Novel drug resistance mechanisms and drug targets in BRAF-mutated peritoneal metastasis from colorectal cancer.

BACKGROUND: Patients with peritoneal metastasis from colorectal cancer (PM-CRC) have inferior prognosis and respond particularly poorly to chemotherapy. This study aims to identify the molecular explanation for the observed clinical behavior and suggest novel treatment strategies in PM-CRC. METHODS: Tumor samples (230) from a Norwegian national cohort undergoing surgery and hyperthermic intraperitoneal chemotherapy (HIPEC) with mitomycin C (MMC) for PM-CRC were subjected to targeted DNA sequencing, and associations with clinical data were analyzed. mRNA sequencing was conducted on a subset of 30 samples to compare gene expression in tumors harboring BRAF or KRAS mutations and wild-type tumors. RESULTS: BRAF mutations were detected in 27% of the patients, and the BRAF-mutated subgroup had inferior overall survival compared to wild-type cases (median 16 vs 36 months, respectively, p < 0.001). BRAF mutations were associated with RNF43/RSPO aberrations and low expression of negative Wnt regulators (ligand-dependent Wnt activation). Furthermore, BRAF mutations were associated with gene expression changes in transport solute carrier proteins (specifically SLC7A6) and drug metabolism enzymes (CES1 and CYP3A4) that could influence the efficacy of MMC and irinotecan, respectively. BRAF-mutated tumors additionally exhibited increased expression of members of the novel butyrophilin subfamily of immune checkpoint molecules (BTN1A1 and BTNL9). CONCLUSIONS: BRAF mutations were frequently detected and were associated with particularly poor survival in this cohort, possibly related to ligand-dependent Wnt activation and altered drug transport and metabolism that could confer resistance to MMC and irinotecan. Drugs that target ligand-dependent Wnt activation or the BTN immune checkpoints could represent two novel therapy approaches.

Molecular characteristics of tubo-ovarian carcinosarcoma at different anatomic locations.

Carcinosarcoma (CS) is an uncommon and clinically aggressive malignancy. The objective of the present study was to characterize the molecular features of CS at various anatomic locations, including serous effusions. Specimens (n = 32) consisted of 25 biopsies/surgical resection specimens and 7 serous effusions (6 peritoneal, 1 pleural) from 25 patients. Fresh-frozen cell pellets and surgical specimens underwent targeted next-generation sequencing covering 50 unique genes. A total of 31 mutations were found in 25 of the 32 tumors studied, of which 1 had 3 mutations, 4 had 2 different mutations, and 20 had a single mutation. The most common mutations were in TP53 (n = 25 in 24 tumors; 1 tumor with 2 different mutations), with less common mutations found in RB1 (n = 2), MET (n = 1), KRAS (n = 1), PTEN (n = 1), and KIT (n = 1). Patient-matched specimens harbored the same TP53 mutation. Tumors with no detected mutations were more common in serous effusion specimens (3/7; 43%) compared with surgical specimens (4/25; 16%). In conclusion, the molecular landscape of CS is dominated by TP53 mutations, reinforcing the observation that the majority of these tumors develop from high-grade serous carcinoma. Whether CS cells in serous effusions differ from their counterparts in solid lesions remains uncertain.

Different Frequency and Clinical Role for MTAP Loss in Pleural and Peritoneal Mesothelioma.

The objective of this study was to analyze the expression and prognostic role of methylthioadenosine phosphorylase (MTAP) in mesothelioma. MTAP protein expression by immunohistochemistry was analyzed in 113 mesotheliomas (60 pleural and 53 peritoneal), consisting of 36 effusions and 77 surgical specimens. MTAP expression was fully lost in 38 tumors and partially lost in 8 tumors. Loss of expression was significantly more common in effusions compared with biopsies/surgical resection specimens (20/36 vs. 26/77; P =0.017), and in pleural compared with peritoneal mesotheliomas (35/60 vs. 11/53; P <0.001). MTAP performed less robustly than BAP1 in comparative analysis of 57 tumors previously analyzed for expression of the latter protein (46 vs. 25 cases with loss of expression). In survival analysis for 69 patients with partial clinical data, male gender was significantly associated with shorter overall survival (OS; P =0.042), whereas loss of MTAP was associated with a trend for shorter OS ( P =0.058), with no prognostic role for patient age ( P =0.379) or anatomic site ( P =0.381). The association between loss of MTAP and poor OS became significant when survival analysis was limited to patients with pleural mesothelioma ( P =0.018). In conclusion, loss of MTAP expression is more frequent in pleural compared with peritoneal mesothelioma and has limited diagnostic relevance at the latter anatomic site. More frequent loss in effusion specimens suggests a role for this marker in effusion cytology. MTAP loss in pleural mesothelioma is associated with poor survival.

Determining a minimum data set for reporting clinical and radiologic data for pseudomyxoma peritonei.

Objectives: Pseudomyxoma peritonei (PMP) is a rare cancer currently affecting over 11,736 patients across Europe. Since PMP is so uncommon, collaboration between scientific centers is key to discovering the mechanisms behind the disease, efficient treatments, and targets pointing to a cure. To date, no consensus has been reached on the minimum data that should be collected during PMP research studies. This issue has become more important as biobanking becomes the norm. This paper begins the discussion around a minimum data set that should be collected by researchers through a review of available clinical trial reports in order to facilitate collaborative efforts within the PMP research community. Content: A review of articles from PubMed, CenterWatch, ClinicalTrials.gov and MedRxiv was undertaken, and clinical trials reporting PMP results selected. Summary: There is a core set of data that researchers report, including age and sex, overall survival, peritoneal cancer index (PCI) score, and completeness of cytoreduction, but after this, reports become variable. Outlook: Since PMP is a rare disease, it is important that reports include as large of a number of standardised data points as possible. Our research indicates that there is still much ground to cover before this becomes a reality.

Molecular characteristics of low-grade serous carcinoma in effusions.

OBJECTIVE: The molecular characteristics of low-grade serous carcinoma (LGSC) in serous effusions have not been studied previously. The present study analysed the molecular profile of LGSC at this anatomical site. METHODS: Specimens consisted of a series of 17 serous effusions (15 peritoneal, 2 pleural) from 16 patients, of which 15 were LGSC and 2 serous borderline tumour (SBT) who later progressed to LGSC. For comparative purposes, 9 surgical specimens from 6 patients with LGSC were analysed. Fresh-frozen cell pellets and surgical specimens underwent targeted next-generation sequencing covering 50 unique genes. RESULTS: Mutations were found in tumours from 14 of the 22 patients, of whom 4 had 2 different mutations and 10 had a single mutation. Overall, the most common mutations were in KRAS (n = 3) and BRAF (n = 3), followed by NRAS (n = 2), CDK2NA (n = 2), TP53 (n = 2), ATM (n = 2). Mutations in MET, STK11, ERBB2 and FLT3 were found in one case each. Patient-matched specimens had the same molecular profile. Both effusions with TP53 mutation had concomitant ATM mutation, and both stained immunohistochemically with a wild-type pattern. The absence of mutations was associated with a trend for shorter overall survival in univariate analysis (p = 0.072). CONCLUSIONS: The molecular alterations in LGSCs in serous effusions are consistent with those found in solid tumours, with frequent alterations in the mitogen-activated protein kinase pathway. Mutations in LGSC may be a marker of better outcomes.

A comprehensive framework for analysis of microRNA sequencing data in metastatic colorectal cancer.

Although microRNAs (miRNAs) contribute to all hallmarks of cancer, miRNA dysregulation in metastasis remains poorly understood. The aim of this work was to reliably identify miRNAs associated with metastatic progression of colorectal cancer (CRC) using novel and previously published next-generation sequencing (NGS) datasets generated from 268 samples of primary (pCRC) and metastatic CRC (mCRC; liver, lung and peritoneal metastases) and tumor adjacent tissues. Differential expression analysis was performed using a meticulous bioinformatics pipeline, including only bona fide miRNAs, and utilizing miRNA-tailored quality control and processing. Five miRNAs were identified as up-regulated at multiple metastatic sites Mir-210_3p, Mir-191_5p, Mir-8-P1b_3p [mir-141-3p], Mir-1307_5p and Mir-155_5p. Several have previously been implicated in metastasis through involvement in epithelial-to-mesenchymal transition and hypoxia, while other identified miRNAs represent novel findings. The use of a publicly available pipeline facilitates reproducibility and allows new datasets to be added as they become available. The set of miRNAs identified here provides a reliable starting-point for further research into the role of miRNAs in metastatic progression.

T cell receptor repertoire sequencing reveals chemotherapy-driven clonal expansion in colorectal liver metastases.

BACKGROUND: Colorectal liver metastasis (CLM) is a leading cause of colorectal cancer mortality, and the response to immune checkpoint inhibition (ICI) in microsatellite-stable CRC has been disappointing. Administration of cytotoxic chemotherapy may cause increased density of tumor-infiltrating T cells, which has been associated with improved response to ICI. This study aimed to quantify and characterize T-cell infiltration in CLM using T-cell receptor (TCR) repertoire sequencing. Eighty-five resected CLMs from patients included in the Oslo CoMet study were subjected to TCR repertoire sequencing. Thirty-five and 15 patients had received neoadjuvant chemotherapy (NACT) within a short or long interval, respectively, prior to resection, while 35 patients had not been exposed to NACT. T-cell fractions were calculated, repertoire clonality was analyzed based on Hill evenness curves, and TCR sequence convergence was assessed using network analysis. RESULTS: Increased T-cell fractions (10.6% vs. 6.3%) were detected in CLMs exposed to NACT within a short interval prior to resection, while modestly increased clonality was observed in NACT-exposed tumors independently of the timing of NACT administration and surgery. While private clones made up >90% of detected clones, network connectivity analysis revealed that public clones contributed the majority of TCR sequence convergence. CONCLUSIONS: TCR repertoire sequencing can be used to quantify T-cell infiltration and clonality in clinical samples. This study provides evidence to support chemotherapy-driven T-cell clonal expansion in CLM in a clinical context.

Peptide vaccine targeting mutated GNAS: a potential novel treatment for pseudomyxoma peritonei.

BACKGROUND: Pseudomyxoma peritonei (PMP) is a rare, slow-growing abdominal cancer with no efficacious treatment options in non-resectable and recurrent cases. Otherwise, rare activating mutations in the GNAS oncogene are remarkably frequent in PMP and the mutated gene product, guanine nucleotide-binding protein α subunit (Gsα), is a potential tumor neoantigen, presenting an opportunity for targeting by a therapeutic cancer vaccine. METHODS: Tumor and blood samples were collected from 25 patients undergoing surgery for PMP (NCT02073500). GNAS mutation analysis was performed by next-generation targeted sequencing or digital droplet PCR. Responses to stimulation with Gsα mutated (point mutations R201H and R201C) 30 mer peptides were analyzed in peripheral blood T cells derived from patients with PMP and healthy donors. Fresh PMP tumor samples were analyzed by mass cytometry using a panel of 35 extracellular markers, and cellular subpopulations were clustered and visualized using the visual stochastic network embedding analysis tool. RESULTS: GNAS mutations were detected in 22/25 tumor samples (88%; R201H and R201C mutations detected in 16 and 6 cases, respectively). Strong T cell proliferation against Gsα mutated peptides was observed in 18/24 patients with PMP. Mass cytometry analysis of tumor revealed infiltration of CD3 +T cells in most samples, with variable CD4+:CD8 + ratios. A large proportion of T cells expressed immune checkpoint molecules, in particular programmed death receptor-1 and T cell immunoreceptor with Ig and ITIM, indicating that these T cells were antigen experienced. CONCLUSION: These findings point to the existence of a pre-existing immunity in patients with PMP towards mutated Gsα, which has been insufficient to control tumor growth, possibly because of inhibition of antitumor T cells by upregulation of immune checkpoint molecules. The results form a rationale for exploring peptide vaccination with Gsα peptides in combination with immune checkpoint inhibiton as a possible curative treatment for PMP and other GNAS mutated cancers.

Omics analyses in peritoneal metastasis-utility in the management of peritoneal metastases from colorectal cancer and pseudomyxoma peritonei: a narrative review.

High-throughput "-omics" analysis may provide a broader and deeper understanding of cancer biology to define prognostic and predictive biomarkers and identify novel therapy targets. In this review we provide an overview of studies where the peritoneal tumor component of peritoneal metastases from colorectal cancer (PM-CRC) and pseudomyxoma peritonei (PMP) were analyzed. Most of the available data was derived from DNA mutation analysis, but a brief review of findings from transcriptomic and protein expression analysis was also performed. Studies reporting genomic analysis of peritoneal tumor samples from 1,779 PM-CRC and 623 PMP cases were identified. The most frequently mutated genes in PM-CRC were KRAS, APC, SMAD4, BRAF, and PIK3CA, while in PMP KRAS, GNAS, FAT4, TGFBR1, TP53 and SMAD3/4 mutations were most commonly identified. Analyses were performed by single-gene analyses and to some extent targeted next-generation sequencing, and a very limited amount of broad explorative data exists. The investigated cohorts were typically small and heterogeneous with respect to the methods used and to the reporting of clinical data. This was even more apparent regarding transcriptomic and protein data, as the low number of cases examined and quality of clinical data would not support firm conclusions. Even for the most frequently mutated genes, the results varied greatly; for instance, KRAS mutations were reported at frequencies between 20-57% in PM-CRC and 38-100% in PMP. Such variation could be caused by random effects in small cohorts, heterogeneity in patient selection, or sensitivity of applied technology. Although a large number of samples have been subjected to analysis, cross-study comparisons are difficult to perform, and combined with small cohorts and varying quality and detail of clinical information, the observed variation precludes useful interpretation in a clinical context. Although omics data in theory could answer questions to aid management decisions in PM-CRC and PMP, the existing data does not presently support clinical implementation. With the necessary technologies being generally available, the main challenge will be to obtain sufficiently large, representative cohorts with adequate clinical data and standardized reporting of results. Importantly, studies where the focus is specifically on peritoneal disease are needed, where the study designs are aligned with clearly defined research questions to allow robust conclusions. Such studies are highly warranted if patients with PM-CRC and PMP are to derive benefit from recent advances in precision cancer medicine.

The circulating soluble form of the CD40 costimulatory immune checkpoint receptor and liver metastasis risk in rectal cancer.

BACKGROUND: In colorectal cancer, the inflamed tumour microenvironment with its angiogenic activities is immune- tolerant and incites progression to liver metastasis. We hypothesised that angiogenic and inflammatory factors in serum samples from patients with non-metastatic rectal cancer could inform on liver metastasis risk. METHODS: We measured 84 angiogenic and inflammatory markers in serum sampled at the time of diagnosis within the population-based cohort of 122 stage I-III patients. In a stepwise manner, the statistically strongest proteins associated with time to development of liver metastasis were analysed in the corresponding serum samples from 273 stage II-III rectal cancer patients in three independent cohorts. RESULTS: We identified the soluble form of the costimulatory immune checkpoint receptor cluster of differentiation molecule 40 (sCD40) as a marker of liver metastasis risk across all patient cohorts-the higher the sCD40 level, the shorter time to liver metastasis. In patients receiving neoadjuvant treatment, the sCD40 value remained an independent variable associated with progression to liver metastasis along with the local treatment response. Of note, serum sCD40 was not associated with progression to lung metastasis. CONCLUSIONS: Circulating sCD40 is a marker of liver metastasis risk in rectal cancer and may be developed for use in clinical practice.

Experimental Treatment of Mucinous Peritoneal Metastases Using Patient-Derived Xenograft Models.

Mucinous peritoneal metastases (PM) generally respond poorly to systemic treatment, and there is a clear unmet need for new treatment strategies to improve survival and quality of life for patients with PM. In this work, the growth inhibitory effect of five drugs (oxaliplatin (OXA; 5 mg/kg), irinotecan (IRI; 60 mg/kg), cabazitaxel (CBZ; 15 or 30 mg/kg), regorafenib (REG; 10, 30 or 60 mg/kg), and capecitabine (CAP; 359 or 755 mg/kg) was investigated in three orthotopic patient-derived xenograft models that mimic mucinous PM. Drugs were administered intraperitoneally (i.p.) as monotherapy weekly for 4 weeks (OXA, IRI), as one single i.p. injection (CBZ), or orally (REG, CAP) daily 5 of 7 days per week for four weeks, and i.p. tumor growth and survival were monitored and compared between treatment groups. The i.p. administered drugs (OXA, IRI, CBZ) had the strongest growth inhibitory effect, with OXA being most efficacious, completely inhibiting tumor growth in the majority of the animals. CBZ and IRI also strongly inhibited tumor growth, but with more variation in efficacy between the models. A moderate reduction in tumor growth was observed in all models treated with REG, while CAP had little to no growth inhibitory effect. Targeted next-generation-sequencing has identified mutational profiles typically associated with PM (mutations in KRAS, GNAS, and BRAF oncogenes), supporting the representativeness of the models. The results presented in this work support the continued exploration of i.p. treatment protocols for PM, with OXA remaining and CBZ emerging as particularly interesting candidates for further studies.