of BRCA1 and BRCA2 mutations in an unselected cohort of Indian women with breast cancer.

e13004 Background: Familial breast cancer accounts for 10-15% of breast cancer cases. The prevalence of germline pathogenic mutations in breast cancer susceptibility genes 1 and 2 ( BRCA1/2) among female breast cancer patients remained variable across studies depending on the population/settings studied. The estimate varied between 8-10% of the unselected female breast cancer population. There is a lack of data regarding the prevalence of BRCA1/2 among female breast cancer patients in India. Methods: This prospective observational study included consecutive patients with breast cancer in females, registered at All India Institute of Medical Sciences (AIIMS), New Delhi, India between the period of 1st September 2022 to 25th January 2023(5 months duration). The staging description was done as per anatomic TNM staging according to AJCC 7th edition. Mutation testing was performed in all patients by a standard next-generation sequencing (NGS) assay covering the BRCA1/2 gene. The reflex multiplex ligation by probe amplification method was performed in all cases that were negative by NGS. For patients with detected germline mutations in BRCA1/2, post-test counseling was done, and family screening was also advised. Results: A total of 210 female patients with breast cancer were sequentially included in the study. Young breast cancer (less than 35 years of age) constituted 40 (19.04%) cases. Thirty-eight patients (18.09%) women had a positive significant family history. The median age of the cohort was 46 years (range 18-85 years). Synchronous breast cancer was present in 7 (3.3%) cases. The stage distribution was stage I, 11(5.23%); stage II, 73 (34.76%); stage III, 83 (39.52%); and stage IV, 43 (20.47) %. One hundred and ten patients (52.38%) were hormone-receptor-positive, whereas 66 (31.42%) patients were HER2/neu positive. Triple-negative breast cancer (TNBC) was found in 65 (30.95%) cases. Overall, the prevalence of germline BRCA1/2 mutations was 35/210 (16.6%), which included BRCA 1 and 2 mutations in 22 (10.47%) and 12 (5.71) cases respectively. One case had mutations in both BRCA 1 and BRCA2. Young ( < 35) TNBCs (22 cases/210, 10.47%) have a high propensity for detecting pathogenic BRCA1 mutation (8/22, 36.36%). Conclusions: The prevalence of pathogenic BRCA1/2 in 16.6% of our cohort of unselected consecutive breast cancer patients. It is higher than that reported in the Western population likely due to the younger population and more proportion of triple-negative breast cancer patients in our cohort.

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]

Analytical and clinical validation of oncomine Dx target (ODxT) test and local testing for identifying patients with HER2 (ERBB2)-mutant (HER2m) non–small-cell lung cancer (NSCLC) for treatment with trastuzumab deruxtecan (T-DXd) in DESTINY-Lung01/02 (DL-01/02).

9033 Background: Based on the international DL-01 trial, the US Food and Drug Administration (FDA) approved the ODxT Test as a companion diagnostic (CDx) to identify patients (pts) with NSCLC harboring activating HER2 mutations who may benefit from T-DXd. The ODxT Test uses next-generation sequencing (NGS) to detect genomic alterations in formalin-fixed, paraffin-embedded tumor tissue. The accuracy of the ODxT Test by central testing and its agreement with clinical trial assays (CTAs; NGS, polymerase chain reaction, etc.) by local testing was evaluated using analytical and clinical validation data from DL-01 cohort 2 and DL-02 arm 1. Methods: ODxT Test and Illumina TruSight Tumor170 NGS assay results were compared for analytical accuracy in NSCLC samples from DL-01 and commercial vendors. Diagnostic agreement was assessed for DL-01/02 by concordance between the ODxT Test and CTAs. Clinical accuracy per objective response rate (ORR) and duration of response (DoR) to T-DXd were evaluated in all pts with HER2m NSCLC identified by ODxT Test vs CTA. Activating HER2 mutations included single nucleotide variants and exon 20 insertions. Pts received T-DXd 6.4 mg/kg in DL-01 or 5.4 mg/kg (FDA-approved dose) in DL-02 every 3 weeks. Results: NSCLC samples from DL-01 (n = 91), DL-02 (n = 102), and commercial vendors (n = 129) were individually assessed. The ODxT Test met requirements for analytical accuracy (≥90% agreement when excluding unknown results), with a positive percent agreement (PPA) of 100% and negative percent agreement (NPA) of 99.1% for detecting HER2 mutations. Clinical accuracy of the ODxT Test also met FDA requirements (excluding unknowns) for NPA (≥98%) and PPA (95% CI lower bound ≥85%). DL-01 PPA (95% CI) was 98.0% (89.2%-100%) and NPA was 100.0% (96.6%-100%); for DL-02, PPA was 96.7% (88.7%-99.6%) and NPA was 100% (96.6%-100%). In DL-01, confirmed ORR (95% CI) for pts with HER2m NSCLC identified by central ODxT Test was 58.3% (43.2%-72.4%) vs 54.9% (44.2%-65.4%) of pts identified by local CTA; median DoR (95% CI) was 12.0 mo (5.5 mo-18.2 mo) per the ODxT Test vs 9.3 mo (5.7 mo-14.7 mo) per CTA. In DL-02 (prespecified interim analysis early cohort), ORR (95% CI) was 53.6% (33.9%-72.5%) per ODxT Test vs 53.8% (39.5%-67.8%) per CTA; median DoR (95% CI) was not estimable (NE [NE-NE]) per the ODxT Test and NE (4.2 mo-NE) per CTA. Conclusions: This study demonstrates the analytical and clinical accuracy of the ODxT Test as a CDx for detecting activating HER2 mutations in NSCLC to identify pts who may benefit from T-DXd. A high level of agreement was also shown between the ODxT Test and CTAs. Clinical trials for HER2m NSCLC may successfully use a central testing assay as a CDx while allowing adequately validated local testing assays to expedite patient enrollment. Clinical trial information: NCT03505710 , NCT04644237 .

Liquid biopsy in combination with PET/CT versus PET/CT alone in diagnosis of lung cancer in small nodules.

3072 Background: In the National Lung Screening Trial, almost two-thirds of patients that underwent an invasive procedure for diagnosis had benign findings, suggesting a significant number of patients were exposed to risk that could be avoided. Currently, to help distinguish malignant from benign nodules, Positron Emission Tomography/Computed Tomography (PET/CT) is used. However, PET/CT can be limited in its use for lesions less than 2 cm in size. The detection of circulating tumor DNA (ctDNA) and cell-free DNA (cfDNA) in the blood stream via liquid biopsy has demonstrated potential as a non-invasive approach in both screening and diagnostic tests. The purpose of this study is to assess whether combining liquid biopsy with PET/CT findings can better detect malignancy in small lung nodules 6-20 mm in size compared to PET/CT alone, possibly allowing earlier cancer detection and reduction in unneeded invasive diagnostic testing. Methods: This was a single-center prospective observational study for which the primary objective was to compare liquid biopsy plus PET/CT to PET/CT alone for detection of lung cancer in patients with newly discovered or newly growing solid lung nodules. Patients with nodules 6-20 mm in size who were scheduled to undergo 18F-Fluorodeoxyglucose PET/CT also underwent blood collection for plasma ctDNA/cfDNA testing using an amplicon-based next-generation sequencing assay incorporating molecular barcodes for error suppression. Tumor diagnosis was based on resolution of the nodule or tissue biopsy. For liquid biopsy, a multiomic model previously built using (1) cfDNA methylation frequencies, (2) ctDNA abundance, (3) somatic ctDNA mutations, and (4) cfDNA fragment size ratio was used to predict malignancy. For imaging, lesions with a maximum standardized uptake value of 2 or greater were considered malignant. Results: Between December 2021 and November 2022, 24 patients were enrolled. Of these, 13 had a definitive diagnosis and were included in the analysis with the remainder still undergoing serial imaging follow-up. Of the 13 patients, 9 were biopsy or surgically proven to be malignant, and 4 were benign based on biopsy or resolution. The median (IQR) nodule size was 14 mm (8-15). The sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) for PET/CT were 0.67, 1, 1, and 0.57, respectively. For multiomic liquid biopsy alone, these values were the same. However, combining multiomic liquid biopsy and PET/CT, the sensitivity, specificity, PPV, and NPV were 0.89, 1, 1, and 0.8, respectively. Conclusions: Combining multiomic liquid biopsy with PET/CT results in improved sensitivity for the detection of early-stage malignant solid lung nodules by 33% compared to PET/CT alone with no reduction in specificity. This could result in improved selection of patients requiring biopsy. Further evaluation with a larger cohort is needed to confirm the findings.

Abstract 1908: Clinical genomic profiling identifies lower frequency of therapeutically actionable alterations and lower prognostic value of APC inactivation in colorectal cancer patients of African ancestry

Abstract Intro. We compared molecular profiles and clinical outcomes in a large cohort of colorectal cancer (CRC) patients treated at a single tertiary center to investigate differences in the frequency of therapeutically targetable alterations and the prognostic value of somatic driver alterations across ancestry groups. Methods. We analyzed targeted DNA sequencing data from 4,441 CRC patients treated at Memorial Sloan Kettering between 2014 and 2022. Tumors were sequenced using MSK-IMPACT, a next-generation tumor-normal sequencing assay containing up to 505 genes. Genetic ancestry was estimated using reference populations from the 1000 Genomes Project, including European (EUR), African (AFR), East Asian (EAS), South Asian (SAS), and Native American (NAM). Patients were assigned specific ancestry labels when the predominant ancestry fraction was >80% and considered to have admixed ancestry (ADM) otherwise. Clinical actionability of individual genomic features was determined using the OncoKB knowledgebase. Results. Our cohort included 3,265 EUR, 263 EAS, 245 AFR, 89 SAS, and 15 NAM patients; 564 ADM patients were excluded from subsequent analyses. The AFR group had shorter overall survival (OS) from time of diagnosis (median 45.7 months vs. 67.1 months, p<0.0001). The fraction of patients who qualify for immunotherapy based on FDA guidelines, including microsatellite unstable (MSI) or highly mutated (TMB>10 mut/Mb), was lower in AFR vs. EUR (13.5% vs. 20.4%, p=0.008) patients. Among microsatellite stable (MSS) and not highly mutated (TMB<10 mut/Mb) patients, AFR patients had lower rates of clinically actionable alterations than EUR patients (5.6% vs 11.2%, p=0.01). This difference was driven by a depletion of actionable BRAF mutations in the AFR group (1.8% vs. 5.0%, p=0.04). Somatic APC alterations were associated with longer OS in MSS EUR patients (median 64.6 months in APC altered vs. 45.6 months in APC wildtype, p<0.0001), EAS patients (median 63.1 vs. 35.0 months, p=0.0015), and SAS patients (median not reached vs. 39.4 months, p=0.012). However, APC alterations exhibited no prognostic value for the AFR patients (median OS 45.0 vs. 45.9 months, p=0.91). Multivariate analyses accounting for sex, age, primary tumor location, and stage at diagnosis showed an association between APC status and OS in EUR patients (HR 0.64, CI 0.52-0.79, p<0.001), but not in AFR patients (HR 0.74, CI 0.31-1.7, p=0.492). Conclusions. AFR patients had fewer clinically actionable alterations than patients from other ancestries. The prognostic value of somatic APC alterations was also lower in AFR patients. Our findings provide novel insights into the genomic basis of racial disparities in CRC and highlight the need of ancestry stratification for the analysis of associations between molecular profiles and clinical outcomes. Citation Format: Henry Walch, Anisha Luthra, Kanika S. Arora, Michele Waters, Samantha Chin, Christopher J. Fong, Doori Rose, Hannah Williams, Jeese J. Smith, Nikolaus Schultz, Michael F. Berger, Karuna Ganesh, Julio Garcia-Aguilar, Rona Yaeger, Walid K. Chatila, Francisco Sanchez-Vega. Clinical genomic profiling identifies lower frequency of therapeutically actionable alterations and lower prognostic value of APC inactivation in colorectal cancer patients of African ancestry [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1908.

Calibration of cell-free DNA measurements by next-generation sequencing.

Accurate monitoring of disease burden depends on accurate disease marker quantification. Although next-generation sequencing (NGS) is a promising technology for noninvasive monitoring, plasma cell-free DNA levels are often reported in misleading units that are confounded by non-disease-related factors. We proposed a novel strategy for calibrating NGS assays using spiked normalizers to improve precision and to promote standardization and harmonization of analyte concentrations. In this study, we refined our NGS protocol to calculate absolute analyte concentrations to (1) adjust for assay efficiency, as judged by recovery of spiked synthetic normalizer DNAs, and (2) calibrate NGS values against droplet digital polymerase chain reaction (ddPCR). As a model target, we chose the Epstein-Barr virus (EBV) genome. In patient (n = 12) and mock (n = 12) plasmas, NGS and 2 EBV ddPCR assays were used to report EBV load in copies per mL of plasma. Next-generation sequencing was equally sensitive to ddPCR, with improved linearity when NGS values were normalized for spiked DNA read counts (R2 = 0.95 for normalized vs 0.91 for raw read concentrations). Linearity permitted NGS calibration to each ddPCR assay, achieving equivalent concentrations (copies/mL). Our novel strategy for calibrating NGS assays suggests potential for a universal reference material to overcome biological and preanalytical variables hindering traditional NGS strategies for quantifying disease burden.

Profiling of Microbial Community in Rearing Water of White Shrimp (Litopenaeus vannamei) Infected with White Feces Disease Syndrome

Shrimp farmers have reported mass mortality of white shrimp (Litopenaeus vannamei), reaching 2-3 kg/day in Gerongan Village, Kraton District, Pasuruan Regency in mid-2021. Preliminary analysis suggests that mass mortality was caused by bacterial diseases. Thus, to find out the main pathogen causing the mass mortality, the present study investigated microbial composition in rearing media of white shrimp (Litopenaeus vannamei) infected by the white feces disease (WFD) and healthy white shrimp using next-generation sequencing (NGS) technology. The research was conducted by collecting normal water samples and infected shrimp pond water samples. The results of the NGS assay showed that Vibrio vulnificus was found dominant in WFD-infected shrimp pond water, therefore was suspected to be the main cause of WFD.

Which trial do we need? Plasma metagenomic next-generation sequencing to diagnose infections in patients with haematological malignancies and febrile neutropenia: proposal for a randomized-controlled trial

Which trial do we need? Plasma metagenomic next-generation sequencing to diagnose infections in patients with haematological malignancies and febrile neutropenia: proposal for a randomized-controlled trial

Additional Oncogenic Alterations in RAS-Driven Differentiated Thyroid Cancers Associate with Worse Clinicopathologic Outcomes.

RAS mutations occur across the spectrum of thyroid neoplasms, and more tools are needed for better prognostication. The objective of this study was to evaluate how additional genetic events affecting key genes modify prognosis in patients with RAS-mutant thyroid cancers, and specifically differentiated thyroid cancers (DTC). We performed a clinical-genomic analysis of consecutive patients with DTC, poorly differentiated (PDTC), or anaplastic thyroid cancer (ATC) between January 2014 and December 2021, in whom a custom-targeted next-generation sequencing assay was performed. Patients harboring RAS mutations were included, and we compared their clinical features and outcomes based upon the presence of additional oncogenic alterations. Seventy-eight patients were identified, with 22% (17/78) harboring a driver RAS mutation plus an additional oncogenic alteration. All six (100%) ATCs had an additional mutation. Compared with DTCs harboring a solitary RAS mutation, patients with DTC with RAS and additional mutation(s) were more likely to be classified as American Thyroid Association high-risk of recurrence (77% vs. 12%; P < 0.001) and to have larger primary tumors (4.7 vs. 2.5 cm; P = 0.002) and advanced stage (III or IV) at presentation (67% vs. 3%; P < 0.001). Importantly, over an average 65-month follow-up, DTC-specific-mortality was more than 10-fold higher (20% vs. 1.8%; P = 0.011) when additional mutations were identified. Identification of key additional mutations in patients with RAS-mutant thyroid cancers confers a more aggressive phenotype, increases mortality risk in DTC, and can explain the diversity of RAS-mutated thyroid neoplasia. These data support genomic profiling of DTCs to inform prognosis and clinical decision-making.

Clinical considerations at the intersection of hematopoietic cell transplantation and hereditary hematopoietic malignancy.

In recent years, advances in genetics and the integration of clinical-grade next-generation sequencing (NGS) assays into patient care have facilitated broader recognition of hereditary hematopoietic malignancy (HHM) among clinicians, in addition to the identification and characterization of novel HHM syndromes. Studies on genetic risk distribution within affected families and unique considerations of HHM biology represent exciting areas of translational research. More recently, data are now emerging pertaining to unique aspects of clinical management of malignancies arising in the context of pathogenic germline mutations, with particular emphasis on chemotherapy responsiveness. In this article, we explore considerations surrounding allogeneic transplantation in the context of HHMs. We review pre- and post-transplant patient implications, including genetic testing donor selection and donor-derived malignancies. Additionally, we consider the limited data that exist regarding the use of transplantation in HHMs and safeguards that might be pursued to mitigate transplant-related toxicities.

Identification of Toxic Proteins Encoded by Mycobacteriophage TM4 Using a Next-Generation Sequencing-Based Method.

Mycobacteriophages are viruses that specifically infect mycobacteria and which, due to their diversity, represent a large gene pool. Characterization of the function of these genes should provide useful insights into host-phage interactions. Here, we describe a next-generation sequencing (NGS)-based, high-throughput screening approach for the identification of mycobacteriophage-encoded proteins that are toxic to mycobacteria. A plasmid-derived library representing the mycobacteriophage TM4 genome was constructed and transformed into Mycobacterium smegmatis. NGS and growth assays showed that the expression of TM4 gp43, gp77, -78, and -79, or gp85 was toxic to M. smegmatis. Although the genes associated with bacterial toxicity were expressed during phage infection, they were not required for lytic replication of mycobacteriophage TM4. In conclusion, we describe here an NGS-based approach which required significantly less time and resources than traditional methods and allowed the identification of novel mycobacteriophage gene products that are toxic to mycobacteria. IMPORTANCE The wide spread of drug-resistant Mycobacterium tuberculosis has brought an urgent need for new drug development. Mycobacteriophages are natural killers of M. tuberculosis, and their toxic gene products might provide potential anti-M. tuberculosis candidates. However, the enormous genetic diversity of mycobacteriophages poses challenges for the identification of these genes. Here, we used a simple and convenient screening method, based on next-generation sequencing, to identify mycobacteriophage genes encoding toxic products for mycobacteria. Using this approach, we screened and validated several toxic products encoded by mycobacteriophage TM4. In addition, we also found that the genes encoding these toxic products are nonessential for lytic replication of TM4. Our work describes a promising method for the identification of phage genes that encode proteins that are toxic to mycobacteria and which might facilitate the identification of novel antimicrobial molecules.

Circulating HPV DNA as a Biomarker for Pre-Invasive and Early Invasive Cervical Cancer: A Feasibility Study.

High-risk HPV infection is responsible for >99% of cervix cancers (CC). In persistent infections that lead to cancer, the tumour breaches the basement membrane, releasing HPV-DNA into the bloodstream (cHPV-DNA). A next-generation sequencing assay (NGS) for detection of plasma HPV circulating DNA (cHPV-DNA) has demonstrated high sensitivity and specificity in patients with locally advanced cervix cancers. We hypothesised that cHPV-DNA is detectable in early invasive cervical cancers but not in pre-invasive lesions (CIN). Blood samples were collected from patients with CIN (n = 52) and FIGO stage 1A-1B CC (n = 12) prior to treatment and at follow-up. DNA extraction from plasma, followed by NGS, was used for the detection of cHPV-DNA. None of the patients with pre-invasive lesions were positive for CHPV-DNA. In invasive tumours, plasma from one patient (10%) reached the threshold of positivity for cHPV-DNA in plasma. Low detection of cHPV-DNA in early CC may be explained by small tumour size, poorer access to lymphatics and circulation, and therefore little shedding of cHPV-DNA in plasma at detectable levels. The detection rate of cHPV-DNA in patients with early invasive cervix cancer using even the most sensitive of currently available technologies lacks adequate sensitivity for clinical utility.

The Prevalence of Undiagnosed Salmonella enterica Serovar Typhi in Healthy School-Aged Children in Osun State, Nigeria.

Typhoid fever remains a significant public health concern due to cases of mis-/overdiagnosis. Asymptomatic carriers play a role in the transmission and persistence of typhoid fever, especially among children, where limited data exist in Nigeria and other endemic countries. We aim to elucidate the burden of typhoid fever among healthy school-aged children using the best surveillance tool(s). In a semi-urban/urban state (Osun), 120 healthy school-aged children under 15 years were enrolled. Whole blood and fecal samples were obtained from consenting children. ELISA targeting the antigen lipopolysaccharide (LPS) and anti-LPS antibodies of Salmonella Typhi, culture, polymerase chain reaction (PCR), and next-generation sequencing (NGS) were used to analyze the samples. At least one of the immunological markers was detected in 65.8% of children, with 40.8%, 37.5%, and 39% of children testing positive for IgM, IgG, and antigen, respectively. Culture, PCR, and NGS assays did not detect the presence of Salmonella Typhi in the isolates. This study demonstrates a high seroprevalence of Salmonella Typhi in these healthy children but no carriage, indicating the inability to sustain transmission. We also demonstrate that using a single technique is insufficient for typhoid fever surveillance in healthy children living in endemic areas.

Insight into the metabolic pathway of EAD based on metabolic flux, microbial community, and enzyme activity

Electrochemical anaerobic digestion (EAD) is essential for methane production. In this study, a cell net analyzer (CNA) was used to analyze the EAD metabolic flux and pathway and explore the dynamic changes in microbial community composition and critical enzyme activities by next-generation sequencing (NGS) and enzyme-linked immunosorbent assay (ELISA) to explore the reason for the high methane production rate of EAD. The results showed that EAD was more inclined toward hydrogen reduction of CO2 than conventional anaerobic digestion. Metabolic flux analysis (MFA) confirmed that the Embden–Meyerhof–Parnas (EMP) pathway was the main glycolysis pathway. According to metabolic flux analysis, hydrogen production from the electrodes accounted for 45.04%. Fermentimonas enhances the source of hydrogen, thereby facilitating the pathway of methane production by hydrogen reduction of CO2. Methanobacterium is the only dominant hydrotropic methanogen. Nicotinamide adenine dinucleotide (NAD) upregulates the hydrogen production pathway, whereas methylofuran (MFR) and tetrahydromethanopterin (MH4) are specific enzymes in the hydrogen-reducing CO2 pathway. Therefore, the results of this study help further reveal the electrochemical and biological principles related to EAD and provide theoretical support for the methanogenesis metabolism mechanism of EAD.

Abstract 4294: Bridging the gap between targeted NGS and FISH gene-level CNV detection capabilities in hematologic malignancies

Abstract Background: Copy Number Variations (CNVs) are prominent features of cancer cells. From a clinical standpoint, their accurate detection at a low cost is a priority. With regular increases in the number of markers to be tested, the cost effectiveness and practicality of gold standard techniques like Fluorescence In Situ Hybridization (FISH) are slowly decreasing. Cost-efficient Next Generation Sequencing (NGS) targeted gene panels can be scaled up but accurately detecting CNVs from the resulting data remains challenging. We demonstrate large amounts of data and machine learning can help bridge the gap between the two techniques. Methods: We collected the sequencing data of 6,277 patients tested using a custom amplicon based NGS assay designed to detect somatic alterations in 297 hematological cancer relevant genes such that at least one concurrent FISH test was also performed. FISH results were used to infer the gain, loss, or normality information for each corresponding gene. The annotated genes were then used to curate a training set by extracting 20 features per gene from the alignment results. A 3-class random forest classifier was trained using this dataset. The selected model was evaluated on a distinct set of 2,738 patients. Results: Evaluation results are provided in Table 1 for 8 genes for which the FISH probe used to infer the gain, loss, or normality information directly spanned the gene region. The predicted CNVs are almost a perfect match with FISH for 6 of these genes with a limit of detection at 20% abnormal cells. In most cases, the model reduces discordant calls by over 50% compared to using existing CNV detection software only. Conclusion: We show the CNV detection capabilities of a targeted NGS assay can closely match the gold-standard FISH technique by analytically correcting the biases introduced by the targeting procedures. The model presented here is used to detect CNVs in ALL patients after a successful formal validation in our laboratory. Table 1. Evaluation results. Gene FISH Positive Cases (Gain/Loss) FISH Negative Cases (Normal) All FISH Cases Total Concordant Sensitivity Total Concordant Specificity Total Concordant Accuracy ATM 72 72 100.00% 641 629 98.13% 713 701 98.32% CBFB 23 22 95.65% 517 506 97.87% 540 528 97.78% EGR1 171 169 98.83% 1,541 1,538 99.81% 1,712 1,707 99.71% KMT2A 27 25 92.59% 474 472 99.58% 501 497 99.20% MET 113 113 100.00% 1,593 1,582 99.31% 1,706 1,695 99.36% NF1 15 15 100.00% 908 908 100.00% 923 923 100.00% TERT 10 10 100.00% 1,723 1,709 99.19% 1,733 1,719 99.19% TP53 76 73 96.05% 1,567 1,556 99.30% 1,643 1,629 99.15% Citation Format: Christophe N. Magnan, Hyunjun Nam, Shashikant Kulkarni, Segun C. Jung. Bridging the gap between targeted NGS and FISH gene-level CNV detection capabilities in hematologic malignancies. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4294.

Abstract 6685: Targeted liquid biopsy mutation profiling and genome-wide cfDNA copy-number landscape in 3000 samples from six major cancer indications

Abstract Introduction. PredicineCARE is a CLIA-certified targeted next-generation sequencing (NGS) assay that provides comprehensive, non-invasive genomic profiling of circulating tumor DNA (ctDNA) in blood and urine. Here we present liquid biopsy mutational profiles and genome-wide cell-free DNA (cfDNA) copy-number burden levels for historical cancer cohorts analyzed with PredicineCARE. We include a total of 3,147 samples from six major cancer indications: prostate (n=1681), lung (n=630), breast (n=300), colorectal (n=217), pancreatic (n=190), and bladder (n=129). Methods. PredicineCARE employs a 152 targeted gene panel covering all major guideline-recommended alterations for patient care and clinical drug development including targeted therapies and immunotherapies. The assay detects single nucleotide variants (SNVs), small insertions and deletions, gene-level copy number loss/gain, and targeted re-arrangements/fusions. Concurrent low-pass whole-genome sequencing (without the need for additional sample volume) is performed to measure blood-based genome-wide copy-number burden (bCNB) and tumor fraction, providing important complementary molecular insights to variant detection. Results. We present data from a representative cohort of blood and urine samples processed at Predicine lab in Hayward California (n=3,147). Sequencing was performed using a minimum of 5 ng total cfDNA input from 1-5 mL of plasma or 20-40mL of urine, at one or more clinical timepoints per patient. Samples were sequenced at ~20,000X target panel coverage and the majority also had ~2.5X low-pass whole genome sequencing. Using the Predicine in-house DeepSea bioinformatics pipeline, we report variants as low as 0.05% allele frequency and estimate genome-wide CNVs and bCNB from low-pass data. We compare the mutational and CNV profiles of important genes in each liquid biopsy cancer cohort to variants reported in solid tumor tissues from several large public data sets including Project GENIE (n=71,817 patients) and 136 additional study programs including TCGA (n=38,619 patients, www.cBioPortal.org). Conclusion. PredicineCARE is a highly sensitive and robust assay for detecting cancer variants in blood and urine. The cfDNA mutation profiles are mostly consistent with public tissue datasets. A small but significant portion of the detected ctDNA variants have been shown to be important for treatment selection or are currently being tested for targeted drug development. Increased bCNB provides a useful measure to monitor treatment response and disease progression. PredicineCARE has been CE-marked and received breakthrough device designation from FDA. A harmonized PredicineCARE assay is available in the US and China to support patient testing in the clinic, global clinical trials and CDx development. Citation Format: Billie Gould, Lu Tan, Chao Dai, Tiantian Zheng, Wei Mo, Xianghong Wang, Michael Sneddon, Shidong Jia, Kemin Zhou, Pan Du. Targeted liquid biopsy mutation profiling and genome-wide cfDNA copy-number landscape in 3000 samples from six major cancer indications [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6685.

Abstract 237: Coincidence of InDels with missense mutations: 0, 1 or 2 artifacts

Abstract The purpose of this study was to explain a serendipitously discovered next generation sequencing (NGS) artifact. While reviewing clinical cases, we found a case of a large deletion that also contained multiple missense mutations. This phenotype was unknown to several molecular pathologists in our group. We reviewed 46 clinical cases with short insertions or deletions (InDels) (1-30 bases) or FLT3 internal tandem duplications (ITDs) (6-183 bases) of solid or hematologic malignancy processed with a clinical NGS assay and identified misaligned reads, ranging from 3 to 100% of reads showing mismapped bases. Reads that straddled the InDel with sufficient numbers of bases on both sides were correctly anchored both upstream and downstream of the InDel, and were called correctly. However, reads that ended in the middle of the InDel were incorrectly mapped onto the deletion, thereby producing artifactual missense mutations. The frequency of mismapped bases increased with InDel size, and the VAF of the InDel can be markedly underestimated. The ratio of forward to reverse reads was commonly skewed for the mismapped base compared to the ratio for the wildtype base at that position. ABRA2 was able to correct 41 to 100% of the reads with mismapped bases and led to absolute increases in the VAF from 1 to 61% along with correction of all of the SBSs except for two cases. Knowledge of the various artifacts commonly seen in next generation sequencing is essential to safely sign out molecular pathology cases. Citation Format: Kelly E. Craven, Catherine G. Fischer, LiQun Jiang, Aparna Pallavajjala, Ming-Tseh Lin, James R. Eshleman. Coincidence of InDels with missense mutations: 0, 1 or 2 artifacts [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 237.

Abstract 4542: Development of a one-step molecular classifier for endometrial carcinoma using an amplicon-based gene panel and next generation sequencing technology

Abstract Objectives: Molecular classification of endometrial carcinoma (EC) is now recommended by the WHO, ESGO/ESTRO/ESP and NCCN guidelines. The pragmatic molecular classification tool, ProMisE, identifies four molecular subtypes based on next generation sequencing (NGS) for the detection of somatic pathogenic POLE mutations, and immunohistochemistry for mismatch repair and p53 proteins. ProMisE provides valuable prognostic and predictive information to direct care, however, multiple molecular results are often received from different time periods and/or from different centers which can cause delays. Therefore, we developed a classifier that relies on a single DNA-based NGS test with the goal of recapitulating the prognostic value of ProMisE by producing concordant results. Methods: Formalin fixed paraffin embedded (FFPE) EC tumor DNA was sequenced using the clinically validated Imagia Canexia Health Find ItTM amplicon-based next generation sequencing gene panel assay. The 38 gene panel assessed single nucleotide variants (SNVs), indels (insertions and deletions), gene amplification and microsatellite instability (MSI) using 21 MSI loci. To compare to the original ProMisE classifier, we identified somatic mutations in POLE, TP53 and MSI-High or MS-Stable for molecular classification. Molecular subtypes assigned by both classifiers were assessed for concordance metrics and Kaplan-Meier survival curves. Results: The one-step NGS molecular classifier assessed 165 unique patient FFPE EC samples that had previously been assessed by ProMisE. There were 152/165 cases that were concordant between molecular subtype assignment from the original ProMisE and the one-step NGS assay with a kappa statistic of 0.88 and accuracy of 0.92. There were 13 samples that were discordant (original classifications: 3 POLE, 3 MMRd, 2 p53abn, 5 NSMP/p53wt), which will be reviewed in detail. Results were concordant in 14 of 15 cases (93%) when both diagnostic biopsy and hysterectomy specimens were tested. Molecular subtypes maintained their associations with clinical outcomes (progression free survival, disease specific survival, overall survival). Conclusion: The one-step NGS molecular classifier demonstrates high concordance with the original ProMisE classifier including between biopsy and hysterectomy samples. This shows that reliable molecular testing could be obtained from time of first diagnosis. Prognostic value of this new one-step classification tool is maintained. Appropriate interpretation of results is critical, including limiting POLE mutation assignment to a confirmed list of pathogenic mutations and correct order of segregation for ECs with more than one molecular feature. Further validation is needed in a larger cohort to implement into standard of care testing. Citation Format: Melissa K. McConechy, Amy Jamieson, Amy Lum, Samuel Leung, Adrian Kense, Sonal Brahmbhatt, Ka Mun Nip, Ebru Baran, Dilmi Perera, Kurt Yakimovich, Ruth Miller, C. Blake Gilks, David Huntsman, Jessica N. McAlpine. Development of a one-step molecular classifier for endometrial carcinoma using an amplicon-based gene panel and next generation sequencing technology. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4542.

Abstract 5452: Complimentary use of DNA- and RNA-based NGS assays optimizes detection of clinically relevant translocations for comprehensive genomic profiling

Abstract Introduction: Detection of gene translocations is a key component of clinical diagnostics to enable precision medicine in oncology. Several methods such as fluorescence in situ hybridization or RT-PCR have historically been employed, however, next-generation sequencing (NGS)-based comprehensive genomic profiling (CGP) including DNA and RNA sequencing approaches have been validated for this purpose. Here we explore the complimentary nature of these methods to enable detection of clinically relevant translocations to guide patient care. Methods: We utilized Endeavor, a 505 gene DNA-based CGP assay developed by Personal Genome Diagnostics, to assess single nucleotide variants, insertion/deletions, amplifications, translocations, microsatellite instability, and tumor mutation burden as well as a 53 gene RNA-based Invitae NGS FusionPlex Solid Tumor v1 assay. 151 patients with advanced or metastatic solid tumors, including lung, colorectal, esophageal, breast, brain, bladder, and prostate cancers, were consecutively enrolled and only overlapping target regions were evaluated to compare performance. Results: Of the 151 cases selected for the study, failure rates were 2.0% and 9.9% for Endeavor and FusionPlex, respectively. Translocations were detected in 21/151 (13.9%) cases by Endeavor and 17/105 (11.3%) cases by FusionPlex. For the 133 cases where data was available from both assays, 12 (9.0%) concordant translocation positive cases were detected involving ALK, RET, NTRK1, NTRK3, MET exon 14 skipping, EGFRvIII, and EWSR1 with 109 (82.0%) cases translocation negative by both assays achieving a concordance rate of 91%. For 7 (5.3%) cases, Endeavor detected translocations events in FGFR1, FGFR2, ETV4, ETV6, MYC, and NTRK3 that were not detected by FusionPlex. Conversely, FusionPlex identified 5 (3.8%) cases with translocations in ROS1, NTRK2, and EGFRvIII that were not detected by Endeavor (Table 1). Discrepancies in translocation detection were attributed to variability in assay failure rates, panel design, and underlying biological differences in detectability associated with DNA- and RNA-based methods. Conclusions: In this study, comparison of translocation detection using DNA and RNA-based NGS approaches revealed a high concordance between the two assays and were equally valuable for identifying actionable targets. These findings provide confirmatory support for the complimentary use of DNA- and RNA-based NGS approaches to most accurately identify clinically relevant translocations thereby providing more comprehensive results to help guide cancer treatment strategies. Citation Format: Rongqin Ren, Jennifer Jackson, Jacob Kames, David Riles, Christopher Coldren, Scott Wheeler, Pranil Chandra, Michelle Shiller. Complimentary use of DNA- and RNA-based NGS assays optimizes detection of clinically relevant translocations for comprehensive genomic profiling. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5452.

FGFR2 testing in cholangiocarcinoma: translating molecular studies into clinical practice.

Cholangiocarcinoma (CCA) is a heterogeneous group of neoplasms burdened by a dismal prognosis. Several studies have investigated the genomic profile of CCA and identified numerous druggable genetic alterations, including FGFR2 fusions/rearrangements. Approximately 5-7% of CCAs and 10-20% of intrahepatic iCCAs harbor FGFR2 fusions. With the recent advent of FGFR-targeting therapies into clinical practice, a standardization of molecular testing for FGFR2 alterations in CCA will be necessary. In this review, we describe the technical aspects and challenges related to FGFR2 testing in routine practice, focusing on the comparison between Next-Generation Sequencing (NGS) and FISH assays, the best timing to perform the test, and on the role of liquid biopsy.