CLIA-certified Laboratory Research Articles

Prognostic implications of epidermal and platelet-derived growth factor receptor alterations in 2 cohorts of IDHwt glioblastoma.

BackgroundGlioblastoma remains a deadly brain cancer with dismal prognosis. Genetic alterations, including IDH mutations, 1p19q co-deletion status and MGMT promoter methylation have been proven to be prognostic and predictive to response to treatment in gliomas. In this manuscript, we aimed to correlate other mutations and genetic alterations with various clinical endpoints in patients with IDH-wild-type (IDHwt) glioblastoma.MethodsWe compiled a comprehensive clinically annotated database of IDHwt GBM patients treated at the Ohio State University Wexner Medical Center for whom we had mutational data through a CLIA-certified genomic laboratory. We then added data that is publicly available from Memorial Sloan Kettering Cancer Center through cBioPortal. Each of the genetic alterations (mutations, deletions, and amplifications) served as a variable in univariate and multivariate Cox proportional hazard models.ResultsA total of 175 IDHwt GBM patients with available MGMT promoter methylation data from both cohorts were included in the analysis. As expected, MGMT promoter methylation was significantly associated with improved overall survival (OS). Median OS for MGMT promoter methylated and unmethylated GBM was 26.5 and 18 months, respectively (HR 0.45; P = .003). Moreover, EGFR/ERBB alterations were associated with favorable outcome (HR of 0.37 (P = .003), but only in MGMT promoter unmethylated GBM. We further found that patients with EGFR/ERBB alterations who also harbored PDGFRA amplification had a significantly worse outcome (HR 7.89; P = .025).ConclusionsOur data provide further insight into the impact of genetic alterations on various clinical outcomes in IDHwt GBM in 2 cohorts of patients with detailed clinical information and inspire new therapeutic strategies for IDHwt GBM.

EPEN-25. EXCEPTIONAL CLINICAL AND IMAGING RESPONSE TO TRK-INHIBITION IN A PATIENT WITH SUPRATENTORIAL EPENDYMOMA HARBORING NTRK2 GENE FUSION

BACKGROUNDPatients with metastatic pediatric ependymoma have limited therapeutic options and poor outcomes. Approximately ¾ of supratentorial ependymomas are driven by C11ORF95-RELA fusions, and the remaining by a heterogeneous group of fusion events. We present a six year-old male diagnosed with supratentorial ependymoma with leptomeningeal carcinomatosis harboring an NTRK2-fusion. Local and distant multifocal, intracranial and intraspinal tumor recurrence occurred seven months following gross total resection of the primary lesion and proton beam craniospinal irradiation.METHODSDNA and RNA from FFPE tumor were used for targeted sequencing using an 81-gene fusion panel and 124-gene mutation panel. Separately, capture transcriptome sequencing, exome sequencing, and copy number array were performed as part of the Texas KidsCanSeq study, an NHGRI/NCI-funded Clinical Sequencing Evidence-Generating Research (CSER) consortium project. All sequencing was carried out in CLIA-certified laboratories.RESULTSAn in-frame fusion between 5’ exons 1–3 of KANK1 and 3’ exons 16–21 of NTRK2, predicted to retain the kinase domain, was identified. At tumor recurrence, therapy was initiated with Larotrectinib, an FDA-approved pan-TRK inhibitor. Clinical improvement in cognitive speed, motor strength, and coordination was observed at two weeks with significant tumor response on MRI at two and four months.CONCLUSIONTRK gene fusions have not previously been reported in ependymoma. Further tumor characterization by methylation profiling is underway and will be of diagnostic interest given the apparent discordance between tumor histology and molecular findings. This case highlights the potential impact of clinical genomic analysis for children with CNS tumors.

HLA Genotyping in Synovial Sarcoma: Identifying HLA-A*02 and Its Association with Clinical Outcome.

To determine if a targeted exome panel utilizing matched normal DNA can accurately detect germline and somatic HLA genes in patients with synovial sarcoma (SS) and whether select HLA-A*02 genotypes are prognostic or predictive of outcome in metastatic SS. Patients with metastatic SS consented to HLA typing by a Clinical Laboratory Improvement Amendments (CLIA)-certified test to determine eligibility for a clinical trial of NY-ESO-1-specific engineered T cells restricted to carriers of HLA-A*02:01, -A*02:05, or -A*02:06 (HLA-A*02 eligible). HLA genotype was determined from Memorial Sloan Kettering Integrated Molecular Profiling of Actionable Cancer Targets (MSK-IMPACT), where feasible, and somatic loss of heterozygosity (LOH) in HLA alleles was identified. Overall survival (OS) was estimated and stratified by HLA-A*02 eligibility. A total of 23 patients had HLA genotyping by a CLIA-certified lab and MSK-IMPACT. Ninety percent (108/110) of the sequenced alleles were concordant between IMPACT and the outside lab. LOH of HLA genes was detected in three tumors, one had loss of HLA-A*02:01. In total, 66 patients were screened for T-cell therapy and 20 (30%) were HLA-A*02 eligible on outside testing. Univariate analysis of OS from the time of metastasis found HLA-A*02 eligibility was marginally associated with shorter OS [HR = 1.95; 95% confidence interval (CI), 0.995-3.813; P = 0.052]. On multivariate analysis, older age and larger tumor size, but not HLA-A*02 eligibility, were significantly associated with decreased OS. HLA-A*02 eligibility did not impact OS after chemotherapy or pazopanib in the metastatic setting. Targeted gene panels like MSK-IMPACT may accurately report HLA type and identify loss of somatic HLA alleles. In a multivariable model, HLA-A*02 eligibility was not significantly associated with OS in patients with metastatic SS.

Unrelated Individuals of Self-Reported White/Caucasian Ancestry with the P.Val142Ile (V122I) Variant of Hereditary Transthyretin-Mediated Amyloidosis are Phenotypically Similar to those with Self-Reported Black/African-American Ancestry

Introduction Hereditary transthyretin-mediated (hATTR) amyloidosis is an autosomal dominant disease characterized by extracellular deposition of amyloid fibrils composed of transthyretin (TTR) protein, with the majority of patients developing a mixed phenotype of polyneuropathy and cardiomyopathy. The most common variant in the United States is p.Val142Ile (V122I) with a prevalence of 3-4%, most commonly observed in the Black/African-American population. To facilitate earlier diagnosis, Alnylam Pharmaceuticals, Inc and Invitae have partnered to offer Alnylam Act®, a no-charge sponsored genetic testing and counseling program for individuals ≥18 years old with suspected diagnosis of hATTR amyloidosis or a confirmed family history of hATTR amyloidosis. A novel cohort of self-reported White/Caucasian unrelated individuals with the p.Val142Ile (V122I) variant has been identified by this program. Methods Between April 2017 and March 2020, 21,235 unrelated individuals were tested for TTR variants in a CLIA-certified laboratory. We analyzed all unrelated individuals with the p.Val142Ile (V122I) variant discovered through the Alnylam Act® program, including all reported demographic (e.g., age, ancestry) and symptomatic characteristics available. Results In total, we observed 795 unrelated individuals with the p.Val142Ile (V122I) variant. We identified 29 unrelated individuals of White/Caucasian ancestry and who carry the p.Val142Ile (V122I) variant, which represents 11.4% of the 254 unrelated White/Caucasian individuals with a TTR positive variant found within the Alnylam Act® program to date. The reported symptoms in these individuals are similar to the Black/African-American p.Val142Ile (V122I) population in this cohort (Figure). Conclusions A novel cohort of White/Caucasian unrelated individuals with the p.Val142Ile (V122I) TTR variant has been identified with similar reported clinical features compared to those with Black/African-American ancestry and the same variant. This is the largest single cohort of individuals of White/Caucasian ancestry with the p.Val142Ile (V122I) variant to be reported thus far, demonstrating the importance of TTR testing in individuals with symptoms of hATTR amyloidosis regardless of self-reported ancestry.

Asymptomatic Individuals with Family History of Hereditary Transthyretin-Mediated Amyloidosis can be Detected almost Two Decades Earlier Than Symptomatic Probands

Introduction Hereditary transthyretin-mediated (hATTR) amyloidosis is an autosomal dominant disease characterized by extracellular deposition of amyloid fibrils composed of transthyretin (TTR) protein, with the majority of patients developing a mixed phenotype of polyneuropathy and cardiomyopathy. To facilitate earlier diagnosis, Alnylam Pharmaceuticals, Inc and Invitae have partnered to offer Alnylam Act®, a no-charge sponsored genetic testing and counseling program for individuals ≥18 years old with suspected diagnosis or a confirmed family history of hATTR amyloidosis. We sought to determine if reported family history and/or symptoms correlated to earlier detection of TTR variants in this cohort. Hypothesis We hypothesized that age at time of testing was earlier in individuals with a reported family history compared to probands with reported symptoms and no family history. Methods From April 2017 to March 2020, 21,235 unrelated individuals were tested for TTR variants in a CLIA-certified laboratory. We analyzed three groups: (1) probands with reported symptoms and positive family history, (2) probands with reported symptoms without family history, and (3) asymptomatic individuals with family history of hATTR amyloidosis. Family members who were also tested through the program were excluded from analysis. Results The overall yield of positive TTR genetic tests in symptomatic probands with no reported family history was 778/20134 (3.9%), which was lower compared to those with reported family history (symptomatic 26.7%; asymptomatic 31.5%; Table 1). Among 778 symptomatic probands with no reported family history and positive TTR results, mean age at testing was 70.5 years. In contrast, those with family history were tested at an earlier age (symptomatic 59.3 years, p Conclusions Mean age at testing of TTR positive asymptomatic individuals with a reported familyhistory of hATTR amyloidosis is lower than TTR positive symptomatic probands without a family history by almost two decades, with an 8-fold higher yield (OR 11.4, 95% CI: 9.5-13.8). Here, we demonstrate knowledge of family history alone is a powerful predictor of the presence of TTR variants, emphasizing the importance of family testing once a confirmed case is identified, which may facilitate earlier diagnosis.

Characterization of Clinical Cases of Malignant PEComa via Comprehensive Genomic Profiling of DNA and RNA

Purpose: Perivascular epithelioid cell tumor (PEComa) is a rare mesenchymal soft tissue neoplasm often linked to mTOR pathway activation via TSC2 mutation. We analyzed a series of 31 consecutive metastatic PEComa (mPEComa) cases using a combined DNA/RNA hybrid capture-based comprehensive genomic profiling (CGP) assay to assess the genomic landscape of mPEComa. Patients and Methods: Formalin-fixed, paraffin-embedded (FFPE) blocks or slides were obtained from tumors from 31 unique patients with mPEC­oma. DNA and RNA were extracted and CGP was performed on 405 genes using a targeted next-generation sequencing (NGS) assay in a CLIA-certified lab. Results: All cases had locally advanced or metastatic disease, and 58% of patients were female with a median age of 50 years (range 8–76), and 17 and 14 specimens were from primary and metastatic sites, respectively. One hundred genomic alterations were identified in the cohort, with an average of 3.2 genomic alterations/case including alterations in TSC2 32.3% of cases (10), TSC1 9.6% (3), TFE3 16.1% (5, all fusions), and folliculin (FLCN) 6.4% (2), with all occurring in mutually exclusive fashion. Of TSC2 mutant cases, 70% had biallelic inactivation of this locus, as were 100% of TSC1 mutant cases. Two TSC1/2 wildtype cases harbored truncating mutations in FLCN, both of which were under LOH. Five TFE3 fusion cases were identified including the novel 5′ fusion partner ZC3H4. Conclusions: We describe for the first time mPEComa cases with FLCN mutations under LOH, further characterizing dysregulation of the mTOR pathway as a unifying theme in mPEC­oma. Cumulatively, we demonstrate the feasibility and potential utility of segregating mPEComa by TSC, TFE3, and FLCN status via CGP in clinical care.

133P HER2 transcription status of diverse solid tumours with clinical implications

Utility of HER2 mRNA expression as a potential therapeutic biomarker is unclear. We previously reported the discordant HER2 status between copy number variation (CNV), mRNA and immunohistochemistry (IHC) from a large multi-institute dataset. Herein, we investigated comprehensive HER2 status by cancer (CA) type with clinical outcome among HER2 positive patients (pts) at University of California San Diego (UCSD). By Paradigm Diagnostics (CLIA-certified laboratory), HER2 status was evaluated using IHC, qRT-PCR and NGS between 2015-2019. We evaluated pts who had all three HER2 testing available by tumor type, with UCSD outcomes included. Of 5,305 pts, 723 had CNV, mRNA and IHC evaluated (Table). Among diverse solid tumors, HER2 positivity in at least one test were seen in 76.6% (49/64) of NSCLC and 58.8% (20/34) of pancreas CA. Sole mRNA positivity (CNV-/IHC-) were found ≥35% in NSCLC, pancreatic and colorectal cancers. Among 53 UCSD pts who had all three HER2 testing, 22 pts had ≥ 1 positive HER2 test (including 24.5% [13/53] pts with mRNA+ only). We identified 7 pts that were given Anti-HER2 therapy (Tx). One pt with CNV+/mRNA+/IHC+ metastatic esophageal CA achieved complete response with chemotherapy plus trastuzumab followed by trastuzumab maintenance (progression-free survival [PFS]: 48 + months). One pt with metastatic cholangiocarcinoma mRNA+ (CNV and IHC unable to perform) achieved durable partial response with dual-anti-Her2 Tx (trastuzumab and lapatinib) (PFS: 24+ months). In contrast, 5 pts had poor outcomes despite anti-HER2 Tx, given after 3-5 previous Tx lines.Table 133PHER2 status per cancer typeCancer Type (N>10)TotalCNV + mRNA + IHC +CNV+ mRNA + IHC -CNV+ mRNA- IHC+CNV- mRNA+ IHC+CNV+ mRNA- IHC-CNV- mRNA+ IHC-CNV- mRNA- IHC+Any +NN%N%N%N%N%N%N%N%Multi-institutionBreast2603513.5––124.6–4416.983.19938.1Colon8844.5––22.3–3236.422.34045.5NSCLC6411.6––1625–3046.923.14976.6Pancreas34–––38.8–1647.112.92058.8Ovary3013.3––26.7–62013.31033.3Sarcoma27–CUP2528––141414416936Prostate21–––––14.814.829.5Hepatobiliary20210––––1515420Bladder19421.1––421.1–15.315.31052.6Gastroesophageal16212.5–16.3––16.316.3531.3H&N14–Rectal14214.317.1–––535.7–857.1Appendix11–UCSD5323.8––47.5–1324.535.72241.5 Open table in a new tab We found diverse HER2 expression patterns among solid tumors, including difficult to treat cancer of unknown primary, pancreatic CA, and hepatobiliary CA. Although it is a small cohort, patients with HER2 mRNA overexpression could respond to anti-HER2 regimen. Further clinical investigation is warranted.

Prevalence of Phosphatidylinositol-3-Kinase (PI3K) Pathway Alterations and Co-alteration of Other Molecular Markers in Breast Cancer.

Background: PI3K/AKT signaling pathway is activated in breast cancer and associated with cell survival. We explored the prevalence of PI3K pathway alterations and co-expression with other markers in breast cancer subtypes.Methods: Samples of non-matched primary and metastatic breast cancer submitted to a CLIA-certified genomics laboratory were molecularly profiled to identify pathogenic or presumed pathogenic mutations in the PIK3CA-AKT1-PTEN pathway using next generation sequencing. Cases with loss of PTEN by IHC were also included. The frequency of co-alterations was examined, including DNA damage response pathways and markers of response to immuno-oncology agents.Results: Of 4,895 tumors profiled, 3,558 (72.7%) had at least one alteration in the PIK3CA-AKT1-PTEN pathway: 1,472 (30.1%) harbored a PIK3CA mutation, 174 (3.6%) an AKT1 mutation, 2,682 (54.8%) had PTEN alterations (PTEN mutation in 7.0% and/or PTEN loss by IHC in 51.4% of cases), 81 (1.7%) harbored a PIK3R1 mutation, and 4 (0.08%) a PIK3R2 mutation. Most of the cohort consisted of metastatic sites (n = 2974, 60.8%), with PIK3CA mutation frequency increased in metastatic (32.1%) compared to primary sites (26.9%), p < 0.001. Other PIK3CA mutations were identified in 388 (7.9%) specimens, classified as “off-label,” as they were not included in the FDA-approved companion test for PIK3CA mutations. Notable co-alterations included increased PD-L1 expression and high tumor mutational burden in PIK3CA-AKT1-PTEN mutated cohorts. Novel concurrent mutations were identified including CDH1 mutations.Conclusions: Findings from this cohort support further exploration of the clinical benefit of PI3K inhibitors for “off-label” PIK3CA mutations and combination strategies with potential clinical benefit for patients with breast cancer.

Abstract 787: Comparable clinical benefits between low and high variant allelic frequency in NSCLC patients treated with targeted therapies based on molecular alterations from ctDNA NGS

Abstract Background: Tumor genomic information from tissue has been the standard of practice for identifying actionable molecular alterations. The same genomic profiling is also widely available by a non-invasive blood test from circulating tumor DNA (ctDNA). We hypothesized that patients with advanced non-small cell lung cancer (NSCLC) with actionable oncogenic driver mutations with either low or high variant allelic frequency (%VAF) identified by ctDNA would have similar clinical benefits after treatment with targeted therapies. Methods: In this retrospective study (UTMDACC IRB-approved protocol PA16-0061), patients with a targetable mutation identified by ctDNA NGS assay who received an FDA-approved therapy were identified in our GEMINI database. All potential patients were individually reviewed and information confirmed with review of their electronic medical records. Blood based genomic profiling was completed by NGS of ctDNA with a panel of up to 70 cancer-related genes at a CLIA-certified lab (Guardant360, Guardant Health, Redwood City, CA). The %VAF was reported for each mutation identified. We defined low group with VAF of 0.01% to 0.99% and high group with VAF &amp;gt; 1%. Tumor measurements were assessed using RECIST V1.1. For comparisons analysis, Kaplan-Meier estimator was used to estimate progression free survival (PFS) with Mantel-Cox and un-paired t-test was used for tumor responses. Results: Eighty-nine patients with advanced NSCLC treated with an FDA approved therapy based on ctDNA genomic profiling were identified and the results are summarized in table below: LOW %VAFHIGH %VAFP valuegroup (n=39)group (n=50)Gender (Female/Male)20/1928/22Age (median, range)54(27-85)63(29-89)Targetable mutations:EGFR2246ALK114MET exon 1420ROS120BRAF20Line of therapy (median, range)2(1-7)2(1-7)ALL PatientsTumor response (mean, range)-37.23% (-100 to +38)-31.08% (-100 to +59)0.42PFS (median, range)215 (27 – 848) days280 (43 – 894) days0.70EGFR onlyTumor response (mean, range)-24.86% (-100 to +16)-23.54% (-100 to +59)0.88PFS (median, range)189 (57 – 848) days293 (43-894) days0.23 Conclusions: In this analysis of patients who were treated with an FDA approved therapy based on genomic results from ctDNA profiling, there were no differences in either tumor response (P value = 0.42) or PFS (P value = 0.70) between those patients with low and high ctDNA as assessed by %VAF of identified targetable mutation. Similar findings were also observed in the analysis of the EGFR only subgroup. This study reveals patients with low %VAF received comparable clinical benefits as those with high %VAF and no deleterious harm was observed from treating patients with low %VAF with appropriate targeted therapy. Citation Format: Hai T. Tran, Vincent K. Lam, Mayra Vasquez, Yasir Elamin, Victoria Raymond, Lingzhi Hong, George Blumenschein, Brett Carter, Richard Lanman, Mehmet Altan, Ann Tsao, Lauren Byers, Don Gibbons, Jianjun Zhang, John V. Heymach. Comparable clinical benefits between low and high variant allelic frequency in NSCLC patients treated with targeted therapies based on molecular alterations from ctDNA NGS [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 787.

Abstract 3272: Is PD-L1 expression a decision-making biomarker for immunotherapy: a meta-analysis and cross-sectional study

Abstract Background: PD-L1 expression has been approved by FDA as a diagnosis for anti-PD-(L)1 therapy in several types of cancer, however, there were still discrepancies of the predictive value of PD-L1 expression. Here we aimed to systematically assess the value of PD-L1 expression in determining cancer patients who should be offered anti-PD-(L)1 therapy and to characterize PD-L1 expression in Chinese cancer population. Methods: Cochrane library, PubMed and Embase databases were systematically searched to include relevant studies published between January 2000 and June 2019. We also reviewed the reports from all major conference proceedings. All randomized controlled trials investigating anti-PD-(L)1 efficacy in solid tumors were initially included if overall survival (OS) data were stratified by PD-L1 expression. Differences in OS benefits between PD-L1-positive and PD-L1-negative patients were analyzed. Effect size derived from each study was pooled using a random-effects model. Data regarding PD-L1 expression in Chinese patients were obtained from a CAP-accredited and CLIA-certified laboratory. Results: Twenty-five RCTs involving 14,471 patients were included. OS benefit was observed in patients with either positive (HR=0.68 [0.63-0.73], P&amp;lt;0.00001) or negative (HR=0.86 [0.78-0.95], P=0.003) PD-L1 expression, though significant longer OS was achieved in patients that are PD-L1 positive (P interaction=0.0001). Superior PFS were observed in patients with positive PD-L1 expression (HR=0.69 [0.57-0.82]) while not with negative PD-L1 expression (HR=1.00 [0.85-1.19]). PD-L1-positive patients exhibited significantly improved ORR than those who tested negative for PD-L1 when treated with immunotherapy (OR=0.50 [0.39-0.65], P&amp;lt;0.00001), while no differences were observed among patients given SOC (OR=1.02 [0.86-1.21], P=0.81). Analysis of Chinese patients PD-L1 status revealed that thymoma and small cell lung cancer expressed the highest and the lowest levels of PD-L1, respectively. Conclusion: PD-L1 expression alone is not determinative in patient selection for anti-PD-(L)1 treatment. Multiple modality biomarkers should be considered for cancer patients especially for those with PD-L1-negative tumors. Citation Format: Zhijie Wang, Jianchun Duan, Longgang Cui, Shangli Cai, Jie Wang. Is PD-L1 expression a decision-making biomarker for immunotherapy: a meta-analysis and cross-sectional study [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3272.

Abstract LB-245: Validating models of imputing germline versus somatic status for variants detected by tumor-only genomic profiling using germline clinical testing data

Abstract The goal of tumor genomic profiling is to identify somatic mutations. However, most implementations lack patient-matched control DNA sequence to enable determination of variant source (somatic or germline) and resolution of allelic status, including possible loss of heterozygosity (LOH), without additional analyses based on accurate estimates of specimens' tumor purity. Here, we present the validation of an information-theoretic algorithm for identifying germline variants detected in tumor-only sequencing using cancer patient clinical germline test data. 1,631 adult patients consented for the PROFILE study at DFCI between 2014 and 2018, and also underwent clinical germline testing. DNA was isolated from tissue containing &amp;gt;20% tumor nuclei and analyzed using Agilent SureSelect hybrid capture kit. All exons and 191 introns in 447 genes were interrogated for single nucleotide variants, small indels, copy number alterations, and structural variants using standard pipelines. Germline testing was performed at CLIA-certified laboratories. Specimen tumor purity was estimated using computational approaches developed at DFCI and Rutgers with further manual curation. LOHGIC (LOH-Germline Inference Calculator), developed on a model-selection scheme using Akaike Information Criterion weighting, was applied to individual variants to infer the most consistent germline-v-somatic model and LOH status, incorporating biases in clinical sequencing and purity estimation. 427 patients had 676 variants detected in high-penetrance cancer predisposition genes: BRCA1, BRCA2, MLH1, MSH2, MSH6 and PMS2. Comparison of LOHGIC's results with germline test data showed 91.4% accuracy, 67.6% precision, and 67.0% recall, with other performance measures reported in Table 1. LOHGIC showed evidence of LOH for 35% of variants with concordant inference and germline testing results. Special challenges with TP53 variants will also be discussed. Our results indicate that tumor-only sequencing can provide excellent power for statistical approaches to identify likely germline mutations and infer LOH. Our analyses may be confounded by the presence of reversion mutations, poor specimen quality, inaccurate purity estimates, low sequencing depths, and/or germline call variability. This work further demonstrates the need for a systematic effort to interpret clinical-grade sequencing results. Table 1.Summary of resultsGeneBRCA1BRCA2MLH1MSH2MSH6PMS2OverallGermline Test: Positive/Negative28/10445/1753/6010/6514/12112/39112/564Germline Inference: Positive/Negative/Ambiguous24/94/1448/138/347/46/1013/51/1115/105/154/37/10111/471/94Positive Predictive Value (PPV)79.270.842.969.246.775.067.6Negative Predictive Value (NPV)95.798.6100.0100.096.286.596.9True Positive Rate (TPR)67.975.6100.090.050.025.067.0False Omission Rate (FOR)4.31.40.00.03.813.53.1Sensitivity82.694.4100.0100.063.637.583.3Specificity94.790.792.092.792.797.092.8Accuracy92.491.492.593.890.085.491.4 Citation Format: Israel Gomy, Nahed Jalloul, Samantha Stokes, Alexander Gusev, Shridar Ganesan, Judy Garber, Hossein Khiabanian. Validating models of imputing germline versus somatic status for variants detected by tumor-only genomic profiling using germline clinical testing data [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr LB-245.

Abstract 1312: Identifying the genomic correlates of clinical benefit (CB) from immunotherapies (IO) and vascular endothelial growth factor-tyrosine kinase inhibitors (VEGF-TKI) in metastatic renal cell carcinoma (mRCC)

Abstract Background: Along with the expanding treatment landscape of mRCC, a substantial need for ways to predict individual patients' (pts) benefit from different therapies has emerged. Recent studies highlight the clinical significance of tumor-specific genomic alterations, revealing a prognostic and/or predictive role of PBRM1 mutations with IO in mRCC (Miao et al Nature 2018). Employing a large institutional database, we aimed to identify genomic correlates of CB from IO and VEGF-TKIs in mRCC. Methods: Consecutive pts who underwent genomic profiling (GP) as part of routine clinical care at the City of Hope Comprehensive Cancer Center were retrospectively identified. GP included the GEM ExTra assay, a clinical tumor-normal whole exome sequencing and tumor whole transcriptome sequencing test performed at Ashion Analytics (Phoenix, AZ), a CAP-accredited, CLIA-certified laboratory. The clinical database included detailed information regarding demographics, treatment, response and survival outcomes. Pts who had complete response (CR), partial response (PR) or stable disease (SD) for &amp;gt;6 months were considered as having CB. Progressive disease (PD) as best response was considered no clinical benefit (NCB). Genomic findings were compared between pts with CB and NCB in the IO and VEGF-TKI cohorts. Results: Among the 58 (45:13 M:F) pts, 17 received sequencing treatment involving both a VEGF-TKI and IO, resulting in 32 pts in the IO cohort and 43 pts in the VEGF-TKI cohort. The most commonly used IO and VEGF-TKIs were nivolumab (59%) and sunitinib (40%). CB rate and median progression free survival were 59.4% (CR: 3.1%, PR: 18.8%, SD: 37.5%) and 15.6 months (95%CI NR-NR) in the IO cohort, and 86% and 14.2 months (95%CI 9.0 - 18.5) in the VEGF-TKI cohort. The most frequently detected alterations in the overall cohort were in VHL (64%), PBRM1 (38%), SETD2 (24%), KDM5C (17%) and TERT (12%). Interestingly, TERT promoter mutations and PBRM1 mutations were found to be mutually exclusive. In both IO and VEGF-TKI cohorts, tumor mutational burden did not differ between CB and NCB pts. No single genes were significantly associated with CB from VEGF-TKIs. While PBRM1 loss of function (LOF) was more common in IO pts with CB, and SETD2, KDM5C, TP53 alteration were more common in IO pts with NCB, there was no statistical significance observed (p values &amp;gt; 0.05). TERT promoter mutations were associated with NCB in the IO cohort (p=0.038). TERT promoter mutant tumors did not have CB with IO. Conclusion: Our analysis found that TERT promoter mutations are enriched in pts with NCB from IO and were mutually exclusive with PBRM1 LOF which had previously been shown to be an indicator of IO sensitivity. These results suggest that TERT promoter mutations may be a negative predictor of IO outcomes, which warrants future investigations with a larger sample size. Citation Format: Nazli Dizman, Sara Byron, Yung Lyou, Paulo Gustavo Bergerot, JoAnn Hsu, Andre-Philippe Sam, Denise Phan Trieu, Jeffrey Trent, Sumanta Kumar Pal. Identifying the genomic correlates of clinical benefit (CB) from immunotherapies (IO) and vascular endothelial growth factor-tyrosine kinase inhibitors (VEGF-TKI) in metastatic renal cell carcinoma (mRCC) [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1312.

Abstract 5742: Comparison of variants identified by next-generation sequencing panels: A 48-gene panel for myeloid neoplasms and a 35-gene panel for hematologic neoplasms

Abstract Background: Identification of genomic variants by molecular testing plays an important role in classification, risk stratification, and management of myeloid neoplasms. Next-generation sequencing (NGS) assays that target frequently mutated genes are commonly used in clinical settings. Owing to the complexity of NGS workflow, the performance requirements of each NGS assay must be validated for the intended use. However, the scarcity of optimal validation samples challenges efficient validation of NGS assays. We previously reported development and validation of a 48-gene NGS panel for molecular profiling of myeloid neoplasms including acute myeloid leukemia (AML), myelodysplastic syndrome (MDS), and myeloproliferative neoplasms (MPN). In this study, we compared the variant calls of a clinically validated 48-gene NGS panel for myeloid neoplasms to those of an independently developed and clinically validated 35-gene NGS panel for hematologic neoplasms. Method: The 48-gene NGS assay is a hybrid capture-based NGS assay using an Illumina NextSeq 500; it was developed to detect single nucleotide variants (SNVs), insertions/deletions (indels), and FLT3 internal tandem duplications (FLT3-ITD). The 35-gene NGS assay HemeSEQ (performed by med fusion, Lewisville, TX) uses Illumina TruSeq custom-amplicon library preparation chemistry, which is sequenced on an Illumina MiSeq. A total of 33,812 bp in 26 genes overlapped between the 2 assays; genes included ASXL1, CALR, FLT3, IDH1, IDH2, JAK2, NPM1, RUNX1, SF3B1, TET2, and TP53. For this study, 141 specimens harboring at least 1 pathogenic mutation within the overlapping genes were selected and sequenced by the 2 assays in CLIA-certified and CAP-accredited laboratories. Detected variants included those for which a targeted therapy or an experimental drug is available. Results: In total, 4.8 million individual base calls (33,812 bp X 141 specimens) were compared. A total of 1,094 variants (278 unique) were concordantly detected by both assays: 1,007 (219 unique) SNVs, and 87 (59 unique) indels; no calls were discordant. Thus, sensitivity was 100% (95% CI: 99.7-100) and specificity was 100% (95% CI: 99.9-100) for overlapping genes. In addition, agreement in variant allele frequency between the 2 assays was good (R2=0.986). The 48-gene assay detected an additional 36 pathogenic mutations from 26 specimens in 14 non-overlapping genes including PTPN11, ATM, BCOR, NF1, and STAG2. Conclusions: Of 4.8 million base calls compared, variant calls were 100% concordant between the 2 NGS assays, and variant allele frequency was in good agreement. The 48-gene assay detected an additional 36 pathogenic mutations in 14 myeloid neoplasm related genes. This study highlights the reliability of robustly validated NGS assays in detection of potentially actionable mutations in clinical settings. Citation Format: Sun Hee Rosenthal, Hansook Chong, Charles Ma, Daniel Sugganth, Renius Owen, Joseph Catanese, Christopher Elzinga, Andrew Grupe, Tracey Freitas, Jennifer Graham, Kristen Champion, Amanda Larsen, Frederick Racke, Felicitas Lacbawan. Comparison of variants identified by next-generation sequencing panels: A 48-gene panel for myeloid neoplasms and a 35-gene panel for hematologic neoplasms [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5742.

Abstract 2295: Bladder care epigenetic test allows the sensitive early detection of bladder cancer from urine samples

Abstract Introduction: Bladder cancer (BC) is the 5th most common cancer in the USA, with 81,190 new cases and 17,240 deaths in 2018. The high recurrence rate of BC requires a lifelong patient follow-up with cystoscopy. However, the invasiveness of cystoscopy and the high costs associated to this procedure, poses a significant burden on patients as well as on the healthcare system. Several tests for the non-invasive detection of BC have been developed in the past years however, because of relatively low sensitivities and/or specificities (especially for low-grade tumors), these tests have not been adopted for the routine clinical use. Therefore, a non-invasive, simple and sensitive test for detection and routine monitoring of BC is needed. Methods: Bladder CARE is a new non-invasive and quantitative test that measures the methylation levels of three BC specific biomarkers and two internal controls in urine samples. Contrary to other epigenetic tests, Bladder CARE analyzes DNA methylation without converting the DNA with sodium bisulfite, thus minimizing the degradation of the target DNA. In this clinical study, we evaluated the performance of this test using voided urine samples collected from 136 healthy individuals and 77 BC patients with no prior history of bladder cancer. Urine specimens were collected and stabilized using Bladder CARE at-home urine sample collection kit and BC detection was performed by a CLIA-certified and CAP-accredited laboratory (Pangea Laboratory LLC) using Bladder CARE. The results were then compared to the clinical information (cystoscopy). Additionally, the test linearity and limit of detection (LOD) were determined by analyzing artificial samples with Bladder CARE test. Results: Bladder CARE was able to discriminate BC from healthy control samples with a sensitivity of 93.5%, a specificity of 92.6%, and a positive and negative predictive value of 87.8% and 96.2%, respectively. Remarkably, low-grade tumors were detected with a sensitivity of 90.0%. We also found that Bladder CARE test has a LOD of 0.046% (the equivalent of detecting 1 cancer cell in a sample containing 2,200 normal cells). Conclusions: While cystoscopy remains the gold standard to confirm the presence of BC, the superior sensitivity of Bladder CARE test, together with its low LOD, make this test an effective early indicator of low-grade tumors and for routine recurrence monitoring of BC patients. In addition, Bladder CARE urine collection kit allows to collect and stabilize the urine specimen comfortably at-home and mail it to the testing laboratory at room temperature, streamlining the patients' detection and monitoring process. Citation Format: Paolo Piatti, Michiko Suwoto, Taikun Jamada, Yap Ching Chew, Gangning Liang, Saum Ghodossipour, Siamak Daneshmand, Xi-Yu Jia. Bladder care epigenetic test allows the sensitive early detection of bladder cancer from urine samples [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2295.

Abstract 818: Organoid based functional test to predict personalized treatment in cholangiocarcinoma

Abstract Introduction: It is difficult to find targeted therapies that work across the genetically diverse Cholangiocarcinoma (CCA) patient population; genomic sequencing alone has not resulted in substantial changes in treatment choices for this rare cancer. We reasoned that CCA patients would benefit from a highly personalized diagnostic test, where patient-derived tumor organoids are tested with a library of targeted drugs in a high-throughput functional assay. Methods: Seventeen patients with CCA were enrolled in an IRB-approved ongoing study from 2017 to 2019; biopsies were collected from surgical resection and shipped on ice overnight to a CLIA-certified laboratory (SEngine Precision Medicine) for organoid derivation and high throughput drug testing. Tumor organoids were challenged with up to 120 FDA-approved and investigational drugs to obtain 6-dose, 3-log response curves. Drug responses were benchmarked against an in-house database of pan-cancer patients to concurrently assess both sensitivity and the uniqueness of each patient's response. Clinical history and genomic information were collected from each patient for retrospective and prospective analysis. Results: 14/19 (74%) samples were successfully derived from 17 patients and screened. Patient age ranged from 36 to 73 (mean=51). The 120-drug library was adapted based on total viable tumor cells per sample, resulting in average of 71 drugs per screen. Top scoring drugs were Ceritinib, MK2206, Belinostat, Taselisib, AZD4547, Everolimus and Poziotinib. In general, the drug sensitivity highlighted the following pathways: AKT, ALK, FGFR, PI3K and EGFR. Drugs were selected per patient and classified as either good or exceptional response and correlated with mutations in known genomic biomarkers in 7 of 12 drug/target pairs. Retrospective analysis demonstrated 100% concordance (n=3) between ex vivo drug resistance and prior treatments. We observed multiple mutation/drug pairs which showed resistance in our functional screen. We achieved a high success rate and clinically relevant turnaround times, from biopsy to final report generation average 17 days (range 9 to 35). Conclusion: This study shows the feasibility of functional testing of organoids derived from CCA patients in a CLIA-certified diagnostic test. While these results correlate well with genomically predicted drug sensitivities, all patients showed additional drug sensitivities beyond those predicted by genomics offering patients additional potential treatment options. This study highlights the importance of functional data in a genetically heterogenous tumor type such as CCA. Each patient represents a mosaic of drug sensitivities, reflecting unique combinations of genetic and epigenetic alterations, and this test can identify the personalized drug combination for them. Citation Format: Astrid Margossian, Franz X. Schaub, Annie Richardson, Michael Churchill, Rachele Rosati, Alexandra Dullea, G Adam Whitney, Hallie Swan, Robert Diaz, Lauren Appleyard, Vijaykrishna Gadi, Ali Zarrinpar, Milind Javle, Christopher Kemp, Shalini Pereira, Carla Grandori. Organoid based functional test to predict personalized treatment in cholangiocarcinoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 818.

Abstract 5903: Clinical immune and genomic profiling of STK11 mutant non-squamous non-small cell lung cancer

Abstract Background: The use of immune PD-1/PD-L1 checkpoint inhibitors (ICI) has dramatically altered the treatment of advanced Non-Small Cell Lung Cancer (NSCLC). However, a large proportion of patients with NSCLC do not derive clinical benefit from ICI treatment. Recent studies have identified STK11 mutations leading to loss of LKB1 in NSCLC as drivers of primary resistance to ICI treatment. Using a clinical cohort of patients whose tumors underwent comprehensive genomic and immune transcriptomic analysis, we characterized the immune gene expression profile of STK11 mutant non-squamous NSCLC tumors and identified TRIM29 as a potential therapeutic target in STK11 mutant non-squamous NSCLC. Methods: We performed comprehensive analysis of the genomic and immunological landscape of 199 formalin-fixed, paraffin-embedded tumor samples (obtained prior to ICI treatment) from advanced stage non-squamous NSCLC patients treated at Roswell Park Comprehensive Cancer Center using a CLIA-certified laboratory test that included targeted NGS genomic sequencing, gene fusion analysis, and RNA-seq of 394 immune transcripts. Differential gene expression analysis was performed using R/Bioconductor package limma with Benjamini-Hochberg adjusted p-values reported. This study was approved by Roswell Park internal review board review (protocol BDR 091817) according to institutional policy for nonhuman subjects research. Results: Among the cohort of 199 cases, 32 contained truncating STK11 mutations. STK11 mutant cases showed an immunosuppressive environment with decreased PD-L1 expression (p=0.01) and decreased T-cell inflammation gene expression signature (p=0.0029) consistent with prior published cohorts. Analysis of differentially expressed immune genes signatures showed significant decreases in antigen presentation (p=0.015) and processing (p=0.044), NK cell activation (p=0.039), chemokine and cytokine signaling (p=0.044), immune checkpoint pathways (p=0.015), innate immune response (p=0.04), and interferon signaling (p=0.039). The top upregulated gene in STK11 mutant cases was TRIM29 (p=0.03). Other overexpressed targets included ID3 (p=0.009), KRT7 (p=0.02), and NOTCH3 (p=0.05). Interrogation of TCGA LUAD dataset also demonstrated elevated TRIM29 expression (p=0.006) in STK11-mutant compared to STK11-wild-type samples and we also confirmed elevated TRIM29 RNA and protein expression in a panel of STK11 mutant NSCLC human cell lines. Immunohistochemical staining of a cohort of 68 KRAS mutant NSLCLC patient samples also showed higher levels of TRIM29 protein staining in STK11 mutant samples. Knockdown of TRIM29 using shRNA inhibited growth of STK11 mutant tumor xenografts. TRIM29 is a E3 ubiquitin ligase and we evaluated known targets of TRIM29 and found decreased TAB2 expression in STK11 mutant NSCLC. Notably, TAB2 is also regulated by SIK1/3, which have been recently identified as signaling partners with STK11. TRIM29 has been implicated as a mediator of processes also involved in STK11 tumorigenesis such as cellular metabolism, DNA damage repair, innate immunity, and squamous differentiation. Conclusion: Using a large clinical cohort of non-squamous NSCLC patients we characterized the immunosuppressive environment of STK11 mutant tumors and identified the E3 ubiquitin ligase TRIM29 as a potential therapeutic target. Citation Format: Edwin Yau, Sarabjot Pabla, Sean Glenn, Antonios Papanicolau-Sengos, Mary Nesline, Grace Dy, Carl Morrison. Clinical immune and genomic profiling of STK11 mutant non-squamous non-small cell lung cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5903.

Oncotype DX Breast Recurrence Score®: A Review of its Use in Early-Stage Breast Cancer.

Oncotype DX Breast Recurrence Score® is a 21-gene prognostic and predictive assay indicated for use in patients with hormone receptor-positive, human epidermal growth factor receptor 2-negative, lymph node (LN)-negative or up to three LN-positive, early-stage breast cancer. The assay is used worldwide and is conducted at a CLIA-certified central laboratory in the USA. The 21-gene assay generates a Recurrence Score for each tumour sample, based on expression levels of 16 breast cancer-related genes, normalized to five reference genes. The Recurrence Score is a continuous variable that provides an individualized estimate of 9-year distant recurrence risk and the likelihood of adjuvant chemotherapy benefit. The 21-gene Recurrence Score assay is extensively validated in clinical studies, which are well supported by real-world registry studies. The assay informs treatment decisions and reduces adjuvant chemotherapy use in routine clinical practices, and is estimated to be cost-effective. The 21-gene Recurrence Score assay is included in all major international treatment guidelines. Currently, it is the only multigene assay validated for prediction of chemotherapy benefit, as well as for prognosis.

Inefficacy of chromogranin a assays as neuroendocrine tumor diagnostic tools compared to the NETest.

4612 Background: Chromogranin A (CgA) remains a commonly used diagnostic and monitoring tool for neuroendocrine tumor disease despite NCCN guidelines identifying it as a category 3 (major concerns about utility) biomarker. Several commercial assays have been developed to measure this protein (or its fragments) and are available both at CLIA-certified laboratories (USA) as well as in NET Centres of Excellence (CoEs - Europe). CgA is typically reimbursed by insurance companies and appears in several guidelines (e.g., ENETS). We sought to directly evaluate the accuracy of detecting NET disease using two different CgA assays, one in the USA (NEOLISA, EuroDiagnostica, IBL-America, CLIA-certified laboratory) and one in an ENETS CoE (CgA ELISA, Demeditec Diagnostics, Germany). We compared the results to the NETest, a circulating mRNA assay, recently validated as an IVD for NETs. Methods: Patients: NETs ( n=258) including lung: n=43; duodenum n=9; gastric: n=44; pancreas: n=67; small bowel: n=40; appendix: n=10; rectum: n=45. No image-evidence of disease ( n=122) (IND) and image-positive disease (IPD) ( n=136). CgA assays (plasma): NEOLISA, ULN &gt;108ng/ml, DD: ULN&gt;99ng/ml. Data mean±SEM. NETest (whole blood): qRT-PCR - multianalyte algorithmic analyses, CLIA-laboratory. All samples de-identified and assessed blinded. Statistics: Mann-Whitney U-test, Pearson correlation &amp; McNemar-test. Results: In the entire group ( n=258), NEOLISA assay CgA levels were significantly ( p&lt;0.0001) higher (216±91ng/ml) vs. the DD-assay (76±8ng/ml). The assays exhibited a high concordance in output (Pearson r=0.81, p&lt;0.0001), but there were 10.9% ( n=31) discordant results. This reflected the NEOLISA assay detecting more CgA-positive samples. IPD group: CgA-positives were detected in 48/136 (35%, NEOLISA) vs. 28 (21%, DD-assay). McNemar’s Chi2=15.04, p&lt;0.001 OR: 11.0, indicating the NEOLISA was significantly better than the DD-assay. The NETest, in contrast, was positive in 135/136 (99%; OR: 87-106, p&lt;0.0001). IND group: CgA-positives were detected in 12/122 (10%, NEOLISA) vs. 9 (7%, DD-assay; p=NS). The majority (75%) of positives were associated with gastric NETs. The NETest was positive in 7 (6%); 4 were gastric NETs and 3 exhibited elevated CgA. Conclusions: Two standard CgA assays used for NET management (one accepted by Medicare in the USA, the second used at a CoE in Europe) only detect NET disease in 21-35% of cases. In contrast, a circulating mRNA fingerprint, the NETest, is ~99% accurate for detecting NET disease.

Comprehensive analysis of HER2 status through genomic, transcription, and translation among 5,305 patients with diverse malignancies.

3632 Background: HER2 alterations is a predictive biomarker for anti-HER2 regimen. Success in breast and gastric cancers with anti-HER2 therapies translated to explore their efficacy in other tumors. Currently, measurement of HER2 can be done by assessing the expression of protein [e.g. immunohistochemistry (IHC)] or gene amplification of copy number variation (CNV) [e.g. fluorescence in situ hybridization or next-generation sequencing (NGS)]. But little is known about the transcription level (mRNA) of HER2. Herein, we investigated HER2 mRNA expression and its association with gene and protein expressions among diverse cancer types. Methods: Between 2015-2019, HER2 status was evaluated using IHC, qRT-PCR and NGS by Paradigm Diagnostics (CLIA-certified laboratory). All tumors in the database were included for analysis. Correlations between all 3 tests were done. An illustrative patient who was treated with anti-HER2 therapy base on the mRNA testing is presented. Results: HER2 testing was performed on 5305 patients (pts) with diverse cancers including NSCLC (n=1175), breast (n=1040) and colon (n=566); 4.1% (161/3926) had amplification through NGS, 33.3% (615/1848) had mRNA overexpression and 9.3% (236/2533) had overexpression by IHC. Of 723 pts who had all three tests performed, we found 7.5% (54/723) of pts with all three HER2 markers being positive (CNV [+]/mRNA [+]/ IHC [+]). Meanwhile, variety of amplification/ expression patterns were seen (see Table). CNV positivity translated to protein expression in 95% of cases. While only 4% of pts were IHC positive when CNV and mRNA were negative. 20% (144/723) of pts had mRNA overexpression alone among diverse cancer types. Representative case of 70yo female with metastatic cholangiocarcinoma harboring mRNA overexpression (but negative for CNV, IHC unclear due to sample insufficiency) who had near complete response to anti-HER2 therapy with progression-free survival of 24+ months is presented. Conclusions: HER2 status can be discordant with different assays but NGS positivity has excellent correlation with mRNA and protein expression. Of importance, HER2 mRNA can be overexpressed in 20% of pts even when gene amplification and protein expression are negative. Further studies are warranted to determine the clinical utility of mRNA as a biomarker for HER2 and potential use for anti-HER2 targeted therapies. [Table: see text]

Validation of an exome and transcriptome based diagnostic platform enabling clinical cancer therapy selection and emerging composite biomarkers for immunotherapy.

e15583 Background: While immunotherapy has become a pillar of cancer treatment, diagnostic biomarkers that consistently predict patient response to these therapies have remained elusive. There is an increasing need for the development of integrative, composite biomarkers that can model the complex biology driving response and/or resistance to immunotherapy more effectively than existing single-analyte approaches. However, the majority of current cancer diagnostic panels, with their focus on a small set of genes, provide limited ability to support these emerging advanced biomarkers. Methods: To address these limitations, we developed and validated NeXT Dx, a comprehensive enhanced exome and transcriptome based diagnostic platform designed to simultaneously characterize tumor and immune genomics from a single limited FFPE sample. To achieve higher accuracy and sensitivity for an exome scale diagnostic platform, we developed an augmented exome assay that improves uniformity of coverage across all ~20,000 genes, including boosted coverage of 248 clinically-relevant cancer genes. We validated this assay using genomic DNA and RNA extracted from tumor-derived cell-lines, constructs, clinical FFPE samples, and proficiency testing samples. The assay utilizes &gt; = 25ng of co-extracted DNA and RNA which were sequenced using Illumina NovaSeq instruments at our CAP-accredited, CLIA-certified laboratory. Additional assay enhancements for HLA, immune repertoire, and oncoviruses were designed to further optimize the platform for immunotherapy biomarker discovery applications. Results: Validation of NeXT Dx demonstrated a performance of 99.5% sensitivity and 99.8% positive predictive value (PPV) for SNVs with &gt; = 5% AF; 98.7% sensitivity and 97.4% PPV for indels with &gt; = 10% AF; 97.2% sensitivity and 94.6% PPV for CNAs in samples with &gt; = 30% tumor content; 94.9% sensitivity and 94.9% PPV for fusions; and a 2.1% error rate for MSI classification. TMB was calculated using gold-standard whole exome data from SNVs and indels. Typical median coverage depth was &gt; 1,000X for 248 clinically-relevant genes, ~300X for the remaining (whole exome) footprint. Conclusions: With NeXT Dx, we demonstrate a exome/transcriptome scale diagnostic platform that can detect current clinical biomarkers with high sensitivity as well as support emerging, advanced biomarkers that integrate across both tumor and immune features.