Next-generation Sequencing Testing Research Articles

Clinical Utility of Tumor Next-Generation Sequencing Panel Testing to Inform Treatment Decisions for Patients With Advanced Solid Tumors in a Tertiary Care Center.

There is limited information about the clinical utility of targeted next-generation sequencing (NGS) panel testing to inform decision making for patients with advanced solid tumors. The Ontario-wide Cancer Targeted Nucleic Acid Evaluation (OCTANE) is a prospective study that enrolled more than 4,500 patients with solid tumor for NGS panel testing. We performed a retrospective survey of medical oncologists to evaluate the impact of NGS testing on treatment decisions. Patients and treating oncologists were identified at the Princess Margaret Cancer Center between 2016 and 2021. Tumor-only sequencing was performed using a gene panel of either 555 or 161 cancer genes. Oncologists were asked to review testing results and complete a survey indicating whether NGS testing affected treatment decisions. The primary outcome of this study was rate of treatment change on the basis of mutation results. Patient, test, and physician factors were evaluated for association with treatment changes using univariate analyses and a mixed-effects model. Of the 582 surveys sent, 394 (67.7%) were completed. We found that 188 (47.7%) patients had testing results classified as actionable by the oncologist and 62 (15.7%) patients were matched to treatment, of whom 37 (60%) were enrolled in a clinical trial, 13 (21%) received an approved drug, four (6%) were prescribed off-label therapy, and eight (13%) avoided ineffective treatment. Patient, test, and physician characteristics were not significantly associated with treatment change. There was no difference in overall survival between patients who received matched treatment versus those who did not (P = .55, median survival not reached). OCTANE testing led to a change in drug treatment in 15.7% of patients, supporting the clinical utility of NGS panel testing for patients with advanced solid tumors.

Safety and immunological effects of ablative radiotherapy followed by pembrolizumab in patients with advanced adrenocortical carcinoma.

TPS4630 Background: Adrenocortical carcinoma (ACC) is a rare and aggressive cancer with poor prognosis. While pembrolizumab is a NCCN recommended treatment, our prior study showed no benefit in patients with liver metastases (NCT02673333).1 Preclinical evidence suggests combining liver-directed radiation therapy (RT) with pembrolizumab might enhance anti-tumor immunity.2 Our current study will assess the safety and describe preliminary efficacy data for ablative RT to symptomatic liver metastases followed by treatment with pembrolizumab in patients with advanced ACC. Methods: This is a single-center pilot study. Main inclusion criteria include: adult patients with metastatic ACC with symptomatic liver metastases, presence of extrahepatic metastases, ECOG 0-1. Ablative RT in 5 or 10 once daily fractions will be delivered over 1 to 2 weeks; pembrolizumab treatment (200mg Q3W) will begin one week after RT completion. The primary objective is to evaluate the safety of treatment with ablative RT followed by pembrolizumab. The study will include a 6 patient safety lead-in, with 10 to 12 patients enrolled in total. The secondary endpoint, efficacy, is defined as the objective response (partial and complete response) according to RECIST v1.1, in the hepatic and extrahepatic disease. Correlative objectives include evaluation of the innate and adaptive immune response during treatment through analysis of pre- and on-treatment blood and tumor tissue samples, circulating tumor (ct) DNA analyses, and PD-L1 and next-generation sequencing testing in pre-treatment tumor tissue samples to evaluate for biomarkers of response and resistance. This trial began enrollment in 9/27/2023, with 3 patients enrolled to date.

Next-Generation Sequencing Testing Can Save Generations of Lives.

Next-Generation Sequencing Testing Can Save Generations of Lives.

Analytical validation of the Labcorp Plasma Complete test to enable precision oncology through solid tumor liquid biopsy comprehensive genomic profiling.

3063 Background: To help guide treatment decisions and enable clinical trial matching for cancer patients, tumor genomic profiling is an essential precision oncology tool. Liquid biopsy may be used as a complementary approach to assess tumor-specific DNA alterations circulating in the blood. The Labcorp Plasma Complete test is a next-generation-sequencing (NGS), cell-free DNA (cfDNA) comprehensive genomic profiling test that identifies actionable and clinically relevant variants in advanced and metastatic solid cancers across 521 genes. Methods: The Labcorp Plasma Complete test is a hybrid capture based, targeted NGS test, optimized for 25 ng of cfDNA input and is intended for variant detection in all solid cancer indications. The test interrogates all coding exons of 521 genes to comprehensively detect single nucleotide variants (SNVs) and insertion and deletions (indels). The test also reports copy number amplifications (CNAs) and gene translocations in 12 genes, and microsatellite instability (MSI). In total, 631 cancer patient plasma samples were evaluated by the test including lung, breast, colorectal, head and neck, pancreatic, gynecologic, and prostate cancers among other indications. Well-characterized reference samples (Genome in a Bottle) were utilized to assess analytical specificity. Contrived cell lines and clinical samples were utilized to establish analytical sensitivity, accuracy, as well as precision, reproducibility, and repeatability (PRR). Results: The test yielded results for 98.3% (620/631) of cases and identified variants in 94.7% (587/620) with a median of 6 variants per case. Clinically actionable variants were detected in 42.1% of clinical samples. Analytical specificity was ≥99.9999% for SNVs and 100% for indels, CNAs, translocations, and MSI. Analytical sensitivity (limit of detection, 95%) was verified for each variant type, with a median variant allele frequency (VAF) of 1.42% for SNVs and 1.43% for indels, 1.7-fold for amplifications, 0.35% fusion read fraction for translocations, and 0.82% VAF for MSI. The intra-laboratory PRR study resulted in 94.9% average percent agreement (APA) and 99.9% average negative agreement (ANA) for sequence variants (SNVs and indels) and 100% APA and ANA for CNAs, translocations, and MSI. Orthogonal assays including other NGS tests and digital PCR were utilized to assess Labcorp Plasma Complete accuracy which demonstrated an aggregate analytical concordance of 96.28% positive percent agreement and >99.9999% negative percent agreement for all variants. Conclusions: The analytical validation of the Labcorp Plasma Complete test demonstrates that the liquid biopsy approach is highly sensitive, specific, accurate, reproducible, and robust for comprehensive genomic profiling to complement tissue-based testing and inform clinical decision making.

The phase 3 INTerpath-002 study design: Individualized neoantigen therapy (INT) V940 (mRNA-4157) plus pembrolizumab vs placebo plus pembrolizumab for resected early-stage non–small-cell lung cancer (NSCLC).

TPS8116 Background: Pembrolizumab (pembro; anti–PD-1) is indicated as adjuvant monotherapy following resection and adjuvant chemotherapy in patients (pts) with stage IB (T2a ≥4 cm), II, or IIIA NSCLC (per AJCC v7). However, many pts experience disease recurrence, and have limited treatment options available. V940 (mRNA-4157) is a novel individualized neoantigen therapy that encodes up to 34 neoantigens that is specifically tailored and manufactured for each pt derived from their tumor. V940 as monotherapy and in combination with pembro has shown preliminary antitumor activity in the phase 1 KEYNOTE-603 study in several solid tumor types, including NSCLC. Additionally, in pts with completely resected, high-risk stage IIIB/C/D and IV cutaneous melanoma in the KEYNOTE-942 primary analysis, V940 plus pembro demonstrated significant improvements in recurrence-free survival (HR, 0.561 [95% CI, 0.309–1.017]; P = 0.0266) and distant metastasis-free survival (HR, 0.347 [95% CI, 0.145–0.828]; P = 0.0063) vs pembro alone. Here we present the design of the phase 3, randomized, double-blind INTerpath-002 study (NCT06077760) of adjuvant V940 plus pembro vs placebo plus pembro in pts with completely resected and chemotherapy-treated stage II–IIIB (N2) NSCLC (AJCC v8). Methods: Eligible pts aged ≥18 years with completely resected stage II, IIIA, or IIIB (N2) squamous or nonsquamous NSCLC (per AJCC v8.0) where EGFR-directed therapy is not indicated as primary therapy (ie, absence of sensitizing EGFR mutations e.g. DEL19 or L858R), who have received 1–4 doses of adjuvant platinum-doublet chemotherapy, have ECOG PS 0 or 1, and have available tumor tissue for next-generation sequencing and PD-L1 testing will be enrolled. Pts must not have received previous neoadjuvant therapy for NSCLC and must not have received or be candidates for adjuvant radiotherapy. Pts will be randomized 1:1 to receive V940 1 mg IM or placebo Q3W for up to 9 doses plus pembro 400 mg IV Q6W for up to 9 cycles or until disease recurrence, pt withdrawal, unacceptable toxicity, or an additional malignancy requiring treatment. Randomization will be stratified by histology (squamous vs nonsquamous), PD-L1 TPS ( < 1% vs 1%–49% vs ≥50%), disease stage (II vs III), and geographic location (North America/Western Europe/Australia vs rest of world). The primary endpoint is disease-free survival per investigator review. Secondary endpoints include distant metastasis-free survival per investigator assessment, OS, lung cancer‒specific survival, patient-reported outcomes, and safety. Tumor imaging will occur at baseline, every 12 wk until wk 48, every 24 wk through year 3, and every 48 wk thereafter. Enrollment began in October 2023 and is ongoing globally (NCT06077760). The abstract was originally presented and published in: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr CT281. Clinical trial information: NCT06077760 .

Access to germline testing and prevalence of somatic mutations in patients with breast cancer from disadvantaged areas: A single-center study.

e13750 Background: Socioeconomic disparities affect clinical outcomes in several malignancies including breast cancer (BC). The Area Deprivation Index (ADI) is a tool used to classify regions based on socioeconomic factors, including employment, education, housing, and poverty. Here, we investigate ADI among patients (pts) with BC seen at Roswell Park Comprehensive Cancer Center and its association with recurrence free survival (RFS), time to next treatment (TNT), overall survival (OS), and access to somatic mutation via next-generation sequencing (NGS) and germline testing of BC pts. Methods: We conducted a retrospective analysis of 409 pts diagnosed with stages 1-3 and de-novo stage 4 BC from 2014 to 2018. ADI scores were derived from patients' home addresses using www.neighborhoodatlas.medicine.wisc.edu and categorized into four quartiles: Q1 (80-100%), Q2 (60-79%), Q3 (40-59%), and Q4 (0-39%) with Q1 representing areas with highest deprivation. Demographic and clinicopathological characteristics were summarized by ADI. Kruskal-Wallis and Chi-square was used for comparing continuous and categorical variables respectively. Results: Among 409 pts, 92% were female, 77% White, and 10% Black. 30% (n=123/409) lived in the most disadvantaged area Q1, 26% (106/409) Q2, 26% (104/409) Q3, and 12% (51/409) Q4, and 6% (25/409) unknown. 72% pts were hormone receptor positive, 14% HER2+, and 14% triple negative BC. No significant differences were noted in the racial distribution, employment, comorbidities, smoking, screening mammogram, type of insurance, BC stage and subtype, and adherence to therapy by ADI. Access to germline testing was significantly lower among pts from Q1 and Q2: (68/232 or 28%) vs. (65/156 or 42%) from Q3 and Q4, p= 0.03. There was no difference in RFS and OS by ADI. There was a trend towards decreased TNT (9 vs. 11 months, p=0.4) among BC pts living in areas with higher vs. lower ADI (>60% vs <60%). Notably, among stage 4 pts, in disadvantaged areas with ADI >60%, there was higher prevalence of Estrogen Receptor1(ESR1) 72% (8/11) vs. 27% (3/11) and TP53 75% (9/12) vs. 25% (3/12) somatic mutations compared to less disadvantaged areas with ADI <60%, p= 0.08 and 0.04, respectively. Conclusions: Our study shows disparities in access to germline testing, with lower rates among pts from more deprived areas. Moreover, somatic testing results show distinct mutational profiles across different ADI groups, suggesting potential impact of ADI on somatic characteristics. Larger studies are needed to investigate the association of ADI with somatic mutation burden of BC.

Impact of next generation sequencing in high grade glioma management.

e14031 Background: Recent advances in genomic profiling have improved our understanding of the molecular pathogenesis of high grade gliomas. Increased availability of high-throughput Next Generation Sequencing (NGS) allows the identification of genetic alterations which could direct personalized cancer treatments for HGG patients. Methods: All patients with high grade glioma (WHO Grade ≥3 glioma by 2016 or 2022 classification) in our institution, who underwent NGS testing between 2019-2023, were identified from a prospectively maintained electronic database. NGS results, from the Oncomine Precision Assay, a multi-gene panel which tests for 50 different mutations, were collated. Medical records were systematically reviewed to collect demographic, treatment and survival data. Alterations detected on NGS were characterized using the ESMO Scale for Clinical Actionability of Molecular Targets (ESCAT). Results: 53 patient samples underwent NGS testing (34 males [64%]; 19 females [36%]). Median age at diagnosis was 52 years (range: 20-72 years). Thirty patients (57%) had an ECOG PS 1 at first clinical review. 18 tumors (34%) were found to have MGMT promoter hypermethylation. 28 patients (53%) were found to have potentially actionable alterations according to the ESCAT scale; 19 (35%) EGFR mutations (ESCAT IIIA level evidence) were detected, 5 PIK3CA (9%) mutations (ESCAT IIIA), 3 FGFR3 (6%) alterations (ESCAT IIB), 2 patients with CDK 4 (4%) copy number gain (ESCAT IIIA), 2 PTPRZ1-MET (4%) fusions (ESCAT IIIA) and 1 BRAF V600E (2%) mutation (ESCAT IB). 4 patients were found to have multiple mutations on NGS. 1 sample had insufficient tissue for analysis. 2 patients (4%) were considered for targeted therapy based on NGS results (1 BRAF V600E mutation and 1 FGFR3 alteration) but both deteriorated in advance of commencing treatment. One patient received nivolumab for MSI-H recurrent GBM (ESCAT IIIB) but progressed within 3 months of commencing treatment. Conclusions: Despite advances in our understanding of the pathogenesis of these high grade gliomas and improved awareness of molecular targets, the delivery of targeted therapy to patients with HGG remains challenging. Rapid access to targeted therapy via compassionate access programmes and improved availability and access to clinical trials for patients with rare cancers and molecular alterations, is paramount.

Assessing the value of repeat next generation testing in patients with non-small cell lung cancer: A retrospective review.

3071 Background: Analyzing a tumor’s molecular profile in non-small cell lung cancer (NSCLC) by next generation sequencing (NGS) has been essential in the advancement of precision medicine and has led to development of several targeted therapeutic agents. While their use significantly improves patients’ outcomes, patients will inevitably develop progression of disease. Tumor sequencing at the time of progression, whether tissue or liquid biopsy, is important to identify the mechanism of resistance. We sought to investigate which patients benefit from repeat NGS testing which ultimately allowed for changes in their therapeutic management based on its updated molecular profile. Methods: We performed a single institution retrospective study of patients with NSCLC between 2014-2023 who have undergone two or more NGS sequencing. Testing was performed through Foundation One tissue CDx or liquid CDx. Data obtained through chart review included NSCLC stage, NGS dates and results, and treatments received. We evaluated clinically meaningful results from repeat testing if patients were continued on their targeted therapy, were switched to or received additional targeted therapy, detected a target when prior tests were negative, had a change in their systemic therapy, or confirmed recurrence or a second primary. Results: A total of 277 subjects with NSCLC were identified to have had more than one NGS sequencing. The average number of NGS obtained was 2.90. The average number of liquid NGS was 2.15 and tissue based NGS was 0.75. Out of the 277 patients, repeat testing was clinically meaningful in 134 (48%) patients. Of the 134 patients, 67 (50%) harbored EGFR, 16 (11.9%) ALK, 9 (6.7%) KRAS, 6 (4.5%) ROS1, 6 (4.5%) MET exon 14 skipping, 3 (2.2%) ERBB2, 1 (0.7%) BRAF, and 1 (0.7%) RET as their primary driver alteration upon initial testing. 25 patients (18.7%) had other alterations or no alterations. NGS was repeated for early stage localized recurrence in 8 patients (6%), stage I-III who developed new metastasis in 18 patients (13.4%), and recurrent metastasis in 108 patients (80.6%). Of the patients who clinically benefited from repeat testing, 77 patients (57.5%) were continued or resumed on their targeted therapy, 19 (14.2%) were switched to or received additional targeted therapy, 12 (9%) detected a target when prior tests were negative, and 3 (2.2%) had a change in their chemotherapy or immunotherapy. In 23 patients (17.1%), repeat NGS either confirmed recurrence (n=14) or second primary (n=9). Conclusions: Repeat NGS testing in NSCLC can be clinically meaningful to guide management, such as in detecting a target when prior tests were negative, allowing for switch in, continuation of or addition of targeted therapy, or providing confirmation of a recurrence or a second primary. Further research is warranted in this field to evaluate if repeat NGS can ultimately improve clinical outcomes in these patients.

Genomic analysis of recurrent early stage and de novo metastatic colorectal cancer among veterans.

e15593 Background: The Veterans Health Administration (VHA) diagnoses approximately 4,000 cases of colon cancer each year and conducts Next Generation Sequencing (NGS) on recurrent and metastatic cases. The molecular profile of cancers resected for cure that recur is poorly characterized and holds promise to clarify both the risk and mechanisms of recurrence. We sought to characterize these genetic differences. Methods: A national database from the VHA consisting of colon cancer patients was analyzed for those with recurrent disease who have received NGS testing from 2015-2021. Data was analyzed by stage and presence of DNA mismatch repair (MMR) (MLH1, MSH2, PMS2, MSH6) and DNA damage repair (DDR) abnormalities (BRCA1, BRCA2, ATM, BRIP1, BARD1, CDK12, CHEK1, CHEK2, FANCL, PALB2, RAD51B, RAD51C, RAD51D) in addition to receipt of chemotherapy and overall survival with univariate and multivariate cox proportional hazards analyses and multivariate logistic regression analyses. Chi squared analyses were also performed comparing presence of DNA abnormalities between early recurrent and stage IV cancers. Results: From309,318 cases with colon cancer codes, we identified 2,366 colon cancer patients who had NGS and had recurrent or de novo metastatic disease. 1,072 patients had initial staging annotated (I n = 89, II n = 194, III n = 320 , IV n = 453). The average age was 69.41 years; 70.5% white, 21.5% Black and 7.8% other. Among all patients, DNA MMR abnormalities (n = 117) were associated with better overall survival, after controlling for age and race (HR 0.6, 95% CI 0.42-0.85, p < 0.005). Among recurrent early stage (I-III) and stage IV patients, MMR (n = 55 and n = 21, respectively) is associated with improved survival in multivariable models (HR 0.39, 95% CI 0.33-0.46, p < 0.001) . Among the 4 DNA MMR genes, MSH2 mutations had the strongest association with survival (univariate HR 0.31, 0.15-0.62, p < 0.001). Among patients with MMR abnormalities, those who received either only capecitabine or only fluorouracil (without irinotecan or oxaliplatin) had worse overall survival (HR 2.6, 1.34 -5.04, p < 0.005). This effect was more pronounced in early stage recurrent patients versus patients with de novo metastatic disease (HR 5.1, 1.87 – 13.9, p < 0.001). Finally, we also found that early stage patients with recurrent disease are more likely to have MMR abnormalities compared to de novo metastatic (8.8% vs 4.6%, p < 0.01) and confirmed this finding after controlling for race and age. (OR 2.13, p < 0.005). Conclusions: Among Veteranswith colon cancer, resected recurrent colon cancer is more likely to have DNA MMR abnormalities than de novo metastatic disease. Veterans with recurrent and or metastatic disease with DNA MMR have better outcomes regardless of stage at diagnosis. These findings suggest different pathogenesis for those with recurrent disease after curative resection, and for those with de novo metastatic disease.

ICatalog: An open-source, collaborative platform for precision oncology studies.

10020 Background: Genomic profiling of patient (pt) tumors using next-generation sequencing (NGS) has become an integral part of precision oncology (PO). To address the need for a streamlined, interoperable approach to clinical and NGS data management, interpretation, and reporting for a multi-institution pediatric PO study we developed iCatalog (iCat), a clinical decision support system for a multi-institution pediatric PO study. Methods: iCat was developed by the University of Chicago (UChicago) and Dana-Farber Cancer Institute to create clinical interpretation reports for the iCat2/Genomic Assessment Informs Novel Therapy Consortium (GAIN) study. It is a web-based application using the Python Django framework using open-source software and runs on virtual machines (VM) behind a secure firewall. iCat manages user and administrative permissions according to role and site. It securely stores, manages, and integrates genomic and clinical information as well as internal and external genomic knowledge on a patient level. Data types available during interpretation and report generation include pt demographics, specimen-level information such as diagnosis from pathology reports, and molecular data. iCat can host molecular data from different NGS test types. These data are entered through web interfaces and API calls. Genomic knowledge accessible in iCat is a combination of information brought in from external resources and gene and variant-level pediatric-specific curations completed in iCat by curators. Editable study-specific patient reports are generated for each test. Results: As of January 10, 2024, iCat has generated reports for 902 tests and stores information for an additional 217 tests for the 742 pts enrolled in the GAIN study. Of the 217 tests, 25 tests have reports in progress and 192 tests had reports generated manually before iCat was developed. Genomic data are from 11 different test types and clinical data are from patients with 92 rare pediatric solid tumor diagnoses. A total of 108 pts have received iCat clinical interpretation reports integrating genomic data from more than one NGS test type. The knowledge base contains pediatric-specific research-team authored curations for 561 genes, 2114 unique SNVs, 268 unique CNVs, and 227 unique structural variants. Test-specific, variant-level therapeutic recommendations have been made on 666 reports for 502 pts. Diagnostic or hereditary risk associations have been made on 729 reports for 537 pts. Conclusions: iCat is an academically developed open-source platform for PO studies that integrates clinical and genomic data, supports gene and variant interpretation, and facilitates the generation of an individualized genomic report with relevant clinical information for treating physicians. We developed and tested iCat for a pediatric precision oncology study and propose it as a customizable, collaborative platform for future precision oncology research.

CtDNA as a biomarker in phase II study of tepotinib in advanced solid cancers with MET exon 14 skipping mutation or amplification (KCSG AL19-17).

3050 Background: Tepotinib consistently demonstrated antitumor activity in patients with MET exon 14 skipping mutation ( METex14) and promising activity in various cancers with MET amplification, according to previous reports. We assessed plasma ctDNA as a potential biomarker in MET-dysregulated advanced cancer patients with tepotinib treatment. Methods: KM-08 (KCSG AL19-17, NCT04647838) was a phase 2, multicenter study with tepotinib treatment for patients with advanced or metastatic solid cancers harboring either METex14 or MET amplification detected in tissue-based next-generation sequencing (NGS). For exploratory analyses, we analyzed the genetic profile using liquid-based NGS testing with AlphaLiquid-100 panel (IMBdx Inc. Seoul, KR) at baseline (T0), after six weeks during treatment (T1), and at the time of disease progression (T2). Results: Baseline ctDNA NGS data was available in 28 (80%) out of 35 patients enrolled in the trial. Among them, METex14 or MET amplification was detected in 18 patients, with a sensitivity of 64.3%. The most commonly co-mutated gene was TP53, followed b y PIK3CA, ATM, MYCN, and KRAS. The objective response rate (ORR) in plasma MET positive (PM+) patients was higher (81.2%) than the ORR in plasma MET negative (PM−) patients’ group (30.0%). In contrast, PM− group had a longer progression-free survival (PFS) and overall survival (OS) than PM+ group. PFS was 11.0 months (95% CI 8.1, 13.9) vs 6.0 months (95% CI 3.6, 8.3) and OS was NR vs 14.0 months (95% CI 4.7, 23.3), respectively. The molecular responder (MR, MET alteration variant allele frequency [VAF] T1/T0 <50%) was 80.0% (12/15 patients), and the molecular non-responder (MNR, METVAF T1/T0≥50%) was 20.0% (3/15 patients). ORR was higher in the MR group (91.7%) than in the MNR group (33.3%). PFS and OS were longer in the MR group than in the MNR group, 6.0 months (95% CI 0.0, 14.5) vs 3.0 months (95% CI 1.4, 4.6) with P= 0.114 and NR vs 4.0 months (95% CI 2.4, 5.6) with P= 0.067, respectively. Out of the 20 patients with samples available at T2, one had an on-target mutation on MET (D1228N, and Y1230H) while two had off-target emerging oncogenic alterations in KRAS, BRAF, and ERBB2 genes. Conclusions: In the liquid biomarker analysis in the MET-dysregulated cancer patients who were treated with tepotinib, the presence of ctDNA METalteration at baseline was associated with a higher response rate but shorter PFS and OS. The molecular response was well correlated with the radiological response and associated with better outcomes. (Funded by Merck KGaA, Darmstadt, Germany, ClinicalTrials.gov number, NCT04647838.) Clinical trial information: NCT04647838 .

Spectrum of Resistance Mechanisms to ALK TKIs in NSCLC: Largest Single-Center Experience from India

Abstract Introduction Anaplastic lymphoma kinase (ALK) rearranged non-small cell lung carcinoma (NSCLC) has emerged as a distinct entity with growing number of potent ALK tyrosine kinase inhibitors (TKIs). Despite showing durable responses and promising survival rates, resistance to these ensue. This is the largest series of repeat biopsies from patients of ALK-positive NSCLC progressing on ALK-directed therapy from this part of the world. Using a combinatorial approach of genomics and histology, we describe the spectrum of various resistance mechanisms encountered. Methods This is a cross-sectional study recruiting ALK-positive NSCLC cases who have progressed on any line ALK TKI and have undergone repeated biopsies followed by genomic sequencing by next-generation sequencing (NGS). Results Thirty-two ALK-positive NSCLC patients progressed on TKI were enrolled. Median age was 53 years (range: 36–75 years) with a male predilection (male:female 1.3:1). Twenty-seven (84.4%) cases harbored an additional resistance mechanism. Eighteen of these harbored an on-target ALK alteration, with L1196M gatekeeper mutation being the most common, in 11 cases, and G1202 alteration in 3 cases. In 9 cases an off-target alteration was detected, the most frequent being TP53 mutation in 8 cases, KRAS mutation in 4 cases and MET amplification in 3 cases. Four patients underwent sequential NGS testing and allele frequency changes in ALK fusion and resistance mechanisms were demonstrated. Sixteen patients have been offered lorlatinib therapy, the median progression-free survival of which has not yet been reached. Conclusion This is the largest series depicting ALK resistance mechanisms from a single center to date. The SPACEWALK study which demonstrated ALK TKI resistance mechanisms using plasma-based genotyping was a multicentric study. The spectrum encountered in this study is distinct from the rest of the world, thus highlighting heterogeneity within ALK-rearranged tumors. Comprehensive clinical evaluation at disease progression coupled with NGS-based genotyping will pave the way for lucid understanding of disease biology, thus aiding in the institution of optimal therapy.

Diagnostic yield of exome and genome sequencing after non-diagnostic multi-gene panels in patients with single-system diseases

BackgroundThough next-generation sequencing (NGS) tests like exome sequencing (ES), genome sequencing (GS), and panels derived from exome and genome data (EGBP) are effective for rare diseases, the ideal diagnostic approach is debated. Limited research has explored reanalyzing raw ES and GS data post-negative EGBP results for diagnostics. Results: We analyzed complete ES/GS raw sequencing data from Mayo Clinic's Program for Rare and Undiagnosed Diseases (PRaUD) patients to assess whether supplementary findings could augment diagnostic yield. ES data from 80 patients (59 adults) and GS data from 20 patients (10 adults), averaging 43 years in age, were analyzed. Most patients had renal (n=44) and auto-inflammatory (n=29) phenotypes. Ninety-six cases had negative findings and in four cases additional genetic variants were found, including a variant related to a recently described disease (RRAGD-related hypomagnesemia), a variant missed due to discordant inheritance pattern (COL4A3), a variant with high allelic frequency (NPHS2) in the general population, and a variant associated with an initially untargeted phenotype (HNF1A). Conclusion: ES and GS show diagnostic yields comparable to EGBP for single-system diseases. However, EGBP's limitations in detecting new disease-associated genes underscore the necessity for periodic updates.

Thyroid papillary carcinoma combined with primary follicular lymphoma: a case report

BackgroundPapillary thyroid carcinoma (PTC) stands out as the most prevalent epithelial malignant thyroid tumor. Thyroid primary follicular lymphoma (PFL) represents a rare malignant tumor originating from mesenchymal tissues. The concurrent occurrence of PTC and PFL is exceptionally rare, particularly in the context of Hashimoto’s thyroiditis, presenting significant challenges in clinical diagnosis and treatment.Case demonstrationA 44-year-old female patient presented with a neck mass persisting for over 1 month. The patient underwent surgery, and the incised tissues were subjected to pathology examinations, along with immunohistochemistry and next-generation sequencing tests suggestive of an EZH2 gene mutation in the tumor cells. The final pathological diagnosis confirmed the presence of PTC combined with PFL. Following a 27-month follow-up, the patient displayed no signs of recurrence or metastasis.ConclusionsThe concurrent occurrence of PTC and PFL poses notable challenges in clinical practice, requiring careful consideration in diagnosis and treatment. Herein, we present a rare case of PTC combined with PFL featuring an EZH2 gene mutation, which can be easily overlooked in the context of Hashimoto’s thyroiditis. The patient’s favorable response to surgical and radiotherapeutic interventions underscores the importance of accurate diagnosis and tailored treatment strategies in similar cases.

Next generation sequencing panel as an effective approach to genetic testing in patients with a highly variable phenotype of neuromuscular disorders

Neuromuscular disorders (NMDs) include a wide range of diseases affecting the peripheral nervous system. The genetic diagnoses are increasingly obtained with using the next generation sequencing (NGS). We applied the custom-design targeted NGS panel including 89 genes, together with genotyping and multiplex ligation-dependent probe amplification (MLPA) to identify a genetic spectrum of NMDs in 52 Polish patients. As a result, the genetic diagnosis was determined by NGS panel in 29 patients so its diagnostic utility is estimated at 55.8%. The most pathogenic variants were found in CLCN1, followed by CAPN3, SCN4A, and SGCA genes. Genotyping of myotonic dystrophy type 1 and 2 (DM1 and DM2) as a secondary approach has been performed. The co-occurrence of CAPN3 and CNBP mutations in one patient as well as DYSF and CNBP mutations in another suggests possibly more complex inheritance as well as expression of a phenotype. In 7 individuals with single nucleotide variant found in NGS testing, the MLPA of the CAPN3 gene was performed detecting the deletion encompassing exons 2—8 in the CAPN3 gene in one patient, confirming recessive limb-girdle muscular dystrophy type 1 (LGMDR1). Thirty patients obtained a genetic diagnosis (57.7%) after using NGS testing, genotyping and MLPA analysis. The study allowed for the identification of 27 known and 4 novel pathogenic/likely pathogenic variants and variants of uncertain significance (VUS) associated with NMDs.In conclusion, the diagnostic approach with diverse molecular techniques enables to broaden the mutational spectrum and maximizes the diagnostic yield. Furthermore, the co-occurrence of DM2 and LGMD has been detected in 2 individuals.

Investigating somatic variants and pathways in mismatch repair-deficient (dMMR) colorectal carcinoma in South Africa

AimsColorectal carcinoma (CRC) is a common cause of morbidity and mortality worldwide, and an emerging public health problem in sub-Saharan Africa. Several authors have described an increased frequency of mismatch...

Laboratory Considerations for Releasing Next-Generation Sequencing Data to Patients.

Title 45, section 164.524 of the Code of Federal Regulations states that health care systems must provide patient health records upon that patient's request. For complex testing, such as next-generation sequencing (NGS), this raises questions related to what data should be released and the laboratory considerations regarding the release of this data. To describe the laboratory implications of releasing different NGS data files and the limitations for the clinical use of different NGS data files. The College of American Pathologists workgroup, composed of laboratorians with expertise regarding NGS testing, reviewed pertinent literature, including title 45, section 164.524, and the Health and Human Services "Guidance on Individuals' Right to Access Health Information." From an accreditation standpoint, validation of NGS includes both the wet bench and data processing (bioinformatics) portions, and appropriately validated laboratory testing is required to ensure quality patient results. NGS testing generates intermediate data files that have not completed the fully validated process but are often kept by the laboratory. These files may be requested by patients, but most patients will not be aware of the test validation process and the limitations of data that have not gone through a fully validated process. Laboratories should encourage patients to receive their health data and to help individuals understand the content, uses, and limitations of laboratory data they have requested or received. NGS data used in a nonvalidated manner should not be used for clinical purposes without confirmation by a clinically validated method.

Real-World Evidence Study of Patients with KRAS-Mutated NSCLC in Finland.

While KRAS is the most frequently mutated oncogene in non-small cell lung cancer (NSCLC), KRAS-mutant tumors have long been considered difficult to treat and thus, an unmet need still remains. Partly due to the lack of targeted treatments, comprehensive real-world description of NSCLC patients with KRAS mutation is still largely missing in Finland. In this study, all adult patients diagnosed with locally advanced and unresectable or metastatic NSCLC from 1 January 2018 to 31 August 2020 at the Hospital District of Helsinki and Uusimaa were first identified in this retrospective registry-based real-world study. The final cohort included only patients tested with next generation sequencing (NGS) and was stratified by the KRAS mutation status. A total of 383 patients with locally advanced and unresectable or metastatic NSCLC and with NGS testing performed were identified. Patients with KRAS mutation (KRAS G12C n = 35, other KRAS n = 74) were younger than patients without KRAS mutations, were all previous or current smokers, and had more often metastatic disease at diagnosis. Also, these patients had poorer survival, with higher age, Charlson comorbidity index (CCI) being 5 or above, and KRAS G12C being the most significant risk factors associated with poorer survival. This suggests that the patients with KRAS mutation have a more aggressive disease and/or tumors with KRAS mutation are more difficult to treat, at least without effective targeted therapies.

Supernatant fluid from endobronchial ultrasound-guided transbronchial needle aspiration for rapid next-generation sequencing

There is an increasing demand to optimize the workflow and maximize tissue available for next-generation sequencing (NGS) for non-small cell carcinoma. We looked at transbronchial needle endobronchial ultrasound-guided bronchoscopy with transbronchial needle aspiration samples and evaluated the performance of supernatant (SN) fluid processed from a dedicated aspirate collected for NGS testing. Nineteen samples were collected and processed using a new workflow. Five aspirates were collected in formalin. One additional dedicated pass was collected fresh and centrifuged. The resulting cell pellet was added to formalin for cell block (CB) processing. DNA and RNA were extracted from concentrated SN for targeted testing using the Oncomine Precision Assay (Thermo Scientific, Waltham, MA). NGS results from the corresponding CB samples were used as "controls" for comparison. Thirty-one mutations were detected in SN (Table1). The most frequently mutated genes were TP53 (35%), EGFR (23%), KRAS (13%), CTNNB1 (6%), and ERBB2 (6%). There was 100% concordance between the mutations detected in SN and corresponding CBs with comparable variant allele frequencies. Turnaround time of NGS results was 1 day for SN compared to 4-10 days for CB. We were able to demonstrate the usefulness of SN for reliable rapid molecular results. We successfully incorporated the workflow for tissue handling and processing among our clinical, cytopathology, and molecular teams. Molecular results were available at the same time as the cytologic diagnosis, allowing for timely reporting of a comprehensive diagnosis. This approach is particularly useful in patients with advanced disease requiring urgent management.

Acute Promyelocytic Leukemia With Torque Teno Mini Virus::RARA Fusion: An Approach to Screening and Diagnosis

Acute promyelocytic leukemia (APL) with variant RARA translocation is linked to over 15 partner genes. Recent publications encompassing 6 cases have expanded the spectrum of RARA partners to torque teno mini virus (TTMV). This entity is likely underrecognized due to the lack of clinician and pathologist familiarity, inability to detect the fusion using routine testing modalities, and informatic challenges in its recognition within next-generation sequencing (NGS) data. We describe a clinicopathologic approach and provide the necessary tools to screen and diagnose APL with TTMV::RARA using existing clinical DNA- or RNA-based NGS assays, which led to the identification of 4 cases, all without other known cytogenetic/molecular drivers. One was identified prospectively and 3 retrospectively, including 2 from custom automated screening of multiple data sets (50,257 cases of hematopoietic malignancy, including 4809 acute myeloid leukemia/myeloid sarcoma/APL cases). Two cases presented as myeloid sarcoma, including 1 with multiple relapses after acute myeloid leukemia-type chemotherapy and hematopoietic stem cell transplant. Two cases presented as leukemia, had a poor response to induction chemotherapy, but achieved remission upon reinduction (including all-trans retinoic acid in 1 case) and subsequent hematopoietic stem cell transplant. Neoplastic cells demonstrated features of APL including frequent azurophilic granules and dim/absent CD34 and HLA-DR expression. RARA rearrangement was not detected by karyotype or fluorescent in situ hybridization. Custom analysis of NGS fusion panel data identified TTMV::RARA rearrangements and, in the prospectively identified case, facilitated monitoring in sequential bone marrow samples. APL with TTMV::RARA is a rare leukemia with a high rate of treatment failure in described cases. The diagnosis should be considered in leukemias with features of APL that lack detectable RARA fusions and other drivers, and may be confirmed by appropriate NGS tests with custom informatics. Incorporation of all-trans retinoic acid may have a role in treatment but requires accurate recognition of the fusion for appropriate classification as APL.