The majority of \u03b2-catenin mutations in colorectal cancer is homozygous

Lynch Syndrome: A Single Hereditary Cancer Syndrome or Multiple Syndromes Defined by Different Mismatch Repair Genes?

Lynch syndrome is caused by variants in DNA mismatch repair (MMR) genes and associated with an increased risk of colorectal cancer (CRC). In patients with Lynch syndrome, CRCs can develop via different pathways. We studied associations between Lynch syndrome-associated variants in MMR genes and risks of adenoma and CRC and somatic mutations in APC and CTNNB1 in tumors in an international cohort of patients. We combined clinical and molecular data from 3 studies. We obtained clinical data from 2747 patients with Lynch syndrome associated with variants in MLH1, MSH2, or MSH6 from Germany, the Netherlands, and Finland who received at least 2 surveillance colonoscopies and were followed for a median time of 7.8 years for development of adenomas or CRC. We performed DNA sequence analyses of 48 colorectal tumors (from 16 patients with mutations in MLH1, 29 patients with mutations in MSH2, and 3 with mutations in MSH6) for somatic mutations in APC and CTNNB1. Risk of advanced adenoma in 10 years was 17.8% in patients with pathogenic variants in MSH2 vs 7.7% in MLH1 (P < .001). Higher proportions of patients with pathogenic variants in MLH1 or MSH2 developed CRC in 10years (11.3% and 11.4%) than patients with pathogenic variants in MSH6 (4.7%) (P= .001 and P= .003 for MLH1 and MSH2 vs MSH6, respectively). Somatic mutations in APC were found in 75% of tumors from patients with pathogenic variants in MSH2 vs 11% in MLH1 (P= .015). Somatic mutations in CTNNB1 were found in 50% of tumors from patients with pathogenic variants in MLH1 vs 7% in MSH2 (P= .002). Noneof the 3 tumors with pathogenic variants in MSH6 had a mutation in CTNNB1, but all had mutations in APC. In an analysis of clinical and DNA sequence data from patients with Lynch syndrome from 3 countries, we associated pathogenic variants in MMR genes with risk of adenoma and CRC, and somatic mutations in APC and CTNNB1 in colorectal tumors. If these findings are confirmed, surveillance guidelines might be adjusted based on MMR gene variants.

Background Clinical trials have shown that cancers originating from different tissues driven by the same oncogene respond differently to targeted anticancer drugs. We aimed to understand different signaling patterns in KRAS mutant cells derived from non-small cell lung cancer (NSCLC), colorectal cancer (CRC) and pancreatic cancer. Materials and methods We optimized a 50 phosphoprotein antibody-based assay on the Luminex 200 platform. We then exposed a panel of 15 KRAS mutant cell lines (5 cell lines each originating in the lung, pancreas and colon) to a DMSO control (n = 3) and clinically significant concentrations (Cmax achieved in humans adjusted for protein binding in culture medium) of a PI3K (GDC-0941), AKT (AZD5363), m-TOR (everolimus), BRAF (vemurafenib), EGFR (gefitinib), MEK (trametinib) and an HSP90 inhibitor (luminespib) for 1 hr. We quantified the change in phosphorylation of proteins for each drug compared to control. Logistic regression analysis was used to analyse differences between KRAS-driven cell lines originating from different anatomical sites. Results There were changes in phosphorylation related to the pharmacodynamic effects of the drug independent of cell line of origin; however, there were interesting differences between KRAS mutant cells originating from different anatomical sites. In NSCLC cell lines, p-EGFR levels changed significantly less when exposed to PI3K, AKT and m-TOR inhibitors (p = 0.047, 0.022 and 0.047, respectively) when compared to cells originating from CRC and pancreatic cancer. CRC cell lines, when compared to NSCLC and pancreatic cancer cell lines, showed significantly less changes in phosphorylation of key cell cycle regulators such as CHK1 when exposed to PI3K, AKT and m-TOR inhibitors, (p = 0.001, 0.047 and 0.047, respectively) and RB when exposed to an AKT and m-TOR inhibitor (p = 0.047 and 0.047, respectively). Interestingly, pancreatic cell lines showed significantly more changes in p-m-TOR compared to CRC and NSCLC cell lines following exposure to PI3K and AKT inhibitors (p = 0.0095 and 0.022, respectively). Of note, drugs not directly targeting the PI3K pathway differentially regulated different nodes in the PI3K pathway, for example, BRAF inhibitors significantly differentially changed levels of phosphorylation at different nodes in the PI3K pathway such as AKT in NSCLC cell lines, p = 0.047, p70S6K in CRC cell lines, p = 0.0472 and PRAS40 in the pancreatic cancer cell lines, p = 0.022. Conclusion These results suggest that there are significant differences in signaling patterns caused by PI3K pathway inhibitors in KRAS mutant NSCLC, CRC and pancreatic cancer cell lines. Our findings shed light on the putative use of PI3K pathway inhibitors in KRAS mutant cancers. They also question the universal application of solely using genetic mutations to stratify patients in ‘basket’ clinical studies. Citation Format: Adam Stewart, Elizabeth Coker, Anna Minchom, Parames Thavasu, Alexandros Georgiou, Anguraj Sadanandam, Timothy A. Yap, Johann S. de Bono, Bissan Al-Lazikani, Udai Banerji. KRAS and clinical context: Differential dynamic signaling output of KRAS mutant lung, colorectal and pancreatic cancer cell lines when exposed to targeted anticancer drugs. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3099.

Truncating SOX9 Alterations Are Heterozygous Null Alleles in Genome-Stable Colorectal Cancer.

Immunotherapy is reportedly effective in colorectal cancers (CRCs) with high microsatellite instability (MSI-H); however, the specific cell types that respond to immune checkpoint therapy are unclear. Herein, we aimed to examine the expression of programmed cell death-ligand 1 (PD-L1) and related proteins in MSI-H and microsatellite-stable (MSS) CRCs to investigate the immune microenvironment at the tumor's invasive front. The MSI status was retrospectively assessed in 499 patients undergoing surgical resection of primary CRC; of these, 48 were classified as MSI-H. Propensity score matching was performed, and tissues from 36 and 37 patients with MSI-H and MSS CRCs, respectively, were immunohistochemically evaluated for PD-L1, PD-1, CD8 and CD68. PD-L1 expression was evaluated separately for tumor cells (PD-L1 [T]) and tumor-infiltrating myeloid cells in the stroma (PD-L1 [I]). PD-L1 (T) was positive in only 5.4% and 36.1% of MSS and MSI-H CRCs, while PD-L1 (I) was positive in 27% and 72.2% of these CRCs, respectively. The PD-L1 (T) and PD-L1 (I) expression levels in MSI-H CRCs significantly correlated with poor differentiation, lymphatic invasion and vascular invasion (p < 0.05), and with early-stage adenocarcinoma and high budding grade (p < 0.05), respectively. Significantly more PD-L1 (I), CD8-positive cells and CD68-positive macrophages were present at the invasive front than in the central tumor in MSI-H CRCs (p < 0.005). PD-L1 was expressed on both tumor cells and CD68/CD163-positive (M2) macrophages at the invasive front of MSI-H CRCs. In conclusion, PD-L1-positive tumor cells and M2-type tumor-associated macrophages may contribute to tumor invasion and immune escape at the invasive front.

High level microsatellite instability (MSI-H) is a hallmark of Lynch syndrome-associated colorectal cancer (CRC). MSI-H CRC express immunogenic tumour antigens as a consequence of DNA mismatch repair deficiency-induced frameshift mutations. Consequently, frameshift antigen-specific immune responses are commonly observed in patients with Lynch syndrome-associated MSI-H CRC. Dendritic cells (DC) and macrophages play a crucial role in the induction and modulation of immune responses. We here analysed DC and macrophage infiltration in MSI-H and microsatellite-stable CRC. Sixty-nine CRC (MSI-H, n=33; microsatellite-stable, n=36) were examined for the density of tumour-infiltrating DC, Foxp3-positive regulatory T cells, and CD163-positive macrophages. In MSI-H lesions, S100-positive and CD163-positive cell counts were significantly higher compared to microsatellite-stable lesions (S100: epithelium P=0.018, stroma P=0.042; CD163: epithelium P<0.001, stroma P=0.046). Additionally, numbers of CD208-positive mature DC were significantly elevated in the epithelial compartment of MSI-H CRC (P=0.027). High numbers of tumour-infiltrating Foxp3-positive T cells were detected in tumours showing a low proportion of CD208-positive, mature DC among the total number of S100-positive cells. Our study demonstrates that infiltration with DC, mature DC, and macrophages is elevated in MSI-H compared to microsatellite-stable CRC. The positive correlation of Foxp3-positive Treg cell density with a low proportion of mature DC suggests that impaired DC maturation may contribute to local immune evasion in CRC. Our results demonstrate that DC and macrophages in the tumour environment likely play an important role in the induction of antigen-specific immune responses in Lynch syndrome. Moreover, impaired DC maturation might contribute to local immune evasion in CRC.

Familial Mutations in PMS2 Can Cause Autosomal Dominant Hereditary Nonpolyposis Colorectal Cancer

Introduction: Advanced endometrial carcinoma (EC) carries a poor prognosis, especially for tumors with CTNNB1 somatic mutations. GOG-86P recently demonstrated that treatment of CTNNB1 -mutated tumors with the anti-VEGFA antibody bevacizumab (bev) resulted in a better outcome, however, the mechanism for this response is unknown. We tested the hypothesis that overexpression of MMP proteins increases angiogenesis through VEGFA downstream signaling leading to improved clinical response in CTNNB1 -mutated tumors. Methods: Data from the endometrial TCGA and CPTAC projects were used to examine the relationship between exon 3 CTNNB1 mutations and VEGFA expression. We used 3 EC cell lines with wildtype (WT) CTNNB1 , Ishikawa, HEC59 and JHUEM7, and a single EC cell line with a mutated CTNNB1 , HEC108. We compared our results to a colorectal cancer (CRC) model by using the WT cell line RKO and mutated cell line HCT116. We measured VEGFA , CTNNB1, MMP2, MMP7, MMP9 RNA and protein levels in these cell lines using RT-PCR, western blots and ELISA. We transiently and stably overexpressed CTNNB1 in WT CRC and EC cell lines and downregulated CTNNB1 using shRNA transduction in mutated cell lines. Results: An analysis of TCGA microsatellite stable, TP53 -wildtype, endometrioid EC showed a 1.4-fold increase in VEGFA gene expression in tumors with CTNNB1 mutations compared to those without (P=0.01). CPTAC endometrial cancer proteomic data did not identify greater VEGFA protein expression in CTNNB1 -mutated tumors. An analysis of known β-catenin targets in TCGA and CPTAC revealed significantly increased MMP7 and MMP9 gene and protein expression in CTNNB1 -mutated tumors. Transient overexpression of CTNNB1 in WT EC and CRC cell lines increased VEGFA gene expression of 2-3 fold. Stable overexpression of physiologic levels of mutated CTNNB1 in WT EC and CRC lines resulted in significantly increased VEGFA and MMP7 gene expression and increased levels of secreted MMP7. Knockdown of CTNNB1 induced a significant decrease in VEGFA gene expression in HCT116, but not HEC108. MMP7 gene expression was paradoxically increased in CTNNB1 -knockdown HCT116, but decreased in CTNNB1- knockdown HEC108. Conclusions: We have shown in silico and in vitro that CTNNB1 exon 3 mutations are associated with increased VEGFA and MMP7 expression. Knockdown experiments in mutated tumors in EC and CRC models suggest that control of angiogenesis and mechanism of action for bev may vary between tumor types supporting the role of CTNNB1 mutations as a biomarker in EC. Ongoing experiments will study the role of MMP7 in CTNNB1- mutated tumors. These findings are being translated into biomarker stratified clinical trials and individualized treatment for women with advanced EC. Citation Format: Amnon A. Berger, Douglas A. Levine, Emily Kawaler. The role of matrix metalloproteinases (MMPs) and CTNNB1 mutations in endometrial carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 184.

In sporadic colorectal tumours the BRAFV600E is associated with microsatellite instability (MSI-H) and inversely associated to KRAS mutations. Tumours from hereditary non-polyposis colorectal cancer (HNPCC) patients carrying germline mutations in hMSH2 or hMLH1 do not show BRAFV600E, however no consistent data exist regarding KRAS mutation frequency and spectrum in HNPCC tumours. We investigated KRAS in 158 HNPCC tumours from patients with germline hMLH1, hMSH2 or hMSH6 mutations, 166 MSI-H and 688 microsatellite stable (MSS) sporadic carcinomas. All tumours were characterized for MSI and 81 of 166 sporadic MSI-H colorectal cancer (CRCs) were analysed for hMLH1 promoter hypermethylation. KRAS mutations were observed in 40% of HNPCC tumours, and the mutation frequency varied upon the mismatch repair gene affected: 48% (29/61) in hMSH2, 32% (29/91) in hMLH1 and 83% (5/6) in hMSH6 (P = 0.01). KRAS mutation frequency was different between HNPCC, MSS and MSI-H CRCs (P = 0.002), and MSI-H with hMLH1 hypermethylation (P = 0.005). Furthermore, HNPCC CRCs had more G13D mutations than MSS (P < 0.0001), MSI-H (P = 0.02) or MSI-H tumours with hMLH1 hypermethylation (P = 0.03). HNPCC colorectal and sporadic MSI-H tumours without hMLH1 hypermethylation shared similar KRAS mutation frequency, in particular G13D. In conclusion, we show that depending on the genetic/epigenetic mechanism leading to MSI-H, the outcome in terms of oncogenic activation may be different, reinforcing the idea that HNPCC, sporadic MSI-H (depending on the hMLH1 status) and MSS CRCs, may target distinct kinases within the RAS/RAF/MAPK pathway.

Polymerase Slippage Restoration of Frameshifted TGFBR2 in Colorectal Cancer: A Novel Paradigm

American Gastroenterological Association Technical Review on the Diagnosis and Management of Lynch Syndrome

Immune Response Against Frameshift-Induced Neopeptides in HNPCC Patients and Healthy HNPCC Mutation Carriers

Immune Response Against Frameshift-Induced Neopeptides in HNPCC Patients and Healthy HNPCC Mutation Carriers

Introduction: Advanced endometrial carcinoma (EC) carries a poor prognosis, especially for tumors with CTNNB1 somatic mutations. GOG-86P recently demonstrated that treatment of CTNNB1-mutated tumors with the anti-VEGFA antibody bevacizumab (bev) resulted in a better outcome, however, the mechanism for this response is unknown. We tested the hypothesis that overexpression of MMP proteins increases angiogenesis through VEGFA downstream signaling leading to improved clinical response in CTNNB1-mutated tumors. Methods: Data from the endometrial TCGA and CPTAC projects were used to examine the relationship between exon 3 CTNNB1 mutations and VEGFA expression. We used 3 EC cell lines with wildtype (WT) CTNNB1, Ishikawa, HEC59 and JHUEM7, and a single EC cell line with a mutated CTNNB1, HEC108. We compared our results to a colorectal cancer (CRC) model by using the WT cell line RKO and mutated cell line HCT116. We measured VEGFA, CTNNB1, MMP2, MMP7, MMP9 RNA and protein levels in these cell lines using RT-PCR, western blots and ELISA. We transiently and stably overexpressed CTNNB1 in WT CRC and EC cell lines and downregulated CTNNB1 using shRNA transduction in mutated cell lines. Results: An analysis of TCGA microsatellite stable, TP53-wildtype, endometrioid EC showed a 1.4-fold increase in VEGFA gene expression in tumors with CTNNB1 mutations compared to those without (P=0.01). CPTAC endometrial cancer proteomic data did not identify greater VEGFA protein expression in CTNNB1-mutated tumors. An analysis of known β-catenin targets in TCGA and CPTAC revealed significantly increased MMP7 and MMP9 gene and protein expression in CTNNB1-mutated tumors. Transient overexpression of CTNNB1 in WT EC and CRC cell lines increased VEGFA gene expression of 2-3 fold. Stable overexpression of physiologic levels of mutated CTNNB1 in WT EC and CRC lines resulted in significantly increased VEGFA and MMP7 gene expression and increased levels of secreted MMP7. Knockdown of CTNNB1 induced a significant decrease in VEGFA gene expression in HCT116, but not HEC108. MMP7 gene expression was paradoxically increased in CTNNB1-knockdown HCT116, but decreased in CTNNB1-knockdown HEC108. Conclusions: We have shown in silico and in vitro that CTNNB1 exon 3 mutations are associated with increased VEGFA and MMP7 expression. Knockdown experiments in mutated tumors in EC and CRC models suggest that control of angiogenesis and mechanism of action for bev may vary between tumor types supporting the role of CTNNB1 mutations as a biomarker in EC. Ongoing experiments will study the role of MMP7 in CTNNB1-mutated tumors. These findings are being translated into biomarker stratified clinical trials and individualized treatment for women with advanced EC. Citation Format: Amnon A. Berger, Douglas A. Levine, Emily Kawaler. The role of matrix metalloproteinases (MMPs) and CTNNB1 mutations in endometrial carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 184.

We hypothesized that patterns of CTNNB1 (β-catenin) mutations would affect the outcome of conservative therapy in patients with desmoid tumors. This study aimed to determine the significance of CTNNB1 (β-catenin) mutations in predicting the treatment outcome in patients with desmoid tumors treated with meloxicam, a cyclooxygenase-2 (COX-2) selective inhibitor. Between 2003 and 2012, consecutive thirty-three patients with extra-peritoneal sporadic desmoid tumors were prospectively treated with meloxicam as the initial systemic medical therapy. The efficacy of meloxicam was evaluated according to Response Evaluation Criteria in Solid Tumors (RECIST). DNA was isolated from frozen tissue or formalin-fixed materials. CTNNB1 mutation analysis was performed by direct sequencing. Positivity of nuclear β-catenin staining by immunohistochemistry was compared with the status of CTNNB1 mutations. The correlation between the efficacy of meloxicam treatment and status of CTNNB1 mutations was analyzed. Of the 33 patients with meloxicam treatment, one showed complete remission (CR), 7 partial remission (PR), 12 stable disease (SD), and 13 progressive disease (PD). The following 3 point mutations were identified in 21 of the 33 cases (64%): T41A (16 cases), S45F (4 cases) and S45P (one case). The nuclear expression of β-catenin correlated significantly with CTNNB1 mutation status (p = 0.035); all four cases with S45F mutation exhibited strong nuclear expression of β-catenin. S45F mutation was significantly associated with a poor response (all cases; PD) (p = 0.017), whereas the other mutations had no impact on efficacy. The CTNNB1 mutation status was of significant prognostic value for meloxicam treatment in patients with sporadic desmoid tumors.