Comprehensive Tumor Profiling Research Articles

Comparative analysis of actionable gene reporting in targeted panels versus comprehensive NGS testing for solid tumor samples.

11185 Background: The landscape of actionable genes significantly influences clinical decisions in cancer diagnosis, prognosis, and treatment planning. In a clinical context, the selection of NGS panels hinges on striking a balance among informative data, insurance coverage, and the preferences of patients and healthcare providers. Molecular pathology reference laboratories commonly employ large-scale next-generation sequencing (NGS) testing, curating smaller, disease-specific targeted panels. This study investigates the prevalence of unreported actionable genes in real-world clinical samples subjected to disease-specific panels. Methods: We analyzed SNV/InDel DNA variants in 795 clinical solid tumor samples using the Illumina TSO 500 platform for comprehensive NGS panel testing of 517 genes. Comprehensive NGS panel-tested samples were intersected with disease-specific targeted NGS panels for breast (54 genes), brain (62 genes), colorectal (36 genes), and lung (44 genes). An artificial filter was applied to align targeted panels with patient disease. Variants were classified based on pathogenicity, and benign variants were filtered out. Detected variants were assessed for actionable mutations as defined by the FDA recognized OncoKB actionable gene database criteria. Additionally, 1484 lung patient samples tested with a lung-specific targeted panel were de-identified and unmasked to the comprehensive NGS tumor profiling to investigate the prevalence of actionable alteration beyond those covered by the disease-specific panel. Results: The study revealed that 3.2% of breast, 0% of brain, 66.2% of colorectal, and 2.7% of lung samples had additional actionable mutations undisclosed by the targeted panels. The number of actionable genes beyond those included in the targeted panels were 14 for breast, 2 for brain, 25 for lung, and 41 for colorectal cancer. This underscores the importance of comprehensive testing to fully capture each patient tumor’s actionable mutation profile. Conclusions: Our findings detect masked actionable mutations in down-sampled targeted panels and highlight the need for comprehensive testing to accurately identify actionable mutations in various cancer types. Reporting actionable mutations can significantly influence clinical decision-making, emphasizing the importance of NGS test selection in routine molecular diagnostics as well as the impact of integrating comprehensive NGS testing.

Multidimensional tumor profiling and its meaningful impact on personalized therapy outcomes: Insights from a real-world study.

e15064 Background: This real-world study underscores the transformative potential of multidimensional tumor profiling, including tissue and blood-based markers, in guiding personalized oncology treatments. We established a robust link between comprehensive diagnostics and improved clinical outcomes by correlating extensive profiling data with progression-free survival (PFS), the ratio of PFS2 to PFS1, and comparing these with treatment modalities. Methods: Over five years, 44 patients with advanced-stage solid tumors underwent comprehensive tumor profiling, including genetic markers from tissue and blood, mRNA expression, circulating tumor cells, pharmacogenomics, and immunohistochemistry, to tailor therapies. These multidimensional datasets were rigorously analyzed applying machine learning algorithms against responses to target, chemo, and combo therapies, using robust statistical frameworks to elucidate the impact of advanced diagnostics on therapy effectiveness. Results: Multidimensional tumor testing markedly extended progression-free survival, with median PFS1 and PFS2 at 22.5 and 30 weeks, respectively t(43) = 1.76, p = 0.043, and a significant median PFS2/1 ratio of 1.31. Targeted therapies achieved the highest median PFS2 at 61 weeks, surpassing chemo alone (10.5 weeks) and combination therapy (29.5 weeks) significantly. Patients treated based on gene expression, including mRNA profiling and immunohistochemistry/immunocytochemistry (n = 22), had a median PFS of 37.5 weeks, outperforming those treated based on NGS (n = 12) with 27.5 weeks significantly. Those not fully adhering to guideline treatments due to divergent findings had a median PFS2 of 16.5 weeks. Notably, 26 out of 44 patients with critical illness had a PFS2 over 6 months. Conclusions: A clear linkage was established between comprehensive profiling indicators, including genetic, molecular, and cellular markers, and the observed therapeutic outcomes. The study reaffirms the critical importance of multidimensional tumor profiling in the era of precision oncology. However, we proceed monitoring this growing group of patients to establish a clearer picture how to adapt complex diagnostic tools into modern oncological practice.

IMPRESS-Norway: improving public cancer care by implementing precision medicine in Norway; inclusion rates and preliminary results.

In Norway, comprehensive molecular tumour profiling is implemented as part of the public healthcare system. A substantial number of tumours harbour potentially targetable molecular alterations. Therapy outcomes may improve if targeted treatments are matched with actionable genomic alterations. In the IMPRESS-Norway trial (NCT04817956), patients are treated with drugs outside the labelled indication based on their tumours molecular profile. IMPRESS-Norway is a national, prospective, non-randomised, precision cancer medicine trial, offering treatment to patients with advanced-stage disease, progressing on standard treatment. Comprehensive next-generation sequencing, TruSight Oncology 500, is used for screening. Patients with tumours harbouring molecular alterations with matched targeted therapies available in IMPRESS-Norway, are offered treatment. Currently, 24 drugs are available in the study. Primary study endpoints are percentage of patients offered treatment in the trial, and disease control rate (DCR) defined as complete or partial response or stable disease in evaluable patients at 16 weeks (W16) of treatment. Secondary endpoint presented is DCR in all treated patients. Between April 2021 and October 2023, 1,167 patients were screened, and an actionable mutation with matching drug was identified for 358 patients. By the data cut off 186 patients have initiated treatment, 170 had a minimum follow-up time of 16 weeks, and 145 also had evaluable disease. In patients with evaluable disease, the DCR was 40% (58/145). Secondary endpoint analysis of DCR in all treated patients, showed DCR of 34% (58/170). Precision cancer medicine demonstrates encouraging clinical effect in a subset of patients included in the IMPRESS-Norway trial.

What Do German Molecular Tumor Boards Recommend in Patients with PIK3CA-Mutated Tumors? Launch and First Results from the German Transsectoral Molecular Tumor Board Exchange Platform Deutschland

Introduction: Comprehensive molecular tumor profiling is widely used in the management of patients with cancer. Molecular tumor boards devise treatment strategies based on testing results. In this setting, the Transsectoral Molecular Tumor Board exchange platform Deutschland (TEAM-D) aims to drive peer-to-peer exchange to connect experts in the field. Methods: During the first virtual TEAM-D meeting, participants from 16 German universities and 5 nonacademic institutions discussed five cases with PIK3CA hotspot mutations. Furthermore, an illustrative case vignette was presented. Results: Overall, German caregivers show restraint in administering off-label PIK3CA inhibitor and favor clinical trials in this setting. Conclusion: In the setting of precision oncology, TEAM-D enables virtual case discussion across the different sectors of the German healthcare system. Based on the example of PIK3CA hotspot mutations, TEAM-D demonstrated the value of integrating knowledge from different healthcare professionals.

A Retrospective Review and Comprehensive Tumour Profiling of Advanced Non-Melanomatous Cutaneous Spindle Cell Neoplasms Treated with Immune-Checkpoint Inhibitors.

Non-melanomatous cutaneous spindle cell neoplasms are a rare group of malignancies that present a diagnostic challenge, and for which there is a lack of consensus on how to best manage patients with advanced disease and only limited reports of immune-checkpoint inhibitor (ICI) responses. In this study, we performed a single-center retrospective review of treatment outcomes for all advanced non-melanomatous cutaneous spindle cell neoplasms treated with ICIs. Blinded histopathology reviews occurred to confirm each diagnosis. Comprehensive tumour profiling included whole exome sequencing for tumour mutational burden (TMB) and ultraviolet(UV) signatures, and immunohistochemistry for immune-cell infiltration (CD4/CD3/CD8/CD103/CD20) and immune-checkpoint expression (PD-L1/LAG3/TIGIT). Seven patients were identified. The objective response rate was 86% (6/7) with five complete responses (CR). Responses were durable with two patients in CR > 30 months after ICI commencement. All patients had high TMB and UV signatures. One patient had PD-L1 100% (combined positive score) with abundant immune-cell infiltration and LAG3 expression. In advanced non-melanomatous cutaneous spindle cell neoplasms, excellent responses to ICIs with durable disease control were observed. ICIs are worthy of further exploration in these patients. UV signatures and high TMB could be used to help select patients for treatment.

Abstract 5094: Immunotherapy in advanced cutaneous non-melanomatous spindle cell neoplasms

Abstract Background: Non-melanomatous cutaneous spindle cell neoplasms (NMCSpC) are a rare group of malignancies including pleomorphic dermal sarcoma (PDS) and spindle-cell cutaneous squamous cell carcinoma (SpSCC). They present a diagnostic challenge with overlapping morphological features and variable patterns of immunohistochemical (IHC) staining. There is a lack of consensus on how to best manage advanced NMCSpC and limited reports of immunotherapy (IO) responses. This study reviews the efficacy of IO in NMCSpC and performs comprehensive tumor profiling to identify biological rationale for IO use in this rare group of tumors. Methods: We performed a single-center retrospective review of all locally-advanced or metastatic NMCSpCs treated with IO. Blinded histopathology reviews occurred to confirm each diagnosis. Overall response rates (ORR) were collected as per Response Evaluation Criteria in Solid Tumors 1.1 or Positron Emission Tomography Response Criteria 1.0. Comprehensive tumor profiling included whole exome sequencing for tumor mutational burden (TMB) and ultraviolet(UV)-signatures, and IHC for immune-cell infiltration (CD103/CD4/CD3/CD8/CD20) and immune-checkpoint expression (PD-L1/TIGIT/LAG3). Results: 7 patients were identified. Median follow up was 18.1 months (range: 5.3-34.7). ORR 86% (6/7) with five complete responses (CR). One patient who achieved stable disease progressed. Responses were durable with two patients in CR >30 months after IO commencement. Blinded pathological review identified 3 SpSCC, 2 PDS and 2 spindle cell tumors of unclear sub-classification. All patients had high TMB and UV-signatures. One patient had PD-L1 100% (combined positive score) with abundant immune-cell infiltration and LAG3 expression (Table 1). Conclusion: In advanced NMCSpC, excellent responses to IO with durable disease control was observed. IO is worthy of further exploration in these patients. UV-signatures and high TMB could be used to help select patients for treatment. Immunohistochemistry (IHC)^, tumor mutational burden and ultraviolet signatures Patient T-cell markers B-cell marker Immune-checkpoints Tumor mutational burden (mutations/megabase) Ultraviolet-mutational signature Best response assessment# CD103 CD4 CD3 CD8 CD20 PD-L1 TIGIT LAG3 1 1 20 1 1 0 7 0 0 17.94 Yes Stable metabolic response 2 1 20 30 10 0 100 0 10 35.34 Yes Complete metabolic response 3 1 10 10 5 0 5 0 0 104.87 Yes Complete metabolic response 4 1 0 1 0 0 0 0 0 74.04 Yes Partial metabolic response 5* NA NA NA NA NA NA NA NA 29 Yes Complete metabolic response 6 1 0 0 5 0 0 0 4 12.02 Yes Complete metabolic response 7 1 0 5 1 0 0 0 0 41.17 Yes Complete response ^IHC scores are % of positively stained cells, except for PD-L1 which is combined positive score (CPS). *Insufficient tissue for IHC on Patient 5. #As per PERCIST 1.0 on FDG-PET/CT or RECIST1.1 on CT. Citation Format: Luke S. McLean, Annette M. Lim, Christopher Angel, Richard J. Young, Angela Pizzolla, Stuart Archer, Alesha A. Thai, Benjamin Solomon, Jeremy Lewin, Danny Rischin. Immunotherapy in advanced cutaneous non-melanomatous spindle cell neoplasms [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 5094.

Abstract 2498: Assessing the clinical value of ctDNA sequencing for initial tumor profiling in metastatic colorectal cancer patients with sufficient tumor tissue

Abstract Introduction Tumor profiling including RAS, BRAF, HER2, and MSI/MMR status, is required to determine the treatment for patients with metastatic colorectal cancer (mCRC) at the time of diagnosis. While comprehensive tumor profiling using tissue-based next-generation sequencing (NGS) is increasingly adopted for initial profiling in mCRC, the role of circulating tumor DNA (ctDNA) NGS as initial testing in patients with sufficient tumor tissue is not clearly understood. We assessed the clinical value of ctDNA sequencing compared to tumor NGS in patients with newly diagnosed mCRC who have sufficient tumor specimens. Methods We prospectively enrolled consecutive patients with newly diagnosed mCRC at the National Cancer Center Korea. As per the institutional protocol for mCRC, initial tumor profiling was performed on primary tumor tissue using an in-house NGS panel (NCC PCP ver.3), which included 525 genes. For ctDNA sequencing, patients were evaluated using the AlphaLiquid®100 comprehensive cancer panel (IMBdx, Inc.), which included 118 genes, before the initiation of chemotherapy. Additionally, immunohistochemical (IHC) testing for HER2 and polymerase chain reaction (PCR)/IHC testing for MSI and/or MMR were performed to assess the accuracy of HER2 and MSI/MMR status. Results A total of 188 patients were enrolled. In 139 eligible patients, 275 potentially actionable mutations were found in 12 selected CRC-related genes (APC, TP53, KRAS, NRAS, BRAF, FBXW7, PIK3CA, ERBB2, SMAD4, NF1, EGFR, MET). Of these, 32% were found both in ctDNA and tissue; 54% were found in ctDNA only; 12% in tissue only, and 2% were discordant. For RAS/BRAF mutations, which are required for anti-EGFR treatment decisions, the concordance rate between ctDNA and tissue NGS was 83.1%, and the concordance was higher in patients with higher ctDNA concentrations. For 9 patients with potentially actionable copy number variations (CNV) in EGFR, HER2, MET, and FGFR1, 3 cases were found by both assays; 4 were found by ctDNA only, and 2 were found by tissue only. ctDNA NGS correctly predicted MSI/MMR status in 2 out of 4 patients with MSI-H/dMMR; in the 2 other patients, the MSI and MMR statuses were different, suggesting potential false positivity. In addition, the fold changes in ctDNA dynamics during treatment significantly correlated with changes in tumor size and CEA levels, as well as with droplet digital PCR copy number fold changes. In a patient with MET amplification, ctDNA NGS identified MET Y1230H as a potential acquired resistance mutation after crizotinib treatment, which responded to cabozantinib but not to capmatinib. Conclusions Initial tumor profiling using ctDNA NGS yielded outcomes comparable to those of tumor tissue NGS in guiding treatment for patients with newly diagnosed mCRC, thereby suggesting its utility as an initial profiling method in mCRC. Citation Format: Yongjun Cha, Bun Kim, Dong Woon Lee, Hyun Yang Yeo, Chang Won Hong, Kyung Su Han, Byung Chang Kim, Hee Jin Chang. Assessing the clinical value of ctDNA sequencing for initial tumor profiling in metastatic colorectal cancer patients with sufficient tumor tissue [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 2498.

Abstract 3800: Next generation of spatial biology: High-throughput multiplexed Imaging Mass Cytometry™ with whole slide modes

Abstract Gaining spatial insights into the cellular composition of tumor tissue has tremendous potential to inform clinical and translational researchers about mechanisms behind spatial predictors of immunotherapy success, disease etiology and progression. Imaging Mass Cytometry™ (IMC™) is a high-plex spatial biology imaging technique that enables deep characterization of the diversity and complexity of the tumor microenvironment (TME). IMC supports detailed assessment of cell phenotype and function using 40-plus metal-tagged antibodies simultaneously on a single slide without issues associated with fluorescence-based spectral overlap, tissue autofluorescence or implementation of multiple washing and acquisition cycles. Currently, IMC enables user-defined regions of interest (ROI) in tissues to evaluate cellular and structural composition. To enhance the IMC user experience, we developed two new whole slide imaging (WSI) modes which enable streamlined workflows using ultrafast preview mode (PM) and high-throughput tissue mode (TM). PM samples the entire tissue at predefined spacing to rapidly capture a low-resolution image of all expressed markers in the antibody panel. PM generates an image in minutes to enable informed ROI placements while leaving the stained tissue intact for higher-resolution imaging. PM and TM are designed so acquisitions can easily occur on the same slide without additional processing steps. TM rapidly acquires images of the whole tissue at lower resolution (7 μm pixel size) at a quality that can be used for quantitative analysis of the tissue spatial biology. Specifically designed for high-throughput applications, TM in combination with a newly available 40-slide loader for the Hyperion XTi™ Imaging System permits automated and continuous imaging of more than 40 large tissue samples (400 mm2 per tissue) per week. We showcase the application of WSI modes using the newly developed Maxpar® Human Immuno-Oncology IMC Panel Kit. The 31-marker panel was combined with catalog antibodies to create a 40-plus-marker panel that expands the ability to conduct comprehensive high-plex tumor and immune cell profiling. Tumor tissue microarrays (TMA) and whole tumor tissue sections were stained with the expanded panel. Single-cell analysis of selected ROIs, on whole tumor sections and TMAs, guided by PM data successfully provided quantitative analyses of spatial biology at single-cell resolution. In parallel, TM on whole tumor sections followed by pixel-based analysis provided a spatially resolved quantitative assessment of specific tumor and immune components of the TME. This work demonstrates the expanded capabilities of IMC and establishes it as a reliable high-plex spatial biology imaging platform with high-throughput imaging capabilities ideally suited for translational and clinical applications. For Research Use Only. Not for use in diagnostic procedures. Citation Format: Qanber Raza, Thomas D. Pfister, Jyh Yun Chwee, Liang Lim, David Howell, Nikesh Parsotam, David King, Christina Loh. Next generation of spatial biology: High-throughput multiplexed Imaging Mass Cytometry™ with whole slide modes [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 3800.

Analytical Performance Evaluation of a 523-Gene Circulating Tumor DNA Assay for Next-Generation Sequencing–Based Comprehensive Tumor Profiling in Liquid Biopsy Samples

Next-generation sequencing (NGS)-based comprehensive tumor profiling from liquid biopsy samples can significantly improve diagnosis and monitoring of tumors when high-quality tissue material is difficult to obtain. In addition, it offers the potential to capture the entire complexity of the tumor, which is particularly important for highly heterogeneous or metastatic tumors. Here, we report the findings of an analytical performance evaluation of the TruSight Oncology 500 circulating tumor DNA (ctDNA) assay, a 523-gene NGS panel developed for ctDNA-based comprehensive genomic profiling of tumors, using reference and patient samples. Using 30 ng cell-free DNA, the assay showed high sensitivity and low variant detection variability for single-nucleotide variants, insertions and deletions, and fusions down to a variant allele frequency (VAF) of 0.5% in the reference samples and VAFs that were highly concordant with previous digital droplet PCR results in the patient samples. At reduced input amounts (20, 15, and 5 ng) and below VAFs of 0.5%, sensitivity was considerably lower and variant detection variability increased. Covering 523 tumor-associated genes, the assay demonstrated a convincing performance comparable to NGS-based ctDNA assays with smaller gene panels, highlighting its value to screen large numbers of different genes.

Current and Emerging Strategies to Treat Urothelial Carcinoma.

Urothelial cell carcinoma (UCC, bladder cancer, BC) remains a difficult-to-treat malignancy with a rising incidence worldwide. In the U.S., UCC is the sixth most incident neoplasm and ~90% of diagnoses are made in those >55 years of age; it is ~four times more commonly observed in men than women. The most important risk factor for developing BC is tobacco smoking, which accounts for ~50% of cases, followed by occupational exposure to aromatic amines and ionizing radiation. The standard of care for advanced UCC includes platinum-based chemotherapy and programmed cell death (PD-1) or programmed cell death ligand 1 (PD-L1) inhibitors, administered as frontline, second-line, or maintenance therapy. UCC remains generally incurable and is associated with intrinsic and acquired drug and immune resistance. UCC is lethal in the metastatic state and characterized by genomic instability, high PD-L1 expression, DNA damage-response mutations, and a high tumor mutational burden. Although immune checkpoint inhibitors (ICIs) achieve long-term durable responses in other cancers, their ability to achieve similar results with metastatic UCC (mUCC) is not as well-defined. Here, we discuss therapies to improve UCC management and how comprehensive tumor profiling can identify actionable biomarkers and eventually fulfill the promise of precision medicine for UCC patients.

219P Multicentric evaluation of amplicon-based next-generation sequencing solution for local comprehensive molecular tumor profiling

219P Multicentric evaluation of amplicon-based next-generation sequencing solution for local comprehensive molecular tumor profiling

Abstract 1945: Racial disparities in comprehensive cancer tumor profiling testing from real-world data of 100,000 cancer patient records

Abstract Inequities in cancer patient outcomes and healthcare utilization by race and ethnicity are coming to the forefront. Overcoming these inequities requires a better understanding of the extent of such disparities across cancer types and medical interventions. Here, we studied the race and ethnicity of patients receiving tumor profiling for therapy decision support by examining real-world data (RWD) from 100,000 de-identified patient records across major cancer types. Tumors and normal tissue (when available) were sequenced with the Tempus xT assay, a 648-gene next-generation sequencing panel. Since race and ethnicity metadata are frequently incomplete in RWD, we imputed these categories from continental genetic ancestry inferred from genotypes of 654 ancestry informative markers obtained from sequencing data. Using a heuristic that combines East Asian, African, Amerindian, European, and South Asian ancestry proportions, we imputed Asian (A), Non-Hispanic Black (NHB), Hispanic/Latino/Native American (HIS), and Non-Hispanic White (NHW) categories with <2% error rate (based on available metadata), leaving 3% of unclassified patients due to complex admixture. NHW patients are overrepresented in the cohort at 72% as compared to NHB (11%), HIS (9%) and ASN (5%). We compared the distributions of patients by race/ethnicity categories across specific cancer types against the overall cohort race/ethnicity distributions. These distributions were compared to the expected distribution under the null hypothesis of no relationship by the χ2 test of independence. Significantly (p<0.05) over/under-represented categories in each cancer type were compared to ranks of incidence by race/ethnicities in SEER program data at the national level. Differences are of a smaller magnitude in NHW as compared to the other races/ethnicities. We observed several differences that are concordant with SEER; for example, overrepresentations of NHB in breast, colorectal, and endometrial cancers, which correspond to high incidences of these cancers for NHB. Notably, we observed several discordances with SEER incidence rate ranks, including underrepresentation of NHB in pancreatic and urinary tract cancers, underrepresentation of NHW in breast and colorectal cancers, overrepresentation of ASN in gallbladder and biliary cancer, and overrepresentation of HIS in colorectal cancer. These differences can be the result of a complex interplay of factors, such as stage of the disease at sequencing, access to early-stage curative therapies, comorbidities/cofactors, access to care, insurance, socioeconomic status, and others. Nonetheless, our results show that analysis of racial disparities can be performed with RWD, and this information can be useful in studying causes and devising strategies to improve healthcare equity. Citation Format: Francisco M. De La Vega, Brooke Rhead, Yannick Pouliot, Justin Guinney. Racial disparities in comprehensive cancer tumor profiling testing from real-world data of 100,000 cancer patient records [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1945.

Peritoneal metastases from primary appendiceal and colorectal carcinomas demonstrate distinct molecular identities on comprehensive tumor analysis.

Published data comparing peritoneal metastases from appendiceal cancers (pAC) andcolorectal cancers (pCRC) remain sparse. We compared pAC and pCRC using comprehensive tumor profiling (CTP). CTP was performed, including next-generation sequencingand analysis of copy number variation (CNV), microsatellite instability (MSI) and tumor mutational burden (TMB). One hundred thirty-sixpAC and 348 pCRC samples underwent CTP. The cohorts' age and gender were similar. pCRC demonstrated increased pathogenic variants (PATHs) in APC (48% vs. 3%, p < 0.01), ARID1A (12% vs. 2%, p < 0.01), BRAF (12% vs. 2%, p < 0.01), FBXW7 (7% vs. 2%, p < 0.01), KRAS (52% vs. 41%, p < 0.05), PIK3CA (15% vs. 2%, p < 0.01), and TP53 (53% vs. 23%, p < 0.01), and decreased PATHs in GNAS (8% vs. 31%, p < 0.01). There was no difference in CNV, fusion rate, or MSI. Median TMB was higher in pCRC (5.8 vs. 5.0 mutations per megabase, p = 0.0007). Rates of TMB-high tumors were similar (pAC 2.1% vs.pCRC 9.0%, p = 0.1957). pCRC had significantly more TMB-high tumors at lower thresholds. Despite a reduced overall TMB, pAC demonstrated mutations distinct from those seen in pCRC. These may serve as discrete biomarkers for future study.

Molecular profiling of male breast cancer by multigene panel testing: Implications for precision oncology.

Compared with breast cancer (BC) in women, BC in men is a rare disease with genetic and molecular peculiarities. Therapeutic approaches for male BC (MBC) are currently extrapolated from the clinical management of female BC, although the disease does not exactly overlap in males and females. Data on specific molecular biomarkers in MBC are lacking, cutting out male patients from more appropriate therapeutic strategies. Growing evidence indicates that Next Generation Sequencing (NGS) multigene panel testing can be used for the detection of predictive molecular biomarkers, including Tumor Mutational Burden (TMB) and Microsatellite Instability (MSI). In this study, NGS multigene gene panel sequencing, targeting 1.94 Mb of the genome at 523 cancer-relevant genes (TruSight Oncology 500, Illumina), was used to identify and characterize somatic variants, Copy Number Variations (CNVs), TMB and MSI, in 15 Formalin-Fixed Paraffin-Embedded (FFPE) male breast cancer samples. A total of 40 pathogenic variants were detected in 24 genes. All MBC cases harbored at least one pathogenic variant. PIK3CA was the most frequently mutated gene, with six (40.0%) MBCs harboring targetable PIK3CA alterations. CNVs analysis showed copy number gains in 22 genes. No copy number losses were found. Specifically, 13 (86.7%) MBCs showed gene copy number gains. MYC was the most frequently amplified gene with eight (53.3%) MBCs showing a median fold-changes value of 1.9 (range 1.8-3.8). A median TMB value of 4.3 (range 0.8-12.3) mut/Mb was observed, with two (13%) MBCs showing high-TMB. The median percentage of MSI was 2.4% (range 0-17.6%), with two (13%) MBCs showing high-MSI. Overall, these results indicate that NGS multigene panel sequencing can provide a comprehensive molecular tumor profiling in MBC. The identification of targetable molecular alterations in more than 70% of MBCs suggests that the NGS approach may allow for the selection of MBC patients eligible for precision/targeted therapy.

EXTH-20. IDENTIFICATION OF EX VIVO THERAPEUTIC VULNERABILITIES IN DIFFUSE GLIOMAS USING A FUNCTIONAL GENOMICS APPROACH

Abstract Diffuse gliomas are aggressive brain tumors that lack effective therapies. Despite advances in the molecular characterization of gliomas, targeted treatments have failed to improve patient outcomes to date. Here, we employed a functional genomics approach to rapidly identify therapeutic vulnerabilities and provide a strategy to individualize patient treatment. Our process combined comprehensive genomic and transcriptomic tumor profiling, custom algorithms and visualization software for data integration, and preclinical 3-dimensional ex vivo models (mcancers) for drug screening to assess response to therapeutic agents targeting specific molecular alterations. A total of 32 cases, including 5 oligodendrogliomas (1 primary and 4 recurrent), 5 astrocytomas (1 primary and 4 recurrent), and 22 glioblastomas (5 primary and 17 recurrent), were included in the study. Immunohistochemistry and integrated genomics confirmed that mcancers maintain key molecular alterations, including mutations, chromosomal rearrangements and focal amplifications, cellularity, and heterogeneity, faithfully recapitulating the original tumor. A total of 119 single drugs and 183 combinations were tested, including a panel of 26-brain penetrant targeted agents (18 single drugs and 8 combinations) that was curated to target common glioma oncogenic pathways and was tested across all cases. A strong correlation was observed between therapeutic vulnerabilities of glioma mcancers to targeted agents (EGFR, MET, PDGFR, HDAC inhibitors, etc.) and clinical experience. Single agent activity of panel compounds in the ex vivo setting was generally poor, with only 35% mCancers exhibiting &amp;gt; 70% growth inhibition at Cmax. In contrast, combination strategies, especially ones including epigenetic drugs, were significantly more effective (affecting 22 out of 32 mCancers), underscoring the molecular complexity of gliomas and the need for clinical trials testing combination treatments. Our data identified a number of novel combination treatments with significant efficacy across glioma subtypes and molecular profiles, suggesting that our functional genomics pipeline can guide treatment selection and inform clinical trial enrollment for gliomas.

Personalized Therapy based on Several Parameters of Comprehensive Tumour Profiling: a Challenge in NEBC

Primary neuroendocrine breast cancer (NEBC) is a rare entity accounting for &lt;0.1% of all breast carcinomas and &lt;1% of all neuroendocrine tumours. In most cases, treatment strategies in NEBC are empirical in absence of prospective trial data on NEBC cohorts. Herein, we present two case reports diagnosed with anaplastic large and small cell NEBC. After initial therapies analogue to those of breast cancer (BC) or neuroendocrine cancers (NEC) had failed, comprehensive tumour profiling was applied, leading to individualized treatment options for both patients. Due to targetable mutations and important co-alterations, the PIK3-mTOR pathway was highlighted from different aspects in both patients. The epicrisis of the two patients exemplifies how to manage the challenge of rare and difficult to treat cancers and how new diagnostic tools contribute to medical management.

Molecular profiles of endometrial cancer tumors among Black patients

ObjectivesDisparate outcomes exist between Black and White patients with endometrial cancer (EC). One contributing factor is the disproportionately low representation of Black patients in clinical trials and in tumor molecular profiling studies. Our objective was to investigate molecular profiles of ECs in a cohort with a high proportion of tumors from Black patients. MethodsA total of 248 EC samples and self-reported race data were collected from 6 institutions. Comprehensive tumor profiling and analyses were performed by Caris Life Sciences. ResultsTumors from 105 (42%) Black and 143 (58%) White patients were included. Serous histology (58% vs 36%) and carcinosarcoma (25% vs 16%), was more common among Black patients, and endometrioid was less common (17% vs 48%) (p < 0.01). Differences in gene mutations between cohorts corresponded to observed histologic differences between races. Specifically, TP53 mutations were predominant in serous tumors. In endometrioid tumors, mutations in ARID1A were the most common, and high rates of MSI-H, MMRd, and TMB-H were observed. In carcinosarcoma tumors, hormone receptor expression was high in tumors of Black patients (PR 23.4%, ER 30.8%). When stratified by histology, there were no significant differences between tumors from Black and White women. ConclusionsThis cohort had a high proportion of tumors from Black women. Distinct molecular profiles were driven primarily by more aggressive histologic subtypes among Black women. Continued effort is needed to include Black women and other populations under-represented in EC molecular profiling studies as targeted therapies and personalized medicine become mainstream.

Next-Generation Sequencing Concordance Analysis of Comprehensive Solid Tumor Profiling between a Centralized Specialty Laboratory and the Decentralized Personal Genome Diagnostics elio Tissue Complete Kitted Solution

Genomic tumor profiling by next-generation sequencing (NGS) allows for large-scale tumor testing to inform targeted cancer therapies and immunotherapies, and to identify patients for clinical trials. These tests are often underutilized in patients with late-stage solid tumors and are typically performed in centralized specialty laboratories, thereby limiting access to these complex tests. Personal Genome Diagnostics Inc., elio tissue complete NGS solution is a comprehensive DNA-to-report kitted assay and bioinformatics solution. Comparison of 147 unique specimens from >20 tumor types was performed using the elio tissue complete solution and Foundation Medicine's FoundationOne test, which is of similar size and gene content. The analytical performance of all genomic variant types was evaluated. In general, the overall mutational profile is highly concordant between the two assays, with agreement in sequence variants reported between panels demonstrating >95% positive percentage agreement for single-nucleotide variants and insertions/deletions in clinically actionable genes. Both copy number alterations and gene translocations showed 80% to 83% positive percentage agreement, whereas tumor mutation burden and microsatellite status showed a high level of concordance across a range of mutation loads and tumor types. The Personal Genome Diagnostics Inc., elio tissue complete assay is comparable to the FoundationOne test and will allow more laboratories to offer a diagnostic NGS assay in house, which will ultimately reduce time to result and increase the number of patients receiving molecular genomic profiling and personalized treatment.

Abstract 2221: Whole transcriptome sequencing reveals oncogenic fusions in melanoma

Abstract Introduction: Oncogenic gene fusions are frequently identified in different cancers; however, the exact incidence of oncogenic fusions in melanoma is not well defined. Several targeted therapies are approved and considered the standard of care for patients whose solid or hematologic tumors harbor particular gene fusions, but their role as targets in melanoma also remains undelineated. We sought to determine the prevalence of oncogenic fusions in metastatic or locally advanced melanoma. Methods: We retrospectively analyzed data from formalin-fixed paraffin-embedded (FFPE) tumor samples sent to a commercial CLIA-certified laboratory (Caris Life Sciences) from February 2019 to July 2020. Samples were profiled by next-generation sequencing of a 592-gene DNA panel, whole transcriptome sequencing, and immunohistochemistry (IHC). Results: Melanoma specimens were analyzed from 1,255 subjects, of whom 478 (38.1%) were female and 777 (61.9%) were male, with a median age of 67 years. Of these specimens, 780 (63.1%) were from metastatic sites. We identified 33 (2.6%) cases with in-frame oncogenic fusions (14 novel) including 21 BRAF fusions and 4 RAF1 fusions, as well as fusions involving PRKCA (n=4), TERT (n=2), AXL (n=1), and FGFR3 (n=1). PD-L1 expression by IHC (SP142 or 28-8 antibody) was detected in 512 (42.5%) specimens, including 10 (32.3%) of the fusion-positive tumors, while 572 (47.4%) specimens were TMB-High (≥10 mutations/Mb), including 11 (33.3%) of the fusion-positive tumors, suggesting these patients may respond to immunotherapy. We identified 796 (63.4%) cases with RAS/RAF pathogenic or likely pathogenic mutations, including 373 (30.0%) BRAF p.V600X mutations. With the exception of a single BRAF p.S467L (Class 3, “kinase-dead”) mutation, the absence of BRAF mutations in BRAF fusion-positive tumors suggests these fusions are oncogenic drivers. However, tumors harboring PRKCA and TERT fusions were each detected with at least one MAPK pathway co-alteration (NRAS, NF1, or BRAF p.V600E mutation). Fusion transcripts with unknown pathogenicity were also detected in 668 (53.2%) cases. RNA expression analysis of key molecular pathways (including Wnt/β-catenin, PI3K/AKT/MTOR, DNA repair, INFG, and JAK/STAT) showed a high degree of variability among fusion-positive tumors and other mutational subgroups, which may reflect the heterogeneity common to melanoma, prior treatments or development of various resistance mechanisms. Conclusions: Oncogenic gene fusions are rare in melanoma when compared with other genetic variants. We identified potentially actionable fusions as well as co-alterations in fusion-positive cases, with notably mutual exclusivity of BRAF fusions and p.V600X mutations. The data suggest that targetable variants, including oncogenic fusions, may be identified in melanoma with comprehensive tumor profiling and provide patients with personalized treatment or clinical trial options. Citation Format: Sourat Darabi, Andrew Elliott, David R. Braxton, Jia Zeng, Kelsey Poorman, Jeffrey Swensen, Geoffrey T. Gibney, Justin C. Moser, Thuy Phung, Michael B. Atkins, Gino K. In, Wolfgang Michael Korn, Burton L. Eisenberg, Michael J. Demeure. Whole transcriptome sequencing reveals oncogenic fusions in melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2221.

Robust prediction of response to immunotherapy in a mixed cohort of previously treated and immunotherapy-naive melanoma patients.

e21548 Background: While immune checkpoint blockade (ICB) has become a pillar of cancer treatment, biomarkers that consistently predict patient response remain elusive due to the complex mechanisms driving immune response to tumors. We hypothesized that a multi-dimensional approach modeling both tumor and immune-related molecular mechanisms would better predict ICB response than simpler mutation-focused biomarkers. Methods: Pre-treatment samples from 51 unresectable, stage III/IV melanoma patients who underwent anti-PD-1 therapy were characterized to assess factors influencing response. RECIST criteria were used to evaluate tumor response to therapy, with a median follow-up of 24 months. For each patient, a single paired FFPE tumor and normal blood sample was collected and profiled using Personalis’ ImmunoID NeXT Platform; an augmented exome/transcriptome platform and analysis pipeline which produces comprehensive tumor mutation information, gene expression profiling, neoantigen characterization, HLA typing and LOH, TCR repertoire profiling, and tumor microenvironment profiling. These data were then integrated to form a composite neoantigen presentation score (NEOPS) for each patient. Results: We demonstrate increasing predictive power with deeper modeling of neoantigens and immune-related resistance mechanisms to ICB. Neoantigen burden score (NBS) was derived from SHERPA, our exome and transcriptome integrative neoantigen prediction model. We found that NBS more significantly stratified responders and non-responders (P = 0.016) than TMB alone (P = 0.049). A broader score, NEOPS, incorporated DASH, to capture HLA allele-specific loss of heterozygosity and additional antigen presenting machinery resistance mechanisms. NEOPS consequently demonstrated a stronger association with response (p = 0.002). We used area under the curve (AUC) to benchmark NEOPS performance against established mutational and transcriptomic predictive models, demonstrating the model was more predictive of response in both the treatment naive setting, as well as in a mixed cohort of treatment-naive and experienced patients. These findings were confirmed in an independent cohort of patients (n = 110), suggesting that NEOPS is a robust, novel biomarker of ICB response in melanoma. Conclusions: Given the complex nature of resistance to immunotherapy, as well as potential toxicities associated with treatment, there is a need for biomarkers that can more accurately predict response. Here we demonstrate that NEOPS can significantly improve stratification of patient response. We also demonstrate that data intensive biomarkers like NEOPS can be clinically practical, with comprehensive tumor profiling in our clinical cohort achieved using very limited tumor tissue.