Low Concordance Between T-Cell Densities in Matched Primary Tumors and Liver Metastases in Microsatellite Stable Colorectal Cancer

BackgroundThe subtype, density and location of tumor infiltrating T-cells are being explored as prognostic and predictive biomarkers in primary colorectal cancer (pCRC) and colorectal liver metastases (CLM). Very limited data exist comparing findings in pCRC and matched CLM.Patients and methodsFifty-eight patients with available pCRC and matched CLM (57/58 microsatellite stable) were included in this OSLO-COMET substudy. In immunohistochemically stained sections, total (Ttot), helper (TH), cytotoxic (CTL), and regulatory (Treg) T-cells were manually counted in hotspots from the invasive margin (IM), intratumor (IT), and tumor adjacent regions to determine T-cell densities.ResultsA striking accumulation of T-cells was found in IM of both pCRC and CLM with much lower densities in the IT region, exemplified by Ttot of 2838 versus 340 cells/mm2, respectively, in CLM. The correlation at the individual level between T-cell densities in pCRC and corresponding CLM was poor for all regions and T-cell subtypes; for instance, the correlation coefficient (R2) for IM Ttot was 0.07. The IT TH : CTL and Treg : TH ratios were 2.94 and 0.44, respectively, in pCRC, and 1.84 and 0.24, respectively, in CLM.ConclusionThe observed accumulation of T-cells in the IM regions of pCRC and CLM with low penetration to the IT regions, combined with high TH : CTL and Treg : TH ratios, point to the presence of an immune suppressive microenvironment. T-cell densities of CLM differed markedly from the matched pCRC, indicating that to evaluate T-cell biomarkers in metastasis, the commonly available pCRC cannot serve as a surrogate for the metastatic tumor

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 nextgeneration 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 upregulated at multiple metastatic sites Mir-210 3p, Mir191 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-tomesenchymal 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

Molecular and immune landscape of colorectal liver metastases

Colorectal cancer is a common cancer in both genders, and 50% develop metastases which is the dominating cause of death from CRC, most frequently located in the liver (CLM). Only 20% of patients with CLM are eligible for surgical resection. For the first time in a randomised study, the OSLO-COMET trial compared short-term outcome between open and laparoscopic resection in 280 patients. Laparoscopic resection had a significantly lower complication rate (19% vs 31%), shorter hospital stay (53 vs 96 hours) at a similar cost. The postoperative 90-day mortality was low (0.04%). The long-term outcome after CLM resection may be associated with molecular and immunological features. Based on analysis of samples from the OSLO-COMET biobank the most frequent mutations were located in the TP53, APC, KRAS, PIK3CA, SMAD4, and NRAS genes, and the cohort was enriched for consensus molecular subtype 2. Importantly, the identified transcriptomic changes suggested that immune activation had taken place in tumours exposed to neoadjuvant chemotherapy (NACT). Almost all analysed cases were microsatellite stable, which is associated with poor response to immunotherapy. Quantification of T-cell densities by immunohistochemistry in primary CRC and matched CLM showed low concordance. T-helper cell dominated with regulatory T-cells comprising up to 44%, suggesting immune suppression. T-cells accumulated in the invasive tumour margin, particularly in CLM. A short-interval (<9.5 weeks) between NACT exposure and CLM resection was strongly associated with high T-cell density, including cytotoxic T-cells. In conclusion, the short-term results of the OSLO-COMET trial support the use of laparoscopy for CLM resection. Analyses of molecular and immunological features suggested the presence of immune suppression in CLM, and that NACT induces a transient immune activation that could be exploited in future studies in combination with immune therapy

Laparoscopic versus open liver resection in the posterosuperior segments: a sub-group analysis from the OSLO-COMET randomized controlled trial

Background Laparoscopic liver resection in the posterosuperior segments is technically challenging. This study aimed to compare the perioperative outcomes for laparoscopic and open resection of colorectal liver metastases located in the posterosuperior segments. Methods This was a subgroup analysis of the OSLO-COMET randomized controlled trial, where 280 patients were randomly assigned to open or laparoscopic parenchyma-sparing liver resections of colorectal metastases. Patients with tumors in the posterosuperior segments were identified, and perioperative outcomes and health related quality of life (HRQoL) were compared. Results We identified a total of 136 patients, 62 in the laparoscopic and 74 in the open group. The postoperative complication rate was 26% in the laparoscopic and 31% in the open group. The blood loss was less in the open group (500 vs. 250 ml, P = 0.006), but the perioperative transfusion rate was similar. The operative time was similar, while postoperative hospital stay was shorter in the laparoscopic group (2 vs. 4 days, P < 0.001). HRQoL was significantly better after laparoscopy at 1 month. Conclusion In patients undergoing laparoscopic or open liver resection of colorectal liver metastases in the posterosuperior segments, laparoscopic surgery was associated with shorter hospital stay and comparable perioperative outcomes

Impact of KRAS, BRAF and microsatellite instability status after cytoreductive surgery and HIPEC in a national cohort of colorectal peritoneal metastasis patients

Background Patients with metastatic colorectal cancer (mCRC) carrying BRAF (mutBRAF) or KRAS mutation (mutKRAS) have an inferior prognosis after liver or lung surgery, whereas the prognostic role in the context of peritoneal metastasis (PM) after cytoreductive surgery (CRS) and hyperthermic intraperitoneal chemotherapy (HIPEC) has been less investigated. Methods In total, 257 patients with non-appendiceal PM-CRC were included from the Norwegian National Unit for CRS-HIPEC. Results In total, 180 patients received CRS-HIPEC with Mitomycin C, 77 patients received palliative surgery only. In the CRS-HIPEC group, mutBRAF was found in 24.7%, mutKRAS 33.9% and double wild-type 41.4% without differences in survival. MSI was found in 29.3% of mutBRAF cases. Patients with mutBRAF/MSI had superior 5-year survival compared to mutBRAF with MSS (58.3% vs 25.2%, P = 0.022), and better 3-year disease-free survival (DFS) compared to mutKRAS (48.6% vs 17.2%, P = 0.049). Peritoneal Cancer Index and the number of lymph node metastasis were prognostic for OS, and the same two, location and gender prognostic for DFS in multivariate analysis. Conclusions PM-CRC with CRS-HIPEC patients has a surprisingly high proportion of mutBRAF (24.7%). Survival was similar comparing mutBRAF, mutKRAS and double wild-type cases, whereas a small subgroup with mutBRAF and MSI had better survival. Patients with mutBRAF tumours and limited PM should be considered for CRS-HIPEC

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

Impact of KRAS, BRAF and microsatellite instability status after cytoreductive surgery and HIPEC in a national cohort of colorectal peritoneal metastasis patients

Patients with metastatic colorectal cancer (mCRC) carrying BRAF (mutBRAF) or KRAS mutation (mutKRAS) have an inferior prognosis after liver or lung surgery, whereas the prognostic role in the context of peritoneal metastasis (PM) after cytoreductive surgery (CRS) and hyperthermic intraperitoneal chemotherapy (HIPEC) has been less investigated. Methods In total, 257 patients with non-appendiceal PM-CRC were included from the Norwegian National Unit for CRS-HIPEC. Results In total, 180 patients received CRS-HIPEC with Mitomycin C, 77 patients received palliative surgery only. In the CRS-HIPEC group, mutBRAF was found in 24.7%, mutKRAS 33.9% and double wild-type 41.4% without differences in survival. MSI was found in 29.3% of mutBRAF cases. Patients with mutBRAF/MSI had superior 5-year survival compared to mutBRAF with MSS (58.3% vs 25.2%, P = 0.022), and better 3-year disease-free survival (DFS) compared to mutKRAS (48.6% vs 17.2%, P = 0.049). Peritoneal Cancer Index and the number of lymph node metastasis were prognostic for OS, and the same two, location and gender prognostic for DFS in multivariate analysis. Conclusions PM-CRC with CRS-HIPEC patients has a surprisingly high proportion of mutBRAF (24.7%). Survival was similar comparing mutBRAF, mutKRAS and double wild-type cases, whereas a small subgroup with mutBRAF and MSI had better survival. Patients with mutBRAF tumours and limited PM should be considered for CRS-HIPEC

Quality of life from a randomized trial of laparoscopic or open liver resection for colorectal liver metastases

Background Most treatments for cancer cause a decline in patients' health‐related quality of life (HRQoL). Limiting this decline is a universal goal for healthcare providers. Using minimally invasive instead of open surgical techniques might be one way to achieve this. The aim of this study was to compare postoperative HRQoL after open and laparoscopic liver resection. Methods This was a predefined substudy of an RCT comparing open with laparoscopic liver resection. Patients with colorectal liver metastases were assigned randomly to open or laparoscopic parenchyma‐sparing liver resection. HRQoL was assessed with the Short Form 36 questionnaire at baseline, and 1 and 4 months after surgery. Results A total of 280 patients were randomized, of whom 273 underwent surgery (129 laparoscopic, 144 open); 682 questionnaires (83.3 per cent) were available for analysis. One month after surgery, patients in the laparoscopic surgery group reported reduced scores in two HRQoL domains (physical functioning and role physical), whereas those in the open surgery group reported reduced scores in five domains (physical functioning, role physical, bodily pain, vitality and social functioning). Four months after surgery, HRQoL scores in the laparoscopic group had returned to preoperative levels, whereas patients in the open group reported reduced scores for two domains (role physical and general health). The between‐group difference was statistically significant in favour of laparoscopy for four domains after 1 month (role physical, bodily pain, vitality and social functioning) and for one domain after 4 months (role physical). Conclusion Patients assigned to laparoscopic liver surgery reported better postoperative HRQoL than those assigned to open liver surgery. For role limitations caused by physical health problems, patients in the laparoscopic group reported better scores up to 4 months after surgery. Registration number: NCT01516710 ( http://www.clinicaltrials.gov)

Impact of KRAS, BRAF and microsatellite instability status after cytoreductive surgery and HIPEC in a national cohort of colorectal peritoneal metastasis patients

Background - Patients with metastatic colorectal cancer (mCRC) carrying BRAF (mutBRAF) or KRAS mutation (mutKRAS) have an inferior prognosis after liver or lung surgery, whereas the prognostic role in the context of peritoneal metastasis (PM) after cytoreductive surgery (CRS) and hyperthermic intraperitoneal chemotherapy (HIPEC) has been less investigated. Methods - In total, 257 patients with non-appendiceal PM-CRC were included from the Norwegian National Unit for CRS-HIPEC. Results - In total, 180 patients received CRS-HIPEC with Mitomycin C, 77 patients received palliative surgery only. In the CRS-HIPEC group, mutBRAF was found in 24.7%, mutKRAS 33.9% and double wild-type 41.4% without differences in survival. MSI was found in 29.3% of mutBRAF cases. Patients with mutBRAF/MSI had superior 5-year survival compared to mutBRAF with MSS (58.3% vs 25.2%, P = 0.022), and better 3-year disease-free survival (DFS) compared to mutKRAS (48.6% vs 17.2%, P = 0.049). Peritoneal Cancer Index and the number of lymph node metastasis were prognostic for OS, and the same two, location and gender prognostic for DFS in multivariate analysis. Conclusions - PM-CRC with CRS-HIPEC patients has a surprisingly high proportion of mutBRAF (24.7%). Survival was similar comparing mutBRAF, mutKRAS and double wild-type cases, whereas a small subgroup with mutBRAF and MSI had better survival. Patients with mutBRAF tumours and limited PM should be considered for CRS-HIPEC

Neoadjuvant chemotherapy is associated with a transient increase of intratumoral T-cell density in microsatellite stable colorectal liver metastases

Patients with colorectal liver metastases (CLM) commonly receive neoadjuvant chemotherapy (NACT) prior to surgical resection. NACT may induce immunogenic cell death with subsequent recruitment of T-cells to the tumor microenvironment, which could be exploited by immune checkpoint inhibition (ICI). In theory, this could expand the use of ICI to obtain responses also in microsatellite stable colorectal cancer, but evidence to suggest optimal treatment schedules are lacking. In this study, densities of total-, cytotoxic-, helper- and regulatory T-cells were quantified by immunohistochemistry in resected CLM from 92 patients included in the OSLO-COMET trial (NCT01516710). All but one patient had microsatellite stable tumors (91/92). Associations between T-cell densities and clinicopathological parameters were analyzed. Fluoropyrimidine-based NACT (in most cases with addition of oxaliplatin or irinotecan) was administered to 45 patients completed median 8 weeks prior to surgical resection. No overall association was found between NACT administration and intratumoral T-cell densities. However, within the NACT group, a short time interval (<9.5 weeks) between NACT completion and CLM resection was strongly associated with high intratumoral T-cell densities compared to the long-interval and no NACT groups (medians 491, 236, and 292 cells/mm2, respectively; P < .0001). The results from this study suggest that the observed increase in intratumoral T-cells after NACT administration may be transient. The significance of this finding should be further explored to ensure that optimal treatment schedules are chosen for studies combining cytotoxic chemotherapy and ICI