Stefan M. Woerner

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.

Pathogenesis of DNA repair-deficient cancers: a statistical meta-analysis of putative Real Common Target genes

DNA mismatch repair deficiency is observed in about 15% of human colorectal, gastric, and endometrial tumors and in lower frequencies in a minority of other tumors thereby causing insertion/deletion mutations at short repetitive sequences, recognized as microsatellite instability (MSI). Evolution of tumors, including those with MSI, is a continuous process of mutation and selection favoring neoplastic growth. Mutations in microsatellite-bearing genes that promote tumor cell growth in general (Real Common Target genes) are assumed to be the driving force during MSI carcinogenesis. Thus, microsatellite mutations in these genes should occur more frequently than mutations in microsatellite genes without contribution to malignancy (ByStander genes). So far, only a few Real Common Target genes have been identified by functional studies. Thus, comprehensive analysis of microsatellite mutations will provide important clues to the understanding of MSI-driven carcinogenesis. Here, we evaluated published mutation frequencies on 194 repeat tracts in 137 genes in MSI-H colorectal, endometrial, and gastric carcinomas and propose a statistical model that aims to identify Real Common Target genes. According to our model nine genes including BAX and TGFβRII were identified as Real Common Targets in colorectal cancer, one gene in gastric cancer, and three genes in endometrial cancer. Microsatellite mutations in five additional genes seem to be counterselected in gastrointestinal tumors. Overall, the general applicability, the capacity to unlimited data analysis, the inclusion of mutation data generated by different groups on different sets of tumors make this model a useful tool for predicting Real Common Target genes with specificity for MSI-H tumors of different organs, guiding subsequent functional studies to the most likely targets among numerous microsatellite harboring genes.

Immunoselective pressure and human leukocyte antigen class I antigen machinery defects in microsatellite unstable colorectal cancers.

In colorectal cancer, the immune response is particularly pronounced against tumors displaying the high microsatellite instability (MSI-H) phenotype. MSI-H tumors accumulate mutations affecting microsatellites located within protein encoding regions (coding microsatellites, cMS), which lead to translational shifts of the respective reading frames. Consequently, novel tumor-specific frameshift-derived neopeptides (FSP) are generated and presented by MSI-H tumor cells, thus eliciting effective cytotoxic immune responses. To analyze whether the immunoselective pressure was reflected by the phenotype of MSI-H colorectal cancer cells, we compared here the expression of antigen processing machinery (APM) components and human leukocyte antigen (HLA) class I antigen subunits in 20 MSI-H and 20 microsatellite-stable (MSS) colorectal cancer using a panel of newly developed APM component-specific monoclonal antibodies. In addition, we did a systematic analysis of mutations at cMS located within APM genes and beta2-microglobulin (beta2m). Total HLA class I antigen loss was observed in 12 (60.0%) of the 20 MSI-H lesions compared with only 6 (30.0%) of the 20 MSS colorectal cancer lesions. Moreover, total loss of membraneous HLA-A staining was significantly more frequent in MSI-H colorectal cancer (P = 0.0024). Mutations at cMS of beta2m and genes encoding APM components (TAP1 and TAP2) were detected in at least 7 (35.0%) of 20 MSI-H colorectal cancers but in none of the MSS colorectal cancers (P = 0.0002). These data show that defects of HLA class I antigen processing and presentation seem to be significantly more frequent in MSI-H than in MSS colorectal cancer, suggesting that in MSI-H colorectal cancer the immunoselective pressure leads to the outgrowth of cells with defects of antigen presentation.

Microsatellite instability of selective target genes in HNPCC-associated colon adenomas

Microsatellite instability (MSI) occurs in most hereditary nonpolyposis colorectal cancers (HNPCC) and less frequently in sporadic tumors as the result of DNA mismatch repair (MMR) deficiency. Instability at coding microsatellites (cMS) in specific target genes causes frameshift mutations and functional inactivation of affected proteins, thereby providing a selective growth advantage to MMR deficient cells. At present, little is known about Selective Target Gene frameshift mutations in preneoplastic lesions. In this study, we examined 30 HNPCC-associated MSI-H colorectal adenomas of different grades of dysplasia for frameshift mutations in 26 cMS-bearing genes, which, according to our previous model, represent Selective Target genes of MSI. About 30% (8/26) of these genes showed a high mutation frequency (⩾50%) in colorectal adenomas, similar to the frequencies reported for colorectal carcinomas. Mutations in one gene (PTHL3) occurred significantly less frequently in MSI adenomas compared to published mutation rates in MSI carcinomas (36.0 vs 85.7%, P=0.023). Biallelic inactivation was observed in nine genes, thus emphasizing the functional impact of cMS instability on MSI tumorigenesis. Some genes showed a high frequency of frameshift mutations already at early stages of MSI colorectal tumorigenesis that increased with grade of dysplasia and transition to carcinoma. These include known Target Genes like BAX and TGFBR2, as well as three novel candidates, MACS, NDUFC2, and TAF1B. Overall, we have identified genes of potential relevance for the initiation and progression of MSI tumorigenesis, thus representing promising candidates for novel diagnostic and therapeutic approaches directed towards MMR-deficient tumors.

Microsatellite instability in the development of DNA mismatch repair deficient tumors

Microsatellites are highly abundant short repetitive sequences found in the genomes across different species. They have gained increasing interest in recent years because length alterations in several coding as well as non-coding microsatellites are associated with a variety of different disorders. Particularly, microsatellite mutations play an important role in tumorigenesis of DNA mismatch repair deficient tumors that account for up to a 15% of colorectal, endometrial, and various other cancers. The systematic analysis of the distribution and function of affected microsatellite sequences has facilitated to unravel important steps in the selection processes that drive tumorigenesis. Here, we review the role of microsatellite mutations in the development of cancers with DNA mismatch repair deficiency, outlining biostatistical approaches for the identification of MSI target genes with relevance to MSI associated carcinogenesis. Knowledge about the biological impact of microsatellite mutations in these genes will potentially help to develop modified clinical concepts for diagnosis, prevention, and treatment of microsatellite unstable human cancers.

Sensitive detection of rare cancer cells in sputum and peripheral blood samples of patients with lung cancer by preproGRP-specific RT-PCR

RT‐PCR‐based amplification of transcripts expressed in cancer but not in normal non‐neoplastic cells is increasingly used for the sensitive detection of rare disseminated or exfoliated cancer cells to improve cancer staging and early detection protocols. However, these assays are frequently hampered by false‐positive test results due to low‐level transcription of the marker genes in normal cells. To overcome these limitations, target transcripts have to be identified that are tightly suppressed in normal non‐neoplastic tissues, whereas they should be actively transcribed in the respective cancer cells. Here, we tested RT‐PCR assays for 7 neuroendocrine marker transcripts including NCAM, PGP 9.5, gastrin, gastrin receptor, synaptophysin, preprogastrin‐releasing peptide (preproGRP) and GRP‐receptor to detect rare exfoliated tumor cells in peripheral venous blood and sputum samples from patients with lung cancer. Among these preproGRP RT‐PCR was the only assay with which illegitimate transcription in blood or sputum samples from healthy donors or patients with unrelated diseases did not interfere. However, it reproducibly detected up to 10 small‐cell lung cancer cells diluted in either 10 ml blood or 5 ml sputum samples. Single blood and sputum samples were collected directly before diagnostic bronchoscopy from 175 patients suspected to have lung cancer. Twenty‐six of these had small‐cell lung cancer (SCLC). Thereof, 13 patients (50%) tested positive in the blood sample and 5 of 23 patients (22%) tested positive in the sputum sample. Moreover, among 92 patients with non‐small‐cell lung cancer (NSCLC) 25 patients (27%) had disseminated cancer cells in peripheral blood. Amplification of preproGRP transcripts from clinical samples is a sensitive and specific assay to detect disseminated or exfoliated lung cancer cells either in peripheral blood or sputum samples.

The putative tumor suppressor AIM2 is frequently affected by different genetic alterations in microsatellite unstable colon cancers

Mismatch repair (MMR) deficiency is a major mechanism of colorectal tumorigenesis that is observed in 10–15% of sporadic colorectal cancers and those associated with the hereditary nonpolyposis colorectal cancer (HNPCC) syndrome. MMR deficiency leads to the accumulation of mutations mainly at short repetitive sequences termed microsatellites, constituting the high level microsatellite instability (MSI‐H) phenotype. In recent years, several genes have been described that harbor microsatellites in their coding region (coding microsatellites, cMS) and are frequently affected by mutations in MMR‐deficient cancers. However, evidence for a functional role of most of the known cMS‐containing genes is missing, and further analyses are needed for a better understanding of MSI tumorigenesis. Here, we examined in detail alterations of the absent in melanoma 2 (AIM2) gene that shows a high frequency of cMS frameshift mutations in MSI‐H colorectal, gastric, and endometrial tumors. AIM2 belongs to the HIN‐200 family of interferon (IFN)‐inducible proteins, its role in colon carcinogenesis, however, is unknown. Sequencing of the entire coding region of AIM2 revealed a high frequency of frameshift and missense mutations in primary MSI‐H colon cancers (9/20) and cell lines (9/15). Biallelic AIM2 alterations were detected in 8 MSI‐H colon tumors and cell lines. In addition, AIM2 promoter hypermethylation conferred insensitivity to IFN‐γ‐induced AIM2 expression of three MSI‐H colon cancer cell lines. These results demonstrate that inactivation of AIM2 by genetic and epigenetic mechanisms is frequent in MMR‐deficient colorectal cancers, thus suggesting that AIM2 is a mutational target relevant for the progression of MSI‐H colorectal cancers.

SelTarbase, a database of human mononucleotide-microsatellite mutations and their potential impact to tumorigenesis and immunology

About 15% of human colorectal cancers and, at varying degrees, other tumor entities as well as nearly all tumors related to Lynch syndrome are hallmarked by microsatellite instability (MSI) as a result of a defective mismatch repair system. The functional impact of resulting mutations depends on their genomic localization. Alterations within coding mononucleotide repeat tracts (MNRs) can lead to protein truncation and formation of neopeptides, whereas alterations within untranslated MNRs can alter transcription level or transcript stability. These mutations may provide selective advantage or disadvantage to affected cells. They may further concern the biology of microsatellite unstable cells, e.g. by generating immunogenic peptides induced by frameshifts mutations. The Selective Targets database (http://www.seltarbase.org) is a curated database of a growing number of public MNR mutation data in microsatellite unstable human tumors. Regression calculations for various MSI–H tumor entities indicating statistically deviant mutation frequencies predict TGFBR2, BAX, ACVR2A and others that are shown or highly suspected to be involved in MSI tumorigenesis. Many useful tools for further analyzing genomic DNA, derived wild-type and mutated cDNAs and peptides are integrated. A comprehensive database of all human coding, untranslated, non-coding RNA- and intronic MNRs (MNR_ensembl) is also included. Herewith, SelTarbase presents as a plenty instrument for MSI-carcinogenesis-related research, diagnostics and therapy.

High EPHB2 mutation rate in gastric but not endometrial tumors with microsatellite instability

The EPH/EFN family of receptor tyrosine kinases regulates cell adhesion and migration and has an important role in controlling cell positioning in the normal intestinal epithelium. Inactivation of EPHB2 has recently been shown to accelerate tumorigenesis in the colon and rectum, and we have previously demonstrated frequent frameshift mutations (41%) in an A9 coding microsatellite repeat in exon 17 of EPHB2 in colorectal tumors with microsatellite instability (MSI). In this study, we extended these analyses to extracolonic MSI cancers, and found frameshift EPHB2 mutations in 39% (25/64) of gastric tumors and 14% (8/56) of endometrial tumors. Regression analysis of these EPHB2 mutation data on the basis of our previously proposed statistical model identified EPHB2 as a selective target of frameshift mutations in MSI gastric cancers but not in MSI endometrial carcinomas. These results suggest a functional role for EPHB2 in gastric tumor progression, and emphasize the differences between the tumorigenic processes in MSI gastrointestinal and endometrial cancer.

Frameshift mutations in coding repeats of protein tyrosine phosphatase genes in colorectal tumors with microsatellite instability.

BackgroundProtein tyrosine phosphatases (PTPs) like their antagonizing protein tyrosine kinases are key regulators of signal transduction thereby assuring normal control of cellular growth and differentiation. Increasing evidence suggests that mutations in PTP genes are associated with human malignancies. For example, mutational analysis of the tyrosine phosphatase (PTP) gene superfamily uncovered genetic alterations in about 26% of colorectal tumors. Since in these studies tumors have not been stratified according to genetic instability status we hypothesized that colorectal tumors characterized by high-level of microsatellite instability (MSI-H) might show an increased frequency of frameshift mutations in those PTP genes that harbor long mononucleotide repeats in their coding region (cMNR).ResultsUsing bioinformatic analysis we identified 16 PTP candidate genes with long cMNRs that were examined for genetic alterations in 19 MSI-H colon cell lines, 54 MSI-H colorectal cancers, and 17 MSI-H colorectal adenomas. Frameshift mutations were identified only in 6 PTP genes, of which PTPN21 show the highest mutation frequency at all in MSI-H tumors (17%).ConclusionAlthough about 32% of MSI-H tumors showed at least one affected PTP gene, and cMNR mutation rates in PTPN21, PTPRS, and PTPN5 are higher than the mean mutation frequency of MNRs of the same length, mutations within PTP genes do not seem to play a common role in MSI tumorigenesis, since no cMNR mutation frequency reached statistical significance and therefore, failed prediction as a Positive Selective Target Gene.