MSS Colon Cancer Research Articles

The safety and efficacy of immune checkpoint inhibitor in combination with FOLFOX as neoadjuvant/adjuvant therapy in patients with locally advanced colon cancer.

e15621 Background: The recurrence rate of locally advanced colon cancer (T3-4 or N+, M0) after surgery is significantly high. Emerging data indicate that the combination of immunotherapy and chemotherapy demonstrates synergistic effects in other solid tumors. Consequently, a combination of immunotherapy and chemotherapy holds the potential to benefit patients with MSS and pMMR colon cancer. We have designed a prospective trial to investigate whether the immune checkpoint inhibitor (ICI) in combination with chemotherapy represents a safe and beneficial treatment approach for locally advanced colon cancer. Methods: Inclusive Criteria: Patients with locally advanced colon cancer (T3-4 or N+, M0) confirmed through pathological biopsy, with confirmed indications for neoadjuvant/adjuvant chemotherapy by the multidisciplinary team. Patients must be eligible for surgery, and a measurable mass must be identified according to RECIST version 1.1. All individuals aged over 18 years, with an estimated life expectancy of over one year and an ECOG score within 0-1, are eligible. The neoadjuvant chemoimmunotherapy group (NACI) includes mFOLFOX 6 (Q2W) and the anti-PD-1 monoclonal antibody (Toripalimab) (3mg/kg, Q2W), while the neoadjuvant chemotherapy group (NAC) receives only mFOLFOX 6 for neoadjuvant/adjuvant therapy (a total of 12 cycles, 6 cycles pre-surgery and 6 cycles post-surgery). The primary endpoint is the pathological complete response (pCR) rate. Results: From 2019 to 2022, a total of 22 patients were enrolled in the study, with 13 in the NACI group and 9 in the NAC group. The research findings indicate that in the NACI group, over 30% (4/13) of patients achieved a pathological complete response (pCR), whereas none of the patients in the NAC group reached pCR (0/9). Among the pCR patients, one was identified as MSI-H/dMMR, two as MSS/pMMR, and one had an unknown status. Tumor regression grade (TRG) levels also revealed that all patients in the NACI group were below grade 2, significantly outperforming the NAC group (P < 0.05), with statistically significant differences. However, no significant differences were observed between the NAC and NACI groups in terms of CT regression percentage, R0 resection, vascular cancer emboli, and the level of neural invasion. Adverse reactions did not show significant differences between the two groups. Conclusions: The combination of immunotherapy and chemotherapy exhibits a synergistic effect in treating locally advanced colorectal cancer. Tolerable adverse reactions suggest that neoadjuvant chemoimmunotherapy could potentially serve as a novel therapeutic option. Clinical trial information: NCT03985891 .

Regional bias of tumor suppressor gene mutations of STARD8 and WNK2 in colon cancers

StAR-related lipid transfer domain protein 8 (STARD8), encoding a Rho-GTPase-activating protein, and WNK2, encoding a serine/threonine kinase are candidate tumor suppressor genes (TSGs) in human cancers. Inactivation of these genes that would promote cancer pathogenesis is largely unknown in colon cancer (CC). Our study addressed to address whether STARD8 and WNK2 genes are mutated in CC. STARD8 and WNK2 genes possess mononucleotide repeats in their exons, which could be the targets for frameshift mutations in cancers with high microsatellite instability (MSI-H). By single-strand conformation polymorphism (SSCP) analysis, we analyzed the repeated sequences in 140 CCs (95 CCs with MSI-H and 45 CCs with stable MSI (MSS)). By DNA sequencing, we found that five MSI-H CCs (5/95: 5.3%) harbored the frameshift mutations, whereas MSS CCs (0/45) did not. In addition, we detected regional heterogeneous frameshift mutations of these genes in four (25%) of 16 MSI-H CCs. In immunohistochemistry for WNK2, WNK2 expression in the MSI-H CCs was significantly lower than that in the MSS CCs. Our results for the mutation and expression indicate that STARD8 and WNK2 genes are altered at various levels (frameshift mutation, expression, and regional heterogeneity) in MSI-H CCs, which might play a role in the pathogenesis by inactivating their TSG functions.

Identification of a Twelve-microRNA Signature with Prognostic Value in Stage II Microsatellite Stable Colon Cancer.

We aimed to identify and validate a set of miRNAs that could serve as a prognostic signature useful to determine the recurrence risk for patients with COAD. Small RNAs from tumors of 100 stage II, untreated, MSS colon cancer patients were sequenced for the discovery step. For this purpose, we built an miRNA score using an elastic net Cox regression model based on the disease-free survival status. Patients were grouped into high or low recurrence risk categories based on the median value of the score. We then validated these results in an independent sample of stage II microsatellite stable tumor tissues, with a hazard ratio of 3.24, (CI95% = 1.05-10.0) and a 10-year area under the receiver operating characteristic curve of 0.67. Functional analysis of the miRNAs present in the signature identified key pathways in cancer progression. In conclusion, the proposed signature of 12 miRNAs can contribute to improving the prediction of disease relapse in patients with stage II MSS colorectal cancer, and might be useful in deciding which patients may benefit from adjuvant chemotherapy.

Abstract 1359: Selective HDAC6 inhibition by GB-1101 revokes tumor immune privilege and synergizes with immune checkpoint therapies to induce tumor regressions

Abstract HDAC6 is a cytoplasmic class IIB HDAC isoenzyme that is involved in the deacetylation of cytoplasmic proteins. HDAC6 also binds ubiquitinated proteins, facilitating the formation of the aggresome, to remove misfolded proteins. Previously, HDAC6-selective inhibitors were shown to decrease immunosuppression and enhance immune function of melanoma patient T-cells demonstrating an important role for HDAC6 inhibition for cancer immunotherapy. GB-1101 is a novel, orally bioavailable, nanomolar potent inhibitor of HDAC6. GB-1101 tested in vitro did not induce direct cytotoxicity to CD4+/CD8+ or NK cells at concentrations up to 300 nM. RNA-seq on GB-1101 exposed MHC-null human MSS colon cancer cell lines reveal potent induction of MHC Class I/II genes and expression numerous cancer neoantigens. To test the activity of GB-1101 in vivo, we established large (~450mm3) 4T1-luc syngeneic tumors and initiated treatment of GB-1101 given orally once daily alone and combined with anti-PD1, anti-CTLA4, and anti-PD1/anti-CTLA4 combination. The activity of anti-PD1 in this model was minimal (TGI: 3%); GB-1101 as a single agent showed more potent anti-tumor activity (TGI: 50%) compared to anti-PD1. We observed synergistic benefit when GB-1101 was combined with anti-PD1 (Combination TGI: 78%). GB-1101 showed comparable anti-tumor activity to single agent anti-CTLA4 (TGI: 43%) but revealed significant tumor regressions of large and well-established tumors when combined with anti-CTLA4 (Combination TGI: >100%). As a single agent, GB-1101 was superior to anti-PD1+anti-CTLA4 (TGI: 7%) and induced significant tumor regressions when combined with anti-PD1+anti-CTLA4 (Triple Combination TGI: >100%). In the TC-1, HPV-positive syngeneic model, GB-1101 reprogrammed the tumor immune microenvironment reducing the number of M-MDSC and TReg cells and increase the numbers of CD4+/CD8+ and NK cells. Taken together, these data support GB-1101 as an important new targeted epigenetic immunomodulator able to revoke immune privilege to enhance the clinical activity of immune checkpoint therapies. Citation Format: Kari Kotlarczyk, Mario Sepulveda, Tong Wang, Maggie Murray, Paul Gonzales, Stephen Thomas Gately. Selective HDAC6 inhibition by GB-1101 revokes tumor immune privilege and synergizes with immune checkpoint therapies to induce tumor regressions [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1359.

Mutational Alterations of DNA Methylation-related Genes CTCF, ZFP57, and ATF7IP Genes in Colon Cancers.

Deregulations of DNA-methylation-related genes are common in cancers, but frameshift mutation status in colon cancer (CC) is unknown. Our study aims to assess whether CTCF, ZFP57, and ATF7IP genes in this category are mutated in CC. CTCF, ZFP57, and ATF7IP genes have repeat coding sequences, which are frequently deleted or duplicated in CC, harboring the phenotype of unstable or high microsatellite instability (MSI-H). We studied 140 CCs [95 MSI-H CCs and 45 stable MSI (MSS) CCs], and found 7 CCs with MSI-H (6/95: 6.3%) harbored frameshift mutations within the repeats, whereas those with MSS did not. Of note, the CTCF frameshift mutations showed the regional difference in the 2 (12.5%) of 16 MSI-H CCs, indicating there was intratumoral heterogeneity. In the immunohistochemistry for ATF7IP, the MSI-H CC showed low intensity compared to MSS CC. Together, CTCF, ZFP57, and ATF7IP genes, despite the low incidence of the mutations, are altered in several ways (mutation, expression, and intratumoral heterogeneity) and could contribute to MSI-H CC development.

Impact of Tumor Side on Clinical Outcomes in Stage II and III Colon Cancer With Known Microsatellite Instability Status.

BackgroundTumor sidedness as a prognostic factor in advanced stage colon cancer (CC) is well established. The impact of tumor sidedness on the clinical outcomes of stage II and III CC has not been well studied.MethodsThe National Cancer Database (NCDB) was utilized to identify patients with pathological stage II and III primary adenocarcinoma of the colon from 2010 to 2015 using ICD-O-3 morphology and topography codes: 8140-47, 8210-11, 8220-21, 8260-63, 8480-81, 8490 and C18.0, 18.2,18.3, 18.5,18.6, 18.7. Univariate (UVA) and multivariable (MVA) survival analyses and Kaplan–Meier Curves with Log-rank test were utilized to compare overall survival (OS) based on tumor location and treatment received.ResultsA total of 35,071 patients with stage II (n = 17,629) and III (n = 17,442) CC were identified. 51.3% female; 81.5% Caucasian; median age 66 (range, 18–90). Majority of stage II and III tumors were right sided, 61.2% (n = 10,794) and 56.0% (n = 9,763). Microsatellite instability high (MSI-H) was more common in stage II compared to III, 23.3% (n = 4,115) vs 18.2% (n = 3,171) (p < 0.0001). In stage II MSI-H CC right was more common than left, 78.3% (n = 3223) vs 21.7% (n = 892). There was no significant difference in survival between stage II MSI-H left vs right (5-year OS 76.2 vs 74.7%, p = 0.1578). Stage II MSS CC right was more common than left, 56.0% (n = 7571) vs 44.0% (n = 5943), and survival was better in the left vs right (5-year OS 73.2 vs 70.8%, p = 0.0029). Stage III MSI-H CC was more common in the right than in the left, 75.6% (n = 2,397) vs 24.4% (n = 774) and survival was better in the left (5-year OS 62.5 vs 56.5%, p = 0.0026). Stage III MSS CC was more common in the right than in the left, 51.6% (n = 7,366) vs 48.4% (n = 6,905), and survival was better in the left vs right (5-year OS 67.0 vs 54.4%, p < 0.001).ConclusionSurvival was better in left sided tumors compared to right in stage II MSS, stage III MSS, and stage III MSI-H CC.

O-20 Phase I/IB study of regorafenib and nivolumab in mismatch repair proficient advanced refractory colorectal cancer

Regorafenib is a multi-tyrosine kinase inhibitor that blocks many pathways including CSF1R which is a receptor involved in macrophage proliferation. Inhibition of the CSF1R may reduce the recruitment of immune-suppressive tumor-associated macrophages (TAMs) to the tumor bed. In addition, recent preclinical data demonstrated regorafenib blocked IFNɣ-induced PD-L1 and IDO1 expression while maintaining MCH-I expression. Therefore, regorafenib can potentially augment the antitumor immunity of checkpoint inhibitors such as nivolumab. This is a phase I/IB trial using the combination of nivolumab and regorafenib in refractory MMR proficient metastatic colorectal cancer. A 3+3 dose-escalation design was used. Patients received nivolumab 240mg IV q2wk and regorafenib according to dose escalation of 80mg, 120 mg or 160mg 21 days on 7 days off. The primary endpoint was dose-limiting toxicity (DLT) during cycle one (4 weeks) to estimate the MTD and the recommended dose for the expanded cohort. Additional pts were enrolled in the expanded cohort to further establish the safety and determine the preliminary efficacy. At the data cutoff of March 2, 2020, 28 patients were enrolled during phase I (n = 12) and the expanded cohort (n = 16). Baseline demographics are as follows: median age 54.5 years old (31-79), median lines of prior therapy of 2.5 (2-3), location of primary tumor: colon (17) vs rectal (11); metastatic sites: liver (19), lung (17), and both (11); and 8 patients had wild type RAS, 20 patients had RAS mutations (KRAS: 19, NRAS:1). During the dose-escalation, 1 DLT (Grade 3 rash) was observed in 80 mg of regorafenib and 2 DLTs (two grade 3 rash) were observed at 120mg. The MTD of regorafenib was 80 mg. Of 17 evaluable pts defined as more than one baseline imaging, 1 (6%) had a partial response and 9 (53%) had stable disease with a disease control rate of 59%. Five pts are still waiting to be restaged. Median PFS was 5.7mo (95% CI 0-11.9) and OS was not reached with median follow-up of 4.6 months. Dose modifications (reductions or interruptions) for drug-related TEAEs occurred in 4 pts. The most common Gr 3 or 4 treatment-emergent adverse events (TEAEs) were rash (N=3), hypertension (N=1) and decreased lymphocytes (N=1). The combination of regorafenib of 80 mg and nivolumab 240mg IV q2wk has modest anti tumor activity in MSS colon cancer. The plan is to enroll a total of 40 pts in the expanded cohort.

A pilot study of durvalumab/tremelimumab (durva/treme) and radiation (XRT) for metastatic biliary tract cancer (mBTC): Preliminary safety and efficacy.

547 Background: Metastatic biliary tract cancer (mBTC) is a lethal malignancy with median 5 year OS of less than 10%. Immunotherapy, particularly single agent anti-PD-1/PD-L1, has limited efficacy in mBTC with ORR~9-15%. Recently presented data shows responses in metastatic MSS pancreatic or colon cancer with combination anti-PD-1/CTLA-4 and radiation (XRT) to produce systemic response (abscopal effect) (Parikh A, GI ASCO 2019, ASCO 2019.). We evaluate safety and efficacy of dual PD-1/CTLA-4 inhibition with XRT in MSS mBTC. Methods: 15 of a planned 15 mBTC patients were enrolled. Eligible pts had histologically-confirmed mBTC, ECOG-PS 0/1, and must have progressed on at least one line of previous therapy or refused standard therapy. Safety cohort of 6 pts of durva 1500 mg/treme 75 mg q4w was enrolled. If &gt; 2 DLTs, patients were enrolled subsequently to dose level -1 (durva 1125 mg/ treme 75 mg q4w). 3 fractions of 8 Gy of radiation at C2D1 every other day to a single metastatic site. Durva/treme continued for 4 cycles, followed by 4 cycles of maintenance durva until progressive disease, discontinuation or withdrawal. Endpoints include disease control rate (DCR (SD+PR+CR)), PFS and OS and safety. Radiological evaluations were done q2 mo. Results: 15 mBTC pts enrolled and evaluable from May 2018 to March 2019. Median age 63 years (range 48-75), 47% male. DLTs occurred in 3 patients during the safety run-in. One patient experienced DLT at dose level -1 and subsequent expansion. 3 patients did NOT reach radiation therapy. DCR was 27% with a 13% PR and 7% CR. Of those who reached radiation, DCR was 33% with a 17% PR and 8% CR. At time of analysis, median PFS was 54 days for ITT mBTC. Duration of response for 4 patients with DCR was 26, 52, 122, 254+ days. Treatment-related adverse events were reported in 12/15 patients (80%). Grade ≥3 toxicities were seen in 9/15 pts (60%) with lymphopenia (5 grade 3) and elevated LFTs (2 grade 4 and 4 grade 2) being the main adverse events. All patients with disease control were not MSI. Conclusions: Combination of durva/treme XRT is feasible and shows preliminary activity in metastatic BTC. An expansion cohort is being planned to confirm activity. Clinical trial information: NCT03482102.

The prognostic role of PD-L1 expression according to MSI status in stage III colon cancer after curative resection.

555 Background: Tumors expressing PD-L1 can render immune inactivated via triggering of PD-1 receptor on T cells with various pathways. Based on these mechanism, the blockade PD-1/PD-L1 pathway based been used as a therapeutic target for metastatic CRC. In the present study, we evaluated the prognostic role of PD-L1 expression associated with microsatellite status in surgically resected stage III colon cancer patients. Methods: PD-L1 expression was performed by immunohistochemistry from 182 stage III colon cancer patients after curative resection. Using the immunohistochemical stain, percentages of PD-L1 positive tumor cells and staining intensity were evaluated and categorized as ‘strong’ and ‘weak’ positive group. Clinical and histopathologic parameters including of MSI status and survival outcomes were analyzed with IDO expression which stands for the suppressive immune environment. Results: Strong PD-L1 expression was observed in 29% of all patients. PNI and lymphocyte response response were more frequently shown in strong PD-L1 patients. Among these patients, MSI was shown in 23 patients (12%). Although there was no significant difference between MSI and PD-L1 status, strong PD-L1 tended to better OS in MSS colon cancer (P = 0.056). In contrast, strong PD-L1 expression significantly correlated with significantly worse prognosis in disease free survival (P = 0.001) and overall survival (P &lt; 0.001) than weak PD-L1 expression inMSI patients regardless of adjuvant chemotherapy. In MSI patients, the strong IDO expression was tended to be more frequently shown in strong PD-L1 expression patients (36.4%) than weak PD-L1 expression patients (14.3%). Conclusions: The expression of PD-L1 is differently affected on the survival according to the status of microsatellite. There is no significant relationship between the expression of PD-L1 and prognosis in MSS stage III colon cancer patients. However, in MSI colon cancer which has been well known as a highly immunogenic property, strong PD-L1 expression is significantly associated with poor prognosis on survival outcomes reflecting of immunosuppressive microenvironment in curative resected stage III colon cancer patient.

HLA-A24 ligandome analysis of colon and lung cancer cells identifies a novel cancer-testis antigen and a neoantigen that elicits specific and strong CTL responses

ABSTRACTThis study focused on HLA-A24 and comprehensively analyzed the ligandome of colon and lung cancer cells without the use of MHC-binding in silico prediction algorithms. Affinity purification using the antibody specific to HLA-A24 followed by LC–MS/MS sequencing was used to detect peptides, which harbored the known characteristics of HLA-A24 peptides in terms of length and anchor motifs. Ligandome analysis demonstrated the natural presentation of two different types of novel tumor-associated antigens. The ligandome contained a peptide derived from SUV39H2, a gene found to be expressed in a variety of cancers but not in normal tissues (except for the testis). The SUV39H2 peptide is immunogenic and elicits cytotoxic CD8+ T-cell (CTL) responses against cancer cells and is thus a novel cancer-testis antigen. Moreover, we found that microsatellite instability (MSI)-colon cancer cells displayed a neoepitope with an amino-acid substitution, while microsatellite stable (MSS)-colon and lung cancer cells displayed its counterpart peptide without the substitution. Structure modeling of peptide-HLA-A24 complexes predicted that the mutated residue at P8 was accessible to T-cell receptors. The neoepitope readily elicited CTL responses, which discriminated it from its wild-type counterpart, and the CTLs exhibited considerably high cytotoxicity against MSS-colon cancer cells carrying the responsible gene mutation. The specific and strong CTL lysis observed in this study fosters our understanding of immune surveillance against neoantigens.

Associations of defect mismatch repair genes with prognosis and heredity in sporadic colorectal cancer

BackgroundMicrosatellite instability arises due to defect mismatch repair (MMR) and occurs in 10–20% of sporadic colorectal cancer. The purpose was to investigate correlations between defect MMR, prognosis and heredity for colorectal cancer in first-degree relatives. Material and methodsTumour tissues from 318 patients consecutively operated for colorectal cancer were analysed for immunohistochemical expression of MLH1, MSH2 and MSH6 on tissue microarrays. Information on KRAS and BRAF mutation status was available for selected cases. ResultsForty-seven (15%) tumours displayed MSI. No correlation was seen between patients exhibiting MSI in the tumour and heredity (p = 0.789). Patients with proximal colon cancer and MSI had an improved cancer-specific survival (p = 0.006) and prolonged time to recurrence (p = 0.037). In a multivariate analysis including MSI status, gender, CEA, vascular and neural invasion, patients with MSS and proximal colon cancer had an impaired cancer-specific survival compared with patients with MSI (HR, 4.32; CI, 1.46–12.78). The same prognostic information was also seen in distal colon cancer; no recurrences seen in the eight patients with stages II and III distal colon cancer and MSI, but the difference was not statistically significant. ConclusionNo correlation between MSI and heredity for colorectal cancer in first-degree relatives was seen. Patients with MSI tumours had improved survival.

Microsatellite instability is associated with reduced disease specific survival in stage III colon cancer

BackgroundUp to 15% of colorectal cancers exhibit microsatellite instability (MSI), where errors in replication go unchecked due to defects in the mismatch repair system. This study aimed to determine survival in a large single-centre series of 1250 consecutive colorectal cancers subjected to universal MSI testing. MethodsClinical and pathological features of patients with colorectal cancer identified on prospectively maintained colorectal and pathology databases at St. Vincent's University Hospital from 2004 to May 2012 were examined. Mismatch repair (MMR) status was determined by immunohistochemistry. Kaplan–Meier curves, the log-rank test and Cox regression were used to associate survival with clinical and pathological characteristics. ResultsOf the 1250 colorectal cancers in the study period, 11% exhibited MSI (n = 138). Patients with MSI tumours had significantly lower rates of lymph node and distant metastases (MSI N+ rate: 24.8% compared with MSS N+ rate: 46.2%, p < 0.001). For Stage I and II disease MSI was associated with improved disease free survival (DSS) compared with MSS colon cancer. However, patients with Stage III MSI colon cancers had a worse DSS than those with MSS tumours. Stage III MSI tumours exhibited higher rates of lymphovascular invasion and perineural invasion than Stage I/II MSI tumours. ConclusionMSI is associated with a reduced risk of nodal and distant metastases, with an improved DSS in Stage I/II colon cancer. However, when MSI tumours progress to Stage III these patients had worse outcomes and pathological features. New strategies for this cohort of patients may be required to improve outcomes.

565P - A Bar Code of Selected Gene Copy Number Alterations is Associated with Disease-Free Survival in Stage Ii-Iii Microsatellite Stable (Mss) Colon Cancer

ABSTRACT Aim: Genomic quantitative alteration is a backbone event during the carcinogenesis process in microsatellite stable colon cancer. However, the prognostic value of loss or gain of the main genomic regions is still controversial. We conducted a prospective study to assess the prognostic impact of the main genomic quantitative alterations in stage II-III MSS colon cancer (NCT02110329). Methods: Patients treated for stage II-III colon cancer from 01/2002 to 01/2009 with available frozen tissue were included. The Quantitative Multiplex PCR of Short Fluorescent Fragments (QMPSF) method was used for molecular screening (1). The QMPSF was based on the simultaneous amplification, from normal and tumoral tissues, of 8 selected target genomic sequences according to literature data: DCC (18q21); EGFR (7p12); TP53 (17p13.1); BLK (8p23-p22); MYC (8q24.12); APC (5q22.2); ERBB2 (17q12); STK6 (20q13.31); and two control: PCBD2 (5q31.1); HMBS (11q23.3). The primary end-point was to determine the association between molecular alterations and disease-free survival (DSF). Combinations of alterations were defined according to univariate results (p Results: A total of 401 patients were included with a median follow-up of 48 months. There were 236 stage II and 165 stage III and the recurrence rate was 30.2%. Loss of DCC/18q, TP53/17p, BLK/8p, and APC/5q were detected in respectively 33.2%, 26.7%, 22.7%, 9.7% and gain of EGFR/7p, MYC/8q, ERBB2/17q and STK6/20q were observed in 31.7%, 35.7%, 18.7% and 48.9% of cases, respectively. There was no significant interaction between alterations and disease stage. DFS was significantly associated with loss of DCC/18q (57.5 vs 70.6%, p = 0.02) with a trend for BLK/8p loss (59.8 vs 68.6%, p = 0.08) and ERBB2/17q gain (59.2 vs 68.5%, p = 0.14). In multivariate analysis, the combination of loss DCC/18q and/or loss BLK/8p and/or gain ERBB2/17q was significantly associated with DFS. The DFS significantly decreased from 74.2% to 61.7% (HR = 1.78, 95CI: 1.14-2.77) and to 57.2% (HR =2.06, 95CI: 1.26-3.37) in patients with respectively none, 1 and at least 2 of these 3 alterations. Conclusions: Combination of DCC/18q, BLK/8p and ERBB2/17q gene copy number alterations is associated with disease-free survival in stage II-III colon cancer. Disclosure: All authors have declared no conflicts of interest.

Functional characterization of the tumor-suppressor MARCKS in colorectal cancer and its association with survival

The myristoylated alanine-rich C-kinase substrate (MARCKS) acts as a tumor suppressor in a variety of human neoplasms. In colorectal cancers (CRCs), MARCKS has been shown to be a preferential target of mutational inactivation in tumors following the microsatellite instability (MSI-H) pathway but little is known about its impact on intestinal carcinogenesis. To investigate the relevance of MARCKS inactivation in more detail, we analyzed 926 MSI-typed CRCsfor MARCKS expression by immunohistochemistry and studied the functional consequences of MARCKS depletion in colorectal cancer cell lines. We found that loss of MARCKS expression was not restricted to MSI-H cancers but also occurred in microsatellite stable (MSS) tumors, where it was associated with an adverse outcome regarding overall survival, cancer-specific and disease-free survival (P=0.002, P=0.0018, P=0.0001, respectively; univariate analysis). In MARCKS-positive MSScolon cancer cell lines (SW480 andSW707) small interfering RNA (siRNA)-mediated knockdown of MARCKS conferred resistance to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis. This was accompanied by the downregulation of the TRAIL receptors DR4 and DR5 at the cell surface and activation of AKT signaling. Inhibition of AKT signaling and transient overexpression of wild-type MARCKS, but not of MARCKS lacking the effector domain (ED), abolished the anti-apoptotic effect. In conclusion, our data show that inactivation of MARCKS is common in CRCsand is associated with adverse outcome in MSS cancers. The finding that MARCKS acts as a mediator of apoptosis in MSS CRCcells adds a novel tumor-suppressing function to the so far established roles of MARCKS in cell motility and proliferation and can explain the prognostic effect of MARCKS depletion in MSS CRC.

Microsatellite instability and Beta2-Microglobulin mutations as prognostic markers in colon cancer: results of the FOGT-4 trial

Background:High-level microsatellite instability (MSI-H) has been reported as a prognostic marker in colon cancer. We here analysed the prognostic significance of MSI and mutations of the Beta2-Microglobulin (B2M) gene, which occur in about 30% of MSI-H colon cancer, in the cohort of the prospective FOGT-4 (Forschungsruppe Onkologie Gastrointestinale Tumoren, FOGT) trial.Methods:Microsatellite instability status was determined using standard protocols (NCI/ICG-HNPCC panel and CAT25) in 223 colon cancer lesions. Beta2-Microglobulin mutation status was evaluated by exon-wise sequencing in all MSI-H lesions.Results:Patients with MSI-H (n=34) colon cancer presented with a significantly lower risk of relapse after 12 months of follow-up compared with MSS (n=189) colon cancer patients (5 year time to relapse: MSI-H 0.82 vs MSS 0.66, P=0.03). No significant difference in overall survival was detected. Beta2-Microglobulin mutations were identified in 10 (29.4%) out of 34 MSI-H colon cancers and were associated with a complete absence of disease relapse or tumour-related death events (P=0.09).Conclusion:The risk of late disease relapse was significantly lower in patients with MSI-H compared with MSS colon cancer. Moreover, B2M mutations may contribute to the favourable outcome of MSI-H colon cancer patients and should therefore be evaluated as a potential prognostic marker in future clinical trials.

Abstract 4722: A BRAF-mutated gene expression signature identifies BRAF wild type colon cancer patients with poor prognosis

Abstract Background: We previously identified highly conserved gene expression patterns associated with BRAF V600E mutant MSS colon cancer (Tejpar et al, ASCO 2010). Since these tumors have very poor overall survival and are refractory to therapy, a better understanding of their molecular characteristics is relevant. In this study we explored if the observed BRAF-mutated characteristic gene expression patterns are also present in any non BRAF-mutant subgroup of colon cancer and what the prognostic implications might be. Methods: A representative set of 667 stage II and III FFPE colon cancers from the PETACC3 clinical trial with 7 years follow up data were used for this study. Samples were characterized for KRAS (39% of samples) and BRAF (6.5%) mutation status, Microsatellite instability (9.7%) (Roth et al, JCO 2009) and gene expression data was obtained with the ALMAC “Colorectal Cancer DSA(tm)” platform. A BRAF signature was built using top scoring pairs and was validated internally (split-sample and cross-validation) and externally (in two independent data sets). It was then applied to KRAS mutant and KRAS wild type (WT) MSI and MSS colon carcinomas. Prognostic value of the signature for overall survival (OS) and survival after relapse (SAR) was assessed by Cox proportional hazard models and survival distributions were compared using likelihood ratio tests. Results: A novel 78-gene BRAF signature was identified and validated, with independent validation performance measured by area under the ROC curve between 0.83 and 0.95. The BRAF signature had high sensitivity in predicting BRAF mutants (&amp;gt;88% internal and &amp;gt;75% external validation) and, in addition, it identified a subpopulation of ‘BRAF-like’ patients with poor prognosis, consisting of 37% of all KRAS mutants and of 13% of all double WT (WT2 – non BRAF and non KRAS mutants). In the whole population and in the MSS subpopulation the signature was prognostic for OS (whole population: hazard ratio HR=1.63, p=0.001; MSS HR=2.25, p&amp;lt;0.001) and for SAR (whole population: HR=2.33, p&amp;lt;0.001; MSS HR=2.64, p&amp;lt;0.001). In the WT2/MSS subpopulation, the signature was prognostic for OS (HR=2.37, p=0.018) and SAR (HR=4.22, p&amp;lt;0.001), but only for SAR in the whole WT2 subpopulation (HR=2.07, p=0.021). The signature was prognostic in the KRAS mutants subpopulation for SAR (HR=1.92, p=0.006), and for OS (HR=1.75, p=0.014) and SAR (HR=1.93, p=0.007) in KRAS/MSS. Conclusions: Using a BRAF mutated derived gene expression signature, we identified a new subgroup within the BRAF WT population that bears a similar gene expression pattern and displays a similar bad prognosis. The signature identifies patients for whom additional therapeutic interventions would be important and the genes included might provide new therapeutic targets. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4722. doi:10.1158/1538-7445.AM2011-4722

Abstract 4921: Selective promoter methylation of the cell differentiation marker, intestinal alkaline phosphatase (iALP), in microsatellite unstable colon cancer

Abstract Tumorigenesis is characterized by excessive cell growth, resistance to apoptosis and aberrant cellular differentiation. In particular, MSI colorectal cancer is frequently characterized by poorly differentiated histology. Histone Deacetylase inhibitors (HDACi) are a novel class of cancer therapeutics that induce growth arrest, apoptosis and differentiation of a variety of tumour types, including colon cancer cells. Differentiation inducing agents have been used effectively to treat hematopoietic malignancies, prompting us to examine the effects of HDACi treatment on differentiation of colon cancer cells, and to compare the effects induced in MSS and MSI colon cancers. Remarkably, while HDACi treatment robustly and consistently induced differentiation induction as measured by alkaline phosphatise activity and iALP gene expression in multiple MSS colon cancer cell lines, approximately 70% of MSI colon cancer cell lines were highly resistant to HDACi-induced ALP induction. Examination of the coding sequence of the iALP gene failed to identify any significant repeat elements suggesting mutation is unlikely to be responsible for lack of iALP induction. Furthermore, transient transfection of an iALP promoter reporter construct demonstrated robust induction in response to butyrate treatment in both MSS and MSI lines, suggesting loss of a key regulatory factor in MSI cell lines is not likely to be responsible. Conversely, examination of iALP promoter methylation by bisulphite conversion and direct sequencing demonstrated multiple CpG sites, including a Sp1/Sp3 binding site, to be methylated in 4 of 5 MSI lines examined, while no methylation was observed in MSS lines. These findings were confirmed using the sequenom assay. ALP promoter methylation was independent of CIMP status. The importance of Sp1/Sp3 in butyrate-induced iALP induction was demonstrated by the ability of the Sp1/Sp3 inhibitor, mithramycin, to attenuate butyrate-induced ALP activity. Finally, consistent with methylation being a key determinant of iALP expression in MSI lines, azacytidine treatment was able to re-induce iALP expression, and butyrate-induction of ALP expression was markedly enhanced in isogenic HCT116 cells lacking the DNA methyltransferases Dnmt1 and Dnmt3b (DKO), compared to parental cells. These findings demonstrate selective promoter methylation of iALP in MSI colon cancer cell lines. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4921.

Abstract B115: The epithelial to mesenchymal transition is impaired in colon cancer cells with microsatellite instability

Abstract Colorectal cancers (CRC) displaying DNA microsatellite instability (MSI) are associated with a favorable natural history, but the molecular basis for this observation has not been defined. We sought to determine whether the epithelial-to-mesenchymal transition (EMT), a highly conserved process involved in embryogenesis as well as in tumor progression, invasion, and metastasis, is impaired in MSI-positive CRCs that characteristically have a mutant transforming growth factor-β receptor type II (TGFBR2) gene. The induction of EMT by TGF-β1 was analyzed by phase contrast microscopy, immunofluorescence, qRT-PCR, immunoblotting, and cellular migration and invasion assays in MSS (SW480 and HT29) and MSI (DLD1 and HCT116) colon cancer cell lines. Expression of epithelial (E-cadherin) and mesenchymal (vimentin and N-cadherin) markers was evaluated by immunohistochemistry and qRT-PCR in 129 human colorectal tumors. TGF-β1 induced changes in cellular morphology from an epithelial to a fibroblasticlike morphology, changes in gene and protein expression with a reduction in E-cadherin and induction of vimentin, and changes in motility and invasion consistent with the occurrence of EMT only in MSS colon cancer cells. These effects did not require Smad4 but depended upon the recruitment of ERK. Cells with MSI and mutant TGFBR2 failed to exhibit any of these changes in response to TGF-β1. However, tumor cells with MSI but wildtype TGFBR2 underwent EMT in response to TGF-β1, indicating that TGFBR2 genotype is a key determinant of the EMT response in tumors with MSI. In human colorectal tumors, expression of the EMT markers N-cadherin and vimentin was significantly associated with adverse clinicopathologic features and the absence of MSI. These findings define a unique genotype-phenotype relationship between TGFBR2 and EMT that may contribute to the improved prognosis consistently observed in colon cancers with MSI. Furthermore, these results suggest a potential rationale for the therapeutic inhibition of TGF-β signaling in MSS colorectal tumors. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B115.

Lymphocyte recruitment into the tumor site is altered in patients with MSI-H colon cancer

The ability of the host to mount an appropriate immune response to aberrant cells is one factor that determines prognosis in cancer patients. Naturally occurring regulatory T cells (T regs; CD4+ CD25+ cells) are key regulators of peripheral tolerance. It has been suggested that high levels of T regs are detrimental to the patient in some forms of cancer, but the role of these antigen-specific cells in individuals with colorectal cancers with high levels of microsatellite instability is unknown. Herein, we examined the ability of individuals with MSI-H or microsatellite stable colon cancer to recruit lymphocytes to the tumor site. Immunohistochemical staining was performed on archived paraffin-embedded specimens from a total of 38 individuals with MSI-H (n = 25) or MSS (n = 13) colon cancers to determine the proportion of CD3+, CD8+ and CD25+ cells infiltrating the tumor site. Patients with MSI-H colon cancers had increased percentages of CD8+ TILs (cytotoxic T cells) as compared to individuals with MSS colon cancer (47.3 vs. 24.04% of the infiltrate CD8+, respectively). No differences in the levels of CD25+ T cells were observed between individuals with MSI-H colon cancers and MSS colon cancers (0.53 vs. 0.54% CD25+, respectively). Together, these data suggest that the survival advantage enjoyed by patients with MSI-H colorectal cancer may, in part, be attributed to the increased cytolytic response, but not to an antigen-specific immunosuppressive response in MSS patients.

MRNA/microRNA gene expression profile in microsatellite unstable colorectal cancer

BackgroundColorectal cancer develops through two main genetic instability pathways characterized by distinct pathologic features and clinical outcome.ResultsWe investigated colon cancer samples (23 characterized by microsatellite stability, MSS, and 16 by high microsatellite instability, MSI-H) for genome-wide expression of microRNA (miRNA) and mRNA. Based on combined miRNA and mRNA gene expression, a molecular signature consisting of twenty seven differentially expressed genes, inclusive of 8 miRNAs, could correctly distinguish MSI-H versus MSS colon cancer samples. Among the differentially expressed miRNAs, various members of the oncogenic miR-17-92 family were significantly up-regulated in MSS cancers. The majority of protein coding genes were also up-regulated in MSS cancers. Their functional classification revealed that they were most frequently associated with cell cycle, DNA replication, recombination, repair, gastrointestinal disease and immune response.ConclusionThis is the first report that indicates the existence of differences in miRNA expression between MSS versus MSI-H colorectal cancers. In addition, the work suggests that the combination of mRNA/miRNA expression signatures may represent a general approach for improving bio-molecular classification of human cancer.