Sara Michel

Localization and Density of Immune Cells in the Invasive Margin of Human Colorectal Cancer Liver Metastases Are Prognostic for Response to Chemotherapy

Analysis of tumor-infiltrating lymphocytes (TIL) in primary human colorectal cancer (CRC) by in situ immunohistochemical staining supports the hypothesis that the adaptive immune response influences the course of human CRC. Specifically, high densities of TILs in the primary tumor are associated with good prognosis independent of other prognostic markers. However, the prognostic role of TILs in metastatic CRC lesions is unknown, as is their role in response or resistance to conventional chemotherapy. We analyzed the association of TIL densities at the invasive margin of CRC liver metastases with response to chemotherapy and progression-free survival in a set of 101 large section samples. High-resolution automated microscopy on complete tissue sections was used to objectively generate cell densities for CD3, CD8, granzyme B, or FOXP3 positive immune cells. A predictive scoring system using TIL densities was developed in a training set and tested successfully in an independent validation set. TIL densities at the invasive margin of liver metastases allowed the prediction of response to chemotherapy with a sensitivity of 79% and specificity of 100%. The association of high density values with longer progression-free survival under chemotherapy was statistically significant. Overall, these findings extend the impact of the local immune response on the clinical course from the primary tumor also to metastatic lesions. Because detailed quantification of TILs in metastatic lesions revealed a strong association with chemotherapy efficacy and prognosis, we suggest that the developed scoring system may be used as a predictive tool for response to chemotherapy in metastatic CRC.

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.

Immune evasion of microsatellite unstable colorectal cancers.

Colorectal cancers (CRC) develop through 2 major pathways of genetic instability. In contrast to the majority of CRCs, which are characterized by chromosomal instability, high‐level microsatellite unstable (MSI‐H) CRCs arise as a consequence of the loss of DNA mismatch repair (MMR) functions and show accumulation of insertion and deletion mutations particularly in microsatellite sequences. MSI‐H occurs in about 15% of CRCs, and virtually all CRCs occurring in the context of the hereditary cancer‐predisposing Lynch syndrome. These tumors are characterized by a comparably good prognosis and a low frequency of distant metastases. Because of the expression of a defined set of tumor‐specific antigens, MSI‐H CRCs elicit a strong local and systemic antitumoral immune response of the host and therefore use different strategies to evade the control of the immune system. In this review, we will summarize novel molecular mechanisms that at the same time drive pathogenesis, immunogenicity and immune evasion during the development and progression of MSI‐H CRCs. We will focus on the current knowledge about alterations in human leukocyte antigen (HLA) antigen presentation and discuss how immune evasion—while offering protection against local antitumoral immune responses—paradoxically might interfere with the ability of the tumor to form distant organ metastases.

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.

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.

Lack of HLA class II antigen expression in microsatellite unstable colorectal carcinomas is caused by mutations in HLA class II regulatory genes

Colorectal cancers (CRCs) develop on the basis of a deficient DNA mismatch repair (MMR) system in about 15% of cases. MMR‐deficient CRC lesions show high‐level microsatellite instability (MSI‐H) and accumulate numerous mutations located at coding microsatellite loci that lead to the generation of immunogenic neopeptides. Consequently, the hosts antitumoral immune response is of high importance for the course of the disease in MSI‐H CRC patients. Accordingly, immune evasion mediated by impairment of HLA class I antigen presentation is frequently observed in these cancers. In this study, we aimed at a systematic analysis of alterations affecting HLA class II antigen expression in MSI‐H CRC. HLA class II antigens are expressed by only two‐thirds of MSI‐H CRCs. The mechanisms underlying the lack of HLA class II antigens in a subset of MSI‐H CRCs remain unknown. We here screened HLA class II regulatory genes for the presence of coding microsatellites and identified mutations of the essential regulator genes RFX5 in 9 (26.9%) out of 34 and CIITA in 1 (2.9%) out of 34 MSI‐H CRCs. RFX5 mutations were related to lack of or faint HLA class II antigen expression (p = 0.006, Fishers exact test). Transfection with wild‐type RFX5 was sufficient to restore interferon gamma‐inducible HLA class II antigen expression in the RFX5‐mutant cell line HDC108. We conclude that somatic mutations of the RFX5 gene represent a novel mechanism of loss of HLA class II antigen expression in tumor cells, potentially contributing to immune evasion in MSI‐H CRCs.

Coding microsatellite instability analysis in microsatellite unstable small intestinal adenocarcinomas identifies MARCKS as a common target of inactivation

Approximately 15% of small intestinal adenocarcinomas show inactivation of DNA‐mismatch repair (MMR) and display high‐level microsatellite instability (MSI‐H). MSI‐H tumors progress as a result of mutations affecting coding microsatellites (coding microsatellite instability, cMSI) that may result in a functional inactivation of the encoded proteins and provide a selective growth advantage for the affected cell. To investigate the cMSI selection in small intestinal carcinogenesis 56 adenocarcinomas were tested for MSI. Eleven MSI‐H carcinomas (19.6%) were identified and subjected to cMSI analysis in 24 potentially tumor relevant genes. Mutation frequencies were similar to those observed in colorectal cancer (CRC). Beside high frequencies of cMSI in TGFβR2, ACVR2, and AIM2 we detected MARCKS mutations in 10 out of 11 (91%) tumors with a 30% share of biallelic mutations. Since little is known about MARCKS expression in the intestine, we analyzed MARCKS protein expression in 31 carcinomas. In non‐neoplastic mucosa, MARCKS was found to be expressed with a concentration gradient along the crypt–villus axis. In line with cMSI induced functional inactivation of MARCKS, 8 out of 11 MSI‐H adenocarcinomas showed regional or complete loss of the protein. In microsatellite stable (MSS) small bowel adenocarcinoma, loss of MARCKS expression was seen in 2 out of 20 tumors (10%). In conclusion, we herein present a cMSI profile of MSI‐H small intestinal adenocarcinomas identifying MARCKS as a frequent target of mutation. Loss of MARCKS protein expression suggests a significant role of MARCKS inactivation in the pathogenesis of small intestinal adenocarcinomas.

Molekulare pathogenese. Ihre bedeutung für die zielgerichtete therapie beim kolorektalen karzinom

ZusammenfassungDas kolorektale Karzinom steht in Deutschland an zweiter Stelle der Mortalitätsrate aller Krebserkrankungen. Trotz verbesserter chirurgischer und chemotherapeutischer Verfahren besteht ein großer Bedarf an der Entwicklung effizienter neuer Therapieansätze. In den letzten Jahren wurden durch neue zielgerichtete Therapiestrategien (targeted therapies), die im Gegensatz zur Chemotherapie möglichst gezielt in veränderte Signalwege maligner Zellen eingreifen, wichtige Fortschritte erzielt. Da kolorektale Karzinome eine heterogene Gruppe von Tumoren darstellen, ist ihr Ansprechen auf zielgerichtete Therapien unterschiedlich. Etwa 15 % der kolorektalen Karzinome sind durch Defekte im DNA-Mismatch-Reparatursystem und durch Mikrosatelliteninstabilität (MSI) gekennzeichnet. Diese MSI-Tumoren scheinen eine geringe Chemotherapiesensitivität zu besitzen und zeigen häufig Zeichen einer starken Immunantwort gegen den Tumor, die durch eine hohe Zahl tumorspezifischer Antigene bedingt zu sein scheint. Interventionen, die auf diese Antigene abzielen, könnten die Grundlage für neue therapeutische Ansätze bei kolorektalen Karzinomen mit MSI bilden und stehen vor der Evaluierung in klinischen Studien.AbstractColorectal cancer has the second highest mortality of all cancers in Germany. In spite of advances in surgical and chemotherapeutic treatment, efficient new therapies need to be developed. In recent years, advances have been achieved by novel targeted therapies that are specifically directed against altered signaling pathways of malignant cells. Colorectal cancers represent a heterogeneous tumor entity, and response to targeted therapies varies individually. About 15% of colorectal carcinomas are characterized by a deficient DNA mismatch repair system and microsatellite instability (MSI). These MSI cancers apparently have a decreased sensitivity to chemotherapy and frequently show evidence of a pronounced anti-tumoral immune response of the host. This immune response is likely to be mediated by a high number of tumor-specific antigens generated during MSI tumorigenesis. Interventions specifically targeting these antigens may be the basis for novel therapeutic strategies in MSI colorectal cancer and will be evaluated in clinical trials.

Molekulare Pathogenese@@@Molecular pathogenesis: Ihre Bedeutung für die zielgerichtete Therapie beim kolorektalen Karzinom@@@Its importance in targeted therapy in colorectal cancer

ZusammenfassungDas kolorektale Karzinom steht in Deutschland an zweiter Stelle der Mortalitätsrate aller Krebserkrankungen. Trotz verbesserter chirurgischer und chemotherapeutischer Verfahren besteht ein großer Bedarf an der Entwicklung effizienter neuer Therapieansätze. In den letzten Jahren wurden durch neue zielgerichtete Therapiestrategien (targeted therapies), die im Gegensatz zur Chemotherapie möglichst gezielt in veränderte Signalwege maligner Zellen eingreifen, wichtige Fortschritte erzielt. Da kolorektale Karzinome eine heterogene Gruppe von Tumoren darstellen, ist ihr Ansprechen auf zielgerichtete Therapien unterschiedlich. Etwa 15 % der kolorektalen Karzinome sind durch Defekte im DNA-Mismatch-Reparatursystem und durch Mikrosatelliteninstabilität (MSI) gekennzeichnet. Diese MSI-Tumoren scheinen eine geringe Chemotherapiesensitivität zu besitzen und zeigen häufig Zeichen einer starken Immunantwort gegen den Tumor, die durch eine hohe Zahl tumorspezifischer Antigene bedingt zu sein scheint. Interventionen, die auf diese Antigene abzielen, könnten die Grundlage für neue therapeutische Ansätze bei kolorektalen Karzinomen mit MSI bilden und stehen vor der Evaluierung in klinischen Studien.AbstractColorectal cancer has the second highest mortality of all cancers in Germany. In spite of advances in surgical and chemotherapeutic treatment, efficient new therapies need to be developed. In recent years, advances have been achieved by novel targeted therapies that are specifically directed against altered signaling pathways of malignant cells. Colorectal cancers represent a heterogeneous tumor entity, and response to targeted therapies varies individually. About 15% of colorectal carcinomas are characterized by a deficient DNA mismatch repair system and microsatellite instability (MSI). These MSI cancers apparently have a decreased sensitivity to chemotherapy and frequently show evidence of a pronounced anti-tumoral immune response of the host. This immune response is likely to be mediated by a high number of tumor-specific antigens generated during MSI tumorigenesis. Interventions specifically targeting these antigens may be the basis for novel therapeutic strategies in MSI colorectal cancer and will be evaluated in clinical trials.