Mutant Metastatic Colorectal Cancer Research Articles

The Genomic Environment of BRAF Mutated and BRAF/PIK3CA Double Mutated Colorectal Cancers.

Background: Colorectal cancer represents the most prevalent gastrointestinal malignancy. Prognosis of metastatic disease has improved in recent years with the introduction of effective systemic therapies, but mean survival remains in the range of two to three years. Targeted therapies based on specific molecular alterations in sub-sets of colorectal cancers have the potential of contributing to therapeutic progress. BRAF and PIK3CA are oncogenic kinases commonly mutated in colorectal cancers and can be targeted through small molecule kinase inhibitors. Methods: Clinical and genomic data from two extensive series of colorectal cancers were interrogated to define the molecular characteristics of cancers with BRAF mutations with and without concomitant mutations in PIK3CA. Results: Colorectal cancers that are BRAF and PIK3CA double mutants represent a small minority of about 5% of colorectal cancers in the two examined series of mostly localized disease. They also represent about one third of all BRAF mutated colorectal cancers. Most mutations in BRAF are classic V600E mutations. A high prevalence of MSI and CIMP is observed in BRAF mutated colorectal cancers with or without PIK3CA mutations. Mutations in tumor suppressors FBXW7 and ATM display a higher prevalence in BRAF mutated cancers. The prognosis of BRAF mutated colorectal cancers with or without PIK3CA mutations is not significantly different than counterparts with wild type BRAF. This contrasts with the known adverse prognostic effect of BRAF in metastatic disease and relates to the different prevalence of MSI in mutant BRAF localized versus metastatic colorectal cancers. Conclusions: BRAF mutations are the defining molecular alterations in double mutant BRAF and PIK3CA colorectal cancers as determined by increased MSI and CIMP in BRAF subsets with and without PIK3CA mutations. Moreover, BRAF mutated cancers with and without PIK3CA mutations are characterized by the absence of KRAS mutations and a lower prevalence of APC mutations than BRAF wild type counterparts. Mismatch-repair-associated gene mutations display higher frequencies in BRAF mutated colorectal cancers. Despite the absence of prognosis implications of BRAF mutations in the studied cohorts of mostly localized cancers, such mutations could be prognostic in certain subsets. The presence of mutations in other genes, such as ATM and high MSI status present opportunities for combination therapies.

WJOG13219G: The Efficacy and Safety of FOLFOXIRI or Doublet plus Anti-VEGF Therapy in Previously Untreated BRAFV600E Mutant Metastatic Colorectal Cancer: A Multi-Institutional Registry-Based Study (BRACELET Study)

BackgroundThe real-world survival benefit of FOLFOXIRI (fluorouracil, leucovorin, oxaliplatin, and irinotecan) plus anti-VEGF therapy (Triplet) over doublet chemotherapy (Doublet) remains controversial in patients with BRAFV600E mutant metastatic colorectal cancer (mCRC). Patients and MethodsWJOG13219G was a multicenter, retrospective, registry-based study of patients with BRAFV600E mutant mCRC who received first-line triplet or doublet chemotherapy from January 2014 to December 2019 in Japan. Inverse probability of treatment weighting (IPTW) was used to adjust for patient background. ResultsThe analysis included 79 and 91 patients in the Triplet and Doublet groups, respectively. The Triplet group was significantly younger and had better performance status. No statistical difference was noted in progression-free survival (PFS; HR, 0.82; 95% CI, 0.60–1.13; P = .22) and overall survival (OS; HR, 0.88; 95% CI, 0.62–1.25; P = .48) between both groups. IPTW analysis also showed no difference between the 2 groups in PFS (HR, 0.86; 95% CI, 0.69–1.08; P = .20) and OS (HR, 0.93; 95% CI, 0.73–1.20; P = .59). The Triplet and Doublet groups had an objective response rate of 53% and 41%, respectively (P = .10). At least one grade 3 or 4 adverse event was seen in 51 (65%) and 43 (47%) patients in the Triplet and Doublet groups, respectively, with the incidence of neutropenia being significantly higher in the former. ConclusionTriplet therapy had no survival benefit versus doublet therapy in the overall and IPTW cohorts or specific subgroups for real-world patients with BRAFV600E mutant mCRC.

P41-7 Two cases of BRAF mutant mCRC with intestinal stenosis responded to encorafenib + binimetinib + cetuximab: A case report

BRAF mutant metastatic Colorectal Cancer (mCRC) has a poor prognosis and response to chemotherapy. The oral agents, encorafenib and binimetinib in combination with cetuximab (BEACON-3) was approved as 2nd-line therapy for BRAF mutant mCRC in Japan. However, we often experience mCRC patients with intestinal stenosis. These patients may not be eligible for oral medication due to decline of drug absorption. We report two cases of BRAF mutant mCRC that developed intestinal stenosis during 1st-line therapy and responded to BEACON-3.

RAS co-mutation and early onset disease represent an aggressive phenotype of atypical (non-V600) BRAF mutant metastatic colorectal cancer.

3592 Background: While BRAFV600E accounts for the majority of BRAF mutations in mCRC, non-V600 BRAF variants (a BRAF) have emerged in recent years as a distinct molecular subtype. There are no consensus recommendations regarding management. This study provides a comprehensive profile of a BRAF, their clonalities and co-mutations in mCRC using a large genomic database as well as a prospective treatment cohort of patients with a BRAF and mCRC managed at a single center. Methods: A systematic analysis was performed of patients with mCRC who underwent ctDNA testing (Guardant360 platform, Guardant Health) from September 2014 to May 2021. A variant was defined as clonal if the mutant allele frequency (MAF) was greater than 50% of the highest somatic MAF in the sample; otherwise it was defined as subclonal. Co-mutation analysis was conducted with BRAF, KRAS, NRAS, NF1, ERBB2, PIK3CA and SMAD4. Treatment history and overall survival (OS) for patients with a BRAF mCRC from MD Anderson Cancer Center were included. Results: 1,733 out of 14,742 mCRC patients had at least one BRAF variant, including 6.5% of patients with BRAFV600E variants and 6.2% with a BRAF variants (1.1% with class II, 1.9% with class III, and 3.2% with unclassified variants). 431 unique BRAF variants were identified in a total of 1,905 BRAF variants. BRAF class II and III variants showed a higher rate of co-occurring KRAS mutations (25.6% and 21.5%) and co-occurring NRAS mutations (5.8% and 2.7%) compared with BRAFV600E variants (2.4% for KRAS and 0.1% for NRAS); however, co-occurring KRAS G12C was only noted in one patient. In our MDACC cohort, 38 patients were included in the analysis. The median age was 55, 81% were Caucasian, and 74 % had left sided primary tumors (45% rectal, 24% sigmoid) with 37% being exposed to at least 2 lines of therapy. The most common mutations in clinical practice were class III, D594G (39%), followed by class II G469A (10%), & class III G466E (7%). The median follow-up time was 23.8 months (mo). While there were no survival differences between a BRAF classes II and III, there was a significant difference in OS in patients with RAS co-mutation (28.3 mo vs not reached [NR], p = 0.05) or liver involvement (28.8 mo vs NR, p = 0.02). Patients < 50 years of age had extremely poor survival with OS of 16.3 mo (vs. NR) and HR 7.51 (95% CI 1.82-31.0, p = 0.005). Treatment with anti-EGFR or use of metastasectomy was not associated with improved survival. Conclusions: a BRAF mutations have historically been considered a favorable prognostic marker in mCRC. Co-mutation with RAS is frequent for both classes and portends poor survival in our real-world cohort. Furthermore, early onset a BRAF mCRC is associated with more aggressive disease. These factors highlight the need for dedicated clinical trials for this unique subset of mCRC and may represent an opportunity to improve management in early onset colorectal cancer.

Impact of circulating tumor DNA (ctDNA) mutant allele fraction in response to anti-angiogenic therapy in RAS-mutant metastatic colorectal cancer (mCRC): Clinical data in the first-line setting and correlation in patient-derived xenograft (PDX) models.

3560 Background: Currently, there are no well-established biomarkers available to select mCRC patients (pts) who will benefit most from antiangiogenic therapy. RAS mutant (mt) allele fraction in plasma (plMAF) is an independent prognostic factor in mCRC. Preliminary data from our group suggests the possible predictive role of plMAF in RAS mt pts treated with 1st line chemotherapy (ct) +/- bevacizumab (bev). Methods: Data prospectively/retrospectively collected from RASmt mCRC pts who received 1st line ct+/-bev treatment in our center, selecting the subset of pts with plMAF sample evaluable with digital PCR (BEAMing) at baseline. Pts were stratified as high (≥ 5.8%) or low ( < 5.8%) plMAF, based on a previously established prognostic cutoff (Elez et al, Mol Onc 2019). We investigated the associations between clinic-pathological variables, overall survival (OS), and progression-free survival (PFS) stratified by plMAF RAS levels using Cox regression models. OS and PFS were calculated by Kaplan-Meier method. Murine PDX were developed from mCRC pts including models from patients with KRASG12/G13 mutations to explore recapitulation of the clinical findings. Results: From October ‘17 to December ‘21, 102 basal plasma samples were analyzed by BEAMing. 47 pts (46%) were classified as high, 39 pts (38%) as low, and 16 (16%) as no-mt detected by BEAMing (non-shedding). OS was significant longer in low plMAF pts than in high plMAF pts (median OS 15.9 vs 37.1 months (mo); HR 0.43; p = 0.001). In high plMAF pts, a trend towards a better PFS was observed in those pts treated with ct+bev compared to ct alone (median 9.4 vs 6.1 mo; HR 0.6; p = 0.18). No differences were observed in low plMAF pts treated with ct +/- bev (median 14.5 vs 14.9 mo; HR 1.2; p = 0.58). Results were not modified when adjusted by the presence of liver metastases. The multivariate PFS model showed no association between RAS plMAF and clinicopathological variables, except for treatment benefit with ct+bev and better outcomes in pts with resectable liver metastases. Interestingly, human ctDNA in one murine PDX model from non-shedding pt was not detectable, whereas human ctDNA was present in another PDX model with a plMAF of 36.2%. Conclusions: This study suggests that plMAF could be a promising predictive biomarker of response to bev in 1st line RASmt mCRC, but more pts need to be analyzed to confirm this effect. Our results confirm the prognostic role of RASmt plMAF in mCRC. plMAF could partially depend on tumor cell shedding degree and characteristics on the tumor vasculature architecture, this is being investigated in ongoing imaging studies and PDX models. These models will also help to understand the biology behind tumor response to bev and its connection with ctDNA shedding.

P-68 Regorafenib monotherapy as second-line treatment of patients with RAS-mutant advanced colorectal cancer (STREAM): An academic, multicenter, single-arm, two-stage, phase 2 study

Therapeutic options after first-line treatment in patients with RAS mutant metastatic colorectal cancer (mCRC) are limited by poor clinical outcomes and lack of activity of anti-epidermal growth factor receptor drugs. Maintaining angiogenesis inhibition and switching the chemotherapy backbone represents the current therapeutic strategy of second-line mCRC therapy. Regorafenib, an oral broad-spectrum multikinase inhibitor targeting angiogenesis, has been shown to prolong overall survival (OS) and is approved after progression on chemotherapy. STREAM trial was an academic, multicentric, phase 2, single-arm, Simon’s two-stage design, aimed to establish the activity of regorafenib, defined as the rate of patients alive and progression free after 6months from study entry (6mo-PFS) in patients with RAS mutant mCRC, after progression on fluoropyrimidine, oxaliplatin and bevacizumab.Setting α and β errors at 0.10, defining a 6mo-PFS rate of 30% as p0 and 50% as p1, to consider the study positive ≧8 patients without progression at 6mo, were needed in the first stage (N=22) and ≧18 in the overall population (N=46). Regorafenib was administered orally at the approved schedule of 160mg day1-21 q28. Secondary endpoints were the evaluation of toxicity, objective response rate (ORR), progression-free survival (PFS), OS. Early metabolic response assessed by PET-CT scan after 2 weeks of treatment was an exploratory endpoint. Translational analyses are ongoing and will be presented at a later stage. Between November 2015, and December 2020, 46 patients were enrolled. median age was 67ys; 80.4% had ECOG performance status 0, 32.6% single organ involvement, 26% lung-limited disease. The study did not meet its predefined primary endpoint. Eight of the first 22 patients and 14 in the overall population were 6mo-PFS, as compared to the 18 required by the study protocol. At a median follow-up of 50.2 months (95%CI=24.2-56.3), ORR was 10.9%, Disease Control Rate (DCR) was 54.6%, median (m)PFS was 3.6mo (95%CI=1.9-6.7).Despite the short mPFS, regorafenib did not preclude a subsequent treatment: mPFS2 (from study entry to progression to subsequent treatment line) was 13.3mo (95%CI=8.4-19.7) and mOS was 18.9mo (95%CI=10.3-35.3). No unexpected toxicity was reported. Grade≥3 AEs occurred in 18 patients(39.1%), mostly hand foot syndrome(13%), fatigue and bilirubin increase(6.5%). Early metabolic response with PET/CT was not associated with ORR, mPFS and mOS. Baseline metabolic assessment, not predictive of ORR and mPFS, was significantly associated with mOS. A subgroup of patients, with single organ involvement, mostly lung-limited disease, low baseline PET-CT parameters, a low rate of early progression in first line (<6months=21.4%) and a long mPFS2 (23.3mo 95%CI=13.3-39.9), reported a long disease control with regorafenib: ORR=35.7%; mPFS=10.2mo (95%CI =8.6-13.5), mOS=38.8mo (95%CI=23.9-NR). Conversely, patients with higher radiological and metabolic burden of disease, higher rate of early progression in first-line (<6months=56.3%) and a shorter mPFS2 (8.4mo CI95%=6.1-13.3), reported unfavorable outcomes. Despite the study did not meet its primary endpoint, treatment with regorafenib had no unexpected toxicity and did not preclude efficacy of subsequent treatments. A subgroup of patients characterized by good prognostic features (limited disease, lung metastasis and low metabolic burden) had clinical benefit with regorafenib. Appropriate patients selection might guide the clinical development of regorafenib in an earlier setting, ensuring a chemotherapy-free interval in the treatment sequence.

Encorafenib in Combination With Cetuximab After Systemic Therapy in Patients With BRAFV600E Mutant Metastatic Colorectal Cancer: German Health Technology Assessment-Driven Analyses From the BEACON CRC Study

BackgroundPurpose of this analysis was to report data of the BEACON CRC trial used in the German Health Technology Assessment (HTA) and previously unpublished data focusing on the dual blockade (encorafenib + cetuximab) and appropriate comparative therapy (ACT/control: cetuximab + irinotecan-based chemotherapy) of patients with BRAFV600E-mutant mCRC. Materials and MethodsAnalyses included overall survival (OS) and time-to-event analyses of morbidity and safety. ResultsA total of 220 patients received encorafenib + cetuximab and 221 patients ACT/control.Median OS was 9.3 (encorafenib + cetuximab) versus 5.9 months (ACT/control) (stratified hazard ratio (HRstrat): 0.61 [95% confidence interval: 0.48-0.77]).Time-to-response (TTR) showed a statistically significant advantage for encorafenib + cetuximab compared to ACT/control (HRstrat [95% CI]: 10.46 [3.75; 29.15]; P < .0001). Median progression-free survival 2, ie, PFS after initiation of subsequent treatment after completion of study treatment, was 8.3 (dual blockade) versus 5.3 months (ACT/control), representing a statistically significant benefit for the dual blockade (HRstrat [95% CI]: 0.62 [0.48; 0.78]; P < .0001).The statistically significant advantage for diarrhea (EORTC QLQ-C30) reached clinical relevance (LS-mean [95% CI]; P-value / Hedges'g [95% CI]: -12.61 [-17.75; -7.47]; P < .0001 / -0.53 [-0.74; -0.31]).The time-to-event analyses showed a statistically significant benefit for the dual blockade for serious adverse events (AE), severe AEs and AEs leading to discontinuation. ConclusionIn the HTA, the German G-BA granted a “hint for a considerable additional benefit” of encorafenib + cetuximab compared to the ACT in BRAFV600E-mutant mCRC patients. This treatment is considered the new standard of care for these patients.

Liver Endothelium Promotes HER3-Mediated Cell Survival in Colorectal Cancer with Wild-Type and Mutant KRAS.

This body of work highlighted a potential strategy of using HER3 antibodies in combination with standard chemotherapy agents for treating patients with either KRAS wild-type or KRAS mutant metastatic colorectal cancer.

Combined targeted therapy for BRAF mutant metastatic colorectal cancer: are we there yet?

Combined targeted therapy for BRAF mutant metastatic colorectal cancer: are we there yet?

WJOG13219G: Triplet versus doublet in patients with previously untreated BRAFV600E-mutant metastatic colorectal cancer: A multi-institutional real-world data analysis (BRACELET study).

37 Background: The survival benefit of FOLFOXIRI plus bevacizumab (Triplet) over Doublet in patients (pts) with BRAFV600E-mutant metastatic colorectal cancer (mCRC) remains controversial. We compared Triplet therapy with Doublet and explored the pts subgroups that could benefit from intensive chemotherapy (chemo) using real-world data. Methods: WJOG13219G was a multicenter, retrospective registry-based study of pts with BRAFV600E-mutant mCRC who received first-line doublet or triplet chemo with/out molecular targeted agents in January 2014–December 2019. Primary analysis focused on pts who received VEGF inhibitor-containing chemo. To adjust pts background, the inverse probability of treatment weighting (IPTW) method based on propensity scores calculated by age, ECOG PS, and disease status (recurrent/metastatic) was used. Results: A total of 232 pts from 33 hospitals were registered. After excluding 18 pts treated with anti-EGFR antibody-containing regimen and 44 without any targeted agents, 79 pts with Triplet and 91 with Doublet were analyzed. Baseline pts disposition was as follows: median age, 61 y; male proportion, 51%; PS 0/1/≥2, 63%/32%/5%; recurrent/metastatic, 26%/74%; and right/left primary, 68%/32%. Significant differences were noted in age and PS between the two groups. At median follow-up of 24.0 months, no statistical difference was noted in progression-free survival (PFS) (median 9.7 months of Triplet vs. 7.8 months of Doublet, HR = 0.89, P = 0.49) and overall survival (OS) (median 18.7 vs. 18.3 months, HR = 0.87, P = 0.52). The objective response rate was 53% in the Triplet group and 41% in Doublet (P = 0.10). Curative surgery after chemo was more frequent in the Triplet group than in Doublet (13% vs. 3%, P = 0.02). Two pts (3%) in the Triplet group and 6 (7%) in Doublet received immunotherapy as subsequent chemo; 13 (16%) and 6 (7%) also received BRAF inhibitor-containing therapy. IPTW analysis showed no difference between the two groups in PFS (HR = 0.82, P = 0.07) and OS (HR = 0.92, P = 0.57). In the subgroup analysis, pts with right-sided primary tumor in the Triplet group showed favorable trends of PFS (HR, 0.87; 95% CI, 0.65–1.16) and OS (HR, 0.71; 95%CI, 0.50–1.01), whereas pts with left-sided tumor in the Triplet group showed the reverse trends of PFS (HR, 1.17; 95% CI, 0.77–1.78) and OS (HR, 1.68; 95% CI, 0.97–2.91). Conclusions: Some baseline characteristics were significantly different between real-world pts in the Triplet and Doublet groups. Although the Triplet group did not show any survival benefit compared with Doublet in the original and IPTW cohorts, pts with right-sided BRAFV600E-mutant mCRC could benefit from Triplet therapy.

Options beyond BRAF targeted therapy in second-line treatment of patients with BRAFV600E mutant (BRAFmt) metastatic colorectal cancer (mCRC).

47 Background: BRAFmt is a negative prognostic factor in mCRC but also identifies a patient population that may benefit from BRAF targeted therapy. Results from recent trials (BEACON and SWOG1406) demonstrate improved survival outcomes in second- and third-line settings when combining a BRAF inhibitor, an EGFR inhibitor (EGFRi) +/- a MEK inhibitor. In both trials, irinotecan and cetuximab was the control arm with a dismal response rate of 2-4% and progression free survival (PFS) of only 2 months. This suggests chemotherapy plus an EGFRi may not be the optimal approach where BRAF-targeted therapies are not available or have failed. Methods: Data from July 2009 to September 2021 was analysed from TRACC, a multi-site Australian mCRC comprehensive prospective registry enrolling consecutive patients. Patient characteristics, treatment and survival outcomes were examined for patients treated with chemotherapy (CT) alone, with bevacizumab (BEV) or with an EGFRi. Results: Of 2046 registry patients, 256 (13%) harboured a BRAFmt. 72 BRAFmt patients had received second-line (28%) treatment, including CT alone (n = 28), CT plus BEV (n = 26), and CT plus EGFRi (n = 18). Baseline characteristics are shown in the table. Median second-line PFS was 3.3, 4.7 and 1.8 months, for CT alone, CT plus BEV and CT plus EGFRi respectively. Median overall survival (OS) was 8.7, 7.9 and 2.5 months respectively. In multivariate analysis, PFS when treated with CT plus EGFRi trended inferior to CT alone (p = 0.054) and CT plus BEV (p = 0.061), whereas for OS, treatment with CT plus EGFRi was inferior to CT alone (p = 0.038) and CT plus BEV (p = 0.015). Poor PFS was associated with age ≥ 65 years (HR 3.03, p &lt; 0.001) and ECOG ≥ 2 (HR 2.62, p = 0.004), but not associated with a right side primary (p = 0.17), mismatch repair (MMR) status (p = 0.86), or ≥ 3 organs with metastases (p = 0.32). Poor OS was associated with age ≥ 65 years (HR 3.11, p &lt; 0.001), ECOG ≥ 2 (HR 7.32, p &lt; 0.001), right side primary (HR 3.03, p = 0.002) and proficient MMR status (HR 3.57, p = 0.018), but not associated with ≥ 3 organs with metastases (p = 0.17). Conclusions: Less than one-third of BRAFmt mCRC patients received second-line therapy in a real-world setting, indicating an urgency to explore activity of BRAF targeted therapy in the first line setting. Treated patients received limited benefit, with CT plus EGFRi PFS outcomes comparable to BEACON control arm (1.8 vs 2.0 months) and trending inferior to other options. The best OS outcomes were achieved with CT alone or CT plus BEV.[Table: see text]

Monitoring of RAS mutant clones in plasma of patients with RAS mutant metastatic colorectal cancer

PurposeSome patients with histologically confirmed primary mCRC and mutated RAS reported undetectable RAS mutant clones in plasma after receiving anti-VEGF treatment. The aim was to prospectively assess it with its potential therapeutic implications.MethodsRAS mutant genes in solid biopsy (before first-line treatment: FOLFOX/CAPOX + bevacizumab) were compared in liquid biopsy (before second-line treatment: panitumumab + FOLFIRI), using Idylla™ system. Discordant results between solid/liquid biopsies were assessed by the next-generation sequencing (NGS) test (solid/liquid biopsies).ResultsTwenty-three patients were assessed (seven had RAS mutant discrepancies between solid/liquid biopsies). The NGS test confirmed that 3/23 (13%) patients had undetectable RAS mutant clones in liquid biopsy and 3/23 (13%) presented discrepancies in solid biopsy (Idylla™ system vs. NGS test).ConclusionThirteen percentage of patients had undetectable RAS mutant clones in liquid biopsy after first-line treatment. However, some discrepancies between solid and liquid biopsies have been observed. These results suggest a need to improve accuracy of RAS analyses, especially in solid biopsies.

382O Inhibition of WEE1 is effective in TP53 and RAS mutant metastatic colorectal cancer (mCRC): A randomised phase II trial (FOCUS4-C) comparing adavosertib (AZD1775) with active monitoring

Outcomes in RAS-mutant metastatic colorectal cancer (mCRC) remain poor and patients have limited therapeutic options. Adavosertib is the first small molecule inhibitor of WEE1 kinase. We hypothesised that aberrations in DNA replication seen in mCRC with both RAS and TP53 mutations would sensitise tumours to WEE1 inhibition.

440P Response to BRAF inhibitors combined with anti-EGFR after previous anti-EGFR exposure for BRAF V600E mutant metastatic colorectal cancer patients

Encorafenib and cetuximab is efficient in anti-EGFR-naïve patients (pts) with BRAFV600E mutated (BRAFm) metastatic colorectal cancer (mCRC) after failure of one or two prior lines of treatment (BEACON trial). However to date, the efficacy of BRAF inhibitors in combination with anti-EGFRs (B+E) in patients (pts) previously treated with an anti-EGFR agent has never been reported.

O-10 ANCHOR CRC: Results from a single-arm, phase 2 study of encorafenib, binimetinib plus cetuximab in previously untreated BRAF V600E–mutant metastatic colorectal cancer

O-10 ANCHOR CRC: Results from a single-arm, phase 2 study of encorafenib, binimetinib plus cetuximab in previously untreated BRAF V600E–mutant metastatic colorectal cancer

Random survival forests identify pathways with polymorphisms predictive of survival in KRAS mutant and KRAS wild-type metastatic colorectal cancer patients

KRAS status serves as a predictive biomarker of response to treatment in metastatic colorectal cancer (mCRC). We hypothesize that complex interactions between multiple pathways contribute to prognostic differences between KRAS wild-type and KRAS mutant patients with mCRC, and aim to identify polymorphisms predictive of clinical outcomes in this subpopulation. Most pathway association studies are limited in assessing gene–gene interactions and are restricted to an individual pathway. In this study, we use a random survival forests (RSF) method for identifying predictive markers of overall survival (OS) and progression-free survival (PFS) in mCRC patients treated with FOLFIRI/bevacizumab. A total of 486 mCRC patients treated with FOLFIRI/bevacizumab from two randomized phase III trials, TRIBE and FIRE-3, were included in the current study. Two RSF approaches were used, namely variable importance and minimal depth. We discovered that Wnt/β-catenin and tumor associated macrophage pathway SNPs are strong predictors of OS and PFS in mCRC patients treated with FOLFIRI/bevacizumab independent of KRAS status, whereas a SNP in the sex-differentiation pathway gene, DMRT1, is strongly predictive of OS and PFS in KRAS mutant mCRC patients. Our results highlight RSF as a useful method for identifying predictive SNPs in multiple pathways.

Yes-activated protein promotes primary resistance of BRAF V600E mutant metastatic colorectal cancer cells to mitogen-activated protein kinase pathway inhibitors.

Most colorectal cancer (CRC) patients with the BRAF V600E mutation display resistance to chemotherapy and targeted medicinal treatments. Thus, exploring new drugs and drug resistance mechanisms for the BRAF V600E mutation has become an urgent clinical priority. MTS experiment, cell cloning experiment, cell scratching experiment, Transwell experiment, chromatin immunoprecipitation (ChIP), quantitative polymerase chain reaction (qPCR) and flow cytometry are used. Detect the transcription and protein expression of YAP in colorectal cancer cell lines, establish a transient cell line with YAP gene overexpression and knockdown, and detect the effect of YAP gene expression on the biological functions of colorectal cancer cells RKO and HT-29. And further study the mechanism of YAP regulating the response of RAF and MEK targeted therapy. In this study, for the first time, we verified that the expression of transcription factor yes-associated protein (YAP) was upregulated in BRAF V600E mutant CRC cells. After knocking down YAP, we observed a reduction in the growth rate, proliferation, and invasion ability of colon cancer cells. We further verified that YAP knockdown increased sensitivity of BRAF V600E mutant CRC cells to mitogen-activated protein kinase (MAPK) pathway inhibitors. In addition, we clarified the mechanism underlying YAP regulation of RAF and MAPK/extracellular signal-regulated kinase (MEK)-targeted therapy response: YAP cooperates with RAF→MEK pathway inhibitors to regulate the cell cycle, increase cell G1/S phase arrest, and increase apoptosis. These results suggest that YAP expression may be related to the primary resistance of MAPK inhibitors in metastatic CRC with the BRAF V600E mutation. Therefore, the combination of YAP and MAPK pathway inhibitors in BRAF V600E mutant metastatic CRC may present a promising treatment method.

Discovery of a potential predictive marker for eribulin treatment and novel target genes in BRAF V600E mutant metastatic colorectal cancer using an AI-driven RNA-seq analysis platform: Translational research of the BRAVERY study (EPOC1701).

e15532 Background: Patients (pts) with BRAF V600E mutant (mt) metastatic colorectal cancer (mCRC) still have a poor prognosis even when treated with encorafenib plus cetuximab. We reported that eribulin that inhibits G2-M cell cycle had limited activity for these pts in the phase II BRAVERY study (Masuishi T, et al. WCGC 2020). Barras D, et al. (2017) reported that BRAF V600E mt mCRCs were classified into BM1 and BM2 characterized by KRAS/AKT pathway activation and deregulation of the cell cycle with G2-M phase activation, respectively, based on gene expression. Methods: Whole transcriptome RNA-seq of FFPE pre-treatment tumor samples was performed using NovaSeq 6000 in the BRAVERY study. Molecular features were extracted using the Txome.ai platform (Ocean Genomics Inc., PA, USA) which included transcript- and gene-level expression quantification, expression-based clustering, and structural variant calling. Efficacy of eribulin was classified as “good” if pts had a tumor reduction and/or progression-free survival (PFS) of more than 6 months, and “poor” otherwise. The differential gene expression analysis was performed between pts with “good” and “poor” using Txome.ai. In addition, BM and consensus molecular subtype (CMS) classification were performed using the model developed by Barras D, et al. (2017) and Guinney J, et al. (2015), respectively. Results: Among 27 pts, 26 tumor samples were available to perform RNA-seq and analyze gene expression despite low mapping rates. Patient characteristics were as follows: median age of 58.5 (range, 33–71) years; ECOG PS of 0/1 (16/10); primary tumor location of right/left (11/15); and all 26 pts had MSS/pMMR. Four and 22 pts were classified into “good” and “poor” groups, respectively. Among 52 differentially expressed genes (GENCODE v31) with false discovery rate-adjusted P- value &lt; 0.05, the top 5 genes with the lowest P-values are provided in the table. All 4 pts in the “good” group were classified into BM2 and pts in the “poor” group were classified into BM1 (8/22) and BM2 (14/22) (p = 0.07). In addition, all but 2 pts were classified into CMS4. These two pts belong to the “poor” group with one of them classified into CMS1 and the other into CMS2. Conclusions: These gene expression analyses suggest that BM2 subtype could be a predictive marker for the efficacy of eribulin and some genes could be novel targets with the goal to improve prognosis of pts with BRAF V600E mt mCRC. This is the first finding for a potential biomarker in this subgroup using RNA-seq analysis tools. These findings will require additional validation. Clinical trial information: UMIN000031552. [Table: see text]

The Evolutionary Landscape of Treatment for BRAFV600E Mutant Metastatic Colorectal Cancer.

Simple SummaryThe BRAFV600E mutation accounts for 8–10% of metastatic colorectal cancer (mCRC) patients and it is an established prognostic factor. Median overall survival of this subset of patients is indeed so poor that it is similar to first line PFS of patients without this molecular alteration. An exception is represented by patients displaying concomitant MSI-H status who can benefit from immunotherapy with checkpoint inhibitors (CPIs). Recently, a targeted therapy with the combination of encorafenib and cetuximab provided for the first time a survival gain and thus translation in the clinic, even though acquired resistance limits the possibility of more than an incremental benefit. Many studies exploiting other different strategies are ongoing. In this review we present current therapies specifically headed to BRAFV600E mutant mCRC and systematically review ongoing clinical trials identifying different approaches under investigations: targeting MAPK pathway (monotherapy or combinations), targeting MAPK pathway combined with cytotoxic agents, intensive cytotoxic regimen combinations, targeted agents combined with CPIs, oxidative stress induction, and cytotoxic agents combined with antiangiogenic drugs and CPIs.The BRAFV600E mutation is found in 8–10% of metastatic colorectal cancer (mCRC) patients and it is recognized as a poor prognostic factor with a median overall survival inferior to 20 months. At present, besides immune checkpoint inhibitors (CPIs) for those tumors with concomitant MSI-H status, recommended treatment options include cytotoxic chemotherapy + anti-VEGF in the first line setting, and a combination of EGFR and a BRAF inhibitor (cetuximab plus encorafenib) in second line. However, even with the latter targeted approach, acquired resistance limits the possibility of more than an incremental benefit and survival is still dismal. In this review, we discuss current treatment options for this subset of patients and perform a systematic review of ongoing clinical trials. Overall, we identified six emerging strategies: targeting MAPK pathway (monotherapy or combinations), targeting MAPK pathway combined with cytotoxic agents, intensive cytotoxic regimen combinations, targeted agents combined with CPIs, oxidative stress induction, and cytotoxic agents combined with antiangiogenic drugs and CPIs. In the future, the integration of new therapeutic strategies targeting key players in the BRAFV600E oncogenic pathways with current treatment approach based on cytotoxic chemotherapy and surgery is likely to redefine the treatment landscape of these CRC patients.

Circulating Methylated DNA to Monitor the Dynamics of RAS Mutation Clearance in Plasma from Metastatic Colorectal Cancer Patients.

Simple SummaryOngoing clinical trials are recently investigating the efficacy of second-line EGFR inhibitors in initially RAS mutant metastatic colorectal cancer patients who convert to RAS wild-type in plasma, as assessed by circulating tumor DNA (ctDNA) analysis. To this purpose, discriminating between patients with a real clearance of RAS mutations in plasma (real wild-type) from those who do not shed ctDNA is mandatory. The aim of the present study was to confirm the presence of ctDNA in patients with RAS mutation clearance in plasma at different time points, using a colon-cancer-specific five-gene methylation panel. The methylation test confirmed the presence of ctDNA in most RAS wild-type samples at the time of disease progression, thus confirming that the negative selection of RAS mutant clones during the clonal evolution of mutant RAS colorectal cancer is not an infrequent event.The clearance of RAS mutations in plasma circulating tumor DNA (ctDNA) from originally RAS-mutant metastatic colorectal cancer (mCRC) has been recently demonstrated. Clinical trials investigating whether RAS mutant mCRC who “convert” to wild-type in plasma might benefit from EGFR blockade are ongoing. Detection of tumor-specific DNA methylation alterations in ctDNA has been suggested as a specific tool to confirm the tumoral origin of cell-free DNA. We monitored RAS clearance in plasma from patients with RAS-mutant mCRC at baseline (pre-treatment) (T0); after 4 months of first-line therapy (T1); at the time of first (T2) and second (T3) progression. A five-gene methylation panel was used to confirm the presence of ctDNA in samples in which RAS mutation clearance was detected. At T1, ctDNA analysis revealed wild-type RAS status in 83% of samples, all not methylated, suggesting at this time point the lack of ctDNA shedding. At T2, ctDNA analysis revealed wild-type RAS status in 83% of samples, of which 62.5% were found methylated. At T3, 50% of wild-type RAS samples were found methylated. Non-methylated samples were found in patients with lung or brain metastases. This five-gene methylation test might be useful to confirm the presence of ctDNA in RAS wild-type plasma samples.