Archival paraffin-embedded tumor specimens offer a wealth of information for both cancer research and for routine clinical applications. However, the use of formalin-fixed, paraffin-embedded specimens for quantitative real-time PCR is not yet a standard diagnostic method in many laboratories, in particular for the quantification of human papillomavirus (HPV). Particularly high-risk HPV types are involved in almost 100% of the carcinogenesis of cervical cancer. We compared the diagnostic applicability and sensitivity of real-time PCR to that of chromogenic tyramide-signal-amplified in situ hybridization and conventional PCR for the detection of HPV from archival tissue in 164 cases of carcinoma in situ and cervical cancer. Furthermore, we examined whether the viral load of HPV is of prognostic relevance. Our findings indicate that patients in tumor stage I with a lower viral load of HPV type 16 (HPV16; up to 1,000 copies/ng of DNA) had a significantly better survival than HPV 16-negative patients (P ؍ 0.037). We observed a greater sensitivity of both real-time PCR and conventional PCR for the detection of HPV16 and -18 compared to signal amplified in situ hybridization. We found a considerable concordance between HPV16 ( ؍ 0.661) and HPV18 ( ؍ 0.781) status as measured by real-time PCR and conventional PCR, indicating similar sensitivities. We recognized an inhibitory effect of formalin fixation and paraffin embedding on the evaluation of real-time PCR quantification.Formalin-fixed, paraffin-embedded (FFPE) tissues retained in pathology archives are an important resource for molecular pathology studies and for diagnostic applications. The use of in situ hybridization (ISH) and PCR assays have both been reported for DNA analysis in formalin-fixed archival tissues (6,7,25,40,42,48). Both assays have the advantage that only small amounts of tissue are required, and both tolerate the degradation of target nucleic acids due to the formalin fixation and paraffin-embedding procedures. Each assay per se has its own advantages and disadvantages.For a pathologist, the retention of morphology and the histological context of a tumor is very important. Especially in the case of cervical carcinoma, the phenomenon of koilocytosis indicates an infection with human papillomavirus (HPV) (4) that can only be detected by microscopic analysis of tissue sections. Moreover, episomal and integrated viral DNA can only be differentiated by ISH due to the staining pattern (8). With the use of chromogenic tyramide-signal-amplified ISH (CISH) it is even possible to detect and localize single or very few HPV copies within infected nuclei (15,36,49). However, this technique is rather cost-intensive and quite involved for routine applications.Conventional PCR is held to be the most sensitive detection method available (9,16,20,27,37,41). Although this is probably true, it has some limitations, as does ISH, in that it cannot quantify viral load in a prognostic-diagnostic significant manner. This restriction can be circumvented by the use o...
The significantly inverse correlation between HER1/EGFR and comedo-type DCIS and the observation that VEGF and the other angiogenic factors tested are independent of HER2 overexpression, suggest that progression of comedo-type DCIS and angiogenesis in breast carcinoma are not regulated via the HER1/EGFR or HER2 pathway.
Angiogenesis is a fundamental component of oncogenesis. Angiogenic factors such as vascular endothelial growth factor (VEGF) and platelet derived-endothelial cell growth factor/thymidine phosphorylase (PD-ECGF/TP) are generated from tumor cells to provide tumor growth and are thought to be regulated via the HER2 oncogene, whose amplification is the most common genetic alteration in breast cancer. The present study aimed to evaluate the immunoreactivity of angiogenic factors (VEGF, PD-ECGF/TP) and microvessel density (MVD) via epidermal growth factor receptor (EGFR) and HER2, and to correlate their expression with clinicopathologic features. Two hundred one invasive human breast cancer specimens were tested immunohistochemically for the expression of these proteins. In addition, MVD was examined using computerized image analysis. VEGF could be an additional interesting prognostic variable, as it was significantly associated with tumor grade (P=0.002), stage (P=0.018), and negative estrogen receptor status (P=0.011). EGFR was significantly related to invasive ductal carcinoma (P=0.030), tumor grade (P=0.009), VEGF expression (P=0.013), PD-ECGF/TP expression (P=0.024), and MVD (P=0.050). The finding that VEGF is not correlated to MVD does not rule out a crucial role of VEGF as a key factor in angiogenesis. HER2 could not be correlated to MVD, VEGF expression, or PD-ECGF/TP expression, indicating that this factor is unlikely to be involved in directly regulating angiogenesis, whereas the significant correlations between EGFR and histologic tumor type, tumor grade, the angiogenic factors VEGF and PD-ECGF/TP, and MVD point out that EGF is the major modulating growth factor for angiogenesis in breast cancer.
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