Michael Linnebacher

High prevalence of Mycobacterium avium subspecies paratuberculosis IS900 DNA in gut tissues from individuals with Crohn’s disease

Background and aims: Conflicting results exist about the presence of Mycobacterium avium subspecies paratuberculosis (MAP) specific IS900 DNA in Crohn’s disease (CD) tissues. Therefore, we examined IS900 in a large number of gut samples from patients with CD (nu200a=u200a100) and ulcerative colitis (UC, nu200a=u200a100), and in non-inflamed control tissues (nIBD, nu200a=u200a100). We hypothesised that IS900 DNA detection might be associated with distinct clinical phenotypic characteristics in CD. Methods: The prevalence of MAP DNA in surgically resected tissues was examined using a mechanical-enzymatic disruption technique and nested IS900 specific polymerase chain reaction (PCR). CD patients were stratified according to the criteria of the Vienna classification and other clinical characteristics. Results: IS900 PCR detection rate was significantly higher in CD tissue samples (52%) than in UC (2%) or nIBD (5%) specimens (p<0.0001). In CD patients, IS900 DNA was detected in samples from both diseased small bowel (47%) as well as from the colon (61%). No firm association between MAP specific IS900 detection rates and clinical phenotypic characteristics in CD could be established. However, corticosteroid medication constituted a factor which tended to have a negative influence on IS900 DNA detection rates in CD (p<0.01). Conclusions: The presence of MAP specific IS900 DNA is a predominant feature of CD. Therapeutic intervention against MAP might represent a potential target for disease mitigation in Crohn’s disease.

Frameshift peptide-derived T-cell epitopes: A source of novel tumor-specific antigens

Microsatellite instability (MSI) caused by defective DNA mismatch repair (MMR) is a hallmark of hereditary nonpolyposis colorectal cancers (HNPCC) but also occurs in about 15% of sporadic tumors. If instability affects microsatellites in coding regions, translational frameshifts lead to truncated proteins often marked by unique frameshift peptide sequences at their C‐terminus. Since MSI tumors show enhanced lymphocytic infiltration and our previous analysis identified numerous coding mono‐ and dinucleotide repeat‐bearing candidate genes as targets of genetic instability, we examined the role of frameshift peptides in triggering cellular immune responses. Using peptide pulsed autologous CD40‐activated B cells, we have generated cytotoxic T lymphocytes (CTL) that specifically recognize HLA‐A2.1‐restricted peptides derived from frameshift sequences. Among 16 frameshift peptides predicted from mutations in 8 different genes, 3 peptides conferred specific lysis of target cells exogenously loaded with cognate peptide. One peptide derived from a (−1) frameshift mutation in the TGFβIIR gene gave rise to a CTL bulk culture capable of lysing the MSI colorectal cancer cell line HCT116 carrying this frameshift mutation. Given the huge number of human coding microsatellites and assuming only a fraction being mutated and encoding immunologically relevant peptides in MSI tumors, frameshift protein sequences represent a novel subclass of tumor‐specific antigens. It is tempting to speculate that a frameshift peptide‐directed vaccination approach not only could offer new treatment modalities for existing MSI tumors but also might benefit asymptomatic at‐risk individuals in HNPCC families by a prophylactic vaccination strategy.

WT1 IS A TUMOR-ASSOCIATED ANTIGEN IN COLON CANCER THAT CAN BE RECOGNIZED BY IN VITRO STIMULATED CYTOTOXIC T CELLS

The Wilms tumor suppressor gene (WT1) has been shown to be overexpressed in acute and chronic leukemias and in a variety of solid human malignancies, including cancers of the breast and lung. In our present study, we investigated the potential role of WT1 gene in human colon cancer. WT1 mRNA and protein expression was analyzed in a panel of human colon cancer cell lines and primary colon carcinomas by RT‐PCR and Western blot analysis, respectively. A mutational screen of WT1′ zinc‐finger region was carried out by sequence analysis. Finally, using peptide‐stimulated cytotoxic T cells it was investigated whether WT1‐expressing colon tumor cells are a potential target for antigen‐specific immunotherapy. Medium to high abundant levels of WT1 mRNA were detected by RT‐PCR in 10 of 12 (83%) colon cell lines and by quantitative, real‐time RT‐PCR in 13 of 15 (87%) primary tumors, whereas only very low levels of expression were found in 2 primary tumors. Interestingly, however, low levels of WT1 mRNA were also detected in all samples derived from normal colon mucosa. When RT‐PCR products were examined by sequence analysis, both +KTS and −KTS splice isoforms but no zinc‐finger mutations were found, suggesting that the wild‐type form of the WT1 gene is expressed. To determine whether the WT1 protein can serve as a target antigen for immunotherapy, 2 HLA‐A2.1‐restricted WT1 peptides (Db126 and WH187) were used for the in vitro induction of WT1‐specific cytotoxic T lymphocytes (CTLs). The WH187‐specific CTLs not only lysed target cells pulsed exogenously with cognate peptide but also WT1‐expressing colon tumor cells in a HLA‐restricted manner. These findings identify the WT1 protein as an attractive target for the development of antigen‐specific immunotherapy in human colon cancer.

Identification of an HLA-A0201-Restricted CTL Epitope Generated by a Tumor-Specific Frameshift Mutation in a Coding Microsatellite of the OGT Gene

Deficient DNA mismatch repair results in microsatellite instability and might induce shifts of translational reading frames of genes encompassing coding microsatellites. These may be translated in truncated proteins, including neo-peptide tails functioning as tumor rejection antigens, when presented in the context of MHC class I. Recently, others and we identified a frameshift mutation in the coding T(10) microsatellite of the O-linked N-acetylglucosamine transferase gene (OGT) occuring in up to 41% of microsatellite unstable colorectal cancers. Here we describe a novel HLA-A0201-restricted cytotoxic T lymphocyte (CTL)-epitope (28-SLYKFSPFPL; FSP06) derived from this mutant OGT-protein. FSP06-specific CTL-clones killed peptide-sensitized target cells and tumor cell lines expressing both HLA-A0201 and mutant OGT proteins. This demonstrates that FSP06 is endogenously expressed and represents a CD8+-T cell epitope. Our data corroborate the concept of frameshift peptides constituting a novel subset of tumor-associated antigens specifically encountered in cancer cells with deficient mismatch repair.

Compound heterozygosity for two MSH6 mutations in a patient with early onset of HNPCC-associated cancers, but without hematological malignancy and brain tumor.

Heterozygous germline mutations in the human mismatch repair (MMR) genes MLH1, PMS2, MSH2 and MSH6 predispose to the hereditary non-polyposis colorectal cancer (HNPCC) syndrome. Biallelic mutations in these genes have been reported for a limited number of cases resulting in hematological malignancies, brain tumors and gastrointestinal tumors early in childhood. These tumor phenotypes are frequently associated with café-au-lait spots (CALS), one of the clinical hallmarks of neurofibromatosis type 1 (NF1). We report the first case of compound heterozygosity for two MSH6 mutations resulting in a nonconservative amino-acid change of a conserved residue and in a premature stop codon in a patient who developed rectal and endometrial cancer at ages 19 and 24 years, respectively, and presented few CALS in a single body segment. Immunohistochemistry and Western blotting revealed only residual expression of the MSH6 protein in the normal cells. The disease history resembles the HNPCC phenotype rather than a phenotype associated with biallelic MMR gene mutations. Therefore, we assume that one or both mutations abolish protein function only partially, further supported by the parents, which are both carriers of one of the mutations each, and not affected by the disease at ages 57 and 58 years. Our data suggest considering biallelic mutations in MMR genes for patients who develop HNPCC-associated tumors at an unusually young age of onset, even without hematological or brain malignancies.

Generation of RAGE-1 and MAGE-9 peptide-specific cytotoxic T-lymphocyte lines for transfer in patients with renal cell carcinoma.

Renal cell carcinomas (RCCs) are supposed to be immunogenic, and several clinical trials of immunotherapy using tumor lysate‐pulsed dendritic cells (DCs) have been performed. We report on the generation of RAGE‐1 and MAGE‐9 peptide‐specific CTL lines. RAGE‐1 and MAGE‐9 are expressed in 56% and 38% of RCCs. Seven MAGE‐9‐ and 13 RAGE‐1‐derived peptides were found to be immunogenic in the context of the HLA‐A*0201 MHC. CTLs were generated by coculture with peptide‐pulsed, activated B cells, which were easily generated in great quantities and displayed functional activity for a prolonged period of time. MAGE‐9 and RAGE‐1 peptide‐specific CTL lines were strictly peptide‐specific and displayed high cytotoxic activity not only against peptide‐loaded T2 cells but also against HLA‐A*0201‐positive RCC lines, which naturally express MAGE‐9, RAGE‐1 or both. Thus, B cells are well suited as APCs for the generation of large numbers of tumor peptide‐specific CTLs for adoptive transfer. MAGE‐9 as well as RAGE‐1 may well provide suitable targets for immunotherapy of RCC.

Recombinant gp100 protein presented by dendritic cells elicits a T-helper-cell response in vitro and in vivo.

Induction of a helper T (TH)‐cell response is a critical element in the generation of anti‐tumor immunity. The majority of immunotherapeutic approaches have so far been concerned with the generation of cytotoxic T lymphocytes (CTLs). This also accounts for gp100, a melanoma‐associated protein which induces a potent CTL response. Because of the high immunogenicity of gp100, we considered it of special interest to explore the feasibility of generating gp100‐specific TH cells. Human dendritic cells (DCs) were loaded with recombinant gp100 protein, and the response of autologous TH cells was evaluated in vitro and in vivo. We have observed that gp100 peptides can be presented by DCs of certain MHC class II haplotypes, which led to proliferation and cytokine production of TH‐1 cells in vitro. Furthermore, transfer of gp100 protein–loaded human DCs into SCID mice also induced proliferation of autologous, unprimed peripheral blood leukocytes (PBLs) and selective expansion of TH cells. When human T cells from the spleen of SCID mice were recovered and restimulated in vitro, they strongly proliferated in response to gp100‐loaded DCs, while showing minimal proliferative activity in response to DCs loaded with a control antigen. Thus, it is possible to induce an efficient MHC class II–restricted TH response by in vitro stimulation or in vivo vaccination with DCs which have been loaded with a purified tumor‐associated antigen. Int. J. Cancer 83:547–554, 1999.

Characterization of FAMPAC, a newly identified human pancreatic carcinoma cell line with a hereditary background

A novel pancreatic carcinoma cell line, FAMPAC, was identified from investigation of poorly differentiated pancreatic adenocarcinoma cells found in a patient with a familial predisposition to pancreatic carcinoma. A gene responsible for familial pancreatic carcinoma has not been identified to date.

Prevention of chemotherapy-related toxic side effects by infection with adeno-associated virus type 2

Drug resistance and toxic side effects are major limiting factors in the clinical use of antineoplastic chemotherapy. Patients with pancreatic cancer generally do not benefit from chemotherapy. The nonpathogenic adeno‐associated virus type 2 (AAV‐2) has been shown to sensitize human tumor cells to γ irradiation and chemotherapeutic drugs. In the present study, we characterized the therapeutic role of AAV‐2 infection in combination with 5‐fluorouracil (5‐FU)–based chemotherapy on pancreatic cancer cells in an animal model. In Lewis rats bearing s.c. implants of syngeneic DSL6A pancreatic cancer cells, intratumoral infection with AAV‐2 (MOI 10E8 i.u.) in combination with 5‐FU (5 or 50 mg/kg body weight) resulted in significantly reduced tumor growth and prolonged survival time compared with 5‐FU single therapy. Most surprisingly, AAV‐2‐infected rats remained in a much better physical condition compared to their noninfected counterparts. While rats treated with 5‐FU single therapy lost weight, were sluggish and died within 4 months after tumor implantation, animals infected with AAV showed much better vigilance, with body weight, leukocyte number and hemoglobin levels similar to healthy rats. In particular, 5‐FU‐related side effects like thrombocytopenia and leukopenia were significantly reduced in animals treated with the combination regimen. By in vitro analysis, human (Capan‐1 and DANG) pancreatic cancer cell lines were shown to be sensitized to 5‐FU chemotherapy to an extent similar to DSL6A cells. AAV‐2 infection enhanced 5‐FU‐induced apoptosis by a factor of 8 to 14 in both human and rat pancreatic cancer cell lines. The data suggest that infection with the nonpathogenic AAV‐2 significantly improves both chemotherapy efficacy and physical appearance and offers a novel strategy in cancer treatment.

Die Applikation von Adeno-assoziierten Viren Typ 2 (AAV-2) führt zur Tumorprotektion in einem syngenen Rattenmodell des Pankreaskarzinoms

The success of cancer gene therapy is likely to require the targeting of multiple antitumor mechanisms. In this study we demonstrate that infection of Lewis rats bearing pancreatic tumors with the Adenoassociated Virus type 2 (AAV-2) induces a systemic anti-tumor response and protects these animals from a subsequent tumor rechallenge.