Longitudinal monitoring of circulating tumor DNA to detect relapse early and predict outcome in early breast cancer.

PURPOSE: Detection of molecular residual disease (MRD) allows for the identification of breast cancer patients at high-risk of recurrence, with the potential that early initiation of treatment at early stages of relapse could improve patient outcomes. The Invitae Personalized Cancer Monitoring™ assay (PCM) is a newly developed next-generation sequencing approach that utilizes up to 50 patient-specific, tumor-informed DNA variants, to detect circulating tumor DNA (ctDNA). The ability of the PCM assay to detect MRD before clinical relapse was evaluated. METHODS: The cohort included 61 female patients with high-risk breast cancer who underwent neoadjuvant chemotherapy. Plasma samples were collected before and during neoadjuvant therapy, after surgery and during monitoring. PCM was used to detect ctDNA at each time point. RESULTS: The sensitivity to detect ctDNA in plasma from patients who relapsed during the monitoring phase was 76.9% (10/13). Specificity and positive predictive values were both 100% with all (10/61, 16%) of the patients who had ctDNA detected during the monitoring phase subsequently relapsing. Detection of ctDNA during monitoring was associated with a high-risk of future relapse (HR 37.2, 95% CI 10.5-131.9, p < 0.0001), with a median lead-time from ctDNA detection to clinical relapse of 11.7 months. CONCLUSION: PCM detected ctDNA in patients who relapsed with a long lead-time over clinical relapse, shows strong association with relapse-free survival and may be used to identify patients at high-risk for relapse, allowing for earlier intervention.

Large-scale analysis of CDH1 mutations defines a distinctive molecular subset with treatment implications in gastric cancer.

Although histological and molecular classifications have been extensively studied for gastric cancer (GC), targeted therapies for GC remain limited. CDH1 mutations (MT) are characteristic of genomically stable GC and are associated with poor prognosis, but lack effective or targeted therapies. Here, we showed the overall mutation frequency of CDH1 was 9.7% (155 of 1596). CDH1-MT GC showed significantly lower rates of PD-L1 positivity (CPS score ≥1) than CDH1-wildtype (WT) GC (56.7% vs. 73.3%, p < 0.05). Compared to CDH1-WT GC, mutations of ARID1A, WRN, POT1, CDK12, and FANCC were significantly higher, while TP53 and APC were significantly lower in CDH1-MT GC (p < 0.05); The rates of KRAS and HER2 amplifications were significantly lower, while CRKL and IGF1R amplifications were significantly higher in CDH1-MT GC, compared to CDH1-WT GC (p < 0.05). Frequently altered genes in CDH1-MT GC were especially enriched in DNA damage repair and cell cycle checkpoint pathways. Inhibition of E-cadherin sensitized GC cell lines to PARP and Wee1 inhibitors by disrupting DNA damage repair pathway and cell cycle checkpoint. This is the largest study to investigate the distinct genomic landscape of CDH1-MT GC. Our data indicated GC patients with CDH1 mutations could potentially benefit from agents targeting PARP and Wee1.

Consensus molecular subtyping of metastatic colorectal cancer expands biomarker-directed therapeutic benefit for patients with CMS1 and CMS2 tumors.

BACKGROUND: We developed a whole transcriptome sequencing (WTS)-based Consensus Molecular Subtypes (CMS) classifier using FFPE tissue and investigated its prognostic and predictive utility in a large clinico-genomic database of CRC patients (n = 24,939). METHODS: The classifier was trained against the original CMS datasets using an SVM model and validated in an independent blinded TCGA dataset (88.0% accuracy). Kaplan-Meier estimates of overall survival (OS) and time-on-treatment (TOT) were calculated for each CMS (p < 0.05 considered significant). RESULTS: CMS2 tumors were enriched on left-side of colon and conferred the longest median OS. In RAS-wildtype mCRC, left-sided tumors and CMS2 classification were associated with longer TOT with anti-EGFR antibodies (cetuximab and panitumumab). When restricting to only CMS2, there was no significant difference in TOT between right- versus left-sided tumors. CMS1 tumors were associated with a longer median TOT with pembrolizumab relative to other CMS groups, even when analyzing only microsatellite stable (MSS) tumors. DISCUSSION: A WTS-based CMS classifier allowed investigation of a large multi-institutional clinico-genomic mCRC cohort, suggesting anti-EGFR therapy benefit for right-sided RAS-WT CMS2 tumors and immune checkpoint inhibitor benefit for MSS CMS1. Routine CMS classification of CRC provides important treatment associations that should be further investigated.

Evaluation of markers of immunity in different metastatic immune microenvironments suggests more suppression within breast to liver metastases in breast cancer.

PURPOSE: Programmed death receptor ligand-1 (PD-L1) expression and tumor mutational burden (TMB) are approved screening biomarkers for immune checkpoint inhibition (ICI) in advanced triple negative breast cancer. We examined these biomarkers along with characterization of the tumor microenvironment (TME) between breast tumors (BrTs), axillary metastases (AxMs), liver metastases (LvMs), non-axillary lymph node metastases, and non-liver metastases to determine differences related to site of metastatic disease. METHODS: 3076 unpaired biopsies from breast cancer patients were analyzed using whole transcriptome sequencing and NextGen DNA depicting TMB within tumor sites. The PD-L1 positivity was determined with VENTANA PD-L1 (SP142) assay. The immune cell fraction within the TME was calculated by QuantiSeq and MCP-counter. RESULTS: Compared to BrT, more LvM samples had a high TMB (≥ 10 mutations/Mb) and fewer LvM samples had PD-L1+ expression. Evaluation of the TME revealed that LvM sites harbored lower infiltration of adaptive immune cells, such as CD4+, CD8+, and regulatory T-cells compared with the BrT foci. We saw differences in innate immune cell infiltration in LvM compared to BrT, including neutrophils and NK cells. CONCLUSIONS: LvMs are less likely to express PD-L1+ tumor cells but more likely to harbor high TMB as compared to BrTs. Unlike AxMs, LvMs represent a more immunosuppressed TME and demonstrate lower gene expression associated with adaptive immunity compared to BrTs. These findings suggest biopsy site be considered when interpreting results that influence ICI use for treatment and further investigation of immune composition and biomarkers expression by metastatic site.

CCR5 and CCL5 gene expression in colorectal cancer: comprehensive profiling and clinical value.

BACKGROUND: The C-C motif chemokine receptor 5 (CCR5)/C-C motif chemokine ligand 5 (CCL5) axis plays a major role in colorectal cancer (CRC). We aimed to characterize the molecular features associated with CCR5/CCL5 expression in CRC and to determine whether CCR5/CCL5 levels could impact treatment outcomes. METHODS: 7604 CRCs tested with NextGen Sequencing on DNA and RNA were analyzed. Molecular features were evaluated according to CCR5 and CCL5 tumor gene expression quartiles. The impact on treatment outcomes was assessed in two cohorts, including 6341 real-world patients and 429 patients from the Cancer and Leukemia Group B (CALGB)/SWOG 80405 trial. RESULTS: CCR5/CCL5 expression was higher in right-sided versus left-sided tumors, and positively associated with consensus molecular subtypes 1 and 4. Higher CCR5/CCL5 expression was associated with higher tumor mutational burden, deficiency in mismatch repair and programmed cell death ligand 1 (PD-L1) levels. Additionally, high CCR5/CCL5 were associated with higher immune cell infiltration in the tumor microenvironment (TME) of MMR proficient tumors. Ingenuity pathway analysis revealed upregulation of the programmed cell death protein 1 (PD-1)/PD-L1 cancer immunotherapy pathway, phosphatase and tensin homolog (PTEN) and peroxisome proliferator-activated receptors (PPAR) signaling, and cytotoxic T-lymphocyte antigen 4 (CTLA-4) signaling in cytotoxic T lymphocytes, whereas several inflammation-related pathways were downregulated. Low CCR5/CCL5 expression was associated with increased benefit from cetuximab-FOLFOX treatment in the CALGB/SWOG 80405 trial, where significant treatment interaction was observed with biologic agents and chemotherapy backbone. CONCLUSIONS: Our data show a strong association between CCR5/CCL5 gene expression and distinct molecular features, gene expression profiles, TME cell infiltration, and treatment benefit in CRC. Targeting the CCR5/CCL5 axis may have clinical applications in selected CRC subgroups and may play a key role in developing and deploying strategies to modulate the immune TME for CRC treatment.

Comparative Genomic Analysis of Pancreatic Acinar Cell Carcinoma (PACC) and Pancreatic Ductal Adenocarcinoma (PDAC) Unveils New Actionable Genomic Aberrations in PACC.

PURPOSE: Pure pancreatic acinar cell carcinomas (PACC) are rare malignancies with no established treatment. PACC demonstrates significant genetic intertumoral heterogeneity with multiple pathways involved, suggesting using targeted cancer therapeutics to treat this disease. We aggregated one of the largest datasets of pure PACC to examine the genomic variability and explore patient-specific therapeutic targets. EXPERIMENTAL DESIGN: PACC specimens (n = 51) underwent next-generation sequencing of DNA (n = 29) or whole exome (n = 22) and RNA (whole transcriptome, n = 29) at a commercial laboratory. We performed comparative analyses of a genomic cohort of pancreatic ductal adenocarcinomas (PDAC; n = 4,205). In parallel, we conducted a retrospective review of patients with PACC treated at Huntsman Cancer Institute (HCI). RESULTS: The real-world dataset included samples from 51 patients with PACC. We found key molecular differences between pure PACC and PDAC, highlighting the unique characteristics of pure PACC. Major differences in PACC include lower MAPK signaling and less stromal cell abundance compared with PDAC. Pure PACC showed genomic loss-of-heterozygosity to largely coincide with mutations in BRCA1, BRCA2, and PALB2. Of the 7 patients treated at HCI, one had a tumor that harbored a BRAF-V600E mutation. Leveraging precision oncology, this patient is being treated with encorafenib plus binimetinib, achieving an exceptionally durable and ongoing complete response of more than 3 years. CONCLUSIONS: There are major differences between PACC and PDAC, including downregulation of the MAPK signaling pathway, and less stromal cell abundance. In addition, genomic characterization of pure PACC revealed frequent targetable alterations, which can guide patient treatment.

Metastatic Prostate Cancers with BRCA2 versus ATM Mutations Exhibit Divergent Molecular Features and Clinical Outcomes.

PURPOSE: In patients with metastatic prostate cancer (mPC), ATM and BRCA2 mutations dictate differences in PARPi inhibitor response and other therapies. We interrogated the molecular features of ATM- and BRCA2-mutated mPC to explain the divergent clinical outcomes and inform future treatment decisions. EXPERIMENTAL DESIGN: We examined a novel set of 1,187 mPCs after excluding microsatellite-instable (MSI) tumors. We stratified these based on ATM (n = 88) or BRCA2 (n = 98) mutations. As control groups, mPCs with mutations in 12 other homologous recombination repair (HRR) genes were considered non-BRCA2/ATM HRR-deficient (HRDother, n = 193), whereas lack of any HRR mutations were considered HRR-proficient (HRP; n = 808). Gene expression analyses were performed using Limma. Real-world overall survival was determined from insurance claims data. RESULTS: In noncastrate mPCs, only BRCA2-mutated mPCs exhibited worse clinical outcomes to AR-targeted therapies. In castrate mPCs, both ATM and BRCA2 mutations exhibited worse clinical outcomes to AR-targeted therapies. ATM-mutated mPCs had reduced TP53 mutations and harbored coamplification of 11q13 genes, including CCND1 and genes in the FGF family. BRCA2-mutated tumors showed elevated genomic loss-of-heterozygosity scores and were often tumor mutational burden high. BRCA2-mutated mPCs had upregulation of cell-cycle genes and were enriched in cell-cycle signaling programs. This was distinct from ATM-mutated tumors. CONCLUSIONS: Tumoral ATM and BRCA2 mutations are associated with differential clinical outcomes when patients are stratified by treatments, including hormonal or taxane therapies. ATM- and BRCA2-mutated tumors exhibited differences in co-occurring molecular features. These unique molecular features may inform therapeutic decisions and development of novel therapies.

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

BACKGROUND AND OBJECTIVES: Published data comparing peritoneal metastases from appendiceal cancers (pAC) and colorectal cancers (pCRC) remain sparse. We compared pAC and pCRC using comprehensive tumor profiling (CTP). METHODS: CTP was performed, including next-generation sequencing and analysis of copy number variation (CNV), microsatellite instability (MSI) and tumor mutational burden (TMB). RESULTS: One hundred thirty-six pAC 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. CONCLUSIONS: Despite a reduced overall TMB, pAC demonstrated mutations distinct from those seen in pCRC. These may serve as discrete biomarkers for future study.

Mutational analysis of microsatellite-stable gastrointestinal cancer with high tumour mutational burden: a retrospective cohort study.

BACKGROUND: Genomic signatures contributing to high tumour mutational burden (TMB-H) independent from mismatch-repair deficiency (dMMR) or microsatellite instability-high (MSI-H) status are not well studied. We aimed to characterise molecular features of microsatellite stable (MSS) TMB-H gastrointestinal tumours. METHODS: Molecular alterations of 48 606 gastrointestinal tumours from Caris Life Sciences (CARIS) identified with next-generation sequencing were compared among MSS-TMB-H, dMMR/MSI-H, and MSS-TMB-low (L) tumours, using χ2 or Fisher's exact tests. Antitumour immune response within the tumour environment was predicted by analysing the infiltration of immune cells and immune signatures using The Cancer Genome Atlas database. The Kaplan-Meier method and the log-rank test were used to evaluate the impact of gene alterations on the efficacy of immune checkpoint inhibitors in MSS gastrointestinal cancers from the CARIS database, a Memorial Sloan Kettering Cancer Center cohort, and a Peking University Cancer Hospital cohort. FINDINGS: MSS-TMB-H was observed in 1600 (3·29%) of 48 606 tumours, dMMR/MSI-H in 2272 (4·67%), and MSS-TMB-L in 44 734 (92·03%). Gene mutations in SMAD2, MTOR, NFE2L2, RB1, KEAP1, TERT, and RASA1 might impair antitumour immune response despite TMB-H, while mutations in 16 other genes (CDC73, CTNNA1, ERBB4, EZH2, JAK2, MAP2K1, MAP2K4, PIK3R1, POLE, PPP2R1A, PPP2R2A, PTPN11, RAF1, RUNX1, STAG2, and XPO1) were related to TMB-H with enhanced antitumour immune response independent of dMMR/MSI-H, constructing a predictive model (modified TMB [mTMB]) for immune checkpoint inhibitor efficacy. Patients with any mutation in the mTMB gene signature, in comparison with patients with mTMB wildtype tumours, showed a superior survival benefit from immune checkpoint inhibitors in MSS gastrointestinal cancers in the CARIS cohort (n=95, median overall survival 18·77 months [95% CI 17·30-20·23] vs 7·03 months [5·73-8·34]; hazard ratio 0·55 [95% CI 0·31-0·99], p=0·044). In addition, copy number amplification in chromosome 11q13 (eg, CCND1, FGF genes) was more prevalent in MSS-TMB-H tumours than in the dMMR/MSI-H or MSS-TMB-L subgroups. INTERPRETATION: Not all mutations related to TMB-H can enhance antitumour immune response. More composite biomarkers should be investigated (eg, mTMB signature) to tailor treatment with immune checkpoint inhibitors. Our data also provide novel insights for the combination of immune checkpoint inhibitors and drugs targeting cyclin D1 or FGFs. FUNDING: US National Cancer Institute, Gloria Borges WunderGlo Foundation, Dhont Family Foundation, Gene Gregg Pancreas Research Fund, San Pedro Peninsula Cancer Guild, Daniel Butler Research Fund, Victoria and Philip Wilson Research Fund, Fong Research Project, Ming Hsieh Research Fund, Shanghai Sailing Program, China National Postdoctoral Program for Innovative Talents, China Postdoctoral Science Foundation, National Natural Science Foundation of China.

Multi-omic Characterization of Pancreatic Ductal Adenocarcinoma Relates CXCR4 mRNA Expression Levels to Potential Clinical Targets.

PURPOSE: Chemokines are essential for immune cell trafficking and are considered to have a major impact on the composition of the tumor microenvironment. CX-chemokine receptor 4 (CXCR4) is associated with poor differentiation, metastasis, and prognosis in pancreatic ductal adenocarcinoma (PDAC). This study provides a comprehensive molecular portrait of PDAC according to CXCR4 mRNA expression levels. EXPERIMENTAL DESIGN: The Cancer Genome Atlas database was used to explore molecular and immunologic features associated with CXCR4 mRNA expression in PDAC. A large real-word dataset (n = 3,647) served for validation and further exploratory analyses. Single-cell RNA analyses on a publicly available dataset and in-house multiplex immunofluorescence (mIF) experiments were performed to elaborate cellular localization of CXCR4. RESULTS: High CXCR4 mRNA expression (CXCR4high) was associated with increased infiltration of regulatory T cells, CD8+ T cells, and macrophages, and upregulation of several immune-related genes, including immune checkpoint transcripts (e.g., TIGIT, CD274, PDCD1). Analysis of the validation cohort confirmed the CXCR4-dependent immunologic TME composition in PDAC irrespective of microsatellite instability-high/mismatch repair-deficient or tumor mutational burden. Single-cell RNA analysis and mIF revealed that CXCR4 was mainly expressed by macrophages and T-cell subsets. Clinical relevance of our finding is supported by an improved survival of CXCR4high PDAC. CONCLUSIONS: High intratumoral CXCR4 mRNA expression is linked to a T cell- and macrophage-rich PDAC phenotype with high expression of inhibitory immune checkpoints. Thus, our findings might serve as a rationale to investigate CXCR4 as a predictive biomarker in patients with PDAC undergoing immune checkpoint inhibition.

Molecular characterization of ESR1 variants in breast cancer.

PURPOSE: Estrogen receptor 1 (ESR1) mutations and fusions typically arise in patients with hormone receptor-positive breast cancer after aromatase inhibitor therapy, whereby ESR1 is constitutively activated in a ligand-independent manner. These variants can impact treatment response. Herein, we characterize ESR1 variants among molecularly profiled advanced breast cancers. METHODS: DNA next-generation sequencing (592-gene panel) data from 9860 breast cancer samples were retrospectively reviewed. Gene fusions were detected using the ArcherDx fusion assay or whole transcriptome sequencing (n = 344 and n = 4305, respectively). Statistical analyses included Chi-square and Fisher's exact tests. RESULTS: An ESR1 ligand-binding domain (LBD) mutation was detected in 8.6% of tumors evaluated and a pathogenic ESR1 fusion was detected in 1.6%. Most ESR1 LBD mutations/fusions were from estrogen receptor (ER)-positive samples (20.1% and 4.9%, respectively). The most common ESR1 LBD mutations included D538G (3.3%), Y537S (2.3%), and E380Q (1.1%) mutations. Among biopsy sites, ESR1 LBD mutations were most observed in liver metastases. Pathogenic ESR1 fusions were identified in 76 samples (1.6%) with 40 unique fusion partners. Evaluating co-alterations, ESR1 variant (mutation/fusion) samples more frequently expressed androgen receptor (78.0% vs 58.6, P < 0.0001) and less frequently immune checkpoint proteins than ESR1 wild-type (PD-1 20.0% vs 53.4, P < 0.05; immune cell PD-L1 10.0% vs 30.2, P < 0.0001). CONCLUSION: We have described one of the largest series of ESR1 fusions reported. ESR1 LBD mutations were commonly identified in ER-positive disease. Limited data exists regarding the clinical impact of ESR1 fusions, which could be an area for future therapeutic exploration.

HER2 in Uterine Serous Carcinoma: Testing platforms and implications for targeted therapy.

OBJECTIVE: HER2 is an important prognostic and therapeutic target in uterine serous carcinoma (USC). Optimal HER2 testing platforms have not been defined and guidelines for testing have changed over time. Our objective is to assess the concordance of HER2 positivity based on chromogenic in situ hybridization (CISH), immunohistochemistry (IHC), and next generation sequencing (NGS) and to determine the rate of downstream mutations that may affect response to HER2 directed therapy. METHODS: Utilizing the Caris tumor registry, 2192 USC tumors were identified and analyzed using NGS (NextSeq, 592 Genes and WES, NovaSEQ), IHC, and CISH. PD-L1 expression was tested by IHC. Microsatellite instability was tested by fragment analysis, IHC, and NGS. Tumor mutational burden (TMB) was measured by totaling somatic mutations per tumor. HER2 positivity through IHC and CISH was determined based on 2007 and 2018 ASCO/CAP HER2 breast cancer guidelines. RESULTS: There was a higher rate of HER2 positivity by IHC when using the 2018 guidelines compared to the 2007 guidelines (16.3% vs 12.3%). Concordance between IHC and CISH was 98.9%. ERBB2 amplification was identified by NGS in 10.5% of tumors. Compared to CISH results, this corresponds to a concordance rate of 91.6% and a positive predictive value (PPV) of 60.3%. Single gene alterations in HER2 amplified tumors that may implicate HER2 therapy resistance included PI3K (33.1%), KRAS (2.5%), and PTEN (1.3%). CONCLUSIONS: There was high concordance between HER2 positivity based on CISH and IHC. Rate of HER2 positivity is the lowest by NGS. Ultimately these testing platforms need to be validated by response to targeted therapy.

Molecular alterations associated with improved outcome in patients with glioblastoma treated with Tumor-Treating Fields.

Background: The genomic and overall biologic landscape of glioblastoma (GB) has become clearer over the past 2 decades, as predictive and prognostic biomarkers of both de novo and transformed forms of GB have been identified. The oral chemotherapeutic agent temozolomide (TMZ) has been integral to standard-of-care treatment for nearly 2 decades. More recently, the use of non-pharmacologic interventions, such as application of alternating electric fields, called Tumor-Treating Fields (TTFields), has emerged as a complementary treatment option that increases overall survival (OS) in patients with newly diagnosed GB. The genomic factors associated with improved or lack of response to TTFields are unknown. Methods: We performed comprehensive genomic analysis of GB tumors resected from 55 patients who went on to receive treatment using TTFields, and compared results to 57 patients who received standard treatment without TTFields. Results: We found that molecular driver alterations in NF1, and wild-type PIK3CA and epidermal growth factor receptor (EGFR), were associated with increased benefit from TTFields as measured by progression-free survival (PFS) and OS. There were no differences when stratified by TP53 status. When NF1, PIK3CA, and EGFR status were combined as a Molecular Survival Score, the combination of the 3 factors significantly correlated with improved OS and PFS in TTFields-treated patients compared to patients not treated with TTFields. Conclusions: These results shed light on potential driver and passenger mutations in GB that can be validated as predictive biomarkers of response to TTFields treatment, and provide an objective and testable genomic-based approach to assessing response.

Phase II Trial of Adjuvant Nivolumab Following Salvage Resection in Patients with Recurrent Squamous Cell Carcinoma of the Head and Neck.

PURPOSE: Locoregional relapse in patients with head and neck squamous cell carcinoma (HNSCC) is common, approaching 50% for some subsites despite multimodality therapy. Salvage surgery is the standard of care, but able to achieve durable control in only a minority of patients. While adjuvant radiotherapy or chemo-radiotherapy is offered to select patients, this approach can be prohibitively toxic. Given the activity and tolerability of programmed death-1 inhibitors in metastatic HNSCC, we investigated the safety and efficacy of adjuvant nivolumab after salvage surgical resection. PATIENTS AND METHODS: This was an open-label, multi-institutional phase II clinical trial (NCT03355560). Patients with recurrent, resectable HNSCC were enrolled within 6 weeks of salvage surgery. Six 28-day cycles of adjuvant nivolumab were planned. The primary endpoint was 2-year disease-free survival (DFS) more than 58%, based on an institutional historical control group of 71 patients with recurrent HNSCC who underwent salvage surgery. RESULTS: Between February 2018 and February 2020, 39 patients were enrolled. At a median follow-up of 22.1 months, 2-year DFS was 71.4% [95% confidence interval (CI), 57.8-88.1] and the 2-year overall survival (OS) was 73% (95% CI, 58-91.8). Three of 39 (8%) patients experienced grade 3 treatment-related adverse events and 3 of 39 (8%) discontinued treatment due to side effects. Ten of 39 had locoregional recurrence, while 2 of 10 also had synchronous metastatic disease. There was no difference in DFS between PD ligand-1 (PD-L1)-positive and PD-L1-negative patients. There was a nonsignificant trend toward improved DFS in patients with high tumor mutational burden (P = 0.083). CONCLUSIONS: Adjuvant nivolumab after salvage surgery in locally recurrent HNSCC is well tolerated and showed improved DFS compared with historical controls.

Identification of potential biomarkers and novel therapeutic targets through genomic analysis of small cell bladder carcinoma and associated clinical outcomes.

OBJECTIVES: Small cell bladder carcinoma (SCBC) represents a rare histologic variant with a poor prognosis and for which no routine biomarkers exist. Limited reports of genomic sequencing in SCBC have demonstrated a high prevalence of TP53 and RB1 gene mutations, though the prognostic value of these and other gene variants in SCBC remains undefined. In this study, we performed targeted genomic sequencing on a cohort of SCBC patients and correlated genomic findings with clinical outcomes to identify potential novel biomarkers. MATERIALS AND METHODS: Thirty-one patients with SCBC and available treatment-naïve tumor specimens were identified from an institutional database (23 limited stage [LS], 8 extensive stage [ES]). Small cell carcinoma specimens were microdissected and subjected to tumor next-generation whole-exon sequencing with a 592 gene panel. Kaplan-Meier techniques and Cox proportional hazards models were used to evaluate genomic aberration association with relapse-free survival (RFS) and overall survival (OS) in the limited stage cohort. RESULTS: The most common pathogenic gene variants included ARID1A (48%), TP53 (48%) and RB1 (48%). Mutations in genes with potential therapeutic targets not routinely evaluated in SCBC included BRCA1/2 (16%), POLE (13%), JAK2 (13%), PDGFB (13%) and FGFR3 (3%). Multiple novel biomarker candidates showed trends for improvements in OS in the LS subset including ERCC2 (HR 0.322, P = 0.122) and RB1 (HR 0.481, P = 0.182), while LS patients with TP53 mutations (HR 2.730, P = 0.056), and MCL1 gene amplification (HR 4.183, P = 0.018) suggested inferior OS. Additionally, gene or copy number variants with potential prognostic benefit included UBR5 and DAXX (P = 0.02, [hazard ratios nonestimable due to zero events in biomarker positive groups]). CONCLUSIONS: These results support the role for tumor genomic profiling in SCBC and identify multiple potential novel biomarkers and therapeutic targets in this rare disease. Efforts to validate these findings should lead to improved decision-making and treatment outcomes in SCBC.

Molecular profiling of signet-ring-cell carcinoma (SRCC) from the stomach and colon reveals potential new therapeutic targets.

Signet ring cell carcinoma (SRCC) is rare: about 10% of gastric cancer (GC) and 1% of colorectal cancer (CRC). SRCC is associated with poor prognosis, however the underlying molecular characteristics are unknown. SRCCs were analyzed using NGS, immunohistochemistry, and in situ hybridization. Tumor mutational burden (TMB) was calculated based on somatic nonsynonymous missense mutations, and microsatellite instability (MSI) was evaluated by NGS of known MSI loci. A total of 8500 CRC and 1100 GC were screened. Seventy-six SRCC were identified from the CRC cohort (<1%) and 98 from the GC cohort (9%). The most frequently mutated genes in CRC-SRCC were TP53 (47%), ARID1A (26%), APC (25%); in GC-SRCC were TP53 (42%), ARID1A (27%), CDH1 (11%). When compared to non-SRCC histology (N = 3522), CRC-SRCC (N = 37) more frequently had mutations in BRCA1 (11% vs 1%, P < 0.001) and less frequently mutations in APC (19% vs 78%, P < 0.001), KRAS (22% vs 51%, P = 0.001) and TP53 (47% vs 73%, P = 0.001). Among the GC cohort, SRCC (N = 54) had a higher frequency of mutations in CDH1, BAP1, and ERBB2, compared to non-SRCC (N = 540). Our data suggest that SRCCs harbor a similar molecular profile, regardless of the tumor location. Tailored therapy may become available for these patients.

Association of TP53 and CDKN2A Mutation Profile with Tumor Mutation Burden in Head and Neck Cancer.

PURPOSE: Head and neck squamous cell carcinoma (HNSCC) is a frequently devastating cancer that affects more than a half million people annually worldwide. Although some cases arise from infection with human papillomavirus (HPV), HPV-negative HNSCC is more common, and associated with worse outcome. Advanced HPV-negative HNSCC may be treated with surgery, chemoradiation, targeted therapy, or immune checkpoint inhibition (ICI). There is considerable need for predictive biomarkers for these treatments. Defects in DNA repair capacity and loss of cell-cycle checkpoints sensitize tumors to cytotoxic therapies, and can contribute to phenotypes such as elevated tumor mutation burden (TMB), associated with response to ICI. Mutation of the tumor suppressors and checkpoint mediators TP53 and CDKN2A is common in HPV-negative HNSCC. EXPERIMENTAL DESIGN: To gain insight into the relation of the interaction of TP53 and CDKN2A mutations with TMB in HNSCC, we have analyzed genomic data from 1,669 HPV-negative HNSCC tumors with multiple criteria proposed for assessing the damaging effect of TP53 mutations. RESULTS: Data analysis established the TP53 and CDKN2A mutation profiles in specific anatomic subsites and suggested that specific categories of TP53 mutations are more likely to associate with CDKN2A mutation or high TMB based on tumor subsite. Intriguingly, the pattern of hotspot mutations in TP53 differed depending on the presence or absence of a cooccurring CDKN2A mutation. CONCLUSIONS: These data emphasize the role of tumor subsite in evaluation of mutational profiles in HNSCC, and link defects in TP53 and CDKN2A to elevated TMB levels in some tumor subgroups.

Molecular profiles of endometrial cancer tumors among Black patients.

OBJECTIVES: Disparate 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. METHODS: A 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. RESULTS: Tumors 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. CONCLUSIONS: This 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.

Comprehensive Analysis of R-Spondin Fusions and RNF43 Mutations Implicate Novel Therapeutic Options in Colorectal Cancer.

PURPOSE: Gene fusions involving R-spondin (RSPOfp) and RNF43 mutations have been shown to drive Wnt-dependent tumor initiation in colorectal cancer. Herein, we aimed to characterize the molecular features of RSPOfp/RNF43 mutated (mut) compared with wild-type (WT) colorectal cancers to gain insights into potential rationales for therapeutic strategies. EXPERIMENTAL DESIGN: A discovery cohort was classified for RSPOfp/RNF43 status using DNA/RNA sequencing and IHC. An independent cohort was used to validate our findings. RESULTS: The discovery cohort consisted of 7,245 colorectal cancer samples. RSPOfp and RNF43 mutations were detected in 1.3% (n = 94) and 6.1% (n = 443) of cases. We found 5 RSPO fusion events that had not previously been reported (e.g., IFNGR1-RSPO3). RNF43-mut tumors were associated with right-sided primary tumors. No RSPOfp tumors had RNF43 mutations. In comparison with WT colorectal cancers, RSPOfp tumors were characterized by a higher frequency of BRAF, BMPR1A, and SMAD4 mutations. APC mutations were observed in only a minority of RSPOfp-positive compared with WT cases (4.4% vs. 81.4%). Regarding RNF43 mutations, a higher rate of KMT2D and BRAF mutations were detectable compared with WT samples. Although RNF43 mutations were associated with a microsatellite instability (MSI-H)/mismatch repair deficiency (dMMR) phenotype (64.3%), and a tumor mutation burden ≥10 mt/Mb (65.8%), RSPOfp was not associated with MSI-H/dMMR. The validation cohort replicated our genetic findings. CONCLUSIONS: This is the largest series of RSPOfp/RNF43-mut colorectal cancers reported to date. Comprehensive molecular analyses asserted the unique molecular landscape associated with RSPO/RNF43 and suggested potential alternative strategies to overcome the low clinical impact of Wnt-targeted agents and immunotherapy.

Molecular Characterization of KRAS Wild-type Tumors in Patients with Pancreatic Adenocarcinoma.

PURPOSE: KRAS mutation (MT) is a major oncogenic driver in pancreatic ductal adenocarcinoma (PDAC). A small subset of PDACs harbor KRAS wild-type (WT). We aim to characterize the molecular profiles of KRAS WT PDAC to uncover new pathogenic drivers and offer targeted treatments. EXPERIMENTAL DESIGN: Tumor tissue obtained from surgical or biopsy material was subjected to next-generation DNA/RNA sequencing, microsatellite instability (MSI) and mismatch repair status determination. RESULTS: Of the 2,483 patients (male 53.7%, median age 66 years) studied, 266 tumors (10.7%) were KRAS WT. The most frequently mutated gene in KRAS WT PDAC was TP53 (44.5%), followed by BRAF (13.0%). Multiple mutations within the DNA-damage repair (BRCA2, ATM, BAP1, RAD50, FANCE, PALB2), chromatin remodeling (ARID1A, PBRM1, ARID2, KMT2D, KMT2C, SMARCA4, SETD2), and cell-cycle control pathways (CDKN2A, CCND1, CCNE1) were detected frequently. There was no statistically significant difference in PD-L1 expression between KRAS WT (15.8%) and MT (17%) tumors. However, KRAS WT PDAC were more likely to be MSI-high (4.7% vs. 0.7%; P < 0.05), tumor mutational burden-high (4.5% vs. 1%; P < 0.05), and exhibit increased infiltration of CD8+ T cells, natural killer cells, and myeloid dendritic cells. KRAS WT PDACs exhibited gene fusions of BRAF (6.6%), FGFR2 (5.2%), ALK (2.6%), RET (1.3%), and NRG1 (1.3%), as well as amplification of FGF3 (3%), ERBB2 (2.2%), FGFR3 (1.8%), NTRK (1.8%), and MET (1.3%). Real-world evidence reveals a survival advantage of KRAS WT patients in overall cohorts as well as in patients treated with gemcitabine/nab-paclitaxel or 5-FU/oxaliplatin. CONCLUSIONS: KRAS WT PDAC represents 10.7% of PDAC and is enriched with targetable alterations, including immuno-oncologic markers. Identification of KRAS WT patients in clinical practice may expand therapeutic options in a clinically meaningful manner.