Comprehensive Molecular Testing Research Articles

Transesophageal ultrasound-guided bronchoscopic Acquire TBNB versus Vizishot2 TBNA needles for neoplastic lesions: A retrospective study

BackgroundLung cancer is often diagnosed at an advanced stage; however, it has shown improved therapeutic efficacy with the introduction of molecularly targeted drugs and immune checkpoint inhibitors, necessitating accurate molecular diagnosis for effective treatment planning. Traditional sampling techniques, including endobronchial ultrasound-guided transbronchial needle aspiration, frequently require multiple biopsies to obtain sufficient tissues for multiplex testing, highlighting the need for more efficient methods. Therefore, we explored the diagnostic utility of endoscopic ultrasound with bronchoscope-guided fine-needle biopsy (EUS-B-FNB) versus fine-needle aspiration (EUS-B-FNA) in patients with lung cancer, focusing on tissue sample collection for molecular testing. The introduction of the Franseen needle in EUS-B-FNB, characterized by three beveled edges, allows for more tissue collection in cylinder form. MethodsWe retrospectively analyzed the data of 97 patients who underwent EUS-B-FNB or EUS-B-FNA at Hakodate Goryoukaku Hospital and evaluated diagnostic yields, safety, and nucleic acid concentrations using collected specimens. ResultsThe diagnostic yields of EUS-B-FNB and EUS-B-FNA were comparable (92.2% vs. 92.3%), with no significant differences in complications. However, EUS-B-FNB provided significantly higher DNA and RNA concentrations (DNA; 41.05 vs. 10.20 ng/mL; P < 0.0001, RNA; 36.80 vs. 11.80 ng/mL; P = 0.0009), essential for comprehensive molecular testing. ConclusionThis study highlights the potential of EUS-B-FNB for enhancing the molecular diagnosis of lung cancer by ensuring adequate tissue sample collection for multiplex testing, paving the way for personalized medicine. This technique is comparable in safety and efficacy to traditional methods while offering a substantial improvement in the quality of molecular diagnostics.

Investigating the prevalence of MFN2 mutations in amyotrophic lateral sclerosis: insights from an Italian cohort.

The MFN2 gene encodes mitofusin 2, a key protein for mitochondrial fusion, transport, maintenance and cell communication. MFN2 mutations are primarily linked to Charcot-Marie-Tooth disease type 2A. However, a few cases of amyotrophic lateral sclerosis and amyotrophic lateral sclerosis/frontotemporal dementia phenotypes with concomitant MFN2 mutations have been previously reported. This study examines the clinical and genetic characteristics of an Italian cohort of amyotrophic lateral sclerosis patients with rare, non-synonymous MFN2 mutations. A group of patients (n = 385) diagnosed with amyotrophic lateral sclerosis at our Neurology Units between 2008 and 2023 underwent comprehensive molecular testing, including MFN2. After excluding pathogenic mutations in the main amyotrophic lateral sclerosis-related genes (i.e. C9orf72, SOD1, FUS and TARDBP), MFN2 variants were classified based on the American College of Medical Genetics and Genomics guidelines, and demographic and clinical data of MFN2-mutated patients were retrieved. We identified 12 rare, heterozygous, non-synonymous MFN2 variants in 19 individuals (4.9%). Eight of these variants, carried by nine patients (2.3%), were either pathogenic, likely pathogenic or variants of unknown significance according to the American College of Medical Genetics and Genomics guidelines. Among these patients, four exhibited a familial pattern of inheritance. The observed phenotypes included classic and bulbar amyotrophic lateral sclerosis, amyotrophic lateral sclerosis/frontotemporal dementia, flail arm, flail leg and progressive muscular atrophy. Median survival after disease onset was extremely variable, ranging from less than 1 to 13 years. This study investigates the prevalence of rare, non-synonymous MFN2 variants within an Italian cohort of amyotrophic lateral sclerosis patients, who have been extensively investigated, enhancing our knowledge of the underlying phenotypic spectrum. Further research is needed to understand whether MFN2 mutations contribute to motor neuron disease and to what extent. Improving our knowledge regarding the genetic basis of amyotrophic lateral sclerosis is crucial both in a diagnostic and therapeutic perspective.

Uterine tumors mimicking ovarian sex cord tumors with rhabdoid differentiation: a clinicopathologic study of 4 cases: A case series analysis.

Uterine tumors resembling ovarian sex cord tumors (UTROSCT) with rhabdoid features are uncommon mesenchymal neoplasms exhibiting diverse histological patterns, including significant rhabdoid morphology. A thorough comprehension of their clinicopathologic features is crucial for precise diagnosis and effective management. This study presents 4 cases of UTROSCT with rhabdoid features, diagnosed in patients aged 31 to 58. Varied recurrence patterns were observed, including similar recurrent lesions to the primary tumors with subsequent mortality, initial invasion and lymph node metastasis, and presence of only primary tumor. Histopathological examination revealed diverse morphological patterns, prominently featuring rhabdoid differentiation. Immunohistochemical analysis showed expression of hormone receptors, sex cord, smooth muscle, and epithelial markers, notably WT1, CD56, and CD99. Molecular analysis identified ESR1-NCOA2 fusions and ESR1 and NCOA2/3 rearrangements, indicating a potential association between these genetic alterations and extensive rhabdoid differentiation. Various treatments were administered post-recurrence, including chemotherapy and targeted therapies. However, poor clinical outcomes were observed in all cases. Despite aggressive treatments, including chemotherapy and targeted therapies, poor clinical outcomes were observed, highlighting the aggressive nature of UTROSCT with significant rhabdoid differentiation. This case series emphasizes the importance of detailed pathological reporting, comprehensive molecular testing, and thorough tumor staging in UTROSCT cases with rhabdoid features. Enhanced understanding of the clinicopathologic characteristics of UTROSCT with rhabdoid differentiation is crucial for accurate diagnosis, prognostication, and management strategies.

Molecular Diagnostic Yield and Safety Profile of Ultrasound-Guided Lung Biopsies: A Cross-Sectional Study.

The recent advances in precision oncology for lung cancer treatment has focused attention on the importance of obtaining appropriate specimens for tissue diagnosis as well as comprehensive molecular profiling. CT scan-guided biopsies and bronchoscopy are currently the main procedures employed for tissue sampling. However, growing evidence suggests that ultrasound-guided biopsies may represent an effective as well as safe approach in this diagnostic area. This study explores the safety and the diagnostic yield for cancer molecular profiling in ultrasound-guided percutaneous lung lesion biopsies (US-PLLB). One hundred consecutive patients with suspected lung cancer, between January 2021 and May 2024, who had ultrasound-guided lung biopsies have been retrospectively analyzed. Molecular profiling was conducted with next-generation sequencing Genexus using Oncomine precision assay or polymerase chain reaction according to specimen quality. Qualitative immunohistochemical assay of programmed death ligand 1 (PD-L1) expression was evaluated by the Dako PD-L1 immunohistochemistry 22C3 pharmDx assay. The co-primary endpoints were the molecular diagnostic yield and the safety profile of US-guided lung biopsies. From January 2021 to May 2024, 100 US-guided lung biopsies were carried out and 95 were considered for inclusion in the study. US-PLLB provided informative tissue for a histological evaluation in 93 of 95 patients with an overall diagnostic accuracy of 96.84% [Sensitivity: 92.63%; Specificity: 96.84%; PPV: 100%; NPV: 100%]. Sixty-Six patients were diagnosed with NSCLC (69.47%) and were considered for molecular diagnostic yield evaluation and PD-L1 testing. Four patients had malignant lymphoid lesions. US-PLLB was not adequate to achieve a final diagnosis in three patients (3.16%). Complete molecular profiling and PD-L1 evaluation were achieved in all patients with adenocarcinoma (molecular diagnostic yield: 100%). PD-L1 evaluation was achieved in 28 of 29 patients (96.55%) with either SCC or NOS lung cancer. The overall complication rate was 9.47% (n = 9). Six patients (6.31%) developed pneumothorax, while three patients (3.16%) suffered mild haemoptysis without desaturation. According to our findings, US-guided lung biopsy is a safe, minimally invasive procedure in patients with suspected lung malignancies, providing an excellent diagnostic yield for both comprehensive molecular profiling and PD-L1 testing. In addition, our results suggest that US-guided biopsy may also be an effective diagnostic approach in patients with suspected lung lymphoma.

Electronic Medical Record-Based Nudge Intervention to Increase Comprehensive Molecular Genotyping in Patients With Metastatic Non-Small Cell Lung Cancer: Results From a Prospective Clinical Trial.

Less than half of the patients with newly diagnosed metastatic non-small cell lung cancer (NSCLC) undergo comprehensive molecular testing. We designed an electronic medical record (EMR)-based "nudge intervention" to prompt plasma-based molecular testing at the time of initial medical oncology consultation. A nonrandomized prospective trial was conducted at the University of Pennsylvania's academic practice and two affiliated community practices. Molecular genotyping was performed by tissue- and/or plasma-based next generation sequencing methods. Comprehensive testing was defined as testing for EGFR, ALK, BRAF, ROS1, MET, RET, KRAS, and NTRK. Guideline-concordant treatment was defined as the use of the appropriate first-line (1L) therapy as per the National Comprehensive Cancer Network (NCCN) guidelines. Proportion of patients with comprehensive molecular genotyping results available at any time, molecular results available before 1L therapy, and guideline-concordant 1L treatment were compared between the preintervention and postintervention cohorts using Fisher's exact test or Pearson's chi-squared test. Five hundred and thirty-three patients were included, 376 in the preintervention cohort and 157 in the postintervention cohort. After implementation of the EMR-based nudge, a higher proportion of patients underwent comprehensive molecular testing in the postintervention versus the preintervention cohort (100% v 88%, P = <.001), had results of comprehensive molecular testing available before initiating 1L treatment (97.3% v 91.6%, P = .026), and received NCCN guideline-concordant care (89.8% v 78.2%, P = .035). Across three practice sites in a large health system, implementation of a provider team-focused EMR-based nudge intervention was feasible, and led to a higher number of patients with NSCLC undergoing comprehensive molecular genotyping. These findings demonstrate that behavioral nudges can promote molecular testing and should be studied further as a tool to improve guideline-concordant care in both community and academic sites.

The 15-gene expression profile test is independent from PRAME and 7-gene next-generation sequencing: Results from 3,267 clinically tested uveal melanomas.

9598 Background: The 15-gene expression profile (15-GEP) test is the gold standard in the US for metastatic risk prediction in uveal melanoma (UM). Studies have shown that the 15-GEP is superior to and independent of other clinicopathologic and molecular risk factors, including PRAME expression status and BAP1 mutation status. Supplemental tests for PRAME and the 7-gene NGS panel ( BAP1, SF3B1, EIF1AX, GNA11, GNAQ, CYSLTR2, PLCB4)are validated and can now be run clinically from the same biopsy specimen. However, the discordance between 15-GEP, PRAME and NGS in clinical specimens is unknown. This study aimed to determine whether PRAME status or DNA mutation status could be used to predict the 15-GEP result. Methods: Data from fine-needle aspiration biopsy or formalin-fixed paraffin-embedded UM specimens submitted for routine comprehensive clinical testing with all three commercial tests (15-GEP, PRAME, and NGS) between February 2018 and December 2023 were collected through an Institutional Review Board-approved protocol. Results: Overall, 3267 tumors with comprehensive molecular testing were analyzed. 15-GEP testing yielded 65.9% (n=2152/3267) Class 1 and 34.1% (n=1115) Class 2 tumors. While Class 2 tumors were more than twice as likely to be PRAME(+) vs Class 1, 54.3% (606/1115) of Class 2 cases were PRAME(–), and 20.9% (450/2152) of Class 1 cases were PRAME(+) resulting in a PRAME/15-GEP discordance rate of 32.3% (10-56/3267). Mutations in genes associated with increasing metastatic risk ( EIF1AX, SF3B1, and BAP1, respectively) were identified in 58.7% (1918/3267) of cases. Most EIF1AX mutations (569/623, 91.3%) occurred in Class 1 tumors that were PRAME(–), while most SF3B1mutations were found in Class 1, PRAME(+) tumors (244/404, 60.4%). Most BAP1 mutations (863/976, 88.4%) occurred in Class 2 (high risk) tumors, of which 53.9% (465/863) were PRAME(–). Importantly, 56.0% (1205/2152) of Class 1 tumors did not harbor detectable EIF1AX or SF3B1 mutations, and 22.6% (252/1115) of Class 2 tumors did not harbor a detectable BAP1 mutation, yielding an NGS/15-GEP discordance rate of 44.6% (1457/3267). Conclusions: The validated PRAME and NGS tests described here, which can be performed from the same initial biopsy sample taken for 15-GEP testing, provide results that are not systematically associated with 15-GEP class. Given that the 15-GEP has been shown to be superior to both PRAME and NGS in predicting metastatic outcomes, it is recommended that these supplemental tests only be considered in the context of a 15-GEP test result to prevent potentially over or under identifying high-risk biology. An ongoing prospective study with long term outcomes for over 1700 UM patients led by the Collaborative Ocular Oncology Group (COOG2) will provide insight into how best to effectively integrate these supplementary biomarkers with the 15-GEP class result.

INUDGE: Integration of liquid biopsy based next generation gene sequencing in newly diagnosed NSCLC—A stepped wedge cluster randomized clinical trial.

TPS8664 Background: The development of targeted therapies has changed the treatment paradigm for non–small cell lung cancer (NSCLC). With the growing number of FDA approved targeted therapies, current NCCN guidelines recommend comprehensive molecular genotyping prior to first line (1L) therapy for all newly diagnosed patients with metastatic non-squamous (mNSq) NSCLC to enable the delivery of personalized therapy. Based on prior work that found improved detection of clinically actionable alterations through concurrent plasma (P) based next-generation gene sequencing (NGS) and tissue NGS, we piloted a behavioral economics (BE) informed electronic health record (EHR)-based “nudge” intervention designed to facilitate P NGS at the new patient medical oncology visit. After implementing across 3 practices within the University of Pennsylvania Health System (UPHS), our findings demonstrated that EHR-based nudges are feasible and can promote guideline concordant diagnostic testing at both community and academic sites (1). Methods: Design: NCT05853887 is a prospective stepped wedge cluster randomized trial that includes 3 “clusters”, representing 6 community-based cancer centers. The primary objective of this trial is to test the effectiveness of a BE informed EHR nudge intervention to increase availability of comprehensive molecular test results before 1L therapy by incorporating concurrent tissue and plasma-based molecular testing. Secondary objectives include evaluation of contextual mechanisms contributing to the adoption, reach, and effectiveness of EHR nudge interventions with a lens for health equity. Intervention: An EHR-based nudge that allows for default order placement of P NGS testing at the time of the first medical oncology visit will be implemented. Subsequently, results detected on the default P NGS order will be conveyed to providers in the form of an electronic clinical decision support notification. Statistical Methods:Sample size is based on estimates of the proportion of patients undergoing comprehensive molecular testing prior to 1L. A sample of 3 clusters in a complete stepped-wedge cluster-randomized design with 4 time periods (including the baseline), 3 steps, 1 cluster(s) switching from control to treatment at each step, and an average of 120 subjects per cluster with an average of 30 subjects per cluster per time period (for a total sample size of 360 subjects) will provide 80% power to detect a difference between proportions of 0.21701. Patients with histological, or cytological diagnosis of Stage IV mNSq NSCLC who have not yet received systemic treatment for metastatic disease are eligible for inclusion. Enrollment is ongoing: Cluster 1 began enrollment in June 2023, Cluster 2 in October 2023, and Cluster 3 is planned for February 2024. 1. Aggarwal C et al, JCO, Vol 40, Num 16, Suppl Jun 2022. Clinical trial information: NCT05853887 .

Real-world performance of the digital drug-assignment system for precision oncology in lung cancer.

e13590 Background: Precision medicine has profoundly transformed lung cancer treatment, an array of molecularly targeted agents (MTAs) are approved and used in clinical practice. Comprehensive molecular testing is key in treatment decisions, nevertheless, interpretation of complex molecular profiles can be challenging and subjective. Previously we demonstrated that digital drug assignment (DDA), an algorithmic computational reasoning model that ranks associated targeted therapies based on the totality of individual tumor genomic data, rather than matching one drug to one biomarker, was predictive of relative benefit of the agents as used in the SHIVA01 trial (1). Here, we collected real-world clinical outcome data from lung cancer patients who received decision support where DDA was integrated to aid a molecular tumor board (MTB) and investigated the effectiveness of administered therapies. Methods: Between 2018 and 2022, 111 lung cancer patients were involved in our precision oncology program. Following molecular diagnostic tests, analysis of the individual molecular profiles and scoring of associated drugs (DDA score) was performed by the DDA system as previously described. The output was assessed by the MTB that provided a strategy to the clinicians who made the final therapy decisions. Treatment courses and outcomes were collected from hospital health records and analyzed. Treatment lines were categorized as the following: high-score MTA (1000≤DDA score), low-score MTA (DDA score&lt;1000), and standard of care (SOC). Results: Overall response rate (ORR) of high-score MTA lines administered following DDA-based decision support (n=13) was 69%, whereas of other treatment lines (low-score MTAs and SOC; n=55) it was 16% (p&lt;0.001); and 26% in low-score MTA lines (n=34; p=0.017). Median progression-free survival (mPFS) of high-score MTA lines administered following DDA-based decision support (n=13) was 63 months (95% CI: 12.0 – 66.0), whereas it was 9 months both for any other treatment lines (low-score MTAs and SOC; n=44; 95% CI: 5.0 – 66.0; p&lt;0.001) and for low-score MTA lines only (n=27; 95% CI: 5.0 – 66.0; p&lt;0.001). Median overall survival (mOS) of patients who received at least one line of high-score MTA after decision support (n=13) was significantly longer than of those who only received non-high score treatments after decision support (patients only treated with low-score MTAs and/or SOC, n=34): not reached vs. 32 months (95% CI 24.0 – not reached), respectively (p=0.012). Conclusions: This study revealed that the DDA system is predictive of relative benefit of the various agents as used in lung cancer care, in line with our previous clinical validation. Validated algorithmic AI-guided drug assignment methods represent a new class of tools that can potentially address challenges in utilizing the results of complex molecular profiles in routine clinical setting. 1. Petak et al., 2021, npj Precis. Onc. 5, 59.

Primary Results of Patients with Genitourinary Malignancies Presented at a Molecular Tumor Board

Introduction: Personalized medicine poses great opportunities and challenges. While the therapeutic landscape markedly expands, descriptions about status, clinical implementation and real-world benefits of precision oncology and molecular tumor boards (MTB) remain sparse, particularly in the field of genitourinary (GU) cancer. Hence, this study characterized urological MTB cases to better understand the potential role of MTB in uro-oncology. Methods: We analyzed patients with complete data sets being reviewed at an MTB from January 2019 to October 2022, focusing on results of molecular analysis and treatment recommendations. Results: We evaluated 102 patients with GU cancer with a mean patient age of 61.7 years. Prostate cancer (PCa) was the most frequent entity with 52.9% (54/102), followed by bladder cancer (18.6%, 19/102) and renal cell carcinoma (14.7%, 15/102). On average, case presentation at MTB took place 54.9 months after initial diagnosis and after 2.7 previous lines of therapy. During the study period, 49.0% (50/102) of patients deceased. Additional MTB-based treatment recommendations were achieved in a majority of 68.6% (70/102) of patients, with a recommendation for targeted therapy in 64.3% (45/70) of these patients. Only 6.7% (3/45) of patients – due to different reasons – received the recommended MTB-based therapy though, with 33% (1/3) of patients reaching disease control. Throughout the MTB study period, GU cancer case presentations and treatment recommendations increased, while the time interval between initial presentation and final therapy recommendation were decreasing over time. Conclusion: Presentation of uro-oncological patients at the MTB is a highly valuable measure for clinical decision-making. Prospectively, earlier presentation of patients at the MTB and changing legislative issues regarding comprehensive molecular testing and targeted treatment approval might further improve patients’ benefits from comprehensive molecular diagnostics.

Microbial aetiology of community-acquired pneumonia in hospitalised adults: A prospective study utilising comprehensive molecular testing

ObjectivesThis study aimed to describe the microbial aetiology of community-acquired pneumonia (CAP) in adults admitted to a tertiary care hospital and assess the impact of syndromic polymerase chain reaction (PCR) panels on pathogen detection. MethodsConducted at Haukeland University Hospital, Norway, from September 2020 to April 2023, this prospective study enrolled adults with suspected CAP. We analysed lower respiratory tract samples using both standard-of-care tests and the BIOFIRE® FILMARRAY® Pneumonia Plus Panel (FAP plus). The added value of FAP Plus in enhancing the detection of clinically relevant pathogens, alongside standard-of-care diagnostics, was assessed. ResultsOf the 3238 patients screened, 640 met the inclusion criteria, with 384 confirmed to have CAP at discharge. In these patients, pathogens with proven or probable clinical significance were identified in 312 (81.3%) patients. Haemophilus influenzae was the most prevalent pathogen, found in 118 patients (30.7%), followed by SARS-CoV-2 in 74 (19.3%), and Streptococcus pneumoniae in 64 (16.7%). Respiratory viruses were detected in 186 (48.4%) patients. The use of FAP plus improved the pathogen detection rate from 62.8% with standard-of-care methods to 81.3%. ConclusionsPathogens were identified in 81% of CAP patients, with Haemophilus influenzae and respiratory viruses being the most frequently detected pathogens. The addition of the FAP plus panel, markedly improved pathogen detection rates compared to standard-of-care diagnostics alone.

Abstract 939: Comprehensive genomic and transcriptomic analysis to guide therapy for patients with metastatic solid tumors

Abstract Introduction: Precision oncology, which aims to profile tumors for identifying actionable alterations to guide therapy has become mainstream in cancer clinics. While most approaches focus on DNA aberrancies on targeted panels, not all patients’ tumors show targetable genomic alterations in these limited gene sets, indicating a need for more comprehensive methodologies combining whole exome (WES) and transcriptome sequencing (RNAseq) to direct a larger fraction of patients to therapies. Here, we aimed to compare the feasibility and utility of BostonGene’s (BG) analytical platform with WES and RNAseq to two targeted exome panels in identifying actionable alterations and therapy selection for patients with metastatic solid tumors. Methods: Study enrolled patients with solid tumors who have had next-generation-sequencing (NGS) testing with one of two tissue-based targeted exome sequencing panels, Oncomine (ThermoFisher, 148 genes), or MDA-MAPP (615 genes) with identification of no College of American Pathologists Tier-1 actionable DNA alterations between August 2022 and November 2023. Patients’ tissue and blood/saliva samples were subjected to BG’s NGS platform. An alteration-level actionability analysis was done upon receipt of WES results to inform treatment decision making, based on functional and therapeutic effects of the alterations detected. Results: A total of 64 patients were enrolled; testing was cancelled for 7 patients (10.9%) with samples showing &amp;lt;10% tumor content, while 5 patients’ reports were pending at the time of data analysis. Median turn-around time from consent to sample acquisition (SA) and from SA to available genomic profile were 8 and 15.5 days, respectively. 52 patients had NGS results representing a diverse group of tumor types, including sarcoma (n=11, 21.2%), head and neck (n=9, 17.3%) and mesothelioma (n=5, 9.6%). BG reported DNA alterations (mean: 3.75; median: 4) not shown by prior testing for 94.2% (49/52) of patients, whereby the most common alteration types included SNVs (43.1%), deletions (23.6%) and amplifications (11.8%). BG identified additional actionable DNA alterations (referred to as “AAs”; mean: 1.9, median: 2) in 32 (61.5%) patients, whereby majority of AAs were deletions (59.7%) and amplifications (27.4%). BG detected AAs in 58.8% (10/17) vs 62.9% (22/35) of patients for whom the same vs different samples were used across different tests compared (P=0.8). Of 32 patients with AAs, 7 (21.9%) were directed to DNA-informed treatments within 6 months based upon the new genomic findings reported by BG, 14 (43.8%) are being followed for treatment matching, while 11 (34.4%) were deceased. Discussion: Our results show the feasibility and utility of comprehensive molecular testing to match patients to WES-informed treatments within a clinically relevant time frame. Assessment of the additional benefit of RNA expression analysis is forthcoming. Citation Format: Burak Uzunparmak, Fei Su, Amber M. Johnson, Krystle Nomie, Nathan Fowler, Lile Kontselidze, Aleksander Bagaev, Anna Butusova, Ecaterina E. Dumbrava, Jordi Rodon-Ahnert, Siqing Fu, David S. Hong, Timothy A. Yap, Aung Naing, Sarina A. Piha-Paul, Daniel D. Karp, Paula R. Pohlmann, Apostolia M. Tsimberidou, Keyur P. Patel, Funda Meric-Bernstam. Comprehensive genomic and transcriptomic analysis to guide therapy for patients with metastatic solid tumors [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 939.

Abstract C074: Clinico-genomic characterization of N=2,460 pancreatic adenocarcinoma identifies KRASMUT dosage as prognostic of overall survival across disease stages

Abstract Background: The majority (~80%) of patients (pts) with pancreatic adenocarcinomas (PDAC) present with advanced disease. Most sequencing efforts to date have focused on early stage disease. Herein, we leverage a large clinical sequencing cohort of N= 2460 pts with PDAC to characterize the clinico-genomic landscape across different disease stages. Methods: Tumors from 2460 pts were sequenced using MSK-IMPACT clinical sequencing assay and mutations called via CLIA-approved pipelines. Detailed clinical descriptors including lines of treatment and response were abstracted for N=1451 (59%) pts. Intratumor heterogeneity estimates including allele-specific copy number were inferred using FACETS. Results: Of N=2460, at diagnosis 34% of pts had resectable tumors, and 24% and 42% had locally advanced and metastatic disease, respectively. 94% of pts had KRASMUT tumors. Expectedly, MAPK pathway alterations were significantly enriched in KRASWT tumors (60% vs. 7% in KRASMUT, p &amp;lt; 0.001) with gain-of-function kinase oncogene fusions exclusively observed in these tumors (p&amp;lt;0.001). In addition, GNAS, PTEN, FOXP1, SMARCB1 and CREBBP were enriched in the KRASWT tumors (q &amp;lt; 0.05). Collectively, pts with KRASWT/MAPK MUT and KRASWT/MAPK WT tumors comprise 4% and 2% of all pts PDAC, respectively. Germline pathogenic variants in high/moderate penetrance genes were more common in pts with KRASWT tumors compared to pts with KRASMUT tumors (20% vs. 10%, p &amp;lt; 0.001). Pathogenic germline alterations in BRCA1/2 and PALB2 were observed in 6% (N=144) of cohort. Biallelic inactivation of BRCA2 specifically was associated with prolonged response of metastatic pts to PARP-inhibitor maintenance therapy with 10.3 months on treatment prior to progression compared to 3.4 months for those with biallelic inactivation of other HRD genes and 2.2 months for those with non-biallelic inactivation. Among KRASMUT tumors assessed for KRAS copy-number allelic imbalance (AI) (N=1097), 41% (N=452) had AI at the KRAS locus with mutant-allele selected for in 94% of tumors with imbalance. Among KRASMUT tumors with normal genome ploidy, overall, 20% had a mutant allele gain ranging from 10% in resectable to 29% of metastatic tumors. We observe significantly lower OS among pts with KRASMUT dosage gain compared to heterozygous KRASMUT across pts with resectable (HR: 0.66, 95% CI: 0.18-1.15, p&amp;lt;0.01) and metastatic (HR: 0.39, 95% CI: 0.16-0.63, p=0.001) diseases with statistical significance not reached among the locally advanced disease (HR: 0.4, 95% CI: -0.01-0.81, p=0.056). Notably, the OS of pts with loss of the wild-type allele and no mutant copy gain was indistinguishable from that of heterozygous KRASMUT tumors (HR: -0.11, 95% CI: -0.37-0.15, p=0.4). Conclusion: In this large cohort of 2460 pts with PDAC, we observed that KRASMUT dosage gain is a significant negative predictor of OS. These findings highlight the importance of incorporating allelic-dosage into comprehensive molecular testing in PDAC given prognostic importance and the emerging era of direct RAS targeting. Citation Format: Anna M. Varghese, Maria A. Perry, Daniel Muldoon, Subhiksha Nandakumar, Amanda Erakky, Bastien Nguyen, Amanda Zucker, Hulya Sahin Ozkan, Olca Basturk, Debyani Chakravarty, David P. Kelsen, Wungki Park, Kenneth H. Yu, Alice C. Wei, Nikolaus Schultz, Michael F. Berger, Christine Iacobuzio-Donahue, Chaitanya Bandlamudi, Eileen M. O'Reilly. Clinico-genomic characterization of N=2,460 pancreatic adenocarcinoma identifies KRASMUT dosage as prognostic of overall survival across disease stages [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr C074.

Rapid molecular testing for the diagnosis of community-acquired pneumonia

Lower respiratory tract infections, including community-acquired pneumonia (CAP), are a leading cause of hospital admissions and mortality. Researchers in the NORCAP project have investigated whether comprehensive molecular testing can be used to improve the microbiological diagnosis of CAP and potentially improve clinical outcomes, as Professor Harleen Grewal explains.

Comprehensive molecular testing prior to first treatment among patient with metastatic non-small cell lung cancer.

431 Background: Nearly 60% of patients with non-small cell lung cancer will have actionable mutations identified via molecular testing. Of these patients, those treated with targeted therapy had longer overall survival than those who did not. Therefore, it is imperative molecular testing is both sent and resulted at time of first treatment. Despite this, multiple barriers remain with reported community rates of &lt;50% with comprehensive molecular testing. We sought to understand testing practices at our institution. Methods: In this retrospective chart review, patients with stage IV, non-small cell, non-squamous cell lung cancer treated at Orlando Regional Medical Center between 1/2021-5/31/2022 were identified. Primary endpoints included rate of comprehensive molecular testing (EGFR, ALK, ROS1, RET, MET, BRAF, KRAS, ErbB2 and NTRK, either tissue or plasma) available prior to first line treatment. Results: Of the 73 patients who were included, 69 patients (94.8%) had comprehensive molecular testing results available prior to first line treatment. 48 (65.8%) patients had tissue testing results prior to first treatment. 44 (60.2%) patients had plasma testing results prior to first treatment. 14 (19.2%) patients had tissue testing sent, without results at time of first treatment. Conclusions: The comprehensive molecular testing rate at a large tertiary hospital is 94.8%, substantially higher than the rates reported in the literature. Despite this, 19.2% of patients had tissue testing sent but not resulted at time of first treatment. Future studies are needed to improve turnaround time of tissue based molecular testing results and evaluate concurrent versus sequential tissue and plasma testing.

Racial and geographic disparities in the use of molecular testing among older Medicare beneficiaries with lung cancer.

125 Background: The National Comprehensive Cancer Network (NCCN) guidelines recommend comprehensive molecular testing for newly diagnosed patients with metastatic non-small cell lung cancer (mNSCLC). However, the uptake of the next-generation sequencing (NGS) tests for NSCLC was low due to lack of insurance coverage. In 2018, the Centers for Medicare &amp; Medicaid Services issued a national coverage determination to cover diagnostic laboratory tests using NGS for Medicare beneficiaries with advanced cancer. It is important to understand the current uptake of the NGS tests among older Medicare beneficiaries with lung cancer and examine the potential racial and geographic disparities in the use of NGS tests. Methods: From the random 20% sample of all Medicare beneficiaries, we selected Medicare beneficiaries who were 66 years and older and newly diagnosed with lung cancer in 2020. We excluded beneficiaries who were ever diagnosed with breast, colorectal, endometrial, lung, or prostate cancer before 2020 and beneficiaries who died in 2020. We further restricted our sample to those with a full year of Medicare Part A and B coverage and no month of Medicare Advantage coverage. We identified molecular testing for our lung cancer cohort using Medicare carrier and outpatient claims. We used two linear probability models to examine the relationship between race and the receipt of any molecular tests among all lung cancer patients (N=9,229), and the relationship between race and the receipt of panel tests among those received any molecular tests (N=1,842). We adjusted for age at the beginning of 2020, sex, Medicaid enrollment (low-income), geographic region, and common pre-existing chronic conditions (Alzheimer’s and related dementia, heart failure, chronic kidney disease, chronic obstructive pulmonary disease, diabetes, depression, ischemic heart disease, and arthritis). Results: Among 9,229 older lung cancer patients newly diagnosed in 2020, 1,842 (19.96%) of them received molecular tests. Among those 1,842 who received molecular tests, 709 (38.49%) of them received NGS tests, and the rest received single-gene tests only. In our regression analyses, we found Asian patients were 12.5-percentage point more likely to receive any molecular tests compared to White patients. Among those received any tests, Hispanic patients were 16.0-percentage point less likely to receive NGS tests over single-gene tests compared to White patients. We also found those residing in the South and the West were less likely to receive NGS tests over single-gene tests than those residing in the Northeast. Conclusions: Given the guideline recommendation and proportion of new mNSCLC cases among all new lung cancers, there was likely underuse of NGS tests. There were also racial and geographic disparities in the use of NGS tests.

Diagnostic algorithm for challenging blue cell sinonasal carcinoma

The newly emerging sinonasal carcinomas have demonstrated diverse morphologies and specific molecular rearrangements along with deviant clinical behavior from conventional counterparts. We aim to propose a diagnostic algorithm that is based on molecular findings of each sinonasal cancer and is considering the new entities has been called upon. Such a diagnostic algorithm should help diagnostic pathologists establish a diagnosis of a challenging sinonasal blue cell carcinomas and researchers performing retrospective analysis of archival cases. Along with consulting our archival cases, literature mining was conducted to retrieve the immunohistochemical and molecular findings regarding the newly emerging entities. Our proposed algorithm distinguishes poorly differentiated (non) keratinizing SNSCC, from anaplastic myoepithelial carcinoma, NUT midline carcinoma, SMARCB1/SMARCA4-deficient teratocarcinosarcoma, SMARCB1/SMARCA4-deficient carcinosarcoma, olfactory neuroblastoma, sinonasal undifferentiated carcinoma, HPV-related multiphenotypic sinonasal carcinoma and other adenocarcinomas. By incorporating morphologic features, immunohistochemical markers, and molecular investigations, the algorithm enhances the accuracy of diagnosis, particularly in cases where comprehensive molecular testing is not readily available. This algorithm serves as a valuable resource for pathologists, facilitating the proper diagnosis of sinonasal malignancies and guiding appropriate patient management.

Novel genomic alterations and transcriptomic-based tumor microenvironment classification of sarcoma and their impact on treatment decision making.

11547 Background: Sarcomas are rare tumors of mesenchymal origin, with over 50 different histological sarcoma subtypes. The heterogeneous molecular and immunologic characteristics of sarcomas present many challenges for diagnostics and treatment plans. We aim to provide a more comprehensive molecular testing and immune profiling of sarcoma patients. Methods: The cohort consisted of samples from 200 patients diagnosed with sarcomas. WES and whole transcriptome analysis were performed on all samples. Tumor Portrait test identified genomic alterations of sarcomas and classified tumor microenvironment (TME) subtypes. The actual response to immune checkpoint inhibitor (ICI) treatment was determined using RECIST criteria and compared to response predictions by the Tumor Portrait test. Results: Across sarcoma subtypes, the most common mutations were in tumor suppressors including TP53, RB1, CDKN2A, and TSC1/2. We report fusions in 20.5% of cases, with commonly described diagnostic fusions accounting for 34/41 cases. In one case, the detection of the ZC3H7B-BCOR fusion suggested a change in diagnosis from uterine leiomyosarcoma to endometrial stromal sarcoma. Several previously unreported fusions were detected, including potentially targetable ( ARID1A-NUDC, MICAL3-MAPK) and prognostic ( YWHE-CIC) fusions. DNA damage response related genes were mutated in 12% of cases. Other less commonly detected targetable alterations were found in the following genes: MDM2, CDK4, SMARCB1, NF1, PIK3CA, NTRK1-3, FGFR1-4. Transcriptomic-based TME classification found that 52% of patients had an Immune-Enriched (IE) TME, with 26.5% and 25.5% grouped in the IE-Fibrotic and IE-non-Fibrotic subtype, respectively, which we previously showed is associated with good response to ICI treatment. In contrast, 48% of the patients presented with a Fibrotic (F, 30%) or Desert (D, 18%) subtype, are predicted to have a poor response to ICI treatment. Our classification of sarcomas based on TME subtypes conforms well with predicted responses of tumors to ICI therapy found in previous reports. Soft-tissue angiosarcomas, undifferentiated pleomorphic sarcoma, and myxofibrosarcomas presented TMEs of the IE subtype in 75% of cases, significantly more compared to 34% of the bone neoplasm cases, which are known for their favorable and unfavorable response to ICI therapy, respectively (Chi-squared test, p = 0.001). In retrospective chart review, we had response data on 24 patients who received ICI treatment. The disease control rate, defined as CR+PR+SD as best response, was significantly higher in the IE subtype compared to the F/D subtype (85.7% vs 20%, Chi-squared test, p &lt; 0.003). Conclusions: Together, our findings identify actionable and diagnostic alterations in diverse mesenchymal tumors and suggest that ICI treatment may be beneficial in immune-enriched sarcomas.

Molecular correlates of invasion pattern in HPV-associated endocervical adenocarcinoma: emergence of two distinct risk-stratified tiers.

The pattern-based (Silva) classification of invasive human papilloma virus (HPV)-associated endocervical adenocarcinomas (HPVA) is an established and reproducible method to predict outcomes for this otherwise stage-dependent group of tumours. Previous studies utilising targeted sequencing have shown a correlation between mutational profiles and an invasive pattern. However, such correlation has not been explored using comprehensive molecular testing. Clinicopathologic data including invasive pattern (Silva groups A, B, and C) was collected for a cohort of invasive HPVA, which previously underwent massive parallel sequencing using a panel covering 447 genes. Pathogenic alterations, molecular signatures, tumour mutational burden (TMB), and copy number alterations (CNA) were correlated with pattern of invasion. Forty five HPVA (11 pattern A, 17 pattern B, and 17 pattern C tumours) were included. Patients with pattern A presented at stage I with no involved lymph nodes or evidence of recurrence (in those with >2 months of follow-up). Patterns B and C patients also mostly presented at stage I with negative lymph nodes, but had a greater frequency of recurrence; 3/17 pattern B and 1/17 pattern C HPVAs harboured lymphovascular space invasion (LVI). An APOBEC mutational signature was detected only in Silva pattern C tumours (5/17), and pathogenic PIK3CA changes were detected only in destructively invasive HPVA (patterns B and C). When cases were grouped as low-risk (pattern A and pattern B without LVI) and high-risk (pattern B with LVI and pattern C), high-risk tumours were enriched in mutations in PIK3CA, ATRX, and ERBB2. There was a statistically significant difference in TMB between low-risk and high-risk pattern tumours (P= 0.006), as well as between Pattern C tumours with and without an APOBEC signature (P= 0.002). CNA burden increased from pattern A to C. Our findings further indicate that key molecular events in HPVA correlate with the morphologic invasive properties of the tumour and their aggressiveness. Pattern B tumours with LVI clustered with pattern C tumours, whereas pattern B tumours without LVI approached pattern A genotypically. Our study provides a biologic foundation for consolidating the Silva system into low-risk (pattern A + B without LVI) and high-risk (pattern B with LVI and pattern C) categories.

Digital droplet PCR TERT promoter mutational screening in fine needle aspiration cytology of thyroid lesions: A highly specific technique for pre-operative identification of high-risk cases.

Despite the advent of comprehensive molecular testing in surgical pathology, most centers still rely on the morphological assessment of fine-needle aspiration cytology (FNAC) to triage patients with thyroid nodules for surgery. Subsets of patients could benefit from the inclusion of molecular testing to increase the diagnostic and/or prognostic properties of the cytology analysis, including the assessment of TERT promoter mutations, an event coupled with thyroid malignancy, and poor prognosis. In this prospective study, preoperative FNAC material from 65 cases was assessed for TERT promoter hotspot mutations C228T and C250T using the digital droplet PCR (ddPCR) technique on frozen pellets and re-evaluated postoperatively. Our cohort consisted of 15 B-III (23%), 26 B-IV (40%), 1 B-V (2%), and 23 (35%) B-VI lesions according to the Bethesda System for Reporting Thyroid Cytopathology. TERT promoter mutations were detected in 7 cases; 4 papillary thyroid carcinomas (all with preoperative B-VI status), two follicular thyroid carcinomas (one B-IV and one B-V status), and one poorly differentiated thyroid carcinoma (with B-VI status). All mutated cases were verified by mutational analysis of tumor tissue derived from postoperative formalin-fixed paraffin-embedded tissue, while all cases identified as wild-type on FNAC remained wild-type postoperatively. Moreover, the occurrence of a TERT promoter mutation was significantly associated with malignant disease and higher Ki-67 proliferation indices. In the present cohort, we found that ddPCR is a highly specific method for detecting high-risk TERT promoter mutations on thyroid FNAC material that could guide different surgical approaches in subsets of indeterminate lesions if reproduced in larger materials.

Inhibition of matrix metalloproteinases by HIV-1 integrase strand transfer inhibitors.

More than fifteen million women with the human immunodeficiency virus type-1 (HIV-1) infection are of childbearing age world-wide. Due to improved and affordable access to antiretroviral therapy (ART), the number of in utero antiretroviral drug (ARV)-exposed children has exceeded a million and continues to grow. While most recommended ART taken during pregnancy suppresses mother to child viral transmission, the knowledge of drug safety linked to fetal neurodevelopment remains an area of active investigation. For example, few studies have suggested that ARV use can be associated with neural tube defects (NTDs) and most notably with the integrase strand transfer inhibitor (INSTI) dolutegravir (DTG). After risk benefit assessments, the World Health Organization (WHO) made recommendations for DTG usage as a first and second-line preferred treatment for infected populations including pregnant women and those of childbearing age. Nonetheless, long-term safety concerns remain for fetal health. This has led to a number of recent studies underscoring the need for biomarkers to elucidate potential mechanisms underlying long-term neurodevelopmental adverse events. With this goal in mind, we now report the inhibition of matrix metalloproteinases (MMPs) activities by INSTIs as an ARV class effect. Balanced MMPs activities play a crucial role in fetal neurodevelopment. Inhibition of MMPs activities by INSTIs during neurodevelopment could be a potential mechanism for adverse events. Thus, comprehensive molecular docking testing of the INSTIs, DTG, bictegravir (BIC), and cabotegravir (CAB), against twenty-three human MMPs showed broad-spectrum inhibition. With a metal chelating chemical property, each of the INSTI were shown to bind Zn++ at the MMP's catalytic domain leading to MMP inhibition but to variable binding energies. These results were validated in myeloid cell culture experiments demonstrating MMP-2 and 9 inhibitions by DTG, BIC and CAB and even at higher degree than doxycycline (DOX). Altogether, these data provide a potential mechanism for how INSTIs could affect fetal neurodevelopment.