Human papilloma virus status of penile squamous cell carcinoma is associated with differences in tumour-infiltrating T lymphocytes.

Meta-analyses show that approximately half of all squamous cell carcinomas (SCCs) of the penis are associated with a human papillomavirus (HPV) infection. As data about the tumour microenvironment of HPV-positive and HPV-negative penile carcinomas is scarce and conflicting, we examined tumour-infiltrating lymphocyte populations in such cases. The HPV status of 28 penile SCCs was determined by polymerase chain reaction, while the number and distribution of different lymphocyte populations were analysed by immunohistochemistry on whole sections of paraffin-embedded tumour specimens. The average number of tumour-infiltrating T cells in HPV-associated SCC was higher than in HPV-negative SCC, and their phenotype showed strong polarization towards a T helper 1 and cytotoxic immune response. In addition, we identified more tumour-infiltrating regulatory T cells in HPV-positive carcinomas, which might represent a mechanism of immune evasion. The present study provides further evidence that the tumour microenvironment of HPV-positive carcinomas differs from that of HPV-negative carcinomas.

Analysis of PIK3CA mutations in breast cancer subtypes.

Phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit α (PIK3CA) is a central element of a signaling pathway involved in cell proliferation, survival, and growth. Certain mutations in this pathway result in enhanced PI3K signaling, which is associated with oncogenic cellular transformation and cancer. The aims of this study were to characterize different types of PIK3CA mutations in exons 9 and 20 in a series of primary breast carcinomas and to correlate the results with clinicopathologic parameters and survival. We used frozen tissue samples and sequenced exons 9 and 20 for a series of 241 patients with a diagnosis of breast carcinoma. We found that 15.8% of the primary breast carcinomas possessed PIK3CA mutations in either exon 9 or exon 20. The rate of PIK3CA mutations was increased in HR(+)/HER2(-) tumors (18.6%), but this difference did not reach a statistical significance. The lowest rate of mutations was observed in HR(+)/HER2(+) tumors (5.3%). No statistically significant association was found between the presence of PIK3CA mutations and the prognostic/clinical features of breast cancer, including histologic subtype, Her2 status, axillary lymph node involvement, tumor grade, and tumor stage. However, the presence of the H1047R mutation in 10 samples was associated with a statistically significantly worse overall survival. PIK3CA mutation was found to be a frequent genetic change in all breast cancer subtypes but occurred with the highest rate in HR(+)/HER2(-) tumors. Further studies are needed to validate the prognostic impact of different PIK3CA mutations.

Applicability of a System for fully automated nucleic acid extraction from formalin-fixed paraffin-embedded sections for routine KRAS mutation testing.

Due to the approval of various new targeted therapies for the treatment of cancer, molecular pathology laboratories with a diagnostic focus have to meet new challenges: simultaneous handling of a large number of samples, small amounts of input material, and fragmentation of nucleic acids because of formalin fixation. As a consequence, fully automated systems for a fast and standardized extraction of high-quality DNA from formalin-fixed paraffin-embedded (FFPE) tissues are urgently needed. In this study, we tested the performance of a fully automated, high-throughput method for the extraction of nucleic acids from FFPE tissues. We investigated the extraction performance in sections of 5 different tissue types often analyzed in routine pathology laboratories (cervix, colon, liver, lymph node, and lung; n=340). Furthermore, we compared the quality, labor input, and applicability of the method for diagnostic purposes with those of a laboratory-validated manual method in a clinical setting by screening a set of 45 colorectal adenocarcinoma for the KRAS mutation. Automated extraction of both DNA and RNA was successful in 339 of 340 FFPE samples representing 5 different tissue types. In comparison with a conventional manual extraction protocol, the method showed an overall agreement of 97.7% (95% confidence interval, 88.2%-99.9%) for the subsequent mutational analysis of the KRAS gene in colorectal cancer samples. The fully automated system is a promising tool for a simple, robust, and rapid extraction of DNA and RNA from formalin-fixed tissue. It ensures a standardization of sample processing and can be applied to clinical FFPE samples in routine pathology.

Core classification of head and neck squamous cell carcinomas: correlations between morphology, DNA ploidy and HPV infection.

AIMS: The study intended to reveal whether HPV infection is reflected by nuclear morphology and DNA cytometry parameters in head and neck squamous cell carcinomas (HNSCC). METHODS: In total, 39 HNSCC were selected for reanalysis by histomorphology applying the core classification, DNA cytometry and HPV detection. For the core classification, HE sections were assessed by a score system to evaluate the nuclear size, the mitosis size, their variabilities and the presence of tripolar or tetrapolar mitoses. HPV was analyzed by consensus PCR followed by a hybridization method for virus typing. Static DNA cytometry was applied on single cell suspension focusing particularly on the parameters DNA modal value, DNA index peak, DNA index mean, 2c deviation index and 5c exceeding rate. Statistical analysis was done by T-test or Fisher's exact test. RESULTS: The analysis revealed that HPV positive HNSCC had significantly smaller nuclei than HPV negative cases. Increasing values of the nuclear size and mitosis size were significantly associated with higher indices of the DNA cytometry analysis. CONCLUSIONS: The study confirms that the core classification can provide information on the ploidy of HNSCC and that HPV positive tumors represent a distinct morphological and genetic carcinoma subtype.

KRAS genotyping of paraffin-embedded colorectal cancer tissue in routine diagnostics: comparison of methods and impact of histology.

KRAS mutation testing before anti-epidermal growth factor receptor therapy of metastatic colorectal cancer has become mandatory in Europe. However, considerable uncertainty exists as to which methods for detection can be applied in a reproducible and economically sound manner in the routine diagnostic setting. To answer this question, we examined 263 consecutive routine paraffin slide specimens. Genomic DNA was extracted from microdissected tumor tissue. The DNA was analyzed prospectively by Sanger sequencing and array analysis as well as retrospectively by melting curve analysis and pyrosequencing; the results were correlated to tissue characteristics. The methods were then compared regarding the reported results, costs, and working times. Approximately 40% of specimens contained KRAS mutations, and the different methods reported concordant results (kappa values >0.9). Specimens harboring fewer than 10% tumor cells showed lower mutation rates regardless of the method used, and histoanatomical variables had no influence on the frequency of the mutations. Costs per assay were higher for array analysis and melting curve analysis when compared with the direct sequencing methods. However, for sequencing methods equipment costs were much higher. In conclusion, Sanger sequencing, array analysis, melting curve analysis, and pyrosequencing were equally effective for routine diagnostic KRAS mutation analysis; however, interpretation of mutation results in conjunction with histomorphologic tissue review and on slide tumor tissue dissection is required for accurate diagnosis.

Prevalence of infectious pathogens in Crohn's disease.

The importance of infectious pathogens in Crohn's disease (CD) is still under debate. Therefore, we examined a panel of potential viral and bacterial pathogens in a large series of CD patients and controls. Archival tissue from 76 patients, 56 with CD and 20 control patients, with normal colon mucosa (n=10) and non-steroid anti-inflammatory drug (NSAID)-induced colitis (n=10) were examined using PCR-based detection methods for human cytomegalovirus (CMV), Epstein-Barr virus (EBV), herpes simplex virus 1, 2 (HSV1,2), adenovirus (AD), varicella-zoster virus (VZV), human herpes virus 6 (HHV6), human herpes virus 8 (HHV8), Mycobacterium tuberculosis complex (Mtbc), atypical mycobacteria (nM/MG1), including Mycobacterium avium (subspecies paratuberculosis, MAP), Stenotrophomonas maltophilia (Sm), and Yersinia enterocolitica (Ye). In CD patients, positive PCR results were achieved in 19 cases (34%). Sm was most frequent in 10 of 56 cases (17.9%) followed by EBV (6/56, 10.7%), nM/MG1 (4/56, 7.1%), including MAP, HHV6, and CMV (2/56, 3.6%), and finally Mtbc and AD (1/56, 1.8%). The control patients showed positive PCR results in 12 patients (12/20, 60%), nine of them with only weak signals, suggesting a persistent infection. In addition, we compared typical pathomorphological features of CD patients with the PCR results and found a significant correlation between EBV infection and mural abscesses (P=0.014). Our data demonstrate that several potential pathogens can be detected in a sizeable fraction of specimens from patients with CD, but also in control patients, suggesting that the analyzed infectious pathogens may be associated with the disease, but do not represent an obligatory cause.

Molecular HPV typing as a diagnostic tool to discriminate primary from metastatic squamous cell carcinoma of the lung.

In this study, typing of human papilloma virus (HPV) was performed in squamous cell carcinomas of the lung (n=26) as well as putative primaries of head and neck (n=21) and female genital tract (n=5) of the same patients, to test whether additional information to discriminate lung primaries from metastases can be gained by a direct comparison of the HPV status in both tumors. In 3 (14.2%) patients with head and neck as well as lung squamous cell carcinoma, an identical HPV subtype could be detected in both tumors suggesting metastatic disease. In 9 (42.9%) cases, discordant HPV status strongly suggested secondary primaries of the lung. In the remaining 9 (42.9%) patients, no HPV was evident in either tumor. In all patients with carcinomas of the cervix uteri an identical HPV subtype was detected in the cervical and in the lung tumor. In conclusion, the results suggest HPV typing, a method routinely used in cervical biopsies for years, as a very useful diagnostic tool to discriminate primary from metastatic squamous cell carcinoma of the lung, which in our cohort in 57.1% of cases allowed for almost definite classification.

Inter-laboratory validation of PCR-based HPV detection in pathology specimens.

The detection and typing of human papilloma virus (HPV) in pathology specimens is gaining increasingly in importance. In the context of the initiative for quality assurance in pathology (QuIP) of the German Society of Pathology and the Professional Association of German Pathologists, four panel laboratories with experience and expertise in polymerase chain reaction (PCR)-based HPV detection were selected to establish an inter-laboratory trial. In a first step, these laboratories performed an internal testing of their own methodologies, which comprised DNA sequencing, multiplex nested PCR and hybridization techniques. Material from 39 samples including paraffin sections and DNA preparations of tissues and plasmids were evaluated by each panel institute according to their own protocols. Despite the different methodologies, a high degree of inter-laboratory reliability was achieved. In this report, we summarise the results. Pretested specimens are available for the external trail and can be ordered from the steering institute via provitro GmbH Berlin ( http://www.provitro.de ). Supplementary data are online available at http://pathologie-ccm.charite.de (rubric "Forschung"), which includes a web-based photo gallery of HPV-associated lesions and their potential association with specific virus types. The initiative is intended to foster the quality assurance of molecular HPV analysis in pathology and its correlation with morphological changes.

HPV typing and CGH analysis for the differentiation of primary and metastatic squamous cell carcinomas of the aerodigestive tract.

Human papilloma virus (HPV) typing and Comparative Genomic Hybridisation (CGH) analysis can be used in the classification of multiple tumours of the aerodigestive tract for the differentiation between secondary malignancy versus metastasis. We present 3 exemplary cases of patients with multiple squamous cell carcinomas, localised within the head and neck region, cervical lymph node and the lung. In two patients, HPV typing identified HPV type 16 in the tonsillar carcinomas and the corresponding cervical lymph node and lung carcinoma indicating that the latter were metastatic spreads. In case 1, CGH confirmed the clonal relationship. Case two showed a peculiar syncytial growth pattern with lymphocytic infiltration which may constitute a potential morphological marker for HPV infection. In case three, a vallecular carcinoma was HPV negative while a lung cancer was positive for HPV type 6 indicating two independent primary tumours. Our case triplet illustrates the variability of HPV infection in squamous cell cancer of the aerodigestive tract and power as well as limitations of morphology, HPV typing and tumour genetics in the classification of multiple tumours.

Inter-laboratory validation of PCR-based detection of Mycobacterium tuberculosis in formalin-fixed, paraffin-embedded tissues.

The present study is based on the initiative for quality assurance in pathology of the German Society of Pathology and the Professional Association of German Pathologists. Four panel laboratories with experience and expertise in polymerase chain reaction (PCR) detection of Mycobacterium tuberculosis were selected to establish the prerequisites for continuous external laboratory trials, in particular, by providing pre-tested specimens and evaluation criteria for participating institutes. In the first step, the four panel laboratories performed an internal trial to test their own reliability and reproducibility. Paraffin sections and DNA preparations from 34 tissues (25 clinical specimens and 9 controls) totalling to 66 samples were evaluated by each panel institute according to their own protocols. The methodologies differed and are described in detail. Despite these differences, a high degree of inter-laboratory reliability was achieved. In this report, we summarise our results including the correlation with the histology and provide recommendations for applying PCR-based methodology for the detection of mycobacterial DNA in surgical specimens. Supplementary data are available online at http://www.charite.de/ch/patho (rubric "Forschung"). Pre-tested specimens are now available for the external trial and can be ordered from the steering institute via Oligene (http://www.oligene.com/). All molecular pathology laboratories are invited to participate in this quality assurance initiative.

Cytomegalovirus is not associated with progression and metastasis of colorectal cancer.

Recently, it was hypothesized that human cytomegalovirus (HCMV) might be associated with colorectal cancer progression. However, the role of HCMV infection in colorectal cancer remains controversial. We aimed to assess whether nucleic acids and/or gene products of HCMV could be detected in primary and metastatic colorectal carcinomas. Fresh-frozen biopsy specimens from 77 primary and metastatic colorectal carcinomas of randomly selected patients were analyzed by PCR and immunohistochemistry. We investigated 57 primary tumors and 20 metastases, comprising 13 tumor pairs from the same patient. In PCR, four primary tumors showed a positive CMV result whereas all investigated metastases were negative including three paired samples from positive primaries. In immunohistochemistry, no specific staining could be determined in all neoplastic epithelial cells. In summary, we think that there is no association between CMV infection and tumor progression or metastasis formation of colorectal cancer.

[Squamous cell carcinoma in lupus vulgaris]. / Carcinoma in lupo.

Lupus vulgaris and carcinoma in lupo have become rare events that take place in the developed countries only under special circumstances. A 53-year-old woman developed such a carcinoma. She suffered from alcoholism, a well known risk factor for tuberculosis. The diagnosis of lupus vulgaris was confirmed by biopsy when an erythematous lesion on her arm that had been present for 25 years enlarged and subsequently ulcerated. Chemotherapy was discontinued because of lack of compliance and the ulcer grew markedly over the following 16 months. Therefore the entire lesion was excised. Histology showed a squamous cell carcinoma within the ulcer. Neither further systemic manifestations of tuberculosis nor metastases of the carcinoma were found. Under continuous combined antituberculous therapy, the patient remained free of symptoms. This case underlines the problems associated with a disease that has been nearly forgotten in the western countries. It also shows that alcoholism is a risk factor for tuberculosis, along with debilitating diseases such as lymphoma and AIDS as well as immunosuppressive therapy.

Nucleolin as activator of human papillomavirus type 18 oncogene transcription in cervical cancer.

High risk human papillomaviruses (HPVs) are central to the development of cervical cancer and the deregulated expression of high risk HPV oncogenes is a critical event in this process. Here, we find that the cell protein nucleolin binds in a sequence-specific manner to the HPV18 enhancer. The DNA binding activity of nucleolin is primarily S phase specific, much like the transcription of the E6 and E7 oncoproteins of HPV18 in cervical cancer cells. Antisense inactivation of nucleolin blocks E6 and E7 oncogene transcription and selectively decreases HPV18(+) cervical cancer cell growth. Furthermore, nucleolin controls the chromatin structure of the HPV18 enhancer. In contrast, HPV16 oncogene transcription and proliferation rates of HPV16(+) SiHa cervical cancer cells are independent of nucleolin activity. Moreover, nucleolin expression is altered in HPV18(+) precancerous and cancerous tissue from the cervix uteri. Whereas nucleolin was homogeneously distributed in the nuclei of normal epithelial cells, it showed a speckled nuclear phenotype in HPV18(+) carcinomas. Thus, the host cell protein nucleolin is directly linked to HPV18-induced cervical carcinogenesis.