NRG1 gene fusions are rare, therapeutically relevant, oncogenic drivers that occur across solid tumor types. To understand the landscape of NRG1 gene fusions, 4397 solid tumor formalin-fixed, paraffin-embedded samples consecutively tested by comprehensive genomic and immune profiling during standard care were analyzed. Nineteen NRG1 fusions were found in 17 unique patients, across multiple tumor types, including non-small-cell lung (n = 7), breast (n = 2), colorectal (n = 3), esophageal (n = 2), ovarian (n = 1), pancreatic (n = 1), and unknown primary (n = 1) carcinomas, with a cumulative incidence of 0.38%. Fusions were identified with breakpoints across four NRG1 introns spanning 1.4 megabases, with a mixture of known (n = 8) and previously unreported (n = 11) fusion partners. Co-occurring driver alterations in tumors with NRG1 fusions were uncommon, except colorectal carcinoma, where concurrent alterations in APC, BRAF, and ERBB2 were present in a subset of cases. The overall lack of co-occurring drivers highlights the importance of identifying NRG1 gene fusions, as these patients are unlikely to harbor other targetable alterations. In addition, RNA sequencing is important to identify NRG1 gene fusions given the variety of fusion partners and large genomic areas where breakpoints can occur.

BackgroundAlthough tumor genomic profiling has aided the advancement of targeted genetic therapy, its clinical integration remains a challenge in pediatric cancers due to lower mutation frequency and less available targeted drugs. There have been multiple novel studies examining molecular sequencing in pediatrics; however, many of these studies primarily utilized large-scale, genome-wide screening applications that limit applicable use due to the availability of testing. This study examined the institutional use of a targeted, clinically available approach to tumor genomic sequencing. MethodsA retrospective chart review was performed on pediatric patients with solid tumors who were managed at Roswell Park Comprehensive Cancer Center and underwent molecular testing of their tumor biopsy via OmniSeq from August 2016 to July 2021. Results were reviewed for mutations considered to be "actionable" by targeted therapy. Patients with actionable mutations were further examined to evaluate treatment course, receival of targeted therapy, and clinical outcomes. ResultsWe identified 64 pediatric patients consisting of 20 (31%) with CNS tumors and 44 (69%) with non-CNS tumors, ranging in age from 9 months to 21 years. Thirty-five total actionable mutations were identified amongst 27 patients (42%). Of these 27, 12 patients (44%) received at least 1 targeted drug against a respective actionable mutation, of which 6 patients (50%) achieved clinical benefit to therapy, including 1 complete response. ConclusionsThe use of a clinically focused and readily available targeted molecular sequencing panel identified actionable mutations at a comparable rate to the large-scale, less readily available sequencing panels utilized in other studies. Half of our patients who received targeted therapy achieved a complete response or clinical benefit from therapy. Although targeted therapy has a role in pediatric cancer treatment, many newer drugs require further research on their safety and efficacy.

Purpose of Study: Cancer testis antigens (CTA) are highly immunogenic genes with the ability to cause cancer-specific immune responses when expressed. Their tumor cell-specific expression makes them a key target of natural T cell response, cancer vaccines, immune checkpoint blockade (ICB), and cell-based immunotherapies in a wide range of tumor types. In this study, we assess the pan-cancer distribution and ICB survival association of CTA burden (CTAB) in real-world solid tumors. Procedure: Three tumor sample cohorts were studied: 1) a pan-cancer discovery cohort to develop a low- and high-CTAB cutoff (n=5450, 39 tumor types), 2) a TCGA cohort (n=19923, 32 tumor types) used to validate the classifier based on CTAB distribution and serve as a non-ICB-treated population, and 3) an ICB-treated retrospective cohort to validate the classification on overall survival (OS) (n=242, 3 tumor types). The expression levels of 17 CTA were measured using targeted RNA-Seq of FFPE tumor samples and then ranked against a pan-cancer reference population. CTAB was calculated for each sample, cohort and tumor type as the sum of the 17 CTA gene expression ranks. The discovery cohort median CTAB of 171 was used to classify all three cohorts into high- and low-CTAB groups. OS analysis was performed on the TCGA and ICB-treated cohorts using a CoxPH regression model to determine the Hazard Ratio (HR). Results: The three cohorts demonstrated overlapping single-peak, left-skewed CTAB distribution curves centered at CTAB values between 170 (discovery cohort) and 256 (retrospective cohort). When grouping by tumor types and ordering by median CTAB, the CTAB distributions for tumor types within all three cohorts were comparable. CoxPH regression analysis revealed an association between the CTAB threshold classifier and OS in both the ICB-treated retrospective and non-ICB TCGA cohorts. However, the direction of this association differed between the two cohorts, with high-CTAB samples having better survival (HR=0.936, p=0.076) in the ICB-treated retrospective cohort and worse survival (HR: 1.007, p=0.084) in the non-ICB-treated cohort. Conclusion: Our studies show that the CTAB distribution was maintained across the discovery and TCGA cohorts and a wide range of tumor types, supporting that the CTAB classifier is valid and histology agnostic. Additionally, when evaluating the ICB and non-ICB-treated cohorts, CTAB demonstrated the ability to predict OS, pointing to the utility of ICB in supporting CTA-specific natural immune response. However, further studies are necessary to verify these mechanisms of response to ICB as well as cancer vaccines and cell-based immunotherapies. Additional validation is needed to establish the predictive utility of CTAB alone and in combination with other immune oncology biomarkers for resistance or response. Citation Format: R.J. Seager, Erik Van Roey, Shuang Gao, Blake Burgher, Paul DePietro, Mary Nesline, Roger Klein, Shengle Zhang, Jeffrey M. Conroy, Sarabjot Pabla. Cancer testis antigen burden: Pan-cancer distribution and survival implications [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 5137.

2623 Background: Immune checkpoint inhibitors (ICIs) have emerged as effective treatments in non-small cell lung cancer (NSCLC). While the clinical utility of single agent ICI or in combination with chemotherapy has been well established, there remains an unmet need for the development of biomarkers that can better predict response. To address this need, we developed and applied a combination genomic and immune biomarker strategy to ICI-treated NSCLC patients which identified distinct patient subgroups with differential benefit among single agent or combination ICI treatment strategies. Methods: A discovery cohort (DC) of 5450 tumors across 37 histologies were evaluated by comprehensive genomic and immune profiling of the tumor immune microenvironment. Individual and combination biomarker assessment included PD-L1 IHC, TMB, tumor inflammation (TIGS), cell proliferation (CP) and cancer testis antigen burden (CTAB). From this cohort, combinations of molecular and immune biomarkers were identified and applied to a retrospective cohort (RC) of 225 metastatic NSCLC patients treated with pembrolizumab + chemo or pembrolizumab alone to correlate with response. Comparison of objective response rates (ORR) was performed using Chi-square test. Kaplan-Meir analysis was performed to test for differences in overall survival (OS) and 1-year OS. Results: Unsupervised analysis of the DC revealed four distinct biomarker combination groups that describe underlying tumor immunobiology: tumor dominant (CTAB, TMB, CP High), proliferative (CP High), inflamed (TIGS High), and checkpoint (PDL1, TIGS and TMB High). Application of these biomarker groups to the RC demonstrated significant differences in response to ICI regimens between groups (p = 0.04). Patients in the proliferative group (35.1%, 79/225; median PD-L1 = 20% TPS) treated with single agent pembrolizumab showed a significantly higher ORR (59%; 16/27) compared to pembrolizumab + chemo (27%; 14/52; p = 0.005), significantly improved 1-yr OS (p = 0.03), and trend towards better OS (p = 0.14). Importantly, patients in the inflamed group (16%, 36/225; median PD-L1 = 1% TPS), suggested that pembrolizumab + chemo (ORR 26.1%; 6/23) was not associated with ORR compared to pembrolizumab (ORR 31%; 4/13, p = 0.76), or OS (p = 0.37) and 1-yr OS (p = 0.57). Conclusions: Comprehensive genomic and immune profiling may identify PD-L1 low NSCLC patients who benefit from single agent pembrolizumab. PD-L1 low NSCLC patients with a proliferative phenotype may benefit from single agent pembrolizumab, whereas PD-L1 low cases with an inflamed phenotype may benefit from both single agent and combination pembrolizumab. Although further clinical validation of these predictive biomarker combinations is required, this data-driven approach demonstrates the potential to provide treatment decision support when selecting an ICI therapeutic strategy in lung cancer.

Background PD-L1 expression by immunohistochemistry (IHC) is associated with HNSCC immunotherapy response.1 The performance of different PD-L1 IHC clones has shown variability and poor concordance for immune vs. tumor cell scoring in HNSCC. Crucially, this leads to poor reproducibility in the combined positive score (CPS) method by the PD-L1 IHC 22C3 companion diagnostic.2 We explored the clinical sensitivity and concordance of CD274 (PD-L1) expression by RNA-sequencing compared to three PD-L1 IHC methods. Methods A retrospective cohort of HNSCC patients (n=258) with FFPE tissue was tested by comprehensive immune profiling3(2017–2022), including CD274 by RNA-seq (normalized percentile rank 0–100). IHC was performed with either the 28–8 or 22C3 PD-L1 clones. 28–8 was scored with% tumor cells stained (TC, n=34), while 22C3 was scored with either tumor proportion score (TPS, n=61) or combined positive score (CPS, n=163). For 22C3, CPS≥1 is low positive, and ≥20 is high positive. For 28–8 TC and 22C3 TPS, ≥1 is low positive, and ≥50 is high positive. ROC models for each IHC method were constructed for 5 sets of patients with different pairwise interpretation groups and used to determine RNA-seq cutoffs based on individual PD-L1 IHC scoring methods and accuracy at those cutoffs. Concordance between standard IHC scoring methods and CD274 by RNA-seq was also assessed. Results PD-L1 IHC results varied depending on the clone and scoring method used. Not surprisingly, CPS had the fewest negative cases (2.7%) and most high cases (47.2%). Tumor cell scoring by TPS (29.5% negative, 24.6% high) and TC (17.6% negative, 26.5% high) was similar for high vs. not high. For all three IHC approaches, PD-L1 RNA-seq classified IHC high v negative, high v low, and high v not high status with at least fair range of AUC (0.758–0.981), sensitivity (0.636–1.00), and specificity (0.785–1.00). RNA-seq could not discern between IHC low v negative status for any method. Pairwise comparisons showed significant concordance between median RNA-seq percentile ranks for IHC high v low and high v negative status for all IHC methods. By RNA-seq, the frequency of PD-L1 high cases for each scoring method increased from 47% to 55% (CPS), 24.6% to 42.6% (TPS) and 26.5% to 47.1% (TC) based on modeled RNA-seq cutoffs. Conclusions RNA-seq accurately discerns PD-L1 high vs. not high HNSCC tumors based on IHC scoring methods and may more reliably identify patients for frontline immunotherapy. References NCCN, 'NCCN Clinical Practice Guidelines in Oncology (NCCN Guidelines®) Head and Neck Cancers, Version 2.2022,' National Comprehensive Cancer Network, Fort Washington, Pennsylvania, Version 2.2022, Mar. J. Ribbat-Idel et al., Performance of Different Diagnostic PD-L1 Clones in Head and Neck Squamous Cell Carcinoma, Front. Med., vol. 8, no. April, pp. 1–8, 2021. J. M. Conroy et al. Analytical Validation of a Next-Generation Sequencing Assay to Monitor Immune Responses in Solid Tumors, J. Mol. Diagnostics, vol. 20, no. 1, pp. 95–109, Jan. 2018.

PURPOSE: The immunohistochemistry companion diagnostic test for pembrolizumab (IHC 22C3 pharmDx) lacks sensitivity, challenging immunotherapy selection for NSCLC patients with lower levels of expression. Unlike IHC 22C3, which restricts assessment of PD-L1 expression to viable tumor cells as a tumor proportion score (% TPS), mRNA next generation sequencing (RNA-seq) measures PD-L1 expression in the tumor microenvironment for both tumor and inflammatory background cells. RNA-seq previously demonstrated concordance with IHC and may be a robust alternative testing method for multiple tumor types. Here, we sought to optimize PD-L1 RNA-seq cutoff values in NSCLC to improve clinical sensitivity. PROCEDURE: NSCLC patients included in the study (n=3,465) were tested for PD-L1 expression by IHC 22C3 and clinically validated RNA-seq, measured as % rank (0-100) relative to a reference population based on normalized reads per million (nRPM). Patients were divided into an RNA-seq cut-off discovery cohort (n=3,168), and a test cohort pembrolizumab treated patients. Principal components analysis (PCA) was used to classify patients based on test results and explore cut-off values in the discovery cohort. Kaplan Meier curves and a Cox proportional hazards regression models assessed overall survival (OS) hazard ratios (HR) for RNA-seq versus standard of care IHC cut-offs in the test cohort. RESULTS: Unsupervised PCA clustering identified three distinct PD-L1 groups separated by combinations of significant over- and under-representation of RNA-seq and IHC result measures from prior testing. The groups were labeled as “low” (rank ≤40), “moderate” (rank 41-73), and “high” (rank ≥74), based on the median RNA-seq rank for each group (+/- 1SD for low and high). Both the low and moderate groups were overrepresented by patients in the PD-L1 IHC low and negative groups. The moderate group was overrepresented by patients with moderately high PD-L1 RNA-seq ranks (median=70), while the low group was overrepresented by patients that were not PD-L1 high by RNA-seq. The high group was overrepresented by patients high for PD-L1 by both IHC and RNA-seq. OS HRs were better for RNA-seq high versus moderate (HR=0.05, CI 0.00-0.63, p=.02), and RNA-seq high versus low (HR=0.16, CI 0.03-0.86, p=.03) groups compared to standard of care IHC 22C3 high versus low groups, (HR=0.21, CI 0.04-1.07, p=.06). Findings were non-significant for the RNA-seq moderate versus low groups, likely due to the limited and disproportionately high number of patients with poor performance status in these groups. CONCLUSIONS: PD-L1 expression by RNA-seq demonstrated improved clinical sensitivity in predicting OS versus standard of care PD-LI IHC in a pembrolizumab treated NSCLC patient cohort. Additional studies are needed to further define cut-offs in the context of performance status, and better understand immune escape mechanisms in the moderate group. Citation Format: Mary K. Nesline, Sarabjot Pabla, Yong Hee Lee, Paul DePietro, Amy Early, Roger Klein, Shengle Zhang, Jeffrey Conroy. PD-L1 expression by RNA-sequencing and survival from pembrolizumab in non-small cell lung cancer (NSCLC) [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 1259.

e21167 Background: CGIP analyzes FFPE tumor tissue by DNA/RNA sequencing for SNVs, indels, copy gain/loss, fusions, splice variants, MSI and TMB, along with PD-L1 IHC. A purported advantage of CGIP in NSCLC is the ability to identify targeted and immunotherapy biomarkers to inform clinical management. However, the extent to which CGIP supports treatment decisions and benefits NSCLC patients in various treatment settings is limited. Methods: A retrospective analysis of OmniSeq CGIP results (June 2017-March 2019) and real-world clinical data (through March 2020) for NSCLC patients (n = 300) was performed to evaluate treatment strategies at Roswell Park Comprehensive Cancer Center. Patient targeted and immunotherapies following CGIP were classified as “matched” to biomarker results (established or potentially clinically significant) at the indication level (single or multi-marker results, histology, treatment line) based on AMP/ASCO/CAP guidance for strength of biomarker clinical evidence. We estimated overall survival (OS) from CGIP report date for patients who first received either matched therapy or chemotherapy (and no subsequent matched therapy), and assessed the predictive value of matched therapy for OS in the first or subsequent line setting, adjusting for clinicopathologic covariates. Results: Most CGIP tested patients were female (55%), stage IIIB/IV (89%), ECOG < 2 (83%), non-squamous (86%), treatment naïve (62%), ever smokers (88%). 74% (228) of patients were treated post-CGIP, with 71% receiving at least one matched therapy. Matched therapies received in the frontline setting were supported by the highest (Tier 1A) category of evidence more often than subsequent line therapies (97% vs. 68%). 90% of patients with oncogenic driver mutations received targeted agents (17% of total) and 57% received matched immunotherapy. In the frontline setting, compared to chemotherapy, OS was highest for patients who first received matched targeted therapy (median = 23.4 mo; HR 0.26; p = .004; 95% CI 0.13-0.68) vs matched immunotherapy (median = 17.9 mo; HR 0.38; p = .001; 95% CI 0.21-0.69). Subsequent line, OS was also highest for patients who first received matched targeted therapy (median not est., mean = 27.5 mo; HR 0.20; p = .063; 95% CI 0.04-1.09) vs matched immunotherapy (median = 17.4 mo; HR 0.20; 95% CI 0.04-1.09), however, these differences were non-significant. Conclusions: CGIP supports evidence-based clinical decision making for NSCLC in the first and subsequent line settings and leads to improved survival for patients who receive matched targeted or immunotherapy compared to chemotherapy. Better predictive markers are needed to identify NSCLC patients who are more likely to respond to immunotherapies. Heterogeneity of patient biomarker profiles and treatment strategies over time in real world practice are a challenge to assessing CGIP efficacy.

Abstract BACKGROUND Non-small cell lung cancer (NSCLC) accounts for ~50% of brain metastases. We present the genomic and immune biomarker landscape for a cohort with metastatic NSCLC to the brain. METHODS We analyzed brain metastases FFPE tissue (n=137; ages 40-85y (mean 65y), 52% female, 48% male) vs. primary sites (n=5533; ages 24-100+y (mean 71y), 51% female, 49% male) for advanced or metastatic NSCLC patients with comprehensive genomic and immune biomarker profiling, including PD-L1 IHC, tumor mutational burden (TMB), Tumor Immunogenic Signature (TIGS), Cell Proliferation (CP), and Cancer Testis Antigen Burden (CTAB). RESULTS Genomic alteration (GA) frequency for NSCLC brain metastasis versus primary sites were similar among the most frequently mutated genes except for KRAS which was significantly higher among brain metastases (39.9% vs 25.5%, p< 0.0005). No significant differences were observed for TP53 (50.7% vs 50.2%), STK11 (11.5% vs 10.9%), CDKN2A (10.1% vs 7%), or EGFR (8.0% vs 11.5%). PD-L1 expression for all cases by IHC was not significantly different (mean TPS 29.9% vs 26.3%); however, brain metastases were more likely to be PD-L1 negative (TPS< 1%) (46.3% vs 33.3%, p< 0.005). Conversely, TMB was higher in brain metastases versus primary sites (12.9 vs 10.2 muts/MB, p< 8x10-10), with more TMB-high cases (10 muts/Mb) (57.0% vs 33.5%, p< 3x10-8). Similarly, brain metastases had a higher proportion of CTAB-high cases (68.6% vs 57.6%, p=0.01). The immune response biomarker score, TIGS, was more often weak in brain metastases (52.6% vs 34.0%, p< 9x10-6). CONCLUSION In NSCLC, metastatic brain lesions have a larger antigen burden, with increased TMB and CTAB, likely due to the immune privileged nature of the brain, which is reflected in the lower TIGS scores and PD-L1 positivity. Despite lower PD-L1 positivity, NSCLC brain metastases with negative PD-L1 IHC may potentially benefit from immunotherapy strategies given the high TMB and CTAB.

Background Cancer of unknown primary (CUP) is a rare tumor type accounting for 2% of solid cancers. In the subset of CUP cases where tumor of origin is posited and treated as such, no clear clinical benefit has been demonstrated. Furthermore, CUP patients treated by empiric platinum-based regimens have low response and survival rates of approximately 20%. 1 2 Support of tissue-agnostic marker-directed immunotherapy is growing because it targets the immune system rather than the tumor, with some efficacy evidence emerging for CUP. 3 Identifying new targets for immunotherapeutic opportunities in this heterogeneous and difficult to treat patient group is a critical unmet need. Methods Comprehensive genomic and immune marker profiling by NGS 4 was performed on FFPE tissue for CUP tumors (n=298) as indicated by physicians’ test orders from >100 clinical practice sites. Histology was verified by a molecular pathologist as part of pre-analytic test quality control, with cases representing tumors with adenocarcinoma (58%), carcinoma (26%), squamous (10%), and neuroendocrine (6%) histologic features. RNA-expression levels of immune genes that are current targets in non-CUP immunotherapy clinical trials (n=36) were ranked against a reference population (≥75th percentile=high), and described by histologic type, along with PD-L1 IHC (22C3) expression, tumor mutational burden (TMB) and genomic variants. Results 90% of all CUP tumors had at least 1 highly expressed immune gene target in active immunotherapy trials, with the most frequent being TGFB1 (47%) and CCL2 (39%). 55% of CUP tumors were PD-L1 IHC 22C3 positive (>=1% TPS), and 21% had high TMB (>=10 mut/Mb) in CUP tumors with neuroendocrine (32%), carcinoma (30%), squamous cell (21%), and adenocarcinoma (17%) histologic features. Overall, 26% of CUP patient tumors, mostly adenocarcinomas (28%) and carcinomas (27%), harbored genomic variants (n=77) with FDA approved targeted therapies in other tumor types. The most frequently immunogenic CUP tumors were carcinomas, showing high RNA-seq expression of 26/36 genes in at least 20% of patients, most represented by CD20, CD27, TLR8, and PD-L1. High expression of CD40, CSF1R, TIM3, and VISTA was most common in adenocarcinomas. Squamous cell carcinomas were relatively immunogenic, with frequent high expression of 17/36 immune genes, uniquely including MAGEA4. Neuroendocrine tumors were the least immunogenic, with frequent high expression in only 4/36 genes, including ADORA2A (42%) and MAGEA1 (37%). Conclusions CUP tumors diversely express both standard marker and novel immunotherapeutic targets based on histology and may benefit from selective access to clinical trials for these therapies. References National Comprehensive Cancer Network. NCCN Clinical Practice Guidelines in Oncology (NCCN Guidelines®) Occult Primary (Cancer of Unknown Primary [CUP]), Version 2.2021. Fort Washington, Pennsylvania: National Comprehensive Cancer Network; 2021. https://www.nccn.org/professionals/physician_gls/pdf/occult.pdf. Laprovitera N, Riefolo M, Ambrosini E, Klec C, Pichler M, Ferracin M. Cancer of unknown primary: Challenges and progress in clinical management. Cancers (Basel) 2021; 13 (3):1–30. doi:10.3390/cancers130304513. Naing A, Meric-Bernstam F, Stephen B, et al. Phase 2 study of pembrolizumab in patients with advanced rare cancers. J Immunother Cancer 2020; 8 (1):e000347. doi:10.1136/jitc-2019-0003474. Conroy JM, Pabla S, Glenn ST, et al. Analytical validation of a next-generation sequencing assay to monitor immune responses in solid tumors. J Mol Diagnostics 2018; 20 (1):95–109. doi:10.1016/j.jmoldx.2017.10.001

BackgroundImmune checkpoint inhibitor-based therapies have achieved impressive success in the treatment of several cancer types. Predictive immune biomarkers, including PD-L1, MSI and TMB are well established as surrogate markers for immune evasion and tumor-specific neoantigens across many tumors. Positive detection across cancer types varies, but overall ~50% of patients test negative for these primary immune markers.1 In this study, we investigated the prevalence of secondary immune biomarkers outside of PD-L1, TMB and MSI.MethodsComprehensive genomic and immune profiling, including PD-L1 IHC, TMB, MSI and gene expression of 395 immune related genes was performed on 6078 FFPE tumors representing 34 cancer types, predominantly composed of lung cancer (36.7%), colorectal cancer (11.9%) and breast cancer (8.5%). Expression levels by RNA-seq of 36 genes targeted by immunotherapies in solid tumor clinical trials, identified as secondary immune biomarkers, were ranked against a reference population. Genes with a rank value ≥75th percentile were considered high and values were associated with PD-L1 (positive ≥1%), MSI (MSI-H or MSS) and TMB (high ≥10 Mut/Mb) status. Additionally, secondary immune biomarker status was segmented by tumor type and cancer immune cycle roles.ResultsIn total, 41.0% of cases were PD-L1+, 6.4% TMB+, and 0.1% MSI-H. 12.6% of cases were positive for >2 of these markers while 39.9% were triple negative (PD-L1-/TMB-/MSS). Of the PD-L1-/TMB-/MSS cases, 89.1% were high for at least one secondary immune biomarker, with 69.3% having ≥3 markers. PD-L1-/TMB-/MSS tumor types with ≥50% prevalence of high secondary immune biomarkers included brain, prostate, kidney, sarcoma, gallbladder, breast, colorectal, and liver cancer. High expression of cancer testis antigen secondary immune biomarkers (e.g., NY-ESO-1, LAGE-1A, MAGE-A4) was most commonly observed in bladder, ovarian, sarcoma, liver, and prostate cancer (≥15%). Tumors demonstrating T-cell priming (e.g., CD40, OX40, CD137), trafficking (e.g., TGFB1, TLR9, TNF) and/or recognition (e.g., CTLA4, LAG3, TIGIT) secondary immune biomarkers were most represented by kidney, gallbladder, and sarcoma (≥40%), with melanoma, esophageal, head & neck, cervical, stomach, and lung cancer least represented (≥15%).ConclusionsOur studies show comprehensive tumor profiling that includes gene expression can detect secondary immune biomarkers targeted by investigational therapies in ~90% of PD-L1-/TMB-/MSS cases. While genomic profiling could also provide therapeutic choices for a percentage of these patients, detection of secondary immune biomarkers by RNA-seq provides additional options for patients without a clear therapeutic path as determined by PD-L1 testing and genomic profiling alone.ReferenceHuang R S P, Haberberger J, Severson E, et al. A pan-cancer analysis of PD-L1 immunohistochemistry and gene amplification, tumor mutation burden and microsatellite instability in 48,782 cases. Mod Pathol 2021;34: 252–263.

Timely and accurate identification of molecular alterations in solid tumors is essential for proper management of patients with advanced cancers. This has created a need for rapid, scalable comprehensive genomic profiling (CGP) systems that detect an increasing number of therapeutically-relevant variant types and molecular signatures. In this study, we assessed the analytical performance of the TruSight Oncology 500 High-Throughput assay for detection of somatic alterations from formalin-fixed paraffin-embedded tissue specimens. In parallel, we developed supporting software and automated sample preparation systems designed to process up to 70 clinical samples in a single NovaSeq 6000(TM) sequencing run with a turnaround time of <7 days from specimen receipt to report. The results demonstrate that the scalable assay accurately and reproducibly detects small variants, copy number alterations, microsatellite instability (MSI) and tumor mutational burden (TMB) from 40ng DNA, and multiple gene fusions, including known and unknown partners and splice variants from 20ng RNA. 717 tumor samples and reference materials with previously known alterations in 96 cancer-related genes were sequenced to evaluate assay performance. All variant classes were reliably detected at consistent and reportable variant allele percentages with >99% overall accuracy and precision. Our results demonstrate that the high-throughput CGP assay is a reliable method for accurate detection of molecular alterations in support of precision therapeutics in oncology. The supporting systems and scalable workflow allow for efficient interpretation and prompt reporting of hundreds of patient cancer genomes per week with excellent analytical performance.

Background Contemporary to the rapidly evolving landscape of cancer immunotherapy is the equally changing understanding of immune tumor microenvironments (TMEs) which is crucial to the success of these therapies. Their reliance on a robust host immune response necessitates clinical grade measurements of immune TMEs at diagnosis. In this study, we describe a stable tumor immunogenic profile describing immune TMEs in multiple tumor types with ability to predict clinical benefit from immune checkpoint inhibitors (ICIs). Methods A tumor immunogenic signature (TIGS) was derived from targeted RNA-sequencing (RNA-seq) and gene expression analysis of 1323 clinical solid tumor cases spanning 35 histologies using unsupervised analysis. TIGS correlation with ICI response and survival was assessed in a retrospective cohort of NSCLC, melanoma and RCC tumor blocks, alone and combined with TMB, PD-L1 IHC and cell proliferation biomarkers. Results Unsupervised clustering of RNA-seq profiles uncovered a 161 gene signature where T cell and B cell activation, IFNg, chemokine, cytokine and interleukin pathways are over-represented. Mean expression of these genes produced three distinct TIGS score categories: strong ( n = 384/1323; 29.02%), moderate ( n = 354/1323; 26.76%), and weak ( n = 585/1323; 44.22%). Strong TIGS tumors presented an improved ICI response rate of 37% (30/81); with highest response rate advantage occurring in NSCLC (ORR = 36.6%; 16/44; p = 0.051). Similarly, overall survival for strong TIGS tumors trended upward (median = 25 months; p = 0.19). Integrating the TIGS score categories with neoplastic influence quantified via cell proliferation showed highly proliferative and strong TIGS tumors correlate with significantly higher ICI ORR than poorly proliferative and weak TIGS tumors [14.28%; p = 0.0006]. Importantly, we noted that strong TIGS and highly [median = not achieved; p = 0.025] or moderately [median = 16.2 months; p = 0.025] proliferative tumors had significantly better survival compared to weak TIGS, highly proliferative tumors [median = 7.03 months]. Importantly, TIGS discriminates subpopulations of potential ICI responders that were considered negative for response by TMB and PD-L1. Conclusions TIGS is a comprehensive and informative measurement of immune TME that effectively characterizes host immune response to ICIs in multiple tumors. The results indicate that when combined with PD-L1, TMB and cell proliferation, TIGS provides greater context of both immune and neoplastic influences on the TME for implementation into clinical practice.

BACKGROUND Molecular genetic testing has raised a variety of policy issues, ranging from privacy to reimbursement. Recently, payment policies have become of paramount importance as Medicare implemented the first significant change to test pricing since 1984 and announced a broad national coverage policy for the use of next-generation sequencing (NGS) in cancer patients that contains significant restrictions. Regulatory and oversight concerns have been important topics for discussion as the US Food and Drug Administration (FDA), Congress, and stakeholders have focused on new approaches to regulation of laboratory-developed tests (LDTs). Patents on gene sequences and relationships between genetic variants and clinical phenotypes have been points of contention since the field's inception. Two Supreme Court cases invalidated patents on gene sequences and biological relationships, ushering in the era of NGS and precision medicine. However, a recent legislative proposal threatens to reverse these gains and restore gene patents as barriers to progress in genetic and genomic testing and the implementation of genomic medicine. CONTENT This review discusses current issues in payment policy, laboratory oversight, and gene patenting and their potential impacts on genetic and genomic testing. SUMMARY Coverage and reimbursement policies present serious challenges to genetic and genomic testing. The potential for FDA regulation of LDTs looms as a significant threat to diagnostic innovation, patient access, and the viability of molecular genetic testing laboratories. Changes in patent law could cause gene patents to reemerge as barriers to the advancement of genomic medicine.

The F9/Yde/Fml pilus, tipped with the FmlH adhesin, has been shown to provide uropathogenic Escherichia coli (UPEC) a fitness advantage in urinary tract infections (UTIs). Here, we used X-ray structure guided design to optimize our previously described ortho-biphenyl Gal and GalNAc FmlH antagonists such as compound 1 by replacing the carboxylate with a sulfonamide as in 50. Other groups which can accept H-bonds were also tolerated. We pursued further modifications to the biphenyl aglycone resulting in significantly improved activity. Two of the most potent compounds, 86 (IC50 = 0.051 μM) and 90 (IC50 = 0.034 μM), exhibited excellent metabolic stability in mouse plasma and liver microsomes but showed only limited oral bioavailability (<1%) in rats. Compound 84 also showed a good pharmacokinetic (PK) profile in mice after IP dosing with compound exposure above the IC50 for 6 h. These new FmlH antagonists represent new antivirulence drugs for UTIs.

BACKGROUND:The detection of mutated epidermal growth factor receptor (EGFR) in non-small cell lung cancer (NSCLC) with residual cell pellets derived from liquid-based cytology (LBC) samples (eg, endoscopic ultrasound-guided fine-needle aspiration) has been validated with allele-specific polymerase chain reaction. The aim of this study was to validate next-generation sequencing (NGS) technology for detecting gene mutations with residual cell pellets from LBC.METHODS:Archived DNA extracted from LBC samples of adenocarcinoma stored in PreservCyt with a known EGFR mutation status was retrieved. Genomic DNA was multiplex-amplified and enriched with Ion AmpliSeq Cancer Hotspot Panel v2 chemistry and the OneTouch 2 instrument; this was followed by semiconductor sequencing on the Ion Personal Genome Machine platform. The mutation hotspots of 6 NSCLC-related genes (BRAF, EGFR, ERBB2, KRAS, MET, and phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit α [PIK3CA]) were analyzed with NextGENe and Torrent Suite bioinformatics tools.RESULTS:The commonly identified EGFR sequence changes, including 4 L858R mutations, 3 exon 19 deletions, and 1 exon 20 insertion, were in 100% concordance between the assay platforms. Less common NSCLC variants were also found in the mutation hotspots of ERBB2, KRAS, MET, and PIK3CA genes.CONCLUSIONS:NSCLC mutation analysis using NGS can be successfully performed on residual cell pellets derived from LBC samples. This approach allows the simultaneous examination of multiple mutation hotspots in a timely manner to improve patient care. Cancer Cytopathol 2017;125:178-187. © 2016 American Cancer Society.

OH. Background: Next Generation Sequencing (NGS) on endoscopic ultrasound-guided fine needle aspiration (EBUS-FNA) samples is useful for facilitating treatment decisions of non-small cell lung cancer (NSCLC) patients. We assessed the effectiveness of detecting driver mutations by NGS in surgical and biopsy tissue versus liquid based cytology (LBC) specimens. Design: Archived extracted DNA from residual cell pellets of LBC specimens (method previously described) with known EGFR and KRAS mutation status were retrieved. For each case, DNA from corresponding formalin-fixed paraffin-embedded (FFPE) tissue was also extracted. Mutation hotspot NGS libraries for BRAF, EGFR, ERBB2, FGFR1, KRAS, MET , and PIK3C were prepared on all DNA samples, sequenced by Illumina MiSeq and analyzed using NextGENe software with reference genome hg19. Each patient’s NGS variant calls were compared between FFPE and LBC results. EGFR and KRAS results were also compared against prior real-time PCR results (Qiagen). Results: Ten patients from June to August 2014 had adequate DNA in both LBC and FFPE specimens as measured by Qubit™ fluorometric quantitation. LBC samples were obtained from EBUS-FNA (9) or bronchial washing (1). Ten corresponding FFPE tissue specimens (6 transbronchial biopsies, 2 lobectomies, 1 each of brain and bone metastases) were selected for sequencing. coverage depth of LBC specimens supports its clinical use. Our initial experience using NGS on 128 clinical lung cancer FNAs detected KRAS mutations in 30 patients and EGFR in 16; qualifying 8 for tyrosine kinase inhibitor therapy. Thus, the clinical utility of NGS on EBUS-FNA derived LBC samples appears promising. Background: Atypical melanocytic tumors that defy classification as benign or malignant using pathological criteria alone are not uncommon. Additional ancillary tests are often needed to enhance diagnostic accuracy. Prior studies have shown that evaluation of copy number variations (CNV) by array comparative genomic hybridization can be used to assist in differentiating nevi from melanomas. The performance of this technique in ambiguous melanocytic lesions and its correlation with clinical outcomes are yet to be fully characterized. More recently, platforms based on single nucleotide polymorphism arrays (SNP) arrays have become available; however, experience with their use in melanocytic lesions is limited. Herein we aim to evaluate the performance of a SNP-array on a cohort of benign, malignant and ambiguous melanocytic tumors with clinical follow-up. Design: Eighty five melanocytic lesions were included. Using histopathological examination and immunohistochemistry, the cases were classified into four categories; benign nevi, atypical nevi, ambiguous lesions and melanomas. Follow-up data was gathered from our institutional electronic health record system and adverse events including sentinel lymph node metastasis, local recurrence, distant metastasis or death were recorded. Tissue from unstained slides or tissue rolls was processed and analyzed for CNV and loss of heterozygosity using the OncoScan V3 SNP-array platform (Affymetrix). Results: Follow-up data was available in 57 patients (mean 5.9 months). 6/6 benign nevi (100%) and 12/15 particular, invasion beyond the skin and metastasis. Therefore, we propose D2-40 IHC as a routine method for evaluation of LVI in all PCMCC cases as a predictive marker for metastasis. Background: Nephrogenic systemic fibrosis occurs in patients with chronic renal disease and it has been linked to previous gadolinium exposure. Microscopically, distinct sclerotic bodies (“elastocollagenous balls”) have also been documented in this clinical setting. To date, only few histopathologic data are available for these peculiar cutaneous deposits. The aim of this study is to determine the specific constituents of cutaneous elastocollagenous deposits in nephrogenic systemic fibrosis using laser microdissection and mass spectrometry based proteomic analysis. Design: Two skin biopsies with characteristic histologic features of nephrogenic systemic fibrosis containing sclerotic bodies were retrieved. In each case, sclerotic bodies were identified and laser microdissected (LMD) using the Leica dissector. These LMD fragments were digested into tryptic peptide and analyzed by nano-flow liquid chromatography electrospray tandem massspectrometry (LC-MS/MS). The informatics analysis was performed using Scaffold (Proteome Software, Portland, OR), where the protein and peptide probability scores were set at 95% and the estimated protein false discovery rate (FDR) was >0.05%. Results: Microscopically, all skin biopsies showed features of nephrogenic systemic fibrosis characterized by pandermal spindle cell proliferation with increased collagen and scattered sclerotic bodies. Irregularly shaped sclerotic bodies, many of which contained elastic fibers, were present in the dermis and superficial subcutis. Proteomic analysis by LMD-LC-MS/MS was able to accurately identify specific constituents of collagen deposits and entrapped elastic fibers. Both cases showed various collagen types, fibrillin, and microfibril-associated glycoprotein; notably, preferential accumulation of type VI collagen (alpha-1 and alpha-3) and fibrillin was noted. One case also revealed elastin. Conclusions: In addition to confirming the presence of collagen and elastic fibers, our pilot LMD-LC-MS/MS -based proteomic analysis demonstrated the specific protein contents in sclerotic bodies of nephrogenic systemic fibrosis. A larger number of cases of sclerotic bodies with additional features, such as osseous metaplasia or calcification, will be necessary to more fully understand their exact nature and significance. Background: Regression in melanoma is defined as the replacement of tumor cells by lymphocytic inflammation, along with attenuation of the epidermis and non-lamellated dermal fibrosis with inflammatory cells, melanophages, and telangiectasia. Whether regression is an adverse prognostic factor in predicting survival in thin (≤1mm) primary cutaneous melanoma (PCM) remains controversial in part because the definition and quantification of regression were not consistent in studies assessing its significance. Here, we examined the correlation between regression and conventional prognostic indicators of outcome in a large series of thin PCM using uniform histologic criteria. Design: We performed a retrospective analysis of 77 patients with thin PCM in the original biopsy (1/09-12/10). We assessed the extent of regression (focal <50% vs. extensive >50%) as well as thickness and correlated these with histologic, demographic and clinical outcome parameters. We measured maximum thickness of regression with a calibrated ocular micrometer from the granular layer of the epidermis (if the lesion was ulcerated from the base of the ulcer) to the deepest point of (1) dermal fibrosis and (2) lymphocytic infiltrate. Results: The extent of regression (focal vs. extensive) was significantly associated with high Clark level (p=0.01), increased Breslow thickness (p=0.001), increased number of mitotic figures (p=0.05), and presence of vertical growth phase (p=0.04). The depth of regression measured by inflammatory infiltrate significantly associated with high Clark level (p=0.01), increased Breslow thickness (p=0.002), presence of ulceration (p=0.02), and presence of lymphovascular invasion (p=0.04). Significantly reduced overall survival was found in the patients with thin PCM with depth of regression (≥0.43 mm) as measured by dermal fibrosis (p=0.02). Conclusions: Our results demonstrate that extent and thickness of regression in thin PCM is significantly associated with adverse clinicopathologic parameters including high Clark level, high Breslow thickness, mitotic figures, ulceration, lymphovascular invasion, vertical growth phase as well as with reduced overall survival. We propose to include regression (both extent and depth of inflammation and fibrosis) as a strong adverse prognostic marker in patients with thin PCM. sarcoma (KS) represents an endothelial cell neoplasm with a rich inflammatory infiltrate of still unclear origin and function. several factors in the tumor microenvironment responsible for changes in myeloid differentiation that can favor tumor immune evasion, angiogenesis and tumor progression. and and evasion, and tumor Background: Tumor-infiltrating lymphocytes (TILs) are classically divided into “brisk”, “non-brisk” and “absent” categories, and this classification bears prognostic significance; in particular the heavy “brisk” category is associated with excellent prognosis, and the “absent” category is associated with thick melanomas. However, little is known about the functional status of TILs or of the antigen-presenting machinery of melanoma cells in these categories, and the prognostic significance of the “non-brisk” category is poorly defined. Nevertheless, the increasing usage of immunomodulatory therapies in disseminated melanoma requires knowledge of the efficacy of the immune response in melanoma. Design: As immune checkpoint-targeted therapies are given to stage IV patients, melanoma metastases were immunohistochemically stained for MHC machinery molecules (HLA ABC, HLA-DR, b2-microglobulin, TAP1, TAP2), T-cell subsets (CD4, CD8, PD-1) and for signs of interferon-gamma (IFNg) induction (CXCL9 and RT-qPCR for Results: The overall correlation between “brisk” TILs and the presence of signs of IFNg production in the melanoma microenvironment (i.e. CXCL9+ melanoma cells or macrophages) was significant (p: 0.009). Furthermore, “brisk” TILs were consistently associated with overexpression of the MHC machinery molecules on melanoma cells. sections All Idylla™ results were compared with INCA Platform results where various reference methods (RM) are performed. Results: Comparing the results of both tests on 239 patients only 1 Wild Type (WT) was detected in our study while

The Center for Medical Technology Policy and the Molecular Evidence Development Consortium gathered a diverse group of more than 50 stakeholders to develop consensus on a core set of data elements and values essential to understanding the clinical utility of molecularly targeted therapies in oncology.