Touchdown General Primer (GP5+/GP6+) PCR and optimized sample DNA concentration support the sensitive detection of human papillomavirus

BACKGROUND: The GP5+/GP6+ PCR assay is a well-established HPV detection technique. This study has examined the effects of incorporating 'hot start' and 'touchdown' steps into the protocol. In addition, dTTP was substituted with dUTP to permit contamination control measures against carry-over PCR product. METHODS: Firstly, HPV-16 was amplified from SiHa cell DNA (0.1 ng–100 ng) diluted in a background of C-33A DNA (100 ng-2 μg). Secondly, the detection of small quantities (15ag-1.5pg) of HPV recombinant plasmids (types 16, 31, 33, 45, 51, 52, and 56) diluted in C-33A DNA was investigated. Thirdly, clinical sample DNA extracts (cervical smears, formalin-fixed vaginal lesions and breast tumors) were tested for HPV. Six different PCR protocols were assessed. HPV was detected by gel electrophoresis, and by Southern and dot blot hybridization. RESULTS: HPV detection sensitivity was dependent on the total amount of DNA in a PCR. Touchdown protocols supported HPV-16 detection from 1 ng or 0.5 ng SiHa cell DNA in a background of 2 μg or 1 μg C-33A DNA respectively, and from 0.1 ng of SiHa cell DNA (~28 copies HPV-16) in 500 ng or 100 ng background DNA. Under standard GP5+/GP6+ annealing conditions, HPV-16 went undetected when the DNA content of a PCR was 2 μg or 1 μg, and with 500 ng C-33A DNA the sensitivity limit was 1 ng SiHa cell DNA. HPV recombinant plasmids were each detected with high (albeit varying) sensitivity by a touchdown protocol. HPV-31 was better amplified under standard annealing conditions (1.5fg in 100 ng background DNA) than by a touchdown approach (15fg detection limit). HPV-52 was not amplified by the standard protocol at the dilutions tested. Seventeen different HPV types were demonstrated in 47/65 (72%) abnormal cytology samples recorded as HPV negative by standard GP5+/GP6+ conditions. Twenty-one different HPV types were recorded in 111/114 (97%) vaginal lesions. Multiple infections were also detectable using a touchdown approach. Of 26 breast tumors, 5 (19%) tested HPV positive by the standard assay and 15/26 (58%) using a touchdown protocol. CONCLUSION: Touchdown modification of the GP5+/GP6+ PCR assay enables the detection of HPV undetected under regular assay conditions. The use of standardized DNA quantities in a PCR rather than standard sample volumes containing arbitrary amounts of DNA is supported. A touchdown approach may be beneficial as an analytical test for the re-evaluation of (apparently) HPV negative abnormal cervical cytological or histological samples, and for investigating the association of HPV with disease conditions at diverse organ sites. The clinical utility of a touchdown approach for HPV detection requires further investigation as increased assay analytical sensitivity may not necessarily equate with improved clinical sensitivity or specificity

Presence of papillomavirus sequences in condylomatous lesions of the mamillae and in invasive carcinoma of the breast

BACKGROUND: Viruses including Epstein–Barr virus (EBV), a human equivalent of murine mammary tumour virus (MMTV) and human papillomavirus (HPV) have been implicated in the aetiology of human breast cancer. We report the presence of HPV DNA sequences in areolar tissue and tumour tissue samples from female patients with breast carcinoma. The presence of virus in the areolar–nipple complex suggests to us a potential pathogenic mechanism. METHODS: Polymerase chain reaction (PCR) was undertaken to amplify HPV types in areolar and tumour tissue from breast cancer cases. In situ hybridisation supported the PCR findings and localised the virus in nipple, areolar and tumour tissue. RESULTS: Papillomavirus DNA was present in 25 of 29 samples of breast carcinoma and in 20 of 29 samples from the corresponding mamilla. The most prevalent type in both carcinomas and nipples was HPV 11, followed by HPV 6. Other types detected were HPV 16, 23, 27 and 57 (nipples and carcinomas), HPV 20, 21, 32, 37, 38, 66 and GA3-1 (nipples only) and HPV 3, 15, 24, 87 and DL473 (carcinomas only). Multiple types were demonstrated in seven carcinomas and ten nipple samples. CONCLUSIONS: The data demonstrate the occurrence of HPV in nipple and areolar tissues in patients with breast carcinoma. The authors postulate a retrograde ductular pattern of viral spread that may have pathogenic significance

Blood-Vessel Invasion in Breast-Cancer - a Comparison of Hematoxylin-Eosin Staining and Immunohistochemical Staining for Factor-Viii Antigen for Their Prognostic Value Concerning Tumor Recurrence

Blood vessel invasion was investigated with haematoxylin-eosin (HE) staining and immuno-histochemical staining for factor VIII antigen (F VIII) in 106 patients with primary carcinoma of the breast, in order to compare their value in prognosticating the probability of recurrence. Blood vessel invasion was diagnosed in 65 cases (61.9%) by HE, but in only 45 (43.4%) by F VIII staining. Lymph-node status and blood vessel invasion correlated positively on HE (r = 0.73; P = 0.0001), but not so on F VIII staining. Multivariate logistic regression showed blood vessel invasion to be a strongly independent prognostic factor for recurrence-free survival with F VIII staining (odds ratio: = 7.19; P = 0.0001), while HE staining was not independent from other prognostic factors. These preliminary data thus suggest that demonstrating vascular invasion by F VIII staining may identify those patients with a very high risk of recurrence, independent of lymph-node status

Tumoral Microvessel Density in Breast-Cancer and its Influence On Recurrence-Free Survival

Angiogenesis quantitation of 106 patients with primary breast cancer and 35 patients with adenofibroma of the breast was compared and examined to its prognostic relevance for five-years disease-free survival in breast cancer patients. Immunocytochemical staining for Factor VIII-related antigen was performed to outline vascular endothelium. We found a significant higher vessel density in breast cancer patients who experienced recurrence (17.4) than in those with no recurrence (9.4) or with adenofibroma (8.7) [p < 0.0001]. The probability of five-years recurrence-free survical for patients with a primary tumor of high vessel density was at 52.3% and 86.4% for tumors of low microvessel density (p < 0.0011). Microvessel density proved to be an independent prognostic factor for breast cancer recurrence in the Cox-Model (relative risk 2.047, p = 0.0002)

Tumoral vascular density in breast tumors and their effect on recurrence-free survival Tumorale Gefässdichte bei Mammatumoren und ihr Einfluss auf das rezidivfreie Uberleben

Angiogenesis quantitation of 106 patients with primary breast cancer and 35 patients with adenofibroma of the breast was compared and examined to its prognostic relevance for five-years disease-free survival in breast cancer patients. Immunocytochemical staining for Factor VIII-related antigen was performed to outline vascular endothelium. We found a significant higher vessel density in breast cancer patients who experienced recurrence (17.4) than in those with no recurrence (9.4) or with adenofibroma (8.7) [p < 0.0001]. The probability of five-years recurrence-free survival for patients with a primary tumor of high vessel density was at 52.3% and 86.4% for tumors of low microvessel density (p < 0.0011). Microvessel density proved to be an independent prognostic factor for breast cancer recurrence in the Cox-Model (relative risk 2.047, p = 0.0002)

Blood vessel invasion in breast cancer. A comparison of haematoxylin-eosin staining and immunohistochemical staining for factor VIII antigen for their prognostic value concerning tumour recurrence TUMORGEFASSINVASION BEIM MAMMAKARZINOM. HAMATOXYLIN-EOSIN- VERSUS IMMUNHISTOCHEMISCHE FARBUNG GEGEN FAKTOR-VIII-ANTIGEN

Blood vessel invasion was investigated with haematoxylin-eosin (HE) staining and immunohistochemical staining for factor VIII antigen (F VIII) in 106 patients with primary carcinoma of the breast, in order to compare their value in prognosticating the probability of recurrence. Blood vessel invasion was diagnosed in 65 cases (61.9%) by HE, but in only 45 (43.4%) by F VIII staining. Lymph-node status and blood vessel invasion correlated positively on HE (r = 0.73; P = 0.0001), but not so on F VIII staining. Multivariate logistic regression showed blood vessel invasion to be a strongly independent prognostic factor for recurrence-free survival with F VIII staining (odds ratio: =7.19; P = 0.00001), while HE staining was not independent from other prognostic factors. These preliminary data thus suggest that demonstrating vascular invasion by F VIII staining may identify those patients with a very high risk of recurrence, independent of lymph-node status