PBRM1 mutations might render a subtype of biliary tract cancers sensitive to drugs targeting the DNA damage repair system.

Polybromo-1 (PBRM1) loss of function mutations are present in a fraction of biliary tract cancers (BTCs). PBRM1, a subunit of the PBAF chromatin-remodeling complex, is involved in DNA damage repair. Herein, we aimed to decipher the molecular landscape of PBRM1 mutated (mut) BTCs and to define potential translational aspects. Totally, 1848 BTC samples were analyzed using next-generation DNA-sequencing and immunohistochemistry (Caris Life Sciences, Phoenix, AZ). siRNA-mediated knockdown of PBRM1 was performed in the BTC cell line EGI1 to assess the therapeutic vulnerabilities of ATR and PARP inhibitors in vitro. PBRM1 mutations were identified in 8.1% (n = 150) of BTCs and were more prevalent in intrahepatic BTCs (9.9%) compared to gallbladder cancers (6.0%) or extrahepatic BTCs (4.5%). Higher rates of co-mutations in chromatin-remodeling genes (e.g., ARID1A 31% vs. 16%) and DNA damage repair genes (e.g., ATRX 4.4% vs. 0.3%) were detected in PBRM1-mutated (mut) vs. PBRM1-wildtype (wt) BTCs. No difference in real-world overall survival was observed between PBRM1-mut and PBRM1-wt patients (HR 1.043, 95% CI 0.821-1.325, p = 0.731). In vitro, experiments suggested that PARP ± ATR inhibitors induce synthetic lethality in the PBRM1 knockdown BTC model. Our findings served as the scientific rationale for PARP inhibition in a heavily pretreated PBRM1-mut BTC patient, which induced disease control. This study represents the largest and most extensive molecular profiling study of PBRM1-mut BTCs, which in vitro sensitizes to DNA damage repair inhibiting compounds. Our findings might serve as a rationale for future testing of PARP/ATR inhibitors in PBRM1-mut BTCs.

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.

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.

Targeting BRCA and DNA Damage Repair Genes in GI Cancers: Pathophysiology and Clinical Perspectives.

Mutated germline alleles in the DNA damage repair (DDR) genes "breast cancer gene 1" (BRCA1) and BRCA2 have originally been identified as major susceptibility genes in breast and ovarian cancers. With the establishment and approval of more cost-effective gene sequencing methods, germline and somatic BRCA mutations have been detected in several cancers. Since the approval of poly (ADP)-ribose polymerase inhibitors (PARPi) for BRCA-mutated cancers, BRCA mutations gained rising therapeutic implications. The impact and significance of BRCA mutations have been evaluated extensively in the last decades. Moreover, other genes involved in the DDR pathway, such as ATM, ATR, or CHK1, have emerged as potential new treatment targets, as inhibitors of these proteins are currently under clinical investigation. This review gives a concise overview on the emerging clinical implications of mutations in the DDR genes in gastrointestinal cancers with a focus on BRCA mutations.

High indoleamine-2,3-dioxygenase 1 (IDO) activity is linked to primary resistance to immunotherapy in non-small cell lung cancer (NSCLC).

BACKGROUND: Metabolic profiling in non-small cell lung cancer (NSCLC) may identify key metabolic vulnerabilities and shows enormous discovery potential. Preclinical studies showed that metabolic rewiring in cancer plays an essential role in modulation of immunotherapy response. However, this situation is understudied in the clinical setting. Therefore, we aimed to evaluate the plasma metabolic profile of immune checkpoint inhibitor (CI) responding versus non-responding NSCLC patients. The aim of this project is to identify potential predictive biomarkers for CI response. METHODS: Plasma samples from CI treated NSCLC patients were analysed at baseline and at the first follow up scan by using a broad targeted metabolomics mass spectrometry panel, and were compared to healthy controls. For further validation of identified key alterations high-performance liquid chromatography (HPLC) for tryptophan (Trp) and kynurenine (Kyn) as indicator of IDO-activity was performed. RESULTS: Sixty-seven metabolites were significantly altered in NSCLC patients compared to healthy controls. The metabolic profile of patients with primary CI resistance showed an increase in indoleamine-2,3-dioxygenase (IDO) and a decrease in branched-chain amino acids (BCAA) compared to baseline concentrations. Deregulated IDO activity was validated by additional HPLC measurements, which revealed that baseline Trp levels were predictive for CI response. According to receiver operating characteristic (ROC)-analysis baseline Trp levels ≥49.3 µmol/L predicted disease control at the first follow up scan with a sensitivity of 89% and a specificity of 71%. CONCLUSIONS: We showed that NSCLC patients are characterized by a distinct metabolic profile compared to healthy controls. Moreover, metabolic changes during CI therapy were observed. Of those IDO metabolism seemed to play an important role in primary CI resistance. Trp as a surrogate parameter of IDO activity is a promising biomarker in patients undergoing treatment with CIs and might be a future marker in trials investigating IDO inhibitors.

Molecular characteristics of BRCA1/2 and PALB2 mutations in pancreatic ductal adenocarcinoma.

INTRODUCTION: Poly-(ADP)-ribose polymerase (PARP) inhibitors are successfully used for treatment of BRCA-mutated (mut) breast cancers and are under extensive evaluation for BRCA- and PALB2-mutated pancreatic ductal adenocarcinoma (PDAC). However, the optimal treatment regimen for BRCA/PALB2-mutated PDCA has yet to be established. Moreover, limited data are available on the association of BRCA/PALB2 gene alterations with other comutations and immunological biomarkers. MATERIAL AND METHODS: Tumour samples of 2818 patients with PDAC were analysed for BRCA1/2 PALB2 mutations and other genes by next-generation sequencing (NGS) (MiSeq on 47 genes, NextSeq on 592 genes). TMB was calculated based on somatic non-synonymous missense mutations. MSI-H/dMMR was evaluated by NGS, and PD-L1 expression was determined using immunohistochemistry. RESULTS: In 4.2% (n=124) of all PDAC samples BRCA mutations have been detected. BRCA2 mutations were more commonly observed than BRCA1 mutations (3.1%(n=89) vs 1.1% [n=35], p<0.0001). BRCA2 mutation was associated with an older age (64 vs 61 years for wild-type (wt), p=0.002) and PALB2 mutation was observed more frequently in female than in male patients. BRCA and PALB2 mutations were associated with MSI-H/dMMR compared with wt (BRCA: 4.8% vs 1.2%, p=0.002; PALB2: 6.7% vs 1.3 %, p=0.18), PD-L1 expression of >1.0% (BRCA: 21.8% vs wt 11.2%, p<0.001, PALB2: 0.0% vs 12.4 %, p=0.38) and high TMB (BRCA: mean 8.7 vs 6.5 mut/MB, p<0.001; PALB2: 10.6 mut/Mb vs 6.6 mut/Mb, p=0.0007). Also, PD-L1 expression and TMB differed between BRCA and PALB2 mutation and wt samples in MSS tumours (p<0.05). BRCA-mutated and PALB2-mutated PDACs were characterised by a different mutational profile than was observed in wt tumours. CONCLUSIONS: BRCA and PALB2 mutations were found in a significant subgroup of PDACs. These mutations were associated with a distinct molecular profile potentially predictive for response to immune-checkpoint inhibitor therapy. Therefore, these data provide a rationale to evaluate PARP inhibitors in combination with immune-checkpoint inhibitors in patients with BRCA/PALB2-mutated PDAC.

WRN-Mutated Colorectal Cancer Is Characterized by a Distinct Genetic Phenotype.

Werner syndrome gene (WRN) contributes to DNA repair. In cancer, WRN mutations (WRN-mut) lead to genomic instability. Thus, WRN is a promising target in cancers with microsatellite instability (MSI). We assessed this study to investigate the molecular profile of WRN-mut in colorectal cancer (CRC). Tumor samples were analyzed using next-generation sequencing (NGS) in-situ hybridization and immunohistochemistry. Tumor mutational burden (TMB) was calculated based on somatic nonsynonymous missense mutations. Determination of tumor mismatch repair (MMR) or microsatellite instability (MSI) status was conducted by fragment analysis. WRN-mut were detected in 80 of 6854 samples (1.2%). WRN-mut were more prevalent in right-sided compared to left-sided CRC (2.5% vs. 0.7%, p < 0.0001). TMB, PD-L1 and MSI-H/dMMR were significantly higher in WRN-mut than in WRN wild-type (WRN-wt). WRN-mut were associated with a higher TMB in the MSI-H/dMMR and in the MSS (microsatellite stable) subgroups. Several genetic differences between WRN-mut and WRN-wt CRC were observed, i.e., TP53 (47% vs. 71%), KRAS (34% vs. 49%) and APC (56% vs. 73%). This is the largest molecular profiling study investigating the genetic landscape of WRN-mut CRCs so far. A high prevalence of MSI-H/dMMR, higher TMB and PD-L1 in WRN-mut tumors were observed. Our data might serve as an additional selection tool for trials testing immune checkpoint antibodies in WRN-mut CRC.

Treatment According to Molecular Profiling in Relapsed/Refractory Cancer Patients: A Review Focusing on Latest Profiling Studies.

In this review we aim to summarize studies investigating the impact of a molecular profiling (MP)-guided treatment approach in heavily pretreated cancer patients. In summary, many independent single- and multicenter studies showed a significant benefit of MP-guided treatment regarding response rates and survival. However, in the only randomized trial conducted so far, no benefit of MP-guided targeted therapy was observed. Notably, various profiling approaches were conducted in the respective studies: some studies used a single analytic approach (i.e. next-generation sequencing), others applied multiple analytic methods to perform comprehensive molecular profiling. It seems that multiplatform profiling analyses, detected an increased number of druggable molecular targets or signaling pathway alterations and that a higher proportion of patients was treated according to the molecular cancer profile. Even though no randomized study has shown a benefit of molecular profiling so far, many studies indicate that MP-guided treatment can be beneficial in patients with relapsed and/or refractory cancer. Currently ongoing large randomized trials (i.e. NCI-MATCH, TAPUR) will add evidence to the role of profiling-guided cancer treatment.

High IDO-1 expression in tumor endothelial cells is associated with response to immunotherapy in metastatic renal cell carcinoma.

Nivolumab belongs to the standard therapy in the second-line setting of metastatic renal cell carcinoma (mRCC). Although deep and long-lasting responses are seen in some patients, the majority of patients will further progress. PD-L1 is still under critical evaluation as a predictive biomarker. Thus, more accurate biomarkers are clearly warranted. Here, we investigated for the first time the predictive role of IDO-1, a negative immune-regulatory molecule, on clear cell RCC tissues of 15 patients undergoing nivolumab therapy. IDO-1 and other immune inhibitory molecules (PD-L1, PD-L2, FOXP3) as well as immune cell subsets (CD3, CD4 and CD8) were measured on formalin-fixed, paraffin-embedded sections of RCC specimens by immunohistochemistry. IDO-1 was predominantly expressed in tumor endothelial cells, and was totally absent from tumor cells itself. IDO-1 overexpression (>10%) could be detected more frequently in responders (100%, n = 6/6) compared to non-responders (33.3%, n = 3/9; P = .028), resulting in a better progression-free survival during immunotherapy (IDO-1 ≤ 10% vs >10%, median: 3.5 vs not estimated (NE) months, P = .01 by log-rank test). In addition, IDO-1 was positively correlated with CD8+ T cell expression (rs = .691, P = .006). PD-L1 expression on tumor cells was negative in 13 (86.7%) of 15 patients, irrespective of therapeutic response (responders vs non-responders: 83.3% vs 88.9%). No differences were noticed in the PD-L1 expression on tumor-infiltrating immune cells (PD-L1 < 1% in 66.7% of both responders and non-responders). In contrast to PD-L1, these results suggest that IDO-1 may be a more promising predictive biomarker for response to immune-based cancer therapy in mRCC.