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Retrospective Analysis of the Treatment Pathways in Patients with BRAF V600E positive, metastatic colorectal cancer

ABSTRACT: Interventions: Group 1: This retrospective data analysis was designed to evaluate the status quo of the current therapeutic approaches and treatment pathways in BRAFmutant mCRC at specialized colon-cancer centers in Germany, Austria and Switzerland. Thereby, this study does not aim at assessing a specific drug. The aim of this project is, however, to assess BRAFV600E-mutated metastatic colorectal cancer treatment routine in general and to document reasons for treatment choice. Primary outcome(s): Evaluation of current treatment practice of BRAF V600E-mutant metastatic colorectal carcinoma Study Design: Allocation: ; Masking: ; Control: ; Assignment: ; Study design purpose: other

DISEASE(S): Malignant Neoplasm Of Rectum,Malignant Neoplasm Of Rectosigmoid Junction,Malignant Neoplasm Of Colon

PROVIDER: 2436703 | ecrin-mdr-crc |

REPOSITORIES: ECRIN MDR

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Project description:We tamoxifen treated 8-12 week old mice that had floxed alleles of the following: 1) both Apc alleles (giving rise to Apc truncation/inactivation); 2) both Cdx2 alleles (giving rise to Cdx2 inactivation; 3) one Braf allele, that upon Cre-mediated recombination gives a Braf V600E mutant allele (details below), and 4) the combination of both the Cdx2 alleles and the BrafV600E allele. All four of those groups also had a CDX2P-CreERT2 transgene that expresses Cre recombinase fused to a tamoxifen-regulated fragment of the estrogen receptor ligand binding domain. CreERT2 expression occurs only in tissues where the Cdx2 gene is expressed, which is almost exclusively in adult mouse cecum and colon epithelium. A fifth group of mice had the floxed Cdx2 alleles, but no CDX2P-CreERT2 gene. Treating the mice having CDX2P-CreERT2 with tamoxifen permits the Cre recombinase to enter the cell nucleus and recombine the Apc, Braf, and/or Cdx2 alleles containing loxP sequence elements. Mice were treated with intraperitoneal injection of tamoxifen dissolved in corn oil. Three mice per group were used. The control mice did not develop tumors or any morphological or histological changes in their epithelium, but their colons were used to create the 3 control samples. To obtain the BrafV600E allele we used a genetically engineered mouse line previously described by Dankort et al. (Genes Dev 2007, 21:379-84) that can express the BrafV600E mutant protein following Cre-mediated recombination. The Braf(CA) (Braf-Cre-activated) allele mice carry a gene-targeted allele of Braf, where Braf sequences from exons 15-18 are present in the normal mouse Braf intron 14, followed by a mutated exon 15 (carrying the V600E mutation). The exon 15-18 sequence element is flanked by loxP sites. In the absence of Cre-mediated recombination, the Braf(CA) allele expresses a wild type Braf protein. Following Cre-mediated recombination, the Braf exon 15-18 element is removed, and the Braf(CA) allele then encodes the Braf V600E protein (from the introduced mutated exon 15). RNA was purified from tumor or normal tissue, and targets for Affymetrix arrays were synthesized from the mRNAs. We used Affymetrix Mouse Gene 2.1 ST arrays, which hold 41345 probe-sets, but we largely analyzed just those 25216 probe-sets that were mapped to Entrez gene IDs. Raw data was processed with the Robust Multi-array Average algorithm (RMA). Data is log2-transformed transcript abundance estimates. We fit a one-way ANOVA model to the five groups of samples. We supply a supplementary excel workbook that holds the same data as the data matrix file, but also holds the probe-set annotation at the time we analyzed the data, and some simple statistical calculations, which selects subsets of the probe-sets as differentially expressed between pairs of groups, as well as significant Cdx2-/- by Braf V600E interactions. It also gives the homologous human gene IDs we used for enrichment testing, which were 1-to-1 best homologs according to build 68 of NCBI's Homologene. A second supplementary sheet shows the data we enrichment tested after collapsing to distinct human homologs, joins of the results of tests with GSE4045 data and of tests with TCGA data to the mouse genes, and the intersections of selected genes in those data set with our gene selections in mouse. Consumers should consider obtaining more up-to-date probe-set annotation for the array platform.

Project description:The mitogen-activated protein kinase (MAPK) pathway is one of the most altered pathways in cancer. It is involved in the control of cell proliferation, invasion, metabolism, and resistance to therapy. A number of aggressive malignancies, including melanoma, colon cancer and glioma, are driven by an activating missense mutation (V600E) in one component of the pathway, BRAF. BRAF V600E mutated cancers may respond initially to MEK inhibition, but may develop resistance mediated by increased reliance on mTOR signaling. We have previously demonstrated that the combination of the MEK inhibitor trametinib with the dual mTORC1/2 inhibitor TAK228 improved survival and decreased vascularization in a BRAFV600E mutant glioma model. To elucidate the mechanism of action of, and the changes in response to, MEK and mTOR inhibition, we performed comprehensive unbiased proteomic and phosphoproteomic characterization of BRAFV600E mutant glioma xenografts after short-course treatment with trametinib and TAK228, alone and in combination. We identified distinct response signatures for each monotherapy and combination therapy and validated that combination treatment inhibited activation of the MAPK and mTOR pathways, increased apoptotic signaling and suppressed angiogenesis signaling. Furthermore, we found that trametinib and TAK228 combination treatment broadly suppressed the activity of the cyclin-dependent kinases and increased the levels of proteins (and their activating phosphorylations) involved in glycolysis, the TCA cycle, nucleotide biosynthesis and DNA replication. We also demonstrated activation of both receptor tyrosine kinase and histone deacetylase proteins. This study reports a detailed (phospho)proteomic analysis of the response of BRAFV600E mutant glioma to combined MEK and mTOR pathway inhibition and identifies a number of targetable upregulated proteins and pathways, providing new avenues for the development of additional rational combination therapies for aggressive BRAF-driven tumors.

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Retrospective Analysis of the Treatment Pathways in Patients with BRAF V600E positive, metastatic colorectal cancer

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