Mirjam Tariverdian

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

BACKGROUND & AIMS Colorectal cancers (CRC) associated with the hereditary nonpolyposis colorectal cancer (HNPCC) syndrome display high-level microsatellite instability (MSI-H) as a consequence of mismatch repair deficiency. HNPCC-associated CRC frequently show features of a pronounced immune response, most likely resulting from the MSI-induced generation of novel tumor-specific carboxy-terminal frameshift peptides (FSPs). However, the role of FSP-specific immune surveillance mechanisms in HNPCC are unknown at present. METHODS The efficacy of tumor-infiltrating T cells isolated from MSI-H CRCs (n = 3) was examined by in vitro killing assays. FSP-specific T-cell responses were measured by enzyme-linked immunospot in the peripheral blood from patients with MSI-H CRC (n = 32), healthy HNPCC mutation carriers (n = 16), patients with microsatellite stable (MSS) CRC (n = 17), and healthy donors (n = 22). RESULTS Tumor-infiltrating T cells isolated from MSI-H CRCs specifically recognized MSI-induced FSPs and showed cytotoxic activity against MSI-H but not MSS CRC cells. FSP-specific T-cell responses were detected in the majority of peripheral blood samples from patients with MSI-H but not MSS CRC. Interestingly, FSP-specific T-cell reactivity was already detectable in the peripheral blood of healthy HNPCC family members with germline mutations but without history of tumor development. CONCLUSIONS These data suggest that FSPs presented by DNA mismatch repair-deficient CRC cells are effectively recognized by the patients immune system and may explain the characteristic clinicopathologic features of HNPCC-associated but also sporadic MSI-H CRCs. These observations are of high relevance for the development of FSP-based vaccination approaches, particularly for the preventive application in HNPCC mutation carriers.

High density of FOXP3-positive T cells infiltrating colorectal cancers with microsatellite instability.

High-level microsatellite instability (MSI-H) in colorectal cancer accounts for about 12% of colorectal cancers and is typically associated with a dense infiltration with cytotoxic CD8-positive lymphocytes. The role of regulatory T cells that may interfere with the hosts antitumoural immune response in MSI-H colorectal cancers has not been analysed yet. Using an antibody directed against the regulatory T-cell marker transcription factor forkhead box P3 (FOXP3), regulatory T cells were examined in 70 colorectal cancers with known MSI status (MSI-H, n=37; microsatellite stable, n=33). In MSI-H colorectal cancers, we found a significantly higher intraepithelial infiltration with FOXP3-positive cells (median: 8.5 cells per 0.25 mm2 vs 3.1 cells per 0.25 mm2 in microsatellite stable, P<0.001), and a significantly elevated ratio of intraepithelial to stromal infiltration (0.05 vs 0.01 in microsatellite stable, P<0.001). CD8-positive cell counts were related positively to the number of FOXP3-positive cells (Spearmans ρ=0.56 and 0.55, respectively). Our results show that the elevated number of CD8-positive lymphocytes found in MSI-H colorectal cancers is paralleled by an enhanced infiltration with CD8-negative FOXP3-positive cells. These data suggest that FOXP3-positive cells may play a role in the regulation of the immune response directed against MSI-H colorectal cancers at the primary tumour site.

Beta2-microglobulin mutations in microsatellite unstable colorectal tumors

Defects of DNA mismatch repair (MMR) cause the high level microsatellite instability (MSI‐H) phenotype. MSI‐H cancers may develop either sporadically or in the context of the hereditary nonpolyposis colorectal cancer (HNPCC) syndrome that is caused by germline mutations of MMR genes. In colorectal cancer (CRC), MSI‐H is characterized by a dense lymphocytic infiltration, reflecting a high immunogenicity of these cancers. As a consequence of immunoselection, MSI‐H CRCs frequently display a loss of human leukocyte antigen (HLA) class I antigen presentation caused by mutations of the β2‐microglobulin (β2m) gene. To examine the implications of β2m mutations during MSI‐H colorectal tumor development, we analyzed the prevalence of β2m mutations in MSI‐H colorectal adenomas (n = 38) and carcinomas (n = 104) of different stages. Mutations were observed in 6/38 (15.8%) MSI‐H adenomas and 29/104 (27.9%) MSI‐H CRCs. A higher frequency of β2m mutations was observed in MSI‐H CRC patients with germline mutations of MMR genes MLH1 or MSH2 (36.4%) compared with patients without germline mutations (15.4%). The high frequency of β2m mutations in HNPCC‐associated MSI‐H CRCs is in line with the hypothesis that immunoselection may be particularly pronounced in HNPCC patients with inherited predisposition to develop MSI‐H cancers. β2m mutations were positively related to stage in tumors without distant metastases (UICC I‐III), suggesting that loss of β2m expression may promote local progression of colorectal MSI‐H tumors. However, no β2m mutations were observed in metastasized CRCs (UICC stage IV, p = 0.04). These results suggest that functional β2m may be necessary for distant metastasis formation in CRC patients.

The molecular basis of EPCAM expression loss in Lynch syndrome-associated tumors

Germline deletions affecting the Epithelial cell adhesion molecule (EPCAM) gene lead to silencing of MSH2 and cause Lynch syndrome. We have recently reported that lack of EPCAM expression occurs in many, but not all tumors from Lynch syndrome patients with EPCAM germline deletions. The differences in EPCAM expression were not related to the localization of EPCAM germline deletions. We therefore hypothesized that the type of the second somatic hit, which leads to MSH2 inactivation during tumor development, determines EPCAM expression in the tumor cells. To test this hypothesis and to evaluate whether lack of EPCAM expression can already be detected in Lynch syndrome-associated adenomas, we analyzed four carcinomas and two adenomas from EPCAM germline deletion carriers for EPCAM protein expression and allelic deletion status of the EPCAM gene region by multiplex ligation-dependent probe amplification. In four out of six tumors we observed lack of EPCAM expression accompanied by biallelic deletions affecting the EPCAM gene. In contrast, monoallelic retention of the EPCAM gene was observed in the remaining two tumors with retained EPCAM protein expression. These results demonstrate that EPCAM expression in tumors from EPCAM deletion carriers depends on the localization of the second somatic hit that inactivates MSH2. Moreover, we report lack of EPCAM protein expression in a colorectal adenoma, suggesting that EPCAM immunohistochemistry may detect EPCAM germline deletions already at a precancerous stage.

Quality of life after restorative proctocolectomy for ulcerative colitis: preoperative status and long-term results.

Background: Restorative proctocolectomy has become the surgical procedure of choice in patients with ulcerative colitis. Only smaller studies have compared postoperative to preoperative quality of life (QoL). Methods: Patients with ulcerative colitis who had undergone restorative proctocolectomy at least 5 years before and who had filled out a disease‐specific validated questionnaire (Gastrointestinal Quality of Life Index, GIQLI) prior to surgery (n = 128) were included into this follow‐up study. Factors potentially influencing QoL at the time of operation were investigated with regard to pre‐ and postoperative QoL in univariate and multivariate analysis. Results: A total of 105 patients responded (82%). QoL at least 5 years after colectomy was significantly improved compared to the preoperative situation (109 versus 75). This improvement was evident in all 5 dimensions (P < 0.0001). The Colitis Activity Index (CAI) (P < 0.00001), a shorter duration of the disease (P < 0.05), and a 3‐staged procedure (<0.001) were negatively correlated with preoperative QoL, whereas neoplasia (P < 0.001) was positively correlated. Colectomy was the reason for most of the increase in QoL. Ileostomy closure resulted in a further improvement in 3 of 5 dimensions but not in overall QoL. Uni‐ and multivariate analysis of the difference in QoL before and 5 years after colectomy revealed CAI, the type of operation (both P < 0.001), and neoplasia as significant factors (P < 0.05). Conclusions: The patients in the worst clinical situation profit the most from restorative proctocolectomy. (Inflamm Bowel Dis 2007)

Mismatch repair-deficient crypt foci in lynch syndrome-molecular alterations and association with clinical parameters

Lynch syndrome is caused by germline mutations of DNA mismatch repair (MMR) genes, most frequently MLH1 and MSH2. Recently, MMR-deficient crypt foci (MMR-DCF) have been identified as a novel lesion which occurs at high frequency in the intestinal mucosa from Lynch syndrome mutation carriers, but very rarely progress to cancer. To shed light on molecular alterations and clinical associations of MMR-DCF, we systematically searched the intestinal mucosa from Lynch syndrome patients for MMR-DCF by immunohistochemistry. The identified lesions were characterised for alterations in microsatellite-bearing genes with proven or suspected role in malignant transformation. We demonstrate that the prevalence of MMR-DCF (mean 0.84 MMR-DCF per 1 cm2 mucosa in the colorectum of Lynch syndrome patients) was significantly associated with patients’ age, but not with patients’ gender. No MMR-DCF were detectable in the mucosa of patients with sporadic MSI-H colorectal cancer (n = 12). Microsatellite instability of at least one tested marker was detected in 89% of the MMR-DCF examined, indicating an immediate onset of microsatellite instability after MMR gene inactivation. Coding microsatellite mutations were most frequent in the genes HT001 (ASTE1) with 33%, followed by AIM2 (17%) and BAX (10%). Though MMR deficiency alone appears to be insufficient for malignant transformation, it leads to measurable microsatellite instability even in single MMR-deficient crypts. Our data indicate for the first time that the frequency of MMR-DCF increases with patients’ age. Similar patterns of coding microsatellite instability in MMR-DCF and MMR-deficient cancers suggest that certain combinations of coding microsatellite mutations, including mutations of the HT001, AIM2 and BAX gene, may contribute to the progression of MMR-deficient lesions into MMR-deficient cancers.

Low density of FOXP3-positive T cells in normal colonic mucosa is related to the presence of beta2-microglobulin mutations in Lynch syndrome-associated colorectal cancer

ABSTRACT Microsatellite instability (MSI-H) is caused by DNA mismatch repair deficiency and occurs in 15% of colorectal cancers. MSI-H cancers generate highly immunogenic frameshift peptide (FSP) antigens, which elicit pronounced local immune responses. A subset of MSI-H colorectal cancers develops in frame of Lynch syndrome, which represents an ideal human model for studying the concept of immunoediting. Immunoediting describes how continuous anti-tumoral immune surveillance of the host eventually leads to the selection of tumor cells that escape immune cell recognition and destruction. Between 30 and 40% of Lynch syndrome-associated colorectal cancers display loss of HLA class I antigen expression as a result of Beta2-microglobulin (B2M) mutations. Whether B2M mutations result from immunoediting has been unknown. To address this question, we related B2M mutation status of Lynch syndrome-associated colorectal cancer specimens (n = 30) to CD3-positive, CD8-positive and FOXP3-positive T cell infiltration in both tumor and normal mucosa. No significant correlation between B2M mutations and immune cell infiltration was observed in tumor tissue. However, FOXP3-positive T cell infiltration was significantly lower in normal mucosa adjacent to B2M-mutant (mt) compared to B2M-wild type (wt) tumors (mean: 0.98% FOXP3-positive area/region of interest (ROI) in B2M-wt vs. 0.52% FOXP3-positive area/ROI in B2M-mt, p = 0.023). Our results suggest that in the absence of immune-suppressive regulatory T cells (Treg), the outgrowth of less immunogenic B2M-mt tumor cells is favored. This finding supports the immunoediting concept in human solid cancer development and indicates a critical role of the immune milieu in normal colonic mucosa for the course of disease.

Abstract A006: Frameshift peptide neoantigens as vaccine targets in microsatellite-unstable cancers

Background: Deficient DNA mismatch repair (MMR) boosts the accumulation of frameshift mutations in genes encompassing coding microsatellites (cMS). This results in translation of proteins with mutation-induced frameshift peptide (FSP) neoantigens rendering MMR-deficient microsatellite-unstable (MSI) cancers highly immunogenic. Patients with MSI cancers and healthy individuals affected by Lynch syndrome, an inherited prediposition for MSI cancers, develop specific immune responses against these neoantigens. MSI cancers are unique in tumor immunology, because they express a defined set of long neoantigens that result from functionally relevant driver mutations and therefore are shared by the majority of MSI cancers. Consequently, MSI cancers in Lynch syndrome are an ideal model to evaluate the concept of cancer vaccines, which, with the increasing knowledge about mutational antigens in a wide variety of cancer types, can potentially be applied to many human cancer types. We here report the results of a clinical phase I/IIa trial as the first step to translate this concept into the clinical application. Methods: The vaccination protocol comprised 3 cycles of 4 subcutaneous applications of FSP antigens (frameshift variants of the coding microsatellite-containing genes AIM2, HT001, TAF1B) mixed with Montanide ISA-51 VG over a 6 month period. Inclusion criteria were history of MSI-H colorectal cancer (UICC stage III or IV) and completion of standard chemotherapy. Phase I of the trial evaluated safety and toxicity as the primary endpoint (6 patients), phase IIa addressed the induction of cellular and humoral immune responses (16 patients). Results: Significant induction of FSP-specific immune responses against one or more FSP antigens was observed in all patients vaccinated per protocol. No vaccination-induced systematic severe adverse events occurred. Few patients had stage IV disease and were evaluable according to RECIST. One heavily pretreated patient with bulky metastases showed a stable disease and stable CEA levels over 7 months under the study treatment. Conclusions: Vaccination with FSPs is well tolerated and leads to the induction of humoral and cellular immune responses. FSP vaccination represents a promising novel approach for treatment of MSI cancer patients and for tumor prevention in Lynch syndrome, allowing the evaluation of the concept of preventive cancer vaccines in an ideal model scenario of a defined high-risk patient population. Note: This abstract was not presented at the conference. Citation Format: Magnus von Knebel Doeberitz, Matthias Kloor, Miriam Reuschenbach, Claudia Pauligk, Mohammad-Reza Rafiyan, Salah-Eddin Al Batran, Julia Karbach, Mirjam Tariverdian, Elke Jaeger. Frameshift peptide neoantigens as vaccine targets in microsatellite-unstable cancers [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr A006.