Next Generation Sequencing Reports Research Articles

Characteristics and predictive modeling of HER2 mutation in patients with non-small cell lung cancer.

e13559 Background: Recent advances in targeted therapies have increased the survival and quality of life for non-small cell lung cancer (NSCLC) patients. Understanding clinical characteristics and predicting HER2 status may facilitate efficient use of next-generation sequencing (NGS) testing and promote precision medicine/targeted therapies. Methods: NSCLC patients ≤65 years old, diagnosed between 01/2011 and 06/2021 who have NGS test results were identified in from two National Cancer Institute designated cancer centers in the Northeastern region of the United States. Demographics, behavioral, and clinical characteristics were extracted from electronic health record (EHR) data and EHR-linked cancer registry. Prevalence prediction modelling of HER2/ERBB2 mutation was performed using machine learning (ML) methods including logistic regression (LR), random forest (RF), naïve Bayes (NB), extreme gradient boosting (XGBoost) and multi-layer perceptron (MLP). 10- fold cross validation and k-nearest neighbor method were used to impute missing values. Under-sampling, oversampling and feature selection methods were tried. Results: Among 761 NSCLC patients with NGS reports available, 353 (46%) were ≤65 years old. Among 353 (mean age 57, 58% female, 88% non-Hispanic, 69% white, 57% had metastasis, 58% prescribed chemotherapy) 25 (7%) had a HER2 mutation. Based on univariate significance tests with LR on the complete data: cerebrovascular disease, BMI between 27.5-29.9, KRAS, EGFR and MET were significant with odds ratios (ORs) ranging from 0.2-3.9. In our prediction models, BMI, cerebrovascular disease, weight loss, hepatic metastases, diabetes, congestive heart failure, and KRAS, EGFR, MET and RET alteration status were significant predictors in multivariate analyses, suggesting the potential usefulness of these features in predicting HER2 mutation among patients with NSCLC. Area under the ROC curve (AUC) varied across different models from 70.9% (RF), 70% (XGB and MLP), 69.5% (LR) to 68.2% (Naïve Bayes). The best performing model with RF yielding an AUC ~71% can provide relative high specificity and acceptable sensitivity depending on the cut off. Conclusions: Our prediction models yielded AUC around 70% with the best performing model’s AUC being 71% in a subgroup aged ≤65. After further validation, our prediction model may be used to identify patients appropriate for repeat NGS testing who did not have HER2 testing in first molecular profiling. Future work is needed to predict status in repeat testing with previously test negative patients who relapse or have progression of disease. Random forest (RF) Model performance across different cutoff for the predicted probability. [Table: see text]

Real world early experience of precision neoadjuvant systemic therapy in patients with resectable NSCLC: Single institution experiences and perspectives.

e20594 Background: The incidence of early-stage NSCLC is increasing with lung cancer screening. However, up to 55% of NSCLC patients (pts) who undergo curative surgery die of recurrence. Neoadjuvant chemoimmunotherapy increases pathological clinical response and reduces tumor recurrences in resectable NSCLC pts without known EGFR/ALK alterations in March 2022. The uptake of this practice is low and there is an unmet need in incorporating effective targeted therapies for driver oncogenes in pts with early-stage NSCLC. As precision neoadjuvant therapy holds the great promise in improving surgical and pathological outcomes and cure rate, the objective of this study was to develop a clinical workflow to timely coordinate the care between oncology specialties and biomarker testing in pts with early-stage NSCLC. Methods: This study included early-stage NSCLC pts with no biomarker testing (Cohort A, 15) and biomarker testing (Cohort B, 19). Driver genomic alterations with targeted therapy in the metastatic setting were identified by next generation sequencing (NGS). Descriptive analysis was used for the type and frequency of each mutation and unpaired t-test was used for time interval analysis. Results: In Cohort A, 10/15 (67%) pts received neoadjuvant chemotherapy (4) or chemoimmunotherapy (6) before surgery, 3 proceeded to surgery, and 2 received concurrent chemoradiation for unresectable tumor. Of 19 pts in Cohort B, 2 did not have biomarker testing due to small tumor size. Of the 17 pts who had NGS testing, 7 (41%) had a targetable oncogene: EGFR l858R (2), EML4-ALK (1), MET amplification (1), RET fusion (1), and EGFR E20in (2). Two received matched targeted therapies on clinical trials. Median (range) dates for NGS testing were 12 (6-44) and for NGS report from pathologic diagnosis were 48 (17-93). Barriers for getting a matched neoadjuvant therapy included no matched targeted therapy (2), tumor progression (1), unresectable tumor (1), and patient preference (1). Of the pts that underwent surgery, there was no statistically significant difference in median time (range) to diagnosis between Cohort A [N = 12, 33 (7-100) days] and Cohort B [N = 8, 34 (9-82) days] (p = 0.74). The median time (range) to surgery from pathologic diagnosis was shorter in Cohort B [78 (29-137)] than Cohort A [166 (56-201)] (p = 0.0002). The difference was due to more pts in the Cohort A receiving neoadjuvant therapy than the biomarker cohort [10/12 (83%) vs 2/7 (25%)]. Conclusions: In this analysis, time to pathological diagnosis were similar between Cohorts A and B. Time for NGS testing was also comparable for standard of care in metastatic disease. However, time to NGS results from pathological diagnosis was longer than anticipated in Cohort B, which is an area of improvement in our workflow in prospective clinical evaluation for precision neoadjuvant treatment of early-stage NSCLC.

Comprehensive multigene profiling impact on clinical decisions in patients with advanced cancers: A multicenter, retrospective analysis.

e15163 Background: Comprehensive molecular profiling (CMP) via next generation sequencing (NGS) is currently applied in clinical practice and has enabled the detection of biomarkers of response to targeted treatment. Given the lack of the data about optimal range of analyzed alterations, the clinical usefulness of CMP has not been established. To address the challenges in incorporating genomics in treatment of oncology patients, we conducted a retrospective study. Methods: Preliminary analysis of NGS reports and clinical data of patients with advanced solid tumors was performed. CMP was conducted in 261 cancer patients (non-small cell lung cancer - 30,6%, colorectal cancer - 24,8%, pancreatic cancer - 7,4%, breast cancer- 6,2%; 59,3% were female, median number of prior systemic treatment lines was 2 (0-12)). In the present study, tumor molecular profile analysis was performed using 160 - 400 gene NGS panels, containing the majority of clinically significant genes for cancer treatment selection. We assessed the percentage of patients who were recommended to receive molecularly-matched therapy (MMT) after CMP, immunotherapy (IT) distribution of recommended therapies according to ESCAT tiers, median progression-free survival (PFS) – ratio ( = PFS after CMP / PFS before CMP, PFS2/PFS1), median PFS2. Results: MMT after CMP was recommended for 55 (21,1%) patients. ESCAT tiers could be assessed in 48 MMT patients (87%). For 31% of patients, administered therapy was considered as ESCAT I tier recommendation (data on mPFS2/1, mPFS2 not available). ESCAT tiers II, III, IV and IT administration were considered in 31, 8, 8 and 22% of patients respectively (detailed data is presented in Table 1). Most commonly altered genes were TP53 (53%), RAS (29%), APC (18%), CDKN2A/B (16%), PIK3CA(14%), NF1 (10%), BRCA1/2 (9%). Positive predictive factors for MMT after CMP were MSI-high status (HR 0,13, 95% CI 0,03 - 0,67), presence of EGFR alteration (HR 0,32, 95% CI 0,12 - 0,86). Presence of RAS mutation was considered a negative predictive factor (HR 2,78, 1,07 - 7,26). Conclusions: Available NGS panels offer a wide range of analyzed alterations that are not feasible in clinical practice. Most of the alterations that can cause impact on treatment plan can be detected with other methods than NGS. Further studies are needed to determine optimal range of alterations depending on tumor site. [Table: see text]

Evaluating the impact of a molecular tumor board in a community oncology center: Results from the Avera Cancer Institute's experience.

e13500 Background: The use of molecular tumor boards (MTB) has been widely documented at large academic medical centers, but there is limited experience and data available in smaller settings like community oncology centers. The Avera Cancer Institute has had a molecular tumor board in place since 2014, but the composition and methodology have changed over time. Our 10 years of hands-on experience and involvement in studies like I-PREDICT showed that in addition to molecular matching, considering factors like tumor type, previous treatment responses, comorbidities, medication coverage, and treatment availability provide crucial insights for determining optimal therapy for patients. Methods: We analyzed next-generation sequencing (NGS) testing results from August 2019 to December 2022. At the time of NGS testing result, providers were offered to have the case reviewed and presented at MTB. Our multidisciplinary team consists of members with backgrounds in bioinformatics, medical oncology, pathology, translational science, trial coordination, and pharmacy. Recommendations from the MTB discussions were provided in three categories: standard of care, clinical trials, and off-label options. Results: From August 2019 to December 2022, 1,514 qualified or resulted NGS reports were returned and considered for analysis. 733 (48%) were presented at the molecular tumor board and from this group, 629 patients were evaluable. Oncologists chose a recommended therapy 66% of the time. Of the remaining 34%, the most common reason for not utilizing recommendations was that patients were unable to receive further lines of systemic therapy. A subset analysis of 202 cases (June 2021 – August 2022) who either received a recommended therapy (n = 88, 43.6%, group 1) or not (n = 114, 56.4%, group 2) showed a survival benefit. Median overall survival was not reached in group 1, as compared with 14 months in group 2 (HR = 0.58; 95% CI, 0.37 – 0.92; p< 0.02). The median PFS was 8.2 months in group 1 versus 5 months in group 2 (HR, 0.67; 95% CI, 0.48 - 0.92; p< 0.017). Conclusions: The results of our study demonstrate the positive impact of a molecular tumor board review in helping oncologists determine the best treatment options for their patients. Our findings show that recommendations made by the board are well received and frequently adopted by providers. While providers generally prefer standard of care options or clinical trials, they are open to considering all available options to make informed therapy decisions.

A Real-World Experience from a Single Center (LPCE, Nice, France) Highlights the Urgent Need to Abandon Immunohistochemistry for ROS1 Rearrangement Screening of Advanced Non-Squamous Non-Small Cell Lung Cancer.

The detection of ROS1 rearrangements in metastatic non-squamous non-small cell lung carcinoma (NS-NSCLC) permits administration of efficient targeted therapy. Detection is based on a testing algorithm associated with ROS1 immunohistochemistry (IHC) screening followed by ROS1 FISH and/or next generation sequencing (NGS) to confirm positivity. However, (i) ROS1 rearrangements are rare (1-2% of NS-NSCLC), (ii) the specificity of ROS1 IHC is not optimal, and (iii) ROS1 FISH is not widely available, making this algorithm challenging to interpret time-consuming. We evaluated RNA NGS, which was used as reflex testing for ROS1 rearrangements in NS-NSCLC with the aim of replacing ROS1 IHC as a screening method. ROS1 IHC and RNA NGS were prospectively performed in 810 NS-NSCLC. Positive results were analyzed by ROS1 FISH. ROS1 IHC was positive in 36/810 (4.4%) cases that showed variable staining intensity while NGS detected ROS1 rearrangements in 16/810 (1.9%) cases. ROS1 FISH was positive in 15/810 (1.8%) of ROS1 IHC positive cases and in all positive ROS1 NGS cases. Obtaining both ROS1 IHC and ROS1 FISH reports took an average of 6 days, while obtaining ROS1 IHC and RNA NGS reports took an average of 3 days. These results showed that systematic screening for the ROS1 status using IHC must be replaced by NGS reflex testing.

Guidance on Therapeutic Strategies for Metastatic Urothelial Cancer

Guidance on Therapeutic Strategies for Metastatic Urothelial Cancer

BRCA mutations detected by tumour next-generation sequencing in non-small cell lung cancer: impact on response to therapy and disease course.

Data regarding the prevalence and clinical relevance of BRCA mutations in non-small cell lung cancer (NSCLC) is limited. Our objective was to evaluate the impact of pathogenic BRCA variants detected by tumour next-generation sequencing (NGS) on disease course and response to therapy. We performed a retrospective analysis of all consecutive NSCLC patients with available NGS reports in a single institution between 01/2015 and 08/2020. Pathogenicity of identified mutations was determined according to American College of Medical Genetics (ACMG) guidelines. Log rank and cox regression analyses were used to determine the association between BRCA mutation status, overall survival (OS) and progression-free survival (PFS) under various front-line treatment modalities for advanced disease. Out of 445 patients with NGS data (54% tissue, 46% liquid), 109 (24.5%) patients had a documented BRCA variant; 5.6% (25/445) had a pathogenic/likely pathogenic variant (pBRCA). Forty percent (10/25) of pBRCA patients had no co-occurring NSCLC driver mutations. Patients with pBRCA NSCLC had a less prominent smoking history [mean 42.6 (29.2) vs. 25.7 (24.0) pack years; P=0.024]. Median PFS with first-line chemo-immunotherapy was significantly prolonged for pBRCA patients (n=7) compared with wild-type BRCA (wtBRCA) patients (n=30) (HR =0.279; P=0.021, 95% CI: 0.094-0.825). pBRCA-mutated NSCLC can represent a specific subtype of pulmonary carcinoma. Patients whose tumours harbor pBRCA mutations present with a less prominent smoking history and exhibit prolonged PFS with chemo-immunotherapy combinations compared with wtBRCA controls. In a subset of these patients, pBRCA is the sole identifiable putative driver mutation, hinting at a significant role for BRCA loss in oncogenesis.

The Rapidly Expanding Genetic Spectrum of Common Variable Immunodeficiency–Like Disorders

The understanding of common variable immunodeficiency disorders (CVID) is in evolution. CVID was previously a diagnosis of exclusion. New diagnostic criteria have allowed the disorder to be identified with greater precision. With the advent of next-generation sequencing (NGS), it has become apparent that an increasing number of patients with a CVID phenotype have a causative genetic variant. If a pathogenic variant is identified, these patients are removed from the overarching diagnosis of CVID and are deemed to have a CVID-like disorder. In populations where consanguinity is more prevalent, the majority of patients with severe primary hypogammaglobulinemia will have an underlying inborn error of immunity, usually an early-onset autosomal recessive disorder. In nonconsanguineous societies, pathogenic variants are identified in approximately 20% to 30% of patients. These are often autosomal dominant mutations with variable penetrance and expressivity. To add to the complexity of CVID and CVID-like disorders, some genetic variants such as those in TNFSF13B (transmembrane activator calcium modulator cyclophilin ligand interactor) predispose to, or enhance, disease severity. These variants are not causative but can have epistatic (synergistic) interactions with more deleterious mutations to worsen disease severity. This review is a description of the current understanding of genes associated with CVID and CVID-like disorders. This information will assist clinicians in interpreting NGS reports when investigating the genetic basis of disease in patients with a CVID phenotype.

The genomic landscape of advanced gastrointestinal (GI) neuroendocrine tumors (NET) and carcinomas (NEC).

656 Background: The impact of specific genomic alterations in advanced GI neuroendocrine malignancies on prognosis and treatment decisions has not been fully characterized. In this study, we seek to evaluate next-generation sequencing (NGS) panels and corresponding clinical outcomes in patients with metastatic GI NET/NEC. Methods: Patients with GI-NET/NEC were identified from a database of NGS reports performed at the Cleveland Clinic, and clinical variables were extracted by chart review through an IRB-approved protocol. We provide an analysis of this cohort. Results: Of a total n=45 patients, 53% were male. Median age at diagnosis was 60 (range: 35-90 years) and 82% were non-Hispanic white, while 9% were black. There were n=25 GI-NETs, mostly intermediate or high grade (11 G3, 7 G2), and n=20 GI-NECs. There was a balanced distribution of primary sites, including colorectal (27%), pancreas (24%), small intestine (18%), unknown primary (24%) and a single case of biliary primary. High grade GI-NETs and GI-NECs were over-represented in the colorectal primary group vs other sites. (n=10 of 12; p=0.021). The majority of patients (73%) had at least one somatic variant, including 12 patients (27%) with one or more mutations deemed actionable, but only one patient (with MSH2 mutation) has received a mutation-matched drug (pembrolizumab). Some recurrent alterations appeared to co-segregate with aggressive histology (NEC vs NET), including 12 of 13 TP53 mutations (p<0.05 by Fisher Exact Test), 7 of 8 RB1 (p<0.05), and 5 of 6 APC (p=0.074). The most common mutations per person based on primary site is shown. Microsatellite instability high was rare (n=1) and median tumor mutational burden (TMB) was 3/mb, with TMB lower in NETs vs NECs. A number of patients (35%) underwent germline testing, with 6 of 16 having a pathogenic germline variant. Conclusions: As 42% of the 12 patients with actionable mutations were alive at time of analysis, it is possible that additional patients could be treated with mutation-matched therapies. More input will be needed from providers as to the role played by alternative effective treatments and barriers such as insurance coverage and prior authorization to implementing mutation-matched therapies. Given the overall low TMB of GI-NETs, with several tumors having zero or 1 reported mutation on targeted NGS, whole genome sequencing may prove useful. Finally, certain mutations such as RB1 and TP53 were associated with more aggressive histology (i.e. NEC). Lastly, only 9% of our cohort with NGS were black, a significant under-representation based on epidemiological data, which raises concerns about disparate accessibility and barriers to NGS testing. Further studies are needed to address any possible racial disparity.[Table: see text]

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.

35. The development, testing, and validation of an automatic LIMS import method for NGS myeloid panel results

For oncology genetic testing, there is a critical need for patient results to be readily available and accessible for the ordering pathologist's review. After an initial genetic testing workup, emergency treatment or follow-up testing may be required, thus accessibility of results is a key feature in providing patient-focused laboratory testing. Ideally, genetic testing results would be incorporated into a LIMS data management system in order to have a permanent record of testing and to allow for the results to be easily accessible, searchable, and transferable by the clinical or pathology team working on behalf of the patient. For maximum impact, Next Generation Sequencing (NGS) reports should be electronically integrated into the patient's electronic health record. NGS reports uploaded as PDF attachments constrain health record searches. Additionally, data in PDF reports often are not able to be transferred to a comprehensive pathology report and may require cumbersome manual entry. Our team has recently completed a project to create an automatic import method for our Myeloid Complete NGS Panel report into our electronic patient data management system. Our data management system is interfaced with our client's LIMS platform enabling the direct and automatic transfer of orders coming in as well as delivery of the resultant NGS report into the patients' health records. We will present the steps taken to create, test, troubleshoot, and validate the automatic upload system. We will also detail critical components of the NGS LIMS report and challenges that were addressed during this project.

44. An international landscape of cancer NGS reporting practices

Multiple guidelines and standards have been issued globally to aid in Next Generation Sequencing (NGS) variant interpretation. The AMP/ASCO/CAP, ACMG/AMP, and ESMO published guidelines are incorporated into cancer NGS reports in different ways around the globe. The Variant Interpretation for Cancer Consortium Virtual Molecular Tumor Board (VICC-VMTB), in collaboration with the Cancer Genomics Consortium (CGC) and the ClinGen Somatic Clinical Domain Working Group (CDWG), distributed a comprehensive survey to membership of each group regarding reporting practices for cancer NGS testing worldwide. Seventy-nine survey responses representing 17 countries in 6 continents were collected. Respondents are involved in reporting cancer NGS tests in clinical laboratories (n=58), academic/research laboratories (n=30), and/or commercial laboratories (n=5). The use of reporting elements both consistently and differentially was evaluated. Consistently reported elements used globally include but are not limited to inclusion of the tissue source, diagnosis, protein-level amino acid sequence-based (p.) variant annotation, and the inclusion of relevant treatment information. Differentially reported elements include but are not limited to the usage of genomic DNA reference sequence-based (g.) nomenclature, the combination of different variant types into a single report, and the practices around reporting mutational signatures. This global survey helps inform communities providing guidance on reporting structure and the necessary components of cancer NGS report. This snapshot of current reporting practices helps further the discussions about differentially utilized report elements and clarify the context of their use on a global scale.

Race in a molecular tumor board compared to cancer registry population.

103 Background: Disparities in cancer care due to race and ethnicity are prevalent in both the care patients receive and patient outcomes. The evaluation of next generation sequencing (NGS) results from patients with advanced cancer by a molecular tumor board (MTB) has become standard practice in many institutions for the identification of additional treatment options and targeted therapies. We sought to compare the racial distribution of patients evaluated by our MTB with our institutional cancer registry (CR). Methods: We tabulated the racial distribution of 560 MTB patients chosen for presentation in a bimonthly case conference based on physician request or clinical interest from more than 2,500 NGS reports of patients with advanced cancer from 2016 through 2020. Self-identified race from patients with stage 4 cancer within our institutional CR from the same time interval was compared to the MTB population from each year using the Chi-Squared test. The Cochran-Mantel-Haenszel test was used to analyze the relationship between race and MTB/CR after controlling for year. Race categories were defined as Asian, Black/African-American (AA), White/Caucasian, and other. Results: We identified 4,151 CR patients with stage 4 cancer from 2016 through 2020, 573 of whom identified as Black/AA (13.8%). Of the 560 MTB patients, 55 were Black/AA (9.8%). When controlling for year, Black/AA patients were less frequently included in the MTB compared to the CR (p = 0.0128). Conclusions: Black/AA patients with advanced cancer are under-represented in our MTB. Larger studies are warranted to examine underlying causes of this discrepancy including implicit bias, generalizability of this finding to other minorities and institutions, and potential remedies to ensure equitable access to state-of-the-art cancer care.[Table: see text]

1679P A comparative analysis of cancer NGS reporting practices between Europe and North America

Results of Next Generation Sequencing (NGS) increasingly incorporated in routine clinical oncology. Multiple guidelines and standards have been issued globally to support clinical NGS variant interpretation and are integrated in different ways into cancer NGS reports around the world. However, a thorough capture of how these guidelines are being translated in real-world laboratory reporting has not yet been clearly studied.

Abstract 4118: Brain metastases (BM) and next-generation sequencing (NGS) timing: Real-world (RW) outcomes for patients (pts) with advanced non-small cell lung cancer (aNSCLC) and MET exon 14 skipping mutations (METex14)

Abstract Background: Treatment (Tx) options for METex14 NSCLC have been limited to chemotherapy (Ctx), immunotherapy (IO), and multikinase inhibitors (MKi) until FDA approvals of selective MET inhibitors (METi) from 2020. BM are a further challenge to treat. We previously reported NGS timing in relation to start of first-line Tx (1L) for pts with METex14 NSCLC in the RW. Here we provide overall survival (OS) data, including subgroup analyses for pts with known BM prior to 1L initiation. Methods: Data originating from ~280 US cancer clinics (~800 sites of care) from the US electronic health record-derived de-identified Flatiron Health Foundation Medicine clinico-genomic database (FH-FMI CGDB) were evaluated. Pts were ≥18 yrs, diagnosed with advanced/metastatic NSCLC from Jan 1, 2011, with NGS-confirmed METex14, ≥1 line of Tx, and ≥90 days follow-up from 1L initiation to cutoff date (Dec 2020). Pts treated with capmatinib or tepotinib were excluded. We examined Tx patterns and OS for pts with and without known BM before starting 1L, and OS for pts with an NGS report before or on/after 1L initiation (time from 1L initiation to death or censoring, analyzed using Kaplan-Meier method with left truncation adjustment to mitigate bias due to the prerequisite for NGS testing). HRs were estimated using Cox regression models with covariates adjustment to estimate risk of death. Results: The analysis included 156 METex14 NSCLC pts: median age 75 yrs, 59% females, 85.3% non-squamous (NSQ), 9% squamous cell carcinoma (SQ), and 64% had history of smoking. A total of 31 pts (20%) had BM before 1L initiation: median age 73 yrs, 61% females, 94% NSQ, 0% SQ, and 71% had history of smoking. Overall, treatment patterns were similar for pts with and without known BM before 1L initiation (Ctx with or without IO, IO monotherapy, and MKi were all used in 1L). Median OS for pts with BM was 8.11 mo (95% CI, 6.0-19.8) and 14.39 mo (11.1-22.1) for pts without BM, corresponding to a 73% increase in risk of death for pts with BM vs pts without BM by univariate analysis (HR 1.73 [95% CI, 1.06-2.82]; P=0.03). Median OS for pts with an NGS report before and on/after 1L initiation was 13.3 mo (8.8-26.2) and 6.8 mo (0.03-19.8), respectively (P=0.45); HR by multivariate analysis was 0.78 (0.45-1.35) when comparing pts with NGS report before vs on/after 1L initiation. Conclusions: Tx patterns in 1L were similar for pts with or without BM at time of 1L initiation; presence of BM prior to 1L was associated with inferior outcomes. Numerically longer OS was observed in pts with NGS report prior to 1L initiation vs pts with NGS report on/after 1L, supporting further investigation of underlying causes. These data underline the ongoing unmet clinical need of METex14 NSCLC BM and the importance of investigating upfront comprehensive genomic testing in NSCLC. Citation Format: Xiuning Le, Stephen Robb, Nydia Caro, Simona Doria, Whitney C. Rhodes, Wen-Hsing Wu, Julia Kim, Vincent Prêtre, Fen Ye. Brain metastases (BM) and next-generation sequencing (NGS) timing: Real-world (RW) outcomes for patients (pts) with advanced non-small cell lung cancer (aNSCLC) and MET exon 14 skipping mutations (METex14) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 4118.

The Use of Next-generation Sequencing in the Diagnosis of Rare Inherited Anaemias: A Joint BSH/EHA Good Practice Paper.

The Use of Next-generation Sequencing in the Diagnosis of Rare Inherited Anaemias: A Joint BSH/EHA Good Practice Paper.

Next-generation Sequencing: For the Present Generation Oncologist

Previously, doctors had to treat this deadly disease based on immunohistochemistry, polymerase chain reaction and fluorescence in situ hybridisation-based diagnostics tests information which used to provide limited information. There was always a requirement of a single test/assay which could give all these information in a single assay and without much loss of precious sample. Comprehensive next-generation sequencing (NGS)-based assays are gaining much importance in treatment decision among clinicians. These present technologies using DNA, RNA, and methylation sequencing have brought a lot of changes in cancer therapeutics. Oncogenic drivers are genomic alteration lead to malignant transformation and progression of cancer. The interpretation of the results of NGS is also very challenging as the results obtained are ‘variants’ are of unknown prognostic significance. Apart from these, NGS helps us in documenting various other genomic signatures such as tumour mutation burden and microsatellite instability. There are now multiple gene panels that are recommended by major international societies such as NCCN, for the treatment of various malignancies. In fact, NGS forms the most important pillar of precision oncology and has brought a paradigm shift in the way major cancers are treated. It is still an evolving field and many times the interpretation of the results of NGS reports is very difficult. NGS-based precision medicine treatment offers true value addition in clinical practice has a positive impact on patient lives in cases of refractory cancer, the role of NGS is the only source of light that helps us in navigating amidst the darkness and hopelessness of a refractory malignancy. In fact, presently not only in refractory malignancies but the role of NGS has come in the management of cancer patients in the first line as it gives us a more comprehensive understanding of the disease. This review article has been written with an idea to make a general practitioner aware of this novel technique, the advantages as well as the pitfalls.

Next-generation sequencing for myeloid malignancies

Over the past two decades, next-generation sequencing (NGS) has revolutionized our understanding of the pathogenesis of myeloid neoplasms (MNs) and their clinical management. While traditional Sanger sequencing allows for the interrogation of single loci, NGS enables the parallel sequencing of multiple genomic locations, ranging from targeted sets of genes to the entire genome. Initially, NGS was used predominantly in research, where the ability to interrogate large regions of the genome facilitated the discovery of genes recurrently mutated in myeloid malignancies. Soon thereafter, NGS entered the clinical realm where it is now routinely utilized in diagnosis, prognostication and treatment decision-making. However, the broad availability of clinical NGS comes with a unique set of challenges. Hematologists must interpret complex molecular reports and appropriately apply the provided mutational information to their patients’ care in real-time. Consequently, a systematic approach to interpreting NGS reports is crucial; the following will outline one such framework.

Frequencies of actionable alterations found by somatic tumor sequencing in veterans with metastatic prostate cancer.

178 Background: Prostate cancer comprises one third of male Veteran cancers and is their second leading cause of cancer death. Metastatic prostate cancer is lethal. Next Generation Sequencing (NGS) of somatic tumors is recommended for metastatic prostate to identify actionable alterations targeted with approved therapies. Veterans with prostate cancers harboring alterations in genes involved in the DNA damage response (e.g. BRCA1/2) or high microsatellite instability (MSI-High) may be eligible for PARP inhibitors or checkpoint blockade immunotherapy, respectively. Potential candidates may be identified for ongoing clinical trials of novel precision oncology approaches. Methods: This is a retrospective analysis of clinical, genomic, demographic data from Veterans with metastatic prostate cancer who underwent somatic NGS using the Foundation Medicine NGS platform from 2019-February 2021. To be included, prostate cancer was submitted diagnosis for the NGS testing and metastatic disease determined by the VINCI natural language processing tool. Variables included demographic, clinical, and pathological characteristics (self-identified race/ethnicity, age, rurality of residence, Gleason score, specimen site, other cancer diagnosis, mutation frequency). Primary outcome was mutation rates in homologous recombination (HR) genes under current FDA approval for olaparib (ATM, BARD1, BRCA1, BRCA2, BRIP1, CDK12, CHEK1, CHEK2, FANCL, PALB2, RAD51B, RAD51C, RAD51D, RAD54L) or MSI-High. Raw variant data, submitted diagnosis, and clinical data were extracted from the NGS reports and harmonized for further variant annotation. Variant data included chromosome, position, reference and alternate allele, total depth, variant allele depth, and quality scores. Variants were annotated using ANNOVAR. Likely oncogenic and oncogenic mutations were identified using OncoKB. Results: 1,597 Veterans with metastatic prostate cancer underwent FMI NGS testing (63% White, 33% African American, 4% other). Median age was 66 years, 78.6% of cases from >60 years. Of the 1,597 who underwent blood or tumor testing, at least one likely oncogenic mutation in an HR gene under FDA approval for olaparib was found in 369 (23.1%) of Veterans (19% of tissue-based tests, 32.9% of blood-based tests). Of 651 liquid biopsy tests with at least one HR gene mutation, 125 of 214 (52%) had mutations at a variant allele frequency (VAF) <0.5% or were found in an MSI-High sample that could indicate a spurious mutation due to clonal hematopoiesis. 33 patients (2.1%) were MSI-High, (21 tissue-based and 12 blood-based). Frequencies of alterations in ATM (3.6%), CDK12 (5.6%), and BRCA2 (4%) in tissue-based tests were not significantly different from those reported in other series. Conclusions: NGS of somatic tumors from Veterans with metastatic prostate cancer identifies alterations that impact management and clinical outcomes.

Abstract P2-01-13: Longitudinal circulating tumor DNA (ctDNA) monitoring by digital droplet PCR (ddPCR) in metastatic breast cancer

Abstract Background: A common challenge in treating patients with metastatic breast cancer (MBC) is ensuring adequate therapeutic efficacy, maintenance of quality of life, and avoidance of drug toxicity by preventing exposure to ineffective treatment. Critical to balancing these goals is the ability to quickly identify progression in order to minimize exposure to ineffective therapies. Imaging is the current standard-of-care for disease response evaluation, but high cost, relative low sensitivity, and tumor flare phenomenon remain significant issues. Circulating tumor DNA (ctDNA) testing based on next generation sequencing (NGS) is emerging as an alternative to imaging-based response assessment, but remains an expensive proposition. Here we evaluate a hybrid monitoring strategy using an initial baseline NGS-based ctDNA analysis to identify mutant alleles, followed by response monitoring with a low-cost, personalized digital droplet PCR (ddPCR) assay. Methods: Blood for ddPCR ctDNA analysis was collected at the same timepoint as a sample for commercial ctDNA analysis by NGS. PCR primer and probe sets were designed against clinically relevant mutations identified from each patient’s commercial ctDNA NGS report. ddPCR was then performed. Variant allele fractions (VAFs) detected by ddPCR were compared to allelic fractions reported by commercial NGS using Bland-Altman analysis and significance was determined by paired t-test. To demonstrate the potential for longitudinal ddPCR-based ctDNA monitoring, serial samples from the same patient were analyzed. Results: In our pilot validation set, 10 paired patient samples were analyzed by NGS and ddPCR. NGS-reported VAFs ranged from 0.30% to 16.20%. High comparative performance was observed between NGS and ddPCR platforms over expected mutant fractional abundance ranges. There was no significant quantitative difference between the two approaches (p=0.45). Longitudinal monitoring of ctDNA by ddPCR was performed on a metastatic hormone-positive breast cancer patient and revealed a significant rise in the clinically-relevant PIK3CA H1047L mutation that preceded imaging-based documentation of progression by 3 months. A final timepoint was taken several months before the patient’s death with a further significant increase in ctDNA burden. Conclusions: Here we demonstrate that highly-sensitive ddPCR-based ctDNA assays provide comparable results to next generation sequencing, but at considerably lower cost. These results indicate that bespoke tracking of select alleles by ddPCR offers an inexpensive and rapid approach to serial ctDNA monitoring in metastatic breast cancer patients. A larger prospective study has been designed and is underway. Citation Format: Nicole Higashiyama, LaTerrica Williams, Bryant McCue, Alphi Kuriakose, Tiffaney Tran, Stephanie Gonzalez, Mayra Licerio, Carol Chenault, Heidi Dowst, Susan Hilsenbeck, Matthew Ellis, Bora Lim, George Miles. Longitudinal circulating tumor DNA (ctDNA) monitoring by digital droplet PCR (ddPCR) in metastatic breast cancer [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P2-01-13.