A Phase 1 Trial of Trebananib, an Angiopoietin 1 and 2 Neutralizing Peptibody, Combined with Pembrolizumab in Patients with Advanced Ovarian and Colorectal Cancer.

Ovarian cancers and microsatellite stable (MSS) colorectal cancers (CRC) are insensitive to anti-PD1 immunotherapy, and new immunotherapeutic approaches are needed. Preclinical data suggests a relationship between immunotherapy resistance and elevated angiopoietin 2 levels. We performed a phase 1 dose-escalation study of pembrolizumab and the angiopoietin 1/2 inhibitor trebananib (NCT03239145). This multicenter trial enrolled patients with metastatic ovarian cancer or MSS CRC. Trebananib was administered intravenously weekly for 12 weeks with 200 mg intravenous pembrolizumab every 3 weeks. The toxicity profile of this combination was manageable, and the protocol-defined highest dose level (trebananib 30 mg/kg weekly plus pembrolizumab 200 mg every 3 weeks) was declared the maximum tolerated dose. The objective response rate for all patients was 7.3% (90% confidence interval: 2.5-15.9%). Three patients with MSS CRC had durable responses for ≥3 years. One responding patient's CRC harbored a POLE mutation. The other two responding patients had left-sided CRCs with no baseline liver metastases, and genomic analysis revealed that they both had KRAS wild-type, ERBB2 amplified tumors. After development of acquired resistance, biopsy of one patient's KRAS wild-type, ERBB2 amplified tumor showed a substantial decline in tumor-associated T cells and an increase in immunosuppressive intratumoral macrophages. Future studies are needed to carefully assess whether clinicogenomic features, such as lack of liver metastases, ERBB2 amplification, and left-sided tumors, can predict increased sensitivity to PD1 immunotherapy combinations.

Single-cell chromatin accessibility reveals malignant regulatory programs in primary human cancers.

To identify cancer-associated gene regulatory changes, we generated single-cell chromatin accessibility landscapes across eight tumor types as part of The Cancer Genome Atlas. Tumor chromatin accessibility is strongly influenced by copy number alterations that can be used to identify subclones, yet underlying cis-regulatory landscapes retain cancer type-specific features. Using organ-matched healthy tissues, we identified the "nearest healthy" cell types in diverse cancers, demonstrating that the chromatin signature of basal-like-subtype breast cancer is most similar to secretory-type luminal epithelial cells. Neural network models trained to learn regulatory programs in cancer revealed enrichment of model-prioritized somatic noncoding mutations near cancer-associated genes, suggesting that dispersed, nonrecurrent, noncoding mutations in cancer are functional. Overall, these data and interpretable gene regulatory models for cancer and healthy tissue provide a framework for understanding cancer-specific gene regulation.

Contemporary Prognostic Signatures and Refined Risk Stratification of Gliomas: An Analysis of 4,400 Tumors.

BACKGROUND: With the significant shift in the classification, risk stratification, and standards of care for gliomas, we sought to understand how the overall survival of patients with these tumors is impacted by molecular features, clinical metrics, and treatment received. METHODS: We assembled a cohort of patients with a histopathologically diagnosed glioma from The Cancer Genome Atlas, Project Genomics Evidence Neoplasia Information Exchange, and Dana-Farber Cancer Institute/Brigham and Women's Hospital. This incorporated retrospective clinical, histological, and molecular data alongside prospective assessment of patient survival. RESULTS: 4,400 gliomas were identified: 2,195 glioblastoma, 1,198 IDH1/2-mutant astrocytoma, 531 oligodendroglioma, 271 other IDH1/2-wildtype glioma, and 205 pediatric-type glioma. Molecular classification updated 27.2% of gliomas from their original histopathologic diagnosis. Examining the distribution of molecular alterations across glioma subtypes revealed mutually exclusive alterations within tumorigenic pathways. Non-TCGA patients had significantly improved overall survival compared to TCGA patients, with 26.7%, 55.6%, and 127.8% longer survival for glioblastoma, IDH1/2-mutant astrocytoma, and oligodendroglioma respectively (all p<0.01). Several prognostic features were characterized, including NF1 alteration and 21q loss in glioblastoma, and EGFR amplification and 22q loss in IDH1/2-mutant astrocytoma. Leveraging the size of this cohort, nomograms were generated to assess the probability of overall survival based on patient age, the molecular features of a tumor, and the treatment received. CONCLUSIONS: By applying modern molecular criteria, we characterize the genomic diversity across glioma subtypes, identify clinically applicable prognostic features, and provide a contemporary update on patient survival to serve as a reference for ongoing investigations.

Velcrin molecular glues induce apoptosis in glioblastomas with high PDE3A and SLFN12 expression.

Background: Velcrins are molecular glues that kill cells by inducing the formation of a protein complex between the RNase SLFN12 and the phosphodiesterase PDE3A. Formation of the complex activates SLFN12, which cleaves tRNALeu(TAA) and induces apoptosis. Velcrins such as the clinical investigational compound, BAY 2666605, were found to have activity across multiple solid tumor cell lines from the cancer cell line encyclopedia, including glioblastoma cell lines. We therefore aim to characterize velcrins as novel therapeutic agents in glioblastoma. Materials and Methods: PDE3A and SLFN12 expression levels were measured in glioblastoma cell lines, the Cancer Genome Atlas (TCGA) tumor samples, and tumor neurospheres. Velcrin-treated cells were assayed for viability, induction of apoptosis, cell cycle phases, and global changes in translation. Transcriptional profiling of the cells was obtained. Xenograft-harboring mice treated with velcrins were also monitored for survival. Results: We identified several velcrin-sensitive glioblastoma cell lines and 4 velcrin-sensitive glioblastoma patient-derived models. We determined that BAY 2666605 crosses the blood-brain barrier and elicits full tumor regression in an orthotopic xenograft model of GB1 cells. We also determined that the velcrins BAY 2666605 and BRD3800 induce tumor regression in subcutaneous glioblastoma PDX models. Conclusions: Velcrins have antitumor activity in preclinical models of glioblastoma, warranting further investigation as potential therapeutic agents.

Molecular profiling of 888 pediatric tumors informs future precision trials and data-sharing initiatives in pediatric cancer.

To inform clinical trial design and real-world precision pediatric oncology practice, we classified diagnoses, assessed the landscape of mutations, and identified genomic variants matching trials in a large unselected institutional cohort of solid tumors patients sequenced at Dana-Farber / Boston Children's Cancer and Blood Disorders Center. Tumors were sequenced with OncoPanel, a targeted next-generation DNA sequencing panel. Diagnoses were classified according to the International Classification of Diseases for Oncology (ICD-O-3.2). Over 6.5 years, 888 pediatric cancer patients with 95 distinct diagnoses had successful tumor sequencing. Overall, 33% (n = 289/888) of patients had at least 1 variant matching a precision oncology trial protocol, and 14% (41/289) were treated with molecularly targeted therapy. This study highlights opportunities to use genomic data from hospital-based sequencing performed either for research or clinical care to inform ongoing and future precision oncology clinical trials. Furthermore, the study results emphasize the importance of data sharing to define the genomic landscape and targeted treatment opportunities for the large group of rare pediatric cancers we encounter in clinical practice.

Clinicogenomic characterization of inflammatory breast cancer.

Background: Inflammatory breast cancer (IBC) is a rare and poorly characterized type of breast cancer with an aggressive clinical presentation. The biological mechanisms driving the IBC phenotype are relatively undefined-partially due to a lack of comprehensive, large-scale genomic studies and limited clinical cohorts. Patients and Methods: A retrospective analysis of 2457 patients with metastatic breast cancer who underwent targeted tumor-only DNA-sequencing was performed at Dana-Farber Cancer Institute. Clinicopathologic, single nucleotide variant (SNV), copy number variant (CNV) and tumor mutational burden (TMB) comparisons were made between clinically confirmed IBC cases within a dedicated IBC center versus non-IBC cases. Results: Clinicopathologic differences between IBC and non-IBC cases were consistent with prior reports-including IBC being associated with younger age at diagnosis, higher grade, and enrichment with hormone receptor (HR)-negative and HER2-positive tumors. The most frequent somatic alterations in IBC involved TP53 (72%), ERBB2 (32%), PIK3CA (24%), CCND1 (12%), MYC (9%), FGFR1 (8%) and GATA3 (8%). A multivariate logistic regression analysis revealed a significant enrichment in TP53 SNVs in IBC; particularly in HER2-positive and HR-positive disease which was associated with worse outcomes. Tumor mutational burden (TMB) did not differ substantially between IBC and non-IBC cases and a pathway analysis revealed an enrichment in NOTCH pathway alterations in HER2-positive disease. Conclusion: Taken together, this study provides a comprehensive, clinically informed landscape of somatic alterations in a large cohort of patients with IBC. Our data support higher frequency of TP53 mutations and a potential enrichment in NOTCH pathway activation-but overall; a lack of major genomic differences. These results both reinforce the importance of TP53 alterations in IBC pathogenesis as well as their influence on clinical outcomes; but also suggest additional analyses beyond somatic DNA-level changes are warranted.

Comprehensive mutational scanning of EGFR reveals TKI sensitivities of extracellular domain mutants.

The epidermal growth factor receptor, EGFR, is frequently activated in lung cancer and glioblastoma by genomic alterations including missense mutations. The different mutation spectra in these diseases are reflected in divergent responses to EGFR inhibition: significant patient benefit in lung cancer, but limited in glioblastoma. Here, we report a comprehensive mutational analysis of EGFR function. We perform saturation mutagenesis of EGFR and assess function of ~22,500 variants in a human EGFR-dependent lung cancer cell line. This approach reveals enrichment of erlotinib-insensitive variants of known and unknown significance in the dimerization, transmembrane, and kinase domains. Multiple EGFR extracellular domain variants, not associated with approved targeted therapies, are sensitive to afatinib and dacomitinib in vitro. Two glioblastoma patients with somatic EGFR G598V dimerization domain mutations show responses to dacomitinib treatment followed by within-pathway resistance mutation in one case. In summary, this comprehensive screen expands the landscape of functional EGFR variants and suggests broader clinical investigation of EGFR inhibition for cancers harboring extracellular domain mutations.

RAS/RAF Comutation and ERBB2 Copy Number Modulates HER2 Heterogeneity and Responsiveness to HER2-directed Therapy in Colorectal Cancer.

PURPOSE: ERBB2-amplified colorectal cancer is a distinct molecular subtype with expanding treatments. Implications of concurrent oncogenic RAS/RAF alterations are not known. EXPERIMENTAL DESIGN: Dana-Farber and Foundation Medicine Inc. Colorectal cancer cohorts with genomic profiling were used to identify ERBB2-amplified cases [Dana-Farber, n = 47/2,729 (1.7%); FMI, n = 1857/49,839 (3.7%)]. Outcomes of patients receiving HER2-directed therapies are reported (Dana-Farber, n = 9; Flatiron Health-Foundation Medicine clinicogenomic database, FH-FMI CGDB, n = 38). Multisite HER2 IHC and genomic profiling were performed to understand HER2 intratumoral and interlesional heterogeneity. The impact of concurrent RAS comutations on the effectiveness of HER2-directed therapies were studied in isogenic colorectal cancer cell lines and xenografts. RESULTS: ERBB2 amplifications are enriched in left-sided colorectal cancer. Twenty percent of ERBB2-amplified colorectal cancers have co-occurring oncogenic RAS/RAF alterations. While RAS/RAF WT colorectal cancers typically have clonal ERBB2 amplification, colorectal cancers with co-occurring RAS/RAF alterations have lower level ERRB2 amplification, higher intratumoral heterogeneity, and interlesional ERBB2 discordance. These distinct genomic patterns lead to differential responsiveness and patterns of resistance to HER2-directed therapy. ERBB2-amplified colorectal cancer with RAS/RAF alterations are resistant to trastuzumab-based combinations, such as trastuzumab/tucatinib, but retain sensitivity to trastuzumab deruxtecan in in vitro and murine models. Trastuzumab deruxtecan shows clinical efficacy in cases with high-level ERBB2-amplified RAS/RAF coaltered colorectal cancer. CONCLUSIONS: Co-occurring RAS/RAF alterations define a unique subtype of ERBB2-amplified colorectal cancer that has increased intratumoral heterogeneity, interlesional discordance, and resistance to trastuzumab-based combinations. Further examination of trastuzumab deruxtecan in this previously understudied cohort of ERBB2-amplified colorectal cancer is warranted.

XRN1 deletion induces PKR-dependent cell lethality in interferon-activated cancer cells.

Emerging data suggest that induction of viral mimicry responses through activation of double-stranded RNA (dsRNA) sensors in cancer cells is a promising therapeutic strategy. One approach to induce viral mimicry is to target molecular regulators of dsRNA sensing pathways. Here, we show that the exoribonuclease XRN1 is a negative regulator of the dsRNA sensor protein kinase R (PKR) in cancer cells with high interferon-stimulated gene expression. XRN1 deletion causes PKR pathway activation and consequent cancer cell lethality. Disruption of interferon signaling with the JAK1/2 inhibitor ruxolitinib can decrease cellular PKR levels and rescue sensitivity to XRN1 deletion. Conversely, interferon-ß stimulation can increase PKR levels and induce sensitivity to XRN1 inactivation. Lastly, XRN1 deletion causes accumulation of endogenous complementary sense/anti-sense RNAs, which may represent candidate PKR ligands. Our data demonstrate how XRN1 regulates PKR and how this interaction creates a vulnerability in cancer cells with an activated interferon cell state.

Genomic and Immunophenotypic Landscape of Acquired Resistance to PD-(L)1 Blockade in Non-Small-Cell Lung Cancer.

PURPOSE: Although immune checkpoint inhibitors (ICI) have extended survival in patients with non-small-cell lung cancer (NSCLC), acquired resistance (AR) to ICI frequently develops after an initial benefit. However, the mechanisms of AR to ICI in NSCLC are largely unknown. METHODS: Comprehensive tumor genomic profiling, machine learning-based assessment of tumor-infiltrating lymphocytes, multiplexed immunofluorescence, and/or HLA-I immunohistochemistry (IHC) were performed on matched pre- and post-ICI tumor biopsies from patients with NSCLC treated with ICI at the Dana-Farber Cancer Institute who developed AR to ICI. Two additional cohorts of patients with intervening chemotherapy or targeted therapies between biopsies were included as controls. RESULTS: We performed comprehensive genomic profiling and immunophenotypic characterization on samples from 82 patients with NSCLC and matched pre- and post-ICI biopsies and compared findings with a control cohort of patients with non-ICI intervening therapies between biopsies (chemotherapy, N = 32; targeted therapies, N = 89; both, N = 17). Putative resistance mutations were identified in 27.8% of immunotherapy-treated cases and included acquired loss-of-function mutations in STK11, B2M, APC, MTOR, KEAP1, and JAK1/2; these acquired alterations were not observed in the control groups. Immunophenotyping of matched pre- and post-ICI samples demonstrated significant decreases in intratumoral lymphocytes, CD3e+ and CD8a+ T cells, and PD-L1-PD1 engagement, as well as increased distance between tumor cells and CD8+PD-1+ T cells. There was a significant decrease in HLA class I expression in the immunotherapy cohort at the time of AR compared with the chemotherapy (P = .005) and the targeted therapy (P = .01) cohorts. CONCLUSION: These findings highlight the genomic and immunophenotypic heterogeneity of ICI resistance in NSCLC, which will need to be considered when developing novel therapeutic strategies aimed at overcoming resistance.

Comprehensive genomic characterization of HER2-low and HER2-0 breast cancer.

The molecular underpinnings of HER2-low and HER2-0 (IHC 0) breast tumors remain poorly defined. Using genomic findings from 1039 patients with HER2-negative metastatic breast cancer undergoing next-generation sequencing from 7/2013-12/2020, we compare results between HER2-low (n = 487, 47%) and HER2-0 tumors (n = 552, 53%). A significantly higher number of ERBB2 alleles (median copy count: 2.05) are observed among HER2-low tumors compared to HER2-0 (median copy count: 1.79; P = 2.36e-6), with HER2-0 tumors harboring a higher rate of ERBB2 hemideletions (31.1% vs. 14.5%). No other genomic alteration reaches significance after accounting for multiple hypothesis testing, and no significant differences in tumor mutational burden are observed between HER2-low and HER2-0 tumors (median: 7.26 mutations/megabase vs. 7.60 mutations/megabase, p = 0.24). Here, we show that the genomic landscape of HER2-low and HER2-0 tumors does not differ significantly, apart from a higher ERBB2 copy count among HER2-low tumors, and a higher rate of ERBB2 hemideletions in HER2-0 tumors.

XRN1 deletion induces PKR-dependent cell lethality in interferon-activated cancer cells.

Emerging data suggest that induction of viral mimicry responses through activation of double-stranded RNA (dsRNA) sensors in cancer cells is a promising therapeutic strategy. One approach to induce viral mimicry is to target molecular regulators of dsRNA sensing pathways. Here, we show that the exoribonuclease XRN1 is a negative regulator of the dsRNA sensor protein kinase R (PKR) in cancer cells with high interferon-stimulated gene (ISG) expression. XRN1 deletion causes PKR activation and consequent cancer cell lethality. Disruption of interferon signaling with the JAK1/2 inhibitor ruxolitinib can decrease cellular PKR levels and rescue sensitivity to XRN1 deletion. Conversely, interferon-ß stimulation can increase PKR levels and induce sensitivity to XRN1 inactivation. Lastly, XRN1 deletion causes accumulation of endogenous complementary sense/anti-sense RNAs, which may represent candidate PKR ligands. Our data demonstrate how XRN1 regulates PKR and nominate XRN1 as a potential therapeutic target in cancer cells with an activated interferon cell state.

Loss of heterozygosity does not occur in BRCA1/2 mutant pediatric solid and central nervous system tumors.

Utilization of tumor-only sequencing has expanded in pediatric cancer patients, which can lead to identification of pathogenic variants in genes that may be germline and/or have uncertain relevance to the tumor in question, such as the homologous recombination (HR) pathway genes BRCA1/2. We identified patients with pathogenic BRCA1/2 mutations from somatic tumor sequencing, and performed additional germline sequencing to assess for the presence of loss of heterozygosity (LOH). Of seven patients identified, four (57.1%) mutations were found in the germline and none had associated LOH. Our data suggest that BRCA1/2 mutations identified in this context are likely incidental findings.

Oncogenic Drivers and Therapeutic Vulnerabilities in KRAS Wild-Type Pancreatic Cancer.

PURPOSE: Approximately 8% to 10% of pancreatic ductal adenocarcinomas (PDAC) do not harbor mutations in KRAS. Understanding the unique molecular and clinical features of this subset of pancreatic cancer is important to guide patient stratification for clinical trials of molecularly targeted agents. EXPERIMENTAL DESIGN: We analyzed a single-institution cohort of 795 exocrine pancreatic cancer cases (including 785 PDAC cases) with a targeted multigene sequencing panel and identified 73 patients (9.2%) with KRAS wild-type (WT) pancreatic cancer. RESULTS: Overall, 43.8% (32/73) of KRAS WT cases had evidence of an alternative driver of the MAPK pathway, including BRAF mutations and in-frame deletions and receptor tyrosine kinase fusions. Conversely, 56.2% of cases did not harbor a clear MAPK driver alteration, but 29.3% of these MAPK-negative KRAS WT cases (12/41) demonstrated activating alterations in other oncogenic drivers, such as GNAS, MYC, PIK3CA, and CTNNB1. We demonstrate potent efficacy of pan-RAF and MEK inhibition in patient-derived organoid models carrying BRAF in-frame deletions. Moreover, we demonstrate durable clinical benefit of targeted therapy in a patient harboring a KRAS WT tumor with a ROS1 fusion. Clinically, patients with KRAS WT tumors were significantly younger in age of onset (median age: 62.6 vs. 65.7 years; P = 0.037). SMAD4 mutations were associated with a particularly poor prognosis in KRAS WT cases. CONCLUSIONS: This study defines the genomic underpinnings of KRAS WT pancreatic cancer and highlights potential therapeutic avenues for future investigation in molecularly directed clinical trials. See related commentary by Kato et al., p. 4527.

Cancer aneuploidies are shaped primarily by effects on tumour fitness.

Aneuploidies-whole-chromosome or whole-arm imbalances-are the most prevalent alteration in cancer genomes1,2. However, it is still debated whether their prevalence is due to selection or ease of generation as passenger events1,2. Here we developed a method, BISCUT, that identifies loci subject to fitness advantages or disadvantages by interrogating length distributions of telomere- or centromere-bounded copy-number events. These loci were significantly enriched for known cancer driver genes, including genes not detected through analysis of focal copy-number events, and were often lineage specific. BISCUT identified the helicase-encoding gene WRN as a haploinsufficient tumour-suppressor gene on chromosome 8p, which is supported by several lines of evidence. We also formally quantified the role of selection and mechanical biases in driving aneuploidy, finding that rates of arm-level copy-number alterations are most highly correlated with their effects on cellular fitness1,2. These results provide insight into the driving forces behind aneuploidy and its contribution to tumorigenesis.

Impact of Aneuploidy and Chromosome 9p Loss on Tumor Immune Microenvironment and Immune Checkpoint Inhibitor Efficacy in NSCLC.

INTRODUCTION: Although gene-level copy number alterations have been studied as a potential biomarker of immunotherapy efficacy in NSCLC, the impact of aneuploidy burden and chromosomal arm-level events on immune checkpoint inhibitor (ICI) efficacy in NSCLC is uncertain. METHODS: Patients who received programmed cell death protein 1 or programmed death-ligand 1 (PD-L1) inhibitor at two academic centers were included. Across all 22 chromosomes analyzed, an arm was considered altered if at least 70% of its territory was either gained or deleted. Among nonsquamous NSCLCs which underwent targeted next-generation sequencing, we retrospectively quantified aneuploidy using the adjusted fraction of chromosomal arm alterations (FAA), defined as the number of altered chromosome arms divided by the number of chromosome arms assessed, adjusted for tumor purity. RESULTS: Among 2293 nonsquamous NSCLCs identified, the median FAA increased with more advanced cancer stage and decreased with higher PD-L1 tumor proportion score (TPS) levels (median FAA in TPS < 1%: 0.09, TPS 1%-49%: 0.08, TPS ≥ 50%: 0.05, p < 0.0001). There was a very weak correlation between FAA and tumor mutational burden when taken as continuous variables (R: 0.07, p = 0.0005). A total of 765 advanced nonsquamous NSCLCs with available FAA values were treated with ICIs. With decreasing FAA tertiles, there was a progressive improvement in objective response rate (ORR 15.1% in upper tertile versus 23.2% in middle tertile versus 28.4% in lowest tertile, p = 0.001), median progression-free survival (mPFS 2.5 versus 3.3 versus 4.1 mo, p < 0.0001), and median overall survival (mOS 12.5 versus 13.9 versus 16.4 mo, p = 0.006), respectively. In the arm-level enrichment analysis, chromosome 9p loss (OR = 0.22, Q = 0.0002) and chromosome 1q gain (OR = 0.43, Q = 0.002) were significantly enriched in ICI nonresponders after false discovery rate adjustment. Compared with NSCLCs without chromosome 9p loss (n = 452), those with 9p loss (n = 154) had a lower ORR (28.1% versus 7.8%, p < 0.0001), a shorter mPFS (4.1 versus 2.3 mo, p < 0.0001), and a shorter mOS (18.0 versus 9.6 mo, p < 0.0001) to immunotherapy. In addition, among NSCLCs with high PD-L1 expression (TPS ≥ 50%), chromosome 9p loss was associated with lower ORR (43% versus 6%, p < 0.0001), shorter mPFS (6.4 versus 2.6 mo, p = 0.0006), and shorter mOS (30.2 versus 14.3 mo, p = 0.0008) to immunotherapy compared with NSCLCs without 9p loss. In multivariable analysis, adjusting for key variables including FAA, chromosome 9p loss, but not 1q gain, retained a significant impact on ORR (hazard ratio [HR] = 0.25, p < 0.001), mPFS (HR = 1.49, p = 0.001), and mOS (HR = 1.47, p = 0.003). Multiplexed immunofluorescence and computational deconvolution of RNA sequencing data revealed that tumors with either high FAA levels or chromosome 9p loss had significantly fewer tumor-associated cytotoxic immune cells. CONCLUSIONS: Nonsquamous NSCLCs with high aneuploidy and chromosome 9p loss have a distinct tumor immune microenvironment and less favorable outcomes to ICIs.