High level microsatellite instability (MSI-H) occurs in about 15% of colorectal cancer (CRCs), either as sporadic cancers or in the context of hereditary non-polyposis cancer (HNPCC) or Lynch syndrome. patients with MSI-H CRC and healthy Lynch syndrome mutation carriers [14], thus confirming that FSPs predicted in fact are relevant tumor antigens in vivo. The detection of humoral immune responses against FSPs may provide the basis for a serological test to identify or monitor patients with MSI-H tumors or Lynch syndrome. Against classical tumor antigens like p53, Her2/neu, or NY-ESO-1, serum antibody frequencies ranged between 10 and 20% [15]. The prevalence of humoral immune responses against FSPs has not been analyzed so far. Up to now, there is only an anecdotal report on antibodies specific for one FSP in a single patient suffering from a Lynch syndrome-associated CRC [16]. MSI-H CRC represents an ideal tumor entity for studying tumor antigen-specific humoral immune responses and designing serum antibody assays because the abundance of FSPs and their predictability by bioinformatics. The present cross-sectional study is the first step towards a comprehensive systematic evaluation of FSP-specific antibodies in MSI-H CRC patients, healthy Lynch syndrome mutation carriers, and healthy controls. Materials and Methods Patients and healthy controls A total number of 152 sera were analyzed for antibodies against FSPs, obtained from 69 Lynch syndrome patients with history of MSI-H CRC (termed MSI-H CRC patients), 31 healthy Lynch syndrome mutation carriers and 52 healthy controls. Controls were age- and gender-matched to the MSI-H CRC patients. The median age of the patients was 50 years, of the mutation carriers 38 years and of the healthy controls 48 years (Table 1). Sera from MSI-H CRC patients and Lynch syndrome mutation carriers were collected at the University Hospitals of Heidelberg and Munich, Germany in the framework of the German HNPCC Consortium funded by the Deutsche Krebshilfe. Time after tumor resection in the MSI-H CRC patients was between 2 months and 16 years with a median of 3.6 years (interquartile range 1.8 to 6.0 years) (Table 1). Healthy Lynch syndrome mutation carriers took part in Rabbit Polyclonal to CCRL1. a clinical surveillance program (yearly colonoscopy and, where applicable, gynecologic examination), and only individuals A-769662 without evidence for any cancer or preneoplastic lesion were included in this group. Healthy control sera were obtained from anonymized specimens of occupational employee examinations performed at the University Hospital of Mannheim, Germany. Sera were stored at ?70C in aliquots to minimize freeze-thaw cycles. All procedures were approved by the institutional ethics committee. Table 1 MSI-H CRC patients, healthy Lynch syndrome mutation carriers and healthy controls. Peptides FSPs derived from six coding microsatellite-containing genes were selected according to the following criteria: high coding microsatellite mutation frequency in MSI-H CRC [12,13] (www.seltarbase.org), and the presence of T cell immune responses in patients [14]. FSP sequences translated from mutated complementary DNA sequences are referred to as gene name (minus number of deleted nucleotides) A-769662 in the text. Based on these criteria, the FSPs AIM2(-1), CASP5(-1), MARCKS(-1), TAF1B(-1), TGFBR2(-1) and ZNF294(-1) were selected. FSPs encompassed wild type and frameshift sequences enabling detection of antibody reactions against the junction regions. In addition to TGFBR2(-1) that covers 49 amino acids, two shorter overlapping peptides were designed (TGFBR2(-1)-N and TGFBR2(-1)-C). A detailed list of FSP sequences is provided in Table 2. Peptides were obtained from the Peptide Synthesis Facility of the German Cancer Research Center (DKFZ) in Heidelberg, Germany, purified by high-performance liquid chromatography (HPLC), and analyzed by mass spectrometry. Peptides were dissolved to 5 mg/ml in DMSO and stored at ?70C. Table 2 Peptide Sequences. Neopeptide sequences are underlined. Peptide ELISA Serum antibodies were detected using a newly developed peptide ELISA. Peptides were coated to 96 well polystyrol microtiter plates Maxisorp (Nunc, Roskilde, Denmark) at a concentration of 40 g/ml in PBS overnight at 4C. After coating, plates were washed 4 times with PBS (0.05% Tween) and blocked for 1 h with 0.5% casein in PBS. Peptide binding to the microtiter plates and optimal saturating peptide concentration were assessed A-769662 using an alkaline phosphatase C peptide competition assay according to a previously published protocol [17]. To monitor individual background reactivity of each serum, a peptide derived from the p16INK4a protein (p16_76C105) was used, against which no antibody reactivity was found.