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Human papillomavirus (HPV) infections in oral carcinomas and normal oral mucosa were studied by consensus primer screening and typing for HPV types 6/11, 16 and 18 DNA. After polymerase chain reaction (PCR) the DNA species of interest were identified by Southern blot hybridization with digoxigenin-labeled oligonucleotide probes. Frozen tissue and scrapings were equally suitable for HPV testing and yielded high HPV detection rates in carcinomas. By comparison, HPV analysis of paraffin-embedded material was much less efficient. HPV were demonstrated in 61.5% (16/26) of oral squamous cell carcinomas, high risk HPV 16 and 18 being the preferential types. The frequency of HPV detection in non-neoplastic mucosa of tumor patients decreased clearly with increasing distance from the tumor (range 26.9-3.8%) suggesting focal HPV infections. In contrast, normal buccal mucosa of a group of healthy volunteers contained HPV DNA only in 1% (1/97).

BackgroundColorectal cancer (CRC) represents a morphologic and molecular heterogenic disease. This heterogeneity substantially impairs drug effectiveness and prognosis. The subtype of mismatch repair deficient (MMR-D) CRCs, accounting for about 15% of all cases, shows particular differential responses up to resistance towards currently approved cytostatic drugs. Pre-clinical in vitro models representing molecular features of MMR-D tumors are thus mandatory for identifying biomarkers that finally help to predict responses towards new cytostatic drugs. Here, we describe the successful establishment and characterization of three patient-derived MMR-D cell lines (HROC24, HROC87, and HROC113) along with their corresponding xenografts.MethodologyMMR-D cell lines (HROC24, HROC87, and HROC113) were established from a total of ten clinicopathological well-defined MMR-D cases (120 CRC cases in total). Cells were comprehensively characterized by phenotype, morphology, growth kinetics, invasiveness, and molecular profile. Additionally, response to clinically relevant chemotherapeutics was examined in vitro and in vivo.Principal FindingsTwo MMR-D lines showing CIMP-H derived from sporadic CRC (HROC24: K-raswt, B-rafmut, HROC87: K-raswt, B-rafmut), whereas the HROC113 cell line (K-rasmut, B-rafwt) was HNPCC-associated. A diploid DNA-status could be verified by flow cytometry and SNP Array analysis. All cell lines were characterized as epithelial (EpCAM+) tumor cells, showing surface tumor marker expression (CEACAM+). MHC-class II was inducible by Interferon-γ stimulation. Growth kinetics as well as invasive potential was quite heterogeneous between individual lines. Besides, MMR-D cell lines exhibited distinct responsiveness towards chemotherapeutics, even when comparing in vitro and in vivo sensitivity.ConclusionsThese newly established and well-characterized, low-passage MMR-D cell lines provide a useful tool for future investigations on the biological characteristics of MMR-D CRCs, both of sporadic and hereditary origin. Additionally, matched patient-derived immune cells allow for comparative genetic studies.

We have examined 118 oral squamous cell carcinomas, 72 oral leukoplakias, 12 cases of cheilitis and 65 of oral lichen planus for the presence of human papillomavirus (HPV) 6/11, 16 and 18 DNA by PCR/Southern blot hybridization. HPV DNA were found in 51/118 carcinomas (43.2%), in 16/72 (22.2%) leukoplakias, 3/12 (25.0%) cheilitic lesions and 10/65 (15.4%) lichen planus cases. These differences were even stronger when analyzing separately for the high-risk types HPV 16 and 18 as compared to low-risk types 6/11. HPV 16 and 18 DNA were present in 41/118 (34.7%) oral carcinomas, 12/72 (16.7%) leukoplakias, 2/12 (16.7%) cheilitic lesions and 6/65 (9.2%) lichen planus. In contrast to this, oral carcinomas displayed the lowest HPV 6/11 detection rate (4.2%), compared with 11.1% for leukoplakias, 8.3% for cheilitic lesions and 7.7% in lichen planus. These results indicate a successive increase of the detection rate of HPV 16 and 18 from low level in non or questionably preneoplastic lesions (lichen planus) to preneoplastic lesions (leukoplakia and cheilitis) and to oral carcinoma. In conclusion, our results suggest an association of oral carcinogenesis and infection with the high-risk HPV types 16 and 18.

BackgroundMolecular heterogeneity of colorectal carcinoma (CRC) is well recognized, forming the rationale for molecular tests required before administration of some of the novel targeted therapies that now are rapidly entering the clinics. For clinical research at least, but possibly even for future individualized tumor treatment on a routine basis, propagation of patients' CRC tissue may be highly desirable for detailed molecular, biochemical or functional analyses. However, complex logistics requiring close liaison between surgery, pathology, laboratory researchers and animal care facilities are a major drawback in this. We here describe and evaluate a very simple cryopreservation procedure for colorectal carcinoma tissue prior to xenografting that will considerably reduce this logistic complexity.MethodsFourty-eight CRC collected ad hoc were xenografted subcutaneously into immunodeficient mice either fresh from surgery (N = 23) or after cryopreservation (N = 31; up to 643 days).ResultsTake rates after cryopreservation were satisfactory (71%) though somewhat lower than with tumor tissues fresh from surgery (74%), but this difference was not statistically significant. Re-transplantation of cryopreserved established xenografts (N = 11) was always successful. Of note, in this series, all of the major molecular types of CRC were xenografted successfully, even after cryopreservation.ConclusionsOur procedure facilitates collection, long-time storage and propagation of clinical CRC specimens (even from different centres) for (pre)clinical studies of novel therapies or for basic research.