Successful Next-generation Sequencing Research Articles

Molecular profiling of metastatic breast cancer and target-based therapeutic matching in an Asian tertiary phase I oncology unit

IntroductionMolecular profiling of metastatic breast cancer (MBC) through the widespread use of next-generation sequencing (NGS) has highlighted actionable mutations and driven trials of targeted therapy matched to tumour molecular profiles, with improved outcomes reported using such an approach. Here, we review NGS results and treatment outcomes for a cohort of Asian MBC patients in the phase I unit of a tertiary centre.MethodsPatients with MBC referred to a phase I unit underwent NGS via Ion AmpliSeq Cancer Hotspot v2 (ACH v2, 2014–2017) prior to institutional change to FoundationOne CDx (FM1; 2017–2022). Patients were counselled on findings and enrolled on matched therapeutic trials, where available. Outcomes for all subsequent treatment events were recorded to data cut-off on January 31, 2022.ResultsA total of 215 patients were enrolled with successful NGS in 158 patients. The PI3K/AKT/PTEN pathway was the most altered with one or more of the pathway member genes PIK3/AKT/PTEN affected in 62% (98/158) patients and 43% of tumours harbouring a PIK3CA alteration. Tumour mutational burden (TMB) was reported in 96/109 FM1 sequenced patients, with a mean TMB of 5.04 mt/Mb and 13% (12/96) with TMB ≥ 10 mt/Mb. Treatment outcomes were evaluable in 105/158 patients, with a pooled total of 216 treatment events recorded. Matched treatment was administered in 47/216 (22%) events and associated with prolonged median progression-free survival (PFS) of 21.0 weeks [95% confidence interval (CI) 11.7, 26.0 weeks] versus 12.1 weeks (95% CI 10.0, 15.4 weeks) in unmatched, with hazard ratio (HR) for progression or death of 0.63 (95% CI 0.41, 0.97; p = 0.034). In the subgroup of PIK3/AKT/PTEN-altered MBC, the HR for progression or death was 0.57 (95% CI 0.35, 0.92; p = 0.02), favouring matched treatment. Per-patient overall survival (OS) analysis (n = 105) showed improved survival for patients receiving matched treatment versus unmatched, with median OS (mOS) of 30.1 versus 11.8 months, HR = 0.45 (95% CI 0.24, 0.84; p = 0.013). Objective response rate (ORR) in the overall population was similar in matched and unmatched treatment events (23.7% versus 17.2%, odds ratio of response 1.14 95% CI 0.50, 2.62; p = 0.75).ConclusionsBroad-panel NGS in MBC is feasible, allowing therapeutic matching, which was associated with improvements in PFS and OS.

Abstract 949: A single stranded DNA library preparation workflow used for hybridization capture based WES assay showed superior uniformity

Abstract Introduction: In many instances in Precision Oncology Medicine fragmented DNA from formalin-fixed paraffin-embedded (FFPE) samples is the only option. The majority of such degradedsamples need to be sequenced using next generation sequencing (NGS) [1]. Standardizedguidelines highlight that a minimum of 500 × coverage depth is required for tissue analyses [2].However, coverage isn’t the only metric required for a successful NGS analysis. Highly uniformDNA libraries allow clinical labs to generate more hits from their screens, saving both time andmoney across the course of diagnosis therefore ultimately benefits patient care. The commerciallyavailable single stranded DNA library prep kit tested in this study renders all input moleculessingle-stranded before ligation therefore it recovers template molecules containing DNA nicks andlabile lesions thus maximizing library complexity. Method: 10 solid tumor FFPE derived DNA samples went through three different NGS workflows:100ng of DNA input in an amplicon-based assay 325 gene panel (Assay A), 60ng input for a 500gene mechanical sheared-based double strand DNA library preparation with hybridization captureassay (~500 gene panel, Assay B), 50ng and 10ng input for a enzymatic shear-based singlestranded DNA library preparation with hybridization capture assay (WES with 1000+ enhancedgene content, Assay C). Data from assays A and C was processed with in-house bioinformaticpipeline, while Assay B’s data was processed with a commercially available bioinformatic pipelinethat was bundled with the assay. NGS QC metrics were compared across the three datasets. Results: This novel single stranded DNA library preparation with hybridization capture workflowshowed superior QC metrics compared to the other two workflows, especially on coverageuniformity and percent target above 100X, with as low as 10ng input. Conclusion: Employing a novel commercially available DNA library preparation workflow in anNGS assay, NeoGenomics was able to rescue poor quality samples that had previously failed tomeet our internal uniformity metric on other NGS assays and generate robust and reportableresults with as low as 10ng DNA input.[1] Nagahashi et al., J Surg Res. 2017 Dec; 220: 125-132.[2] Cappello et al., J Pers Med. 2022 May; 12(5): 750. Citation Format: Jieying Chu, Rachel Schell, Faqiang Wu, Michal Krawczyk, Daniel Whang, Segun Jung, Hyunjun Nam, Kenneth B. Thomas, Fernando J. Lopez-Diaz, Vincent Funari, Julie A. Mayer, Shashi Kulkarni, Jiannan Guo, Steven P. Lau-Rivera. A single stranded DNA library preparation workflow used for hybridization capture based WES assay showed superior uniformity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 949.

Impact of the quality of resected thyroid cancer tissue sample on next-generation sequencing testing.

Activating rearranged during transfection (RET) proto-oncogene alterations can be identified using next-generation sequencing (NGS) of tumor DNA/RNA. We assessed factors associated with NGS (Oncomine Dx Target Test [ODxTT]) success for resected thyroid cancer (TC) specimens, including sample age, processing conditions, and DNA/RNA quality. TC samples were from three Japanese hospitals, with sample age <1-<10 years, fixative 10%/15% neutralized buffered formalin (NBF), and fixation time ≤48 h/>48 h-≤72 h. NGS success rate was defined as the percentage of samples returning validated NGS results (RET fusion-positive/negative [RNA] or RET mutation-positive/negative [DNA], detected using ODxTT). DNA/RNA quality was assessed with indexes based on electrophoresis (DNA/RNA integrity number, DV200 ) and quantitative polymerase chain reaction (DNA/RNA integrity score [ddCq/ΔCq]). NGS success rate (N = 202) was 90%/93% (DNA/RNA) overall, 98%-100% (DNA and RNA) for samples <3 years old, and 91% (DNA and RNA) for samples ≥3-<5 years old fixed in 10% NBF for ≤48 h. Multivariate logistic regression analysis identified ddCq and ΔCq as significant predictors of DNA and RNA NGS success rates, respectively. Quality assessment of nucleic acid extracted from archival tissue samples is important for achieving high NGS success rates in clinical practice, especially for samples ≥3 years old.

Tackling the implementation gap for the uptake of NGS and advanced molecular diagnostics into healthcare systems

Next-generation sequencing (NGS) and liquid biopsy (LB) showed positive results in the fight against different cancer types. This paper aims to assess the uptake of advanced molecular diagnostics/NGS for quick and efficient genetic profiles of tumour cells. For that purpose, the European Alliance for Personalised Medicine conducted a series of expert interviews to ascertain the current status across member states. One stakeholder meeting was additionally conducted to prioritize relevant factors by stakeholders. Seven common pillars were identified, and twenty-five measures were defined based on these pillars. Results showed that a multi-faceted approach is necessary for successful NGS implementation and that regional differences may be influenced by healthcare policies, resources, and infrastructure. It is important to consider different correlations when interpreting the results and to use them as a starting point for further discussion.

Utility of needle biopsy in centrally located lung cancer for genome analysis: a retrospective cohort study

BackgroundIt is essential to collect a sufficient amount of tumor tissue for successful next-generation sequencing (NGS) analysis. In this study, we investigated the clinical risk factors for avoiding re-biopsy for NGS analysis (re-genome biopsy) in cases where a sufficient amount of tumor tissue could not be collected by bronchoscopy.MethodsWe investigated the association between clinical factors and the risk of re-genome biopsy in patients who underwent transbronchial biopsy (TBB) or endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) and required re-genome biopsy in cases enrolled in LC-SCRUM Asia, a prospective nationwide genome screening project in Japan. We also examined whether the frequency of re-genome biopsy decreased between the first and second halves of the enrolment period.ResultsOf the 572 eligible patients, 236 underwent TBB, and 134 underwent EBUS-TBNA. Twenty-four TBBs required re-genome biopsy, and multivariate analysis showed that the risk of re-genome biopsy was significantly increased in lesions where the tumor lesion was centrally located. In these cases, EBUS-TBNA should be utilized even if the lesion is a pulmonary lesion. However, it should be noted that even with EBUS-TBNA, lung field lesions are at a higher risk of re-canalization than mediastinal lymph node lesions. It was also found that even when tumor cells were detected in rapid on-site evaluation, a sufficient amount of tumor tissue was not always collected.ConclusionsFor centrally located pulmonary mass lesions, EBUS-TBNA, rather than TBB, can be used to obtain tumor tissues that can be analyzed by NGS.

BRAF testing modalities in histiocytic disorders: Comparative analysis and proposed testing algorithm.

Understanding of histiocytic disorders has been revolutionized by demonstration of mitogen-activated protein kinase (MAPK) pathway mutations, most commonly BRAFV600E. The optimal testing strategy to assess BRAFV600E is unknown. We aimed to compare performance of testing modalities, to propose a framework for evaluation of BRAFV600E mutation status in histiocytic disorders. We retrospectively reviewed patients with histiocytic disorders and BRAF mutation testing on a lesional tissue specimen. In 120 patients, BRAF assessment included immunohistochemistry (IHC) in 97 (80.2%), polymerase chain reaction (PCR) in 35 (28.9%), and next-generation sequencing (NGS) in 62 (51.2%). Forty-five underwent both NGS and IHC. With NGS as the gold standard, the sensitivity and specificity of IHC were 82.4% and 96.4%. Three false negatives were observed in biopsy specimens with low BRAFV600E variant allele frequency or decalcified tissue. One false-positive IHC was observed in a lung biopsy specimen, likely due to antibody cross-reactivity with respiratory cilia. Among 14 with successful NGS and PCR, a single discordance was observed. Two PCR-to-IHC discrepancies were observed, including one other false-positive IHC. Immunohistochemistry was highly specific for detection of BRAFV600E. Main caveats were false negatives and lack of detection of non-BRAFV600E mutations. We propose the use of IHC as initial screening in general practice with reflex molecular testing if negative.

P-725 Role of Non-Invasive Preimplantation Genetic Testing- Aneuploidy (NIPGT-A) using spent culture media (SCM) and its concordance with Trophoectoderm (TE) biopsy: A Prospective Cohort Study

Abstract Study question What is the ploidy concordance rate between spent culture media (SCM) and trophectoderm biopsy (TE) using Next Generation Sequencing (NGS) and correlation with clinical outcome? Summary answer DNA could be isolated from SCM with successful NGS library sequencing. Ploidy and per-chromosome concordance rate of TE biopsy vs SCM was 68.38% and 85.13%. What is known already Many challenges are associated with TE biopsy, and the possibility of NiPGT-A using cell free DNA (cfDNA) from SCM is very intriguing, as it will be simpler, safer and cheaper alternative to the gold standard TE biopsy. The bottlenecks associated with TE biopsy like additional equipment and trained embryologist, can be substantially reduced. The disparities in reported results in various studies comparing SCM vs TE, could be attributed to culture media contamination (maternal, paternal or environment), different laboratory workflow methodologies used for embryo culture, different whole genome amplification methods, library sequencing methods and different algorithms for analysis. Study design, size, duration Prospective cohort study was conducted in tertiary care centre from August 2021-June 2022. Ethical Approval was taken from Institute Ethics Committee. Couples with female age ≥ 35 years, one or more implantation failures, male factor infertility requiring ICSI, opting for elective single euploid blastocyst transfer, were included in study. Forty four blastocysts were obtained from 14 patients who underwent IVF/ICSI cycles, gave consent for TE biopsy and SCM collection for PGT-A and NiPGT-A respectively. Participants/materials, setting, methods Individualised ovarian stimulation was carried out with GnRH antagonist protocol. ICSI was done for fertilization. Embryos were cultured in sequential medium. On day 3, after thorough washing embryo was cultured separately in 10μl micro-droplets. SCM was collected on day-5, before biopsy. DNA extraction, amplification and library preparation from TE biopsy and SCM was done using Veriseq PGS kit (Illumina) and sequencing was done on Illumina MiSeq system. Euploid embryo transfer was done in FET cycle Main results and the role of chance Out of 44 blastocysts, 31 were day 5 and 13 were day 6. WGA-DNA from TE biopsy and SCM was 100% successful. Average concentration of amplified DNA obtained from TE biopsy and SCM was 34.88 ± 9.56 ng/µl, 32.3 ± 6.84 ng/µl respectively. Quality control parameters for library preparation and sequencing for samples were as per recommended standards. CNV visualization and analysis was done using BlueFuse Multi-Software (Illumina). Ploidy concordance rate could be analysed in 26 embryos, which was 68.38%, per chromosome concordance was 85.13% and sex-chromosome concordance was 73.0%. The sensitivity, specificity, PPV, NPV and diagnostic accuracy of NiPGT-A was 66.6%, 60%, 87.5%, 30% and 65.38% respectively. On comparing day 5 and day 6 blastocysts, day 6 had better concordance rate 72.7% vs 60%, p &amp;gt; 0.05. On comparing good (&amp;gt;BB) and poor morphology (&amp;lt;BB)embryos, &amp;lt;BB had better concordance rate 83.33% vs 50%, p = 0.16. Per chromosome concordance rate was higher for &amp;lt;BB embryos, 90.5% vs 80.5%,p= 0.001. Full maternal cell contamination was suspected when TE was aneuploid/mosaic, XY and SCM was euploid, XX (n = 4/14), assessed only in XY embryos. Single euploid blastocyst transfer had 66.6%clinical pregnancy rate. One resulted in healthy live-birth, two on-going pregnancies, 30weeks and 12weeks period of gestation. Limitations, reasons for caution Before considering NiPGT-A in routine clinical practice, each embryology lab needs standardization, to implement necessary modifications in routine embryology protocol. Labs should ensure that embryo viability is not affected and should try to minimize chances of maternal or external contamination in SCM to reliably predict concordance rates with high confidence. Wider implications of the findings NiPGT-A is useful technique to assess ploidy but presently, NiPGT-A in combination with PGT-A, can help in prioritizing embryos according to their implantation potential. Further studies are required to find out whether NiPGT-A may serve as an alternative to PGT-A and implemented alone in routine clinical practice in future. Trial registration number CTRI/2021/04/033121

Diagnostic utility of DNA integrity number as an indicator of sufficient DNA quality in next-generation sequencing-based genomic profiling.

DNA integrity number (DIN) is a metric for assessing DNA degradation, calculated based on electrophoresis using the Agilent TapeStation System. The utility of DIN as a diagnostic indicator of sufficient DNA quality in clinical next-generation sequencing (NGS) has not been well described. We evaluated the DINs of 166 tumor formalin-fixed, paraffin-embedded (FFPE) tissue samples submitted for 124-gene panel sequencing. We also investigated a new metric on the electropherogram that could improve the predictive accuracy of the DIN. A DIN cutoff of 2.5 discriminated samples with successful analysis (n = 143) from samples with failed analysis (n = 23), with a sensitivity of 0.84 and a specificity of 0.78 (area under the curve [AUC] = 0.88). The DIN was positively correlated with the mean coverage (r = 0.72, P < .0001) but could not discriminate success from failure when the DIN was below 2.5 (negative predictive value, 0.44). We introduced a new metric, the peak/base ratio, that distinguished success from failure with higher accuracy than the DIN (cutoff = 1.6; sensitivity = 0.98, specificity = 0.83, and AUC =0.96). To predict successful NGS, the DNA quality of FFPE tissue can be easily and reliably assessed using the DIN and peak/base ratio.

Next-Generation Sequencing Analysis of Pancreatic Cancer Using Residual Liquid Cytology Specimens from Endoscopic Ultrasound—Guided Fine-Needle Biopsy: A Prospective Comparative Study with Tissue Specimens

This study evaluated the feasibility and clinical utility of liquid-based cytology (LBC) specimens via endoscopic ultrasound-guided fine-needle biopsy (EUS-FNB) for next-generation sequencing (NGS) of pancreatic cancer (PC). We prospectively evaluated the performance of DNA extraction and NGS using EUS-FNB samples obtained from PC. Thirty-three consecutive patients with PC who underwent EUS-FNB at our hospital were enrolled. DNA samples were obtained from 96.8% of the patients. When stratified with a variant allele frequency (VAF) > 10% tumor burden, the NGS success rate was 76.7% (n = 23) in formalin-fixed paraffin-embedded (FFPE), 83.3% (n = 25) in LBC, and 76.7% (n = 23) in frozen samples. The overall NGS success rate was 86.7% (n = 26) using FFPE, LBC, or frozen samples. The detection rates for the main mutated genes were as follows: 86.7% for KRAS, 73.3% for TP53, 66.7% for CDKN2A, 36.7% for SMAD4, and 16.7% for ARID1A. LBC had the highest median value of VAF (23.5%) for KRAS and TP53. PC mutation analysis using NGS was successfully performed using LBC compared with FFPE and frozen samples. This approach provides an alternative and affordable source of molecular testing materials.

Biopsy Method and Needle Size on Success of Next-Generation Sequencing in NSCLC: A Brief Report

IntroductionNext-generation sequencing (NGS) is essential to the care of patients with NSCLC. Nevertheless, NGS is dependent on adequate material from biopsy. We evaluated the impact of biopsy method and needle gauge necessary for optimizing success in tissue NGS. MethodsA total of 1660 formalin-fixed, paraffin-embedded samples were submitted to Caris Life Sciences from 2007 to 2022 for tumor profiling. The results of NGS assays were linked with retrospective biopsy data for patients with lung cancer treated at USC/Norris Cancer Center to create a database with the following parameters: demographics, biopsy method, tumor location (lung mass versus lymph node versus metastasis), needle gauge, number of needle passes, complications, tumor volume, DNA content, and status of NGS. Fisher’s exact test and analysis of variance were performed to determine the impact of biopsy method and needle gauge (G). ResultsIn total, 77 computed tomography (CT)-guided transthoracic core needle (CT-TTCN) biopsies, 74 endobronchial ultrasound (EBUS)-guided transbronchial needle aspirations (TBNAs), 27 bronchial forceps biopsies, and 107 surgical resections were included. Furthermore, 41 of 77 CT-TTCN biopsies (53.2%), 43 of 74 EBUS-TBNAs (58.1%), 22 of 27 bronchial forceps biopsies (81.5%), and 105 of 107 surgical resections (98.1%) underwent successful NGS assays. The probability of successful NGS completion for lung cancers was highest in surgical resections and bronchial forceps biopsies. Needle-based biopsies were more successful when a needle larger than 20G was used. Complication rates were higher for CT-TTCN biopsies compared with EBUS-TBNA (p < 0.0001). Overall, the DNA yield was significantly higher in EBUS-TBNA compared with CT-TTCN biopsies in primary lung sites (p = 0.0002). EBUS-TBNA was found to have higher success rates in NGS compared with CT-TTCN for both primary lung lesions (p = 0.023) and lymph node targets (p = 0.035). ConclusionsThe less invasive EBUS-TBNAs had higher success rates in NGS than CT-TTCN biopsies and resulted in higher DNA concentrations. In CT-TTCN biopsies, use of 20G or smaller needles is associated with a higher risk of obtaining an inadequate specimen regardless of the number of passes taken. Surgical and bronchial forceps biopsies had highest success in achieving NGS.

The Performance of an Extended Next Generation Sequencing Panel Using Endobronchial Ultrasound-Guided Fine Needle Aspiration Samples in Non-Squamous Non-Small Cell Lung Cancer: A Pragmatic Study

Introduction/BackgroundSamples from endobronchial ultrasound-guided fine needle aspiration (EBUS-TBNA) are frequently used for next generation sequencing (NGS) in patients with non-small cell lung cancer (NSCLC) to look for genetic driver mutations. The objective of the current study was to evaluate the performance of extended NGS panels using EBUS-TBNA samples in a real-world setting and identify factors associated with the success of NGS. Materials and MethodsThis study included all patients who underwent EBUS and were diagnosed with non-squamous NSCLC with mediastinal metastasis from 2016 to 2019 at the University of Pennsylvania. We reviewed demographic information, imaging studies, procedure reports, pathology and NGS reports. Logistic regression was used to analyze factors associated with the success of NGS panels. ResultsThe success rates of NGS using EBUS-TBNA samples were 92.5%, and 91.5% for DNA and RNA NGS panels respectively. Samples from higher N stage (N2 and N3 lymph nodes) and with higher tumor cellularity (>25%) resulted in higher success rate for DNA NGS. The effect of tumor cellularity remained borderline significant after entering multivariable logistic regression. The short-axis diameter of the sampled lymph node on CT scan, FDG-avidity on PET CT and >3 EBUS passes per lymph node during the procedure were not associated with NGS success. ConclusionBoth DNA and RNA extended-panel NGS had high performance using EBUS-TBNA samples. Sampling more advanced nodal stations and obtaining samples with higher tumor cellularity were associated with higher success rate of DNA NGS. Other imaging or procedural factors did not affect NGS performance.

Efficacy of MEK Inhibitors in Erdheim Chester Disease

Background Erdheim Chester disease (ECD) is a rare histiocytosis with variable clinical behavior known to be driven by recurrent activating mutations in the MAP kinase pathway. BRAF inhibitors were the first targeted therapy used in this neoplastic disorder given the presence of the BRAFV600E mutation in 50-60% of patients. Increased understanding of additional driver mutations within this pathway resulted in a study of MEK inhibitors (MEKi) in cases not harboring this mutation, ultimately leading to the FDA breakthrough designation of cobimetinib for BRAFV600E negative histiocytic neoplasms. Herein, we present data from our institution on MEKi treatment of ECD, the largest cohort to date. Methods We included all patients with ECD consecutively seen at Mayo Clinic and treated with one of the 3 available MEKi (cobimetinib, trametinib, or binimetinib) between 2019-2021. Tumor MAPK pathway mutation status was determined with next-generation sequencing (NGS) via the Tempus-xT® assay (Chicago, IL). If NGS data was unavailable, immunohistochemistry (IHC) or allele-specific PCR was used to determine BRAF-specific mutation status. Time-to-event analyses were calculated from the time of MEKi initiation to progression, therapy discontinuation due to intolerant side effects, or initiation of systemic non-MEKi therapies. Response assessment was done based on the established positron emission radiography (PET)-response criteria used for clinical trials of targeted therapies in histiocytosis. Adverse events (AEs) were graded using the National Cancer Institute Common Toxicity Criteria v5.0 Results A total of 22 patients with ECD underwent treatment with a MEKi and were included. Median follow up was 19 months (95% CI 10 - 28 months) and 55% (n=12) were female. Median age at diagnosis of ECD was 54 years (IQR 36.8 - 70.8 years). Most common system involvement included osseous (68%), retroperitoneal soft tissue (46%), central nervous system (41%), and cardiovascular (41%). Mutational assessment studies included NGS in 82% (n=18). The four remaining patients had assessment of BRAFV600E by IHC or PCR. Tumor genomic profile included non-BRAFV600E mutations in 15 patients (68%). BRAFV600E was identified by IHC in one patient. All other patients did not have successful NGS (n=2) or had no pathogenic variants identified (n=3). Additional details presented in Table 1. Median time on a MEKi was 6.5 months (IQR 3 - 15 months). It was used as first-line therapy in 72.7% (n=16) of cases. Within the 22 patients, 28 instances of MEKi initiation were observed in our cohort, as 5 patients (23%) received 2 different MEKi throughout follow up and one patient had cobimetinib rechallenge after first progression. MEKi used included: cobimetinib (n=23, 82%), trametinib (n=3, 11%), and binimetinib (n=2, 7%). Response assessment after initiation of first MEKi was not available in 1 patient. In the remaining 21 cases, overall response rate was 81% including complete or near-complete response (CR/nCR) in 5 (24%), partial response (PR) in 12 (57%), stable disease (SD) in 2 (10%), and progressive disease (PD) in 2 (10%). Median EFS was 36 months (95%CI 9 - not reached), respectively. Intolerable AEs with MEKi included G3/4 diarrhea (n=2), G3 lower extremity edema (n=2), G3 acneiform rash (n=1), G2 pericardial effusion (n=1), and "dropped-head syndrome” (n=1). Of the 6 patients who were treated with a second MEKi (Figure 1), five had data for response assessment and had CR/nCR (n=2) or a PR (n=3) as best responses. At time of last follow-up, 21 patients (96%) were alive and one died due to progression of ECD. Of the 22 patients, 10 patients (46%) remained on MEKi at the time of last follow-up. Reasons for therapy discontinuation included intolerable side effects (n=4), drug holiday after response achievement (n=4), disease progression on therapy (n=3), and change to other kinase inhibitors (n=1). Additional details regarding patient follow-up are presented in Figure 1. Conclusion Our study suggests that MEK inhibitors are a highly efficacious treatment in patients with ECD. Main limitation of our study includes its retrospective nature and sample size. While treatment discontinuation due to severe toxicities / intolerance was common, this did not preclude from maintenance of tumor response for extended periods of time. Not all patients will respond to MEKi and further research to guide individualized therapies is required. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Comparison of sampling methods for next generation sequencing for patients with lung cancer.

IntroductionSuccess of next generation sequencing (NGS) analysis is becoming indispensable in the treatment of advanced lung cancer. However, the advantages and disadvantages of each sampling method in the NGS analysis have not yet been clarified.MethodsWe compared the success rates of NGS analysis, and DNA and RNA yields for transbronchial biopsy (TBB), endobronchial ultrasound‐guided transbronchial needle aspiration (EBUS‐TBNA), computed tomography (CT)‐guided biopsy, fluid sample, and surgical biopsy for NGS analysis in patients through the lung cancer genomic screening project for individualized medicine (LC‐SCRUM)‐Asia, a nationwide NGS screening project. In case, sufficient samples could not be collected by TBB and EBUS‐TBNA, re‐biopsy (genome re‐biopsy) was performed.ResultsA total of 223 patients were enrolled and success rates of NGS analysis were not different between samples obtained through TBB, EBUS‐TBNA, and CT‐guided biopsy; however, success rates for fluid samples and surgical biopsy samples were significantly higher than those of other methods. The risk of genome re‐biopsy was higher with TBB for centrally located lesions. CT‐guided biopsy yielded more samples but had a lower success rate for analysis of RNA‐based NGS than TBB.ConclusionsTBB is the mainstay of sampling methods, but for centrally located lesions, EBUS‐TBNA may be a better strategy. For CT‐guided biopsy, the success rate of RNA‐based NGS analysis is low. Fluid samples are expected to yield successful results as surgical biopsy samples, but the latter are better for sample preservation. Determining the optimal method for genome biopsy for each case is important.

Pathogenic variants of homologous recombination repair-related genes in advanced pancreatic cancer and oxaliplatin-based chemotherapy: Prospective multicenter observational study.

555 Background: The latest National Comprehensive Cancer Network Guidelines for pancreatic adenocarcinoma recommended platinum-based chemotherapy for the patients with germline BRCA1/2 or PALB2 variants based on retrospective studies. However, the association between the efficacy of oxaliplatin-based chemotherapy and homologous recombination repair (HRR)-related gene variants has not yet been evaluated in a prospective study. Methods: This was a multicenter, prospective, observational study. Key inclusion criteria were: histologically confirmed pancreatic adenocarcinoma or adenosquamous carcinoma; candidates for systemic chemotherapy or currently under systemic chemotherapy for unresectable disease; age ≥ 20 years; Eastern Cooperative Oncology Group Performance Status 0–2; formalin-fixed paraffin-embedded cancer tissue available for genomic sequencing; and adequate hematological, liver, and renal function. Patients were assessed with the next generation sequencing (NGS)-based ACT-repair panel (ACT genomics; Taipei, Taiwan). ACT-repair panel is accredited by College of American Pathologists and is designed to detect short variants (SVs) including substitutions, insertions, deletions, and copy number variants of 35 genes including 8 HRR-related genes ( ATM, ATR, BRCA1, BRCA2, PALB2, RAD51B, RAD51C, RAD51D). The primary endpoint was the one-year overall survival rate (1yr-OS%) after the initiation of oxaliplatin-based chemotherapy in patients who harbored pathogenic HRR gene variants. On the basis of published retrospective data, expected 1yr-OS% was set at ≥ 60% in this study. Results: Forty patients were enrolled from August 2018 to March 2020. Median age was 67 years (range, 49–81 years). Sequencing data were obtained from 39 patients (NGS success rate = 97.5%). Nine patients (22.5%) harboring HRR gene; ATM SVs (n = 4), BRCA2 loss of heterozygosity (LOH) (n = 3), BRCA2 SVs (n = 1), and PALB2 LOH (n = 1). Three patients received oxaliplatin-based chemotherapy as first-line chemotherapy, while the remaining six patients received it as second- or later-line oxaliplatin-based chemotherapy. The 1yr-OS% was 44.4%, and the median overall survival was 221 days (95% confidence interval, 79–NA days) after the initiation of oxaliplatin-based chemotherapy. In three patients who received oxaliplatin-based chemotherapy as first-line treatment, overall survivals were 703 (alive), 694 (alive), and 405 (dead) days, respectively. Conclusions: Efficacy of oxaliplatin-based chemotherapy on advanced pancreatic cancer harboring HRR-related gene variants did not meet the primary endpoint of 1yr-OS% (≥ 60%). Clinical trial information: UMIN000033655.

The Impact of Core Tissues on Successful Next-Generation Sequencing Analysis of Specimens Obtained through Endobronchial Ultrasound-Guided Transbronchial Needle Aspiration

Simple SummaryNext-generation sequencing (NGS) with specimens obtained through endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) has been used to identify cancer-related genes among patients with lung cancer. However, the reported success rates vary, and the clinical factors associated with successful NGS remain unclear. We retrospectively reviewed consecutive patients with non-small-cell lung cancer who underwent EBUS-TBNA for NGS (OncomineTM Dx Target Test). The numbers of punctures and core tissues, as well as computed tomography (CT) and EBUS findings, were evaluated. The success rate of NGS was 83.3% (130/156). The cut-off value for the number of core tissues was 4, and the sensitivity and specificity for successful NGS were 73.8% and 61.5%, respectively. CT and EBUS findings were not associated with successful NGS. In logistic regression analysis, the number of core tissues (≥4) was the sole predictor of successful NGS. Bronchoscopists should obtain sufficient core tissues for successful NGS using EBUS-TBNA specimens.The success rate of next-generation sequencing (NGS) with specimens obtained through endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) among patients with lung cancer as well as the related clinical factors remain unclear. We aimed to determine the optimal number of punctures and core tissues during EBUS-TBNA for NGS in patients with non-small-cell lung cancer (NSCLC) as well as the association of chest computed tomography (CT) and EBUS findings with successful NGS. We retrospectively reviewed 156 consecutive patients with NSCLC who underwent EBUS-TBNA for NGS (OncomineTM Dx Target Test). Using the receiver operating characteristic curve, we calculated the optimal numbers of punctures and core tissues for NGS and evaluated CT and EBUS findings suggestive of necrosis and vascular pattern within the lesion. The success rate of NGS was 83.3%. The cut-off value for the number of core tissues was 4, and the sensitivity and specificity of successful NGS were 73.8% and 61.5%, respectively. Logistic regression analysis revealed that the number of core tissues (≥4) was the sole predictor of successful NGS. CT and EBUS findings were not associated with successful NGS. Bronchoscopists should obtain sufficient core tissues for successful NGS using EBUS-TBNA specimens.

Next generation sequencing in cytology

The application of next generation sequencing (NGS) technology to cytological samples has significantly modified molecular cytopathology practice. Cytological samples represent a valid source of high‐quality DNA for NGS analysis, especially for predicting patients' response to targeted treatments and for refining the risk of malignancy in indeterminate cytological diagnoses. However, several pre‐analytical factors may influence the reliability of NGS clinical analysis. Here, we briefly review the challenges of NGS in cytology practice, focusing on those pre‐analytical factors that may negatively affect NGS success rates and routine diagnostic applications. Finally, we address the future directions of the field.

Factors Impacting Clinically Relevant RNA Fusion Assays Using Next-Generation Sequencing.

RNA-based next-generation sequencing (NGS) assays are being used with increasing frequency for comprehensive molecular profiling of solid tumors. To evaluate factors that might impact clinical assay performance. A 4-month retrospective review of cases analyzed by a targeted RNA-based NGS assay to detect fusions was performed. RNA extraction was performed from formalin-fixed, paraffin-embedded tissue sections and/or cytology smears of 767 cases, including 493 in-house and 274 outside referral cases. The types of samples included 422 core needle biopsy specimens (55%), 268 resection specimens (35%), and 77 cytology samples (10%). Successful NGS fusion testing was achieved in 697 specimens (90.9%) and correlated positively with RNA yield (P < .001) and negatively with specimen necrosis (P = .002), decalcification (P < .001), and paraffin block age of more than 2 years (P = .001). Of the 697 cases that were successfully sequenced, 50 (7.2%) had clinically relevant fusions. The testing success rates and fusion detection rates were similar between core needle biopsy and cytology samples. In contrast, RNA fusion testing was often less successful using resection specimens (P = .007). Testing success was independent of the tumor percentage in the specimen, given that at least 20% tumor cellularity was present. The success of RNA-based NGS testing is multifactorial and is influenced by RNA quality and quantity. Identification of preanalytical factors affecting RNA quality and yield can improve NGS testing success rates.

Impact of DNA integrity on the success rate of tissue-based next-generation sequencing: Lessons from nationwide cancer genome screening project SCRUM-Japan GI-SCREEN.

In the nationwide cancer genome screening project SCRUM‐Japan GI‐SCREEN, 2590 archival formalin‐fixed paraffin‐embedded (FFPE) tumor tissues from 19 institutions were analyzed with two tissue‐based next‐generation sequencing (NGS) panels at the Clinical Laboratory Improvement Amendments (CLIA)‐certified College of American Pathologists (CAP)‐accredited central laboratory. The Oncomine Cancer Research Panel (OCP; 143 genes) succeeded in producing validated results for only 68.3% of the samples (%OCP‐success). CE‐IVD (25 genes) succeeded in 45.9% of the OCP‐failed samples, leading to an overall NGS success (%combined‐success) rate as high as 82.9%. Among 2573 samples, the DNA‐integrity (ΔC t)‐high (ΔC t < 4.4, n = 1253) samples showed significantly higher %OCP‐ and %combined‐success rates (90.2% and 97.4%, respectively) than the DNA‐integrity‐intermediate (4.4 < ΔC t < 6.3, n = 911; 68.9% and 88.7%) and DNA‐integrity‐low ones (ΔC t > 6.3 or polymerase chain reaction‐failed, n = 409; 5.6% and 24.7%). Other factors associated with NGS success included the FFPE‐sample storage period (<4 years), the specimen type (surgical) and the primary tumor site (colorectal). Multivariable analysis revealed DNA integrity as the factor with the strongest independent association with NGS success, although it was suggested that other institution‐specific factors contribute to the discordance of inter‐institutional NGS success rates. Our results emphasize the importance of DNA quality in FFPE samples for NGS tests and the impact of DNA integrity on quality monitoring of pathology specimens for achieving successful NGS.

Impact of DNA Damage Response and Repair (DDR) Gene Mutations on Efficacy of PD-(L)1 Immune Checkpoint Inhibition in Non-Small Cell Lung Cancer.

DNA damage response and repair (DDR) gene alterations are associated with increased tumor-infiltrating lymphocytes, higher genomic instability, and higher tumor mutational burden (TMB) in cancer. Whether DDR alterations are associated with clinical outcomes to programmed death ligand 1 [PD-(L)1] blockade in non-small cell lung cancer (NSCLC) is unknown. Tumors from patients treated with PD-(L)1 inhibitors were analyzed using targeted next-generation sequencing (NGS). Cancers were categorized on the basis of the presence or absence of deleterious mutations across a panel of 53 DDR genes. Clinical outcomes to PD-(L)1 inhibitors were evaluated according to DDR mutation status. Of 266 patients with successful NGS who received PD-(L)1 inhibitors, 132 (49.6%) were identified as having deleterious DDR mutations (DDR-positive). DDR-positive and DDR-negative groups were similar in terms of baseline clinicopathologic characteristics. The median TMB was significantly higher in the DDR-positive group compared with the DDR-negative group (12.1 vs. 7.6 mutations/megabase; P < 0.001). Compared with DDR-negative patients (N = 134), DDR-positive patients had a significantly higher objective response rate (30.3% vs. 17.2%; P = 0.01), longer median progression-free survival [PFS; 5.4 vs. 2.2 months; HR, 0.58 (95% confidence interval (CI), 0.45-0.76); P < 0.001], and longer median overall survival [OS; 18.8 vs. 9.9 months; HR, 0.57 (95% CI, 0.42-0.77); P < 0.001] with PD-(L)1 therapy. After adjusting for PD-L1, TMB, performance status, tobacco use, and line of therapy, DDR-positive status was associated with a significantly longer PFS [HR, 0.68 (95% CI, 0.51-0.92); P = 0.01] and OS [HR, 0.60 (95% CI, 0.43-0.85); P = 0.004] in multivariate analysis. Deleterious DDR mutations are frequent in NSCLC and are associated with improved clinical outcomes in patients with NSCLC treated with PD-(L)1 blockade.

Improvement strategies for successful next-generation sequencing analysis of lung cancer.

Aim: We aimed to improve the success rate of NGS (next-generation sequencing) analysis through improved strategies of lung cancer sampling. Materials & methods: The improvement strategies are as follows. Surgically resected specimens were preferentially submitted in cooperation with pathologists and surgeons. In bronchoscopic samples, the size of the sample collection device and the number of samples collected was increased. Results: The strategies increased the success rate of NGS analysis of DNA from 69.3 to 91.1%, and that of RNA from 64.6 to 90.0%. Discussion: The introduction of strategies aimed at improving the success of NGS analysis resulted in an improvement in the success rate and brought us closer to the delivery of effective precision medicine in cancer therapy.