BRAF-mutated Colorectal Cancer Patients Research Articles

Preclinical efficacy of carfilzomib in BRAF-mutant colorectal cancer models.

Serine/threonine-protein kinase B-raf (BRAF) mutations are found in 8-15% of colorectal cancer patients and identify a subset of tumors with poor outcome in the metastatic setting. We have previously reported that BRAF-mutant human cells display a high rate of protein production, causing proteotoxic stress, and are selectively sensitive to the proteasome inhibitors bortezomib and carfilzomib. In this work, we tested whether carfilzomib could restrain the growth of BRAF-mutant colorectal tumors not only by targeting cancer cells directly, but also by promoting an immune-mediated antitumor response. In human and mouse colorectal cancer cells, carfilzomib triggered robust endoplasmic reticulum stress and autophagy, followed by the emission of immunogenic-damage-associated molecules. Intravenous administration of carfilzomib delayed the growth of BRAF-mutant murine tumors and mobilized the danger-signal proteins calreticulin and high mobility group box 1 (HMGB1). Analyses of drug-treated samples revealed increased intratumor recruitment of activated cytotoxic T cells and natural killers, concomitant with the downregulation of forkhead box protein P3 (Foxp3)+ T-cell surface glycoprotein CD4 (CD4)+ T cells, indicating that carfilzomib promotes reshaping of the immune microenvironment of BRAF-mutant murine colorectal tumors. These results will inform the design of clinical trials in BRAF-mutant colorectal cancer patients.

BRAF Inhibitors in BRAF-Mutated Colorectal Cancer: A Systematic Review.

Colorectal cancer (CRC) is the second-leading cause of cancer-related deaths globally. BRAF mutation is present in about 10% of CRC patients and is associated with a poor response to chemotherapy. These patients have a relatively poor prognosis. This review aims to assess the efficacy and safety of BRAF inhibitors in BRAF-mutated CRC patients. A literature search was performed on PubMed and Embase, and clinical trials relevant to BRAF inhibitors in CRC were included. Data were extracted for efficacy and safety variables. Two randomized clinical trials (n = 765) and eight non-randomized trials (n = 281) were included based on inclusion criteria. In RCTs, an overall response was reported in 23% of the patients treated with BRAF inhibitor-based regimens compared to 2.5% with control regimens. The hazard ratio of overall survival was also significantly better with triplet encorafenib therapy at 0.52 (95% CI = 0.39-0.70). In single-arm trials, ORR was 17% and 34% in two-drug and three-drug regimens, respectively. BRAF inhibitor-based regimens were safe and effective in the treatment of BRAF-mutated CRC. Large-scale randomized trials are needed to find a suitable population for each regimen. PROSPERO registration No. CRD42023471627.

RNF43 is associated with genomic features and clinical outcome in BRAF mutant colorectal cancer.

Colorectal cancer (CRC) patients with BRAF mutation have very poor prognosis. It is urgent to search for prognostic factors of BRAF mutant CRC. RNF43 is a ENF ubiquitin ligase of Wnt signaling. Mutation of RNF43 has been observed frequently in various types of human cancers. However, few studies have evaluated the role of RNF43 in CRC. The present study aimed to explore the impact of RNF43 mutations on molecular characteristics and prognosis in BRAF mutant CRC. Samples of 261 CRC patients with BRAF mutation were retrospectively analyzed. Tumor tissue and matched peripheral blood samples were collected and subjected to targeted sequencing with a panel of 1021 cancer-related genes. The association of molecular characteristics and survival in patients were then analyzed. 358 CRC patients with BRAF mutation from the cBioPortal dataset were used for further confirmation. This study was inspired by a CRC patient with BRAF V600E and RNF43 co-mutation, who achieved a best remission of 70% and a progression free survival (PFS) of 13 months. Genomic analysis indicated that RNF43 mutation affected the genomic characteristics of patients with BRAF mutation, including microsatellite instability (MSI), tumor mutation burden (TMB) and the proportion of common gene mutations. Survival analysis showed that RNF43 mutation was a predictive biomarker for better PFS and OS in BRAF mutant CRC. Collectively, we identified that RNF43 mutations were correlated with favorable genomic features, resulting in a better clinical outcome for BRAF mutant CRC patients.

Clinicopathologic Features and Prognosis of BRAF Mutated Colorectal Cancer Patients.

Background: BRAFV600E mutation is associated with poor prognosis of colorectal cancer (CRC) patients, but the comparison of clinic-pathologic features between V600E and non-V600E mutation was not well-known in CRC patients. The aim of this study is to evaluate the clinical and pathological features, prognostic value of BRAF mutations in CRC.Methods: We conducted a retrospective study to characterize the clinical and pathological features and survival of patients with BRAF mutated CRC. Patients were classified according to BRAF status as BRAFV600E mutation and non-V600E mutations. Difference of characteristics and survival between the two groups was analyzed.Results: There was no significant difference in gender, family history, location of primary tumor, metastatic sites between patients with BRAF-V600E mutation and non-V600E mutations. Patients with V600E mutation were younger than those with non-V600E mutations (p = 0.002). Patients with BRAFV600E mutation showed a poorer outcome than those with non-V600E mutations (23.1 vs. 49.9 months, respectively, p = 0.0024). Lack of CDX2 expression was associated with worse prognosis (mOS: 9.4 m vs. not reached, respectively, p = 0.016). Status of V600E mutation did not affect the mPFS and ORR of first-line or second-line treatment.Conclusion: BRAFV600E mutation defines a distinct subgroup of CRC with worse prognosis. Lack of CDX2 expression is associated with poor OS. Status of V600E mutation did not affect the mPFS of first-line or second-line treatment.

Connecting Cancer Pathways to Tumor Engines: A Stratification Tool for Colorectal Cancer Combining Human In Vitro Tissue Models with Boolean In Silico Models.

To improve and focus preclinical testing, we combine tumor models based on a decellularized tissue matrix with bioinformatics to stratify tumors according to stage-specific mutations that are linked to central cancer pathways. We generated tissue models with BRAF-mutant colorectal cancer (CRC) cells (HROC24 and HROC87) and compared treatment responses to two-dimensional (2D) cultures and xenografts. As the BRAF inhibitor vemurafenib is—in contrast to melanoma—not effective in CRC, we combined it with the EGFR inhibitor gefitinib. In general, our 3D models showed higher chemoresistance and in contrast to 2D a more active HGFR after gefitinib and combination-therapy. In xenograft models murine HGF could not activate the human HGFR, stressing the importance of the human microenvironment. In order to stratify patient groups for targeted treatment options in CRC, an in silico topology with different stages including mutations and changes in common signaling pathways was developed. We applied the established topology for in silico simulations to predict new therapeutic options for BRAF-mutated CRC patients in advanced stages. Our in silico tool connects genome information with a deeper understanding of tumor engines in clinically relevant signaling networks which goes beyond the consideration of single drivers to improve CRC patient stratification.

Utility of BRAF VE1 Immunohistochemistry as a Screening Tool for Colorectal Cancer Harboring BRAF V600E Mutation.

BackgroundBRAF mutation has been recognized as an important biomarker of colorectal cancer (CRC) for targeted therapy and prognosis prediction. However, sequencing for every CRC case is not cost-effective. An antibody specific for BRAF V600E mutant protein has been introduced, and we thus examined the utility of BRAF VE1 immunohistochemistry for evaluating BRAF mutations in CRC.MethodsFifty-one BRAF-mutated CRCs and 100 age and sexmatched BRAF wild-type CRCs between 2005 and 2015 were selected from the archives of Asan Medical Center. Tissue microarrays were constructed and stained with BRAF VE1 antibody.ResultsForty-nine of the 51 BRAF-mutant CRCs (96.1%) showed more than moderate cytoplasmic staining, except for two weakly stained cases. Six of 100 BRAF wild-type cases also stained positive with BRAF VE1 antibody; four stained weakly and two stained moderately. Normal colonic crypts showed nonspecific weak staining, and a few CRC cases exhibited moderate nuclear reactivity (3 BRAF-mutant and 10 BRAF wild-type cases). BRAF-mutated CRC patients had higher pathologic stages and worse survival than BRAF wild-type patients.ConclusionsBRAF VE1 immunohistochemistry showed high sensitivity and specificity, but occasional nonspecific staining in tumor cell nuclei and normal colonic crypts may limit their routine clinical use. Thus, BRAF VE1 immunohistochemistry may be a useful screening tool for BRAF V600E mutation in CRCs, provided that additional sequencing studies can be done to confirm the mutation in BRAF VE1 antibody-positive cases.

Abstract LB-A34: Modeling convergent therapeutic strategies to overcome the heterogeneity of acquired resistance in BRAF-mutant colorectal cancer

Abstract Clonal heterogeneity associated with the emergence of acquired resistance to therapy presents a critical challenge for therapeutic strategies to overcome resistance. We investigated the molecular landscape of acquired resistance in BRAF-mutant colorectal patients treated with BRAF inhibitor combinations. Through whole-exome sequencing of paired pre-treatment and post-progression tumor biopsies and targeted sequencing of pre-treatment and post-progression plasma circulating tumor DNA (ctDNA), we identified 14 unique alterations in MAPK pathway components driving acquired resistance, affecting KRAS, NRAS, BRAF, MEK1 and MEK2. Analysis of ctDNA at the time of disease progression revealed profound tumor heterogeneity associated with acquired resistance, with multiple concurrent resistance alterations detectable in ctDNA in individual patients, with one patient harboring as many as 8 co-existing resistance alterations. These findings necessitate development of a strategy capable of simultaneously overcoming multiple heterogeneous resistance mechanisms. To evaluate potential strategies for convergent targeting of multiple concurrent resistance alterations, we generated individual resistant models harboring the full spectrum of clinically observed mutations driving acquired resistance. Individually, these alterations drove resistance to BRAF inhibitor combinations currently in clinical trials by maintaining MAPK signaling. However, since acquired resistance is thought to arise from pre-existing clones that emerge during treatment, we developed a novel pooled clone model system to study clonal outgrowth under the selective pressure of therapy. In this system, each resistant clone was pooled at an abundance of 1% in a background of the sensitive parental cells and exposed to an array of potential therapies, both in vitro, and in vivo as xenografts. The change in clonal abundance from baseline to the completion of therapy was assessed for each clone by digital PCR to measure the degree of clonal outgrowth. Moreover, in vitro, we were able to monitor clonal dynamics in real-time by isolating cell-free DNA (cfDNA) from the cell culture media every 3-4 days during therapy. We observed rapid outgrowth of resistant clones during BRAF-EGFR and BRAF-MEK therapy, and delayed, but robust outgrowth during BRAF-MEK-EGFR therapy, all of which are therapies that have been evaluated in recent clinical trials. However, ERK inhibitor alone, and to a greater degree BRAF-ERK and BRAF-ERK-EGFR combinations markedly abrogated the clonal outgrowth of resistant clones. Moreover, in xenograft tumors derived from clonal pools, BRAF-ERK-EGFR triple combination resulted in profound tumor regressions and completely prevented the outgrowth of all resistant clones. In conclusion, we observed the potential for profound heterogeneity of acquired resistance mechanisms in BRAF-mutant colorectal cancer patients, with multiple alterations observed in ctDNA from individual patients. Our data suggest that convergent, upfront therapy with RAF-ERK or RAF-ERK-EGFR inhibitor combinations may suppress outgrowth of clones harboring clinically observed resistant alterations, offering the potential for improved clinical outcome. Citation Format: Mehlika Hazar-Rethinam, Marianna Kleyman, G. Celine Han, David Liu, Leanne G. Ahronian, Heather A. Shahzade, Lifeng Chen, Aparna R. Parikh, Jill N. Allen, Jeffrey W. Clark, Eunice L. Kwak, Jason E. Faris, Janet E. Murphy, Theodore S. Hong, Emily E. Van Seventer, Brandon Nadres, Catriona B. Hong, Joseph M. Gurski Jr., Nicholas A. Jessop, Dora Dias-Santagata, A. John Iafrate, Eli M. Van Allen, Ryan B. Corcoran. Modeling convergent therapeutic strategies to overcome the heterogeneity of acquired resistance in BRAF-mutant colorectal cancer [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr LB-A34.

Abstract LB-055: Clinical acquired resistance to RAF inhibitor combinations in BRAF-mutant colorectal cancer through MAPK pathway alterations

Abstract BRAF V600E mutations occur in ∼10% of colorectal cancer (CRC), and are associated with poor prognosis. RAF inhibition alone has not been an effective treatment in BRAF-mutant (BRAFm) CRC patients, with response rates of only 5%, due to persistence of MAPK signaling. Combined RAF/EGFR, RAF/MEK, or RAF/MEK/EGFR inhibitors have produced improved efficacy in BRAFm CRC patients, yet ultimately resistance develops after an initial treatment response. Understanding the mechanisms of clinical acquired resistance that arise to RAF inhibitor combinations in BRAFm CRC patients may lead to valuable opportunities to overcome resistance and prolong clinical response. We sought to identify clinically relevant mechanisms of acquired resistance to RAF inhibitor combinations by obtaining tumor biopsies from BRAFm CRC patients upon disease progression, after initial response to RAF/EGFR or RAF/MEK inhibitor combinations. Matched pre-treatment, post-progression, and normal DNA were analyzed by whole exome sequencing (WES) and RNA-seq. In one BRAFm CRC patient with prolonged stable disease on a RAF/EGFR combination, WES identified KRAS amplification in a progressing lesion. FISH confirmed the presence of KRAS amplification in the post-progression biopsy, and RNA-seq revealed KRAS transcript overexpression. Interestingly, in resistant clones generated from BRAFm CRC cell lines selected with either RAF/EGFR or RAF/MEK inhibitors, KRAS exon 2 mutations were identified. Either KRAS amplification or KRAS mutation led to sustained MAPK pathway activity and cross-resistance to either RAF/EGFR or RAF/MEK inhibitor combinations. In a second patient with a minor response to a RAF/EGFR inhibitor combination, BRAF amplification was identified in a progressing lesion, which was confirmed by FISH and was not present in a pre-treatment biopsy of the same lesion. BRAF amplification led to cross-resistance to the BRAF/MEK inhibitor combination. In a third patient with a minor response to a RAF/MEK inhibitor combination, WES identified the presence of an ARAF Q489L mutation and a MEK1 F53L mutation in a single progressing lesion, suggesting possible intra-lesional heterogeneity of acquired resistance mechanisms. However, utilizing a cell line derived from the patient's post-progression biopsy, we found that 30 out of 30 single-cell clones harbored both the ARAF and MEK1 mutations, and that MEK1 F53L seemed to function as the primary driver of acquired resistance in these resistant cells. MEK1 F53L expression markedly abrogated the ability of RAF/MEK and RAF/EGFR inhibitor combinations to suppress MAPK signaling. Despite developing resistance to upstream MAPK pathway inhibitors, we found that each of the acquired resistance mechanisms we detected remained sensitive to ERK inhibition, which could effectively suppress MAPK signaling. Our findings demonstrate the central importance of MAPK pathway activity in BRAFm CRC, and highlight the critical need for MAPK pathway inhibition in the prevention of disease progression. Additionally, our work indicates ERK inhibitors may be valuable additions to future therapeutic combinations for BRAFm CRC patients. Further efforts to understand acquired resistance mechanisms will be vital to developing novel therapeutic strategies to overcome resistance and extend clinical benefit in this lethal CRC subtype. Citation Format: Leanne G. Ahronian, Erin M. Sennott, Eliezer M. Van Allen, Nikhil Wagle, Eunice L. Kwak, Jason E. Faris, Jason T. Godfrey, Koki Nishimura, Kerry D. Lynch, Craig H. Mermel, Elizabeth L. Lockerman, Anuj Kalsy, Joseph M. Gurski, Samira Bahl, Kristin Anderka, Lisa M. Green, Niall J. Lennon, Tiffany G. Huynh, Mari Mino-Kenudson, Gad Getz, Dora Dias-Santagata, A. John Iafrate, Jeffrey A. Engelman, Levi A. Garraway, Ryan B. Corcoran. Clinical acquired resistance to RAF inhibitor combinations in BRAF-mutant colorectal cancer through MAPK pathway alterations. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-055. doi:10.1158/1538-7445.AM2015-LB-055

428 Clinical acquired resistance to combined RAF/EGFR or RAF/MEK inhibition in BRAF mutant colorectal cancer (CRC) patients through MAPK pathway alterations

Background: BRAF mutations occur in ~10% of CRC and confer poor prognosis. While RAF inhibitor monotherapy leads to response rates of 60−80% in BRAF mutant (BRAFm) melanoma, response rates in BRAFm CRC are poor (~5%). Promising recent studies with RAF inhibitor-based combinations, including combined RAF/EGFR or RAF/MEK inhibition, have produced higher response rates in BRAFm CRC, but a better understanding of acquired resistance mechanisms will be critical to improving therapy. Methods: Mechanisms of acquired resistance to RAF/EGFR or RAF/MEK inhibition were evaluated in BRAFm CRC cell lines and in clinical biopsies obtained from BRAFm CRC patients following disease progression after initial response or prolonged stable disease to either therapeutic combination. BRAFm CRC cell lines were cultured in the presence of combined RAF/EGFR or RAF/MEK inhibitors until resistant clones emerged. Candidate resistance mutations were identified through exome sequencing. Matched pre-treament, post-progression, and normal DNA from BRAFm CRC patients treated with either RAF/EGFR or RAF/MEK combinations were analyzed by whole exome sequencing to identify clinical acquired resistance mechanisms. Results: In resistant clones generated from BRAFm CRC cell lines selected with either RAF/EGFR or RAF/MEK inhibitor combinations, KRAS exon 2 mutations were identified. KRAS mutation led to sustained MAPK pathway activity and cross-resistance to either RAF/EGFR or RAF/MEK inhibitor combinations. Interestingly, the triple combination of RAF/EGFR/MEK inhibition was able to suppress MAPK activity and overcome resistance. In a BRAFm CRC patient with prolonged stable disease on a RAF/EGFR inhibitor combination, whole exome sequencing identified the presence of KRAS amplification in a progressing lesion. RNA sequencing of the same lesion confirmed KRAS transcript overexpression, and ~25-fold amplification of KRAS in the progressing lesion and absence of amplification in the pre-treatment biopsy was confirmed by FISH. In one patient with a minor response to a RAF/MEK inhibitor combination, whole exome sequencing identified amplification of the c-mer oncogene (MERTK) receptor tyrosine kinase as the likely acquired resistance mechanism. In another patient with a minor response to a RAF/MEK inhibitor combination, whole exome sequencing identified subclonal ARAF and MEK1 mutations in the post-progression biopsy, suggesting the presence of heterogeneous acquired resistance mechanisms in this progressing lesion. Conclusions: The identification of alterations affecting the MAPK pathway in BRAFm CRC patients who have developed clinical acquired resistance to RAF/EGFR or RAF/MEK inhibitor regimens underscores the importance of the MAPK pathway in this cancer. Understanding the mechanisms of acquired resistance can lead to novel combination strategies to overcome or delay resistance. Friday 21 November 2014

The BRAF mutation is associated with the prognosis in colorectal cancer.

Two members of the Ras/Raf signaling pathway, KRAS and B-raf, are suspected to be involved in the stepwise progression of colorectal cancer (CRC) tumorigenesis. We compared the KRAS and BRAF mutation status of CRC patients with their clinicopathological characteristics and examined the effect of mutation status on survival rates. DNA was extracted from 164 samples, and the mutation statuses of KRAS and BRAF were assessed using peptide PNA clamp real-time PCR method. The presences of mutation were compared with clinicopathological factors and 5-year survival rate. Among the 164 CRC cases, KRAS mutation as detected in 71 cases (43.3 %), respectively, with no relationship with clinicopathological factors of the patients. On Kaplan-Meier survival analysis, KRAS mutation was not significantly associated with survival (p = 0.971). BRAF mutation was detected in 26 cases (15.9 %) and not associated with clinicopathological factors of the patients. However, the 5-year survival rate of BRAF mutations was significantly decreased (p = 0.02). The presence of KRAS mutation did not correlate with the various clinicopathological factors of CRC patients or the survival rate. However, the survival rate was reduced in BRAF-mutated CRC patients. Therefore, BRAF mutation could be an important prognostic factor in CRC patients.

Pharmacodynamic and efficacy analysis of the BRAF inhibitor dabrafenib (GSK436) in combination with the MEK inhibitor trametinib (GSK212) in patients with BRAFV600 mutant colorectal cancer (CRC).

3507 Background: TheBRAF V600 mutation occurs in 5-10% of metastatic CRC, predicts poor prognosis, and may predict lack of response to standard therapy. The combination of inhibitors of BRAF (dabrafenib; D) and MEK (trametinib; T) has shown significant efficacy in BRAF-mutant melanoma. The safety, efficacy, and pharmacodynamic effects of this combination were studied in BRAF-mutant CRC patients (pts). Methods: BRAF mutant CRC pts were enrolled to an initial efficacy cohort of 26 pts and a subsequent pharmacodynamic (PD) expansion cohort that included biopsies of 15 pts at screening and at steady state. So far, 36 pts have enrolled, including 10 in the PD cohort. Eligible pts had previously-treated BRAFV600E mutant stage IV CRC. Pts were treated with D (150mg BID) and T (2mg QD). Additional analyses were performed on available archival tissues. Results: Data are available for 36 pts: ECOG performance status 0 (58%) or 1 (42%), 81% had received ≥ 2 prior chemotherapy regimens, 36% had received prior EGFR inhibitor treatment, and 83% had ≥ 1 biologic therapy. Among 34 pts with >1 restaging assessment as of November 2012, 1 (3%) achieved a complete response (confirmed, on study >12m), 3 (9%) achieved a partial response (1 confirmed to date), and 18 (53%) had stable disease (SD). Minor responses were seen in 7/18 pts (39%) with SD. Median PFS was 3.5 mo (95% CI: 1.8-4.9); overall duration on study range: 0.03–15.2 mo 7 pts (24%) remained on study for ≥6 cycles with 9 pts still on study. The most frequent AEs, any grade, included pyrexia (67%), nausea (56%), fatigue (53%), chills (47%), vomiting (39%), headache (31%), peripheral edema (31%), anemia (28%), and decreased appetite (28%). 2 pts (6%) discontinued due to AEs. Decreased pERK staining vs pre-dose samples was seen in all post-dose samples leading to absolute (49% ±29%) and relative (69% ±28%, normalized to total ERK) reduction in pERK. Conclusions: Further investigation is needed, as this combination is tolerable at full monotherapy doses of each drug, with manageable toxicities, and has activity in a subset of BRAF mutant pts. Updated safety, efficacy, and correlative data will be presented. Clinical trial information: NCT01072175.