Prognostic Impact of BRAF and KRAS Mutation in Patients with Colorectal and Appendiceal Peritoneal Metastases Scheduled for CRS and HIPEC

A total of 399 patients were scheduled for CRS and HIPEC between January 2009 and September 2017 at the Department of Surgery, University Hospital, Uppsala, Sweden. Seven patients underwent reoperations, and 58 patients were diagnosed with nonappendiceal or noncolorectal tumors and were excluded from further analysis. All remaining 334 subjects had suspected isolated peritoneal metastases and were judged as potentially curable; i.e., there were no signs of distant tumor spread at the preoperative work-up except for limited and resectable hepatic involvement. The routine work-up consisted of abdominal and thoracic computed tomography (CT) scans, colonoscopy, and routine blood tests including tumor markers, whereas laparoscopy was used in cases where extensive small bowel involvement or other signs of irresectable disease were suspected on preoperative CT scans.

There were no histologically detectable neoplastic cells in the specimens of 39 patients although pre- and intraoperative assessment suggested peritoneal metastases. These patients were also excluded, leaving a total of 295 patients relevant for analysis (Table 1). The primary tumor was colorectal cancer in 178 individuals and appendiceal neoplasms in the remaining 117. KRAS and BRAF mutation status was assessed by pyrosequencing and was performed selectively in 111/295 (38%) of the patients based on clinical indications. A total of 232 patients (79%) received HIPEC, whereas 47 cases were open and close procedures, i.e., judged inoperable, usually because of extensive small bowel involvement. Our policy is to abandon the procedure and refrain from HIPEC if there are definite signs that a completeness of cytoreduction score of 0–1 cannot be achieved.

Sixteen subjects were not treated with HIPEC because of intraoperative complications or doubtful indication. Hepatic Glisson capsulectomy was performed in 24 patients, and formal hepatic resection was performed in 30 cases.

Initially, the abdominal tumor extension was quantified using the PCI, and the ability to perform an R0 resection was assessed by examining the small bowel and other potential failure sites. The technique of cytoreduction consisted of several peritonectomies combined with omentectomy and removal of disease-affected organs, as previously described.12 Briefly, diathermy was used for stripping of the peritoneal layers from the abdominal wall, pelvic walls, and diaphragm. A macroscopically healthy peritoneum was left in situ (i.e., resections were performed depending on the extent of the macroscopic tumor). After CRS, the remaining amount of tumor was graded using the completeness of cytoreduction score.13,14 Immediately after cytoreduction, HIPEC was performed according to the Coliseum method. Briefly, one inflow catheter was placed centrally in the abdomen, and four closed suction drains were inserted through the lateral abdominal wall, allowing for outflow of the chemotherapy solution. Three HIPEC regimens were used: The standard regimen for colorectal primaries was oxaliplatin 460 mg/m² administered over 30 min, preceded by 5-flurouracil 400 mg/m² combined with calcium folinate 30 mg/m² as an IV infusion. As an alternative, e.g., in case of side effects or tumor progress after previous systemic oxaliplatin treatment, irinotecan 460 mg/m² was used as intraperitoneal treatment. Appendiceal tumors with mucinous peritoneal implants were treated with mitomycin 30 mg/m² over 90 min as intraperitoneal treatment.

Solid tumor specimens were collected from all resection sites and fixed in 4% buffered formaldehyde. Paraffin-embedded blocks of tissue were sectioned with a microtome in 3–4-µm sections and stained with hematoxylin–eosin for routine examination. DNA was extracted from paraffin-embedded blocks, and samples with maximum tumor content were obtained by manual microdissection. The PyroMark Q24 BRAF and KRAS version 2.0 assays (Qiagen) were used to detect mutations in BRAF (codon 600) and KRAS (codons 12, 13, and 61 in exons 2 and 3) according to the manufacturer’s recommendations. KRAS and BRAF mutation status was assessed by pyrosequencing (2007–2014) or targeted next-generation sequencing (2015–2016).15,16 Results from mutation anlyses were retrieved from the pathology reports reflecting the clinical routine during the study period.

A sequence library was constructed using a Haloplex™ DiagnPanel_Colon_20160222, and sequencing was performed using a MiSeq instrument (Illumina, San Diego, CA). The analysis was performed on material from the primary tumor in 75 cases and on peritoneal metastases in 36 cases.

Figures are presented as mean (standard deviation, SD), and differences were assessed using Student’s t test. Differences in proportions were assessed by Chi square test or Fisher’s exact test, where appropriate. Overall survival was calculated from date of surgery to date of death from any cause or last follow-up. Survival state was recorded using the Swedish National Population Register as of the end of 2017. Survival curves were constructed according to Kaplan–Meier and differences evaluated by log-rank test. Multivariate analysis was performed using a Cox proportional hazard procedure, and risk estimates are presented as relative risk (RR) with 95% confidence limits of RR. p Value < 0.05 was considered statistically significant. Statistical analyses were performed using Statistica 13 software (Palo Alto, CA). The study was approved by the ethics committee of Uppsala County.

Age (p = 0.97) and gender (p = 0.39) did not affect survival. The most favorable survival was observed for patients with peritoneal metastases from an appendiceal tumor (projected 5-year survival 73%, median survival not reached); intermediate prognosis was observed for peritoneal spread of colon cancer origin (projected 5-year survival 38%, median survival 35 months), and the worst prognosis for rectal cancer origin (projected 5-year survival 22%, median survival 18 moths, p < 0.0001, log-rank test). Signet ring cell differentiation was associated with reduced survival (p = 0.0015), whereas mucinous histology was not (p = 0.57). Those with PCI below 20 experienced longer survival compared with the group with higher PCI (p = 0.0008). Survival outcome in the 25 patients who underwent liver resection as part of CRS did not differ from those without liver resection (p = 0.34).

KRAS mutated tumors were evenly located among the appendix, colon, and rectum, whereas BRAF mutated tumors were predominantly situated in the right colon (Table 2). The proportion undergoing mutation analysis was much smaller among those with an appendiceal primary tumor compared with colorectal tumors (13/117, 11% versus 98/178, 55%, p < 0.0001, Table 1). The 13 patients with a primary in the appendix all had invasive adenocarcinoma, mostly with poor differentiation. Three had mucinous fluid but also solid tumor, whereas 10 had solid peritoneal tumor nodules (Table 3).

Patients whose tumor underwent molecular analysis had shorter survival than those not analyzed (Fig. 1a, p < 0.0001). In all, 101/111 received systemic chemotherapy whereas 10 were not treated. Oxaliplatin-based chemotherapy was used in 55 cases, 5-fluorouracil monotherapy in 9 subjects, whereas 37 patients received both oxaliplatin and irinotecan. In addition, 21 received the anti-EGFR antibody cetuximab and 21 received the VEGF inhibitor bevacizumab. Totally, 36 were treated with one line of chemotherapy, 32 with two lines, 23 with three lines, and 7 with four lines of systemic chemotherapy. Also, after exclusion of patients with appendiceal neoplasms and pseudomyxoma peritonei, there was still a difference between those analyzed and those not analyzed (Fig. 1b, p = 0.019). However, there was no difference in survival related to KRAS mutation (p = 0.126, Fig. 2a), whereas BRAF mutation was associated with shorter survival (Fig. 2b, p = 0.028). These results were also true when only those with CC0 were included in the analyses (data not shown). On multivariate analysis based on all 92 subjects with complete information about age, gender, PCI, CCS, BRAF mutation, and signet cell differentiation, CCS (p = 0.00001), signet ring cell differentiation (p = 0.00001), and BRAF mutation (p = 0.0021) emerged as the statistically significant variables associated with poor survival (Table 4). When the analysis was repeated and stratified based on the open and close procedure, the importance of CCS disappeared and only signet ring cell differentiation (p = 0.000002) and BRAF mutation (p = 0.0049) were predictive of survival.