Use of PCR-PIRA for screening of a point mutation at codon 12 in K-ras oncogene obtained from paraffin embedded tissue sections.

Among various methods which have been developed for facilitating the screening of point mutations in human genomic DNA, PCR-Primer Introduced Restriction Analysis (PCR-PIRA) is of particular interest due to its practicality and short procedure allowing detection of point mutations by simple restriction enzyme digestion directly after PCR amplification. However, one limitation of PCR-PIRA method is the absence of restriction sites in the region of detection, thus creation of the recognition site in primers has been introduced. Detection of a point mutation at codon 12 in K-ras oncogene by BstNI requires one base change in the primer sequence so that only the normal but not mutant PCR product will be digested by the enzyme. However, false positive results generated from undigested normal DNA sequence are always obtained. This effect is compounded when it is used to analyse mixed cell populations in paraffin embedded section of cancer cells. Assay of a mutant band generated from normal DNA by densitometric quantitation enabled the determination of background values and thereby eliminated false positive results. Samples with higher ratios between mutant and normal bands than the background one after the first PCR-PIRA would be subjected to the second PCR-PIRA in order to confirm the results. Screening of such mutations in cervical carcinomas from paraffin embedded sections using the above criteria should reduce misinterpretation of PCR-PIRA results.

Detection of various types of human papillomaviruses in premalignant and malignant cervical lesions using DNA-DNA in situ hybridization.

Detection of human papillomavirus (HPV) in formalin-fixed paraffin embedded tissue sections by in situ hybridization technique employing biotinylated (HPV) DNA probe 6/11, 16/18 and 31/33/35 was done retrospectively in 25 cases of cervical dysplasia, 32 cases of cervical squamous cell carcinoma in situ, 52 cases of invasive cervical squamous cell carcinoma and 7 cases of adenocarcinoma. HPV could be demonstrated in 10 cases (40.00%) of cervical dysplasia, 8 cases (25.00%) of cervical squamous cell carcinoma in situ and 31 cases (59.61%) of invasive cervical squamous cell carcinoma and 4 cases (57.14%) of adenocarcinoma. Among the dysplastic cases, 4 cases showed HPV 31/33/35, 4 cases showed HPV 16/18 together with HPV 31/33/35, 1 case showed mixed typing of HPV 6/11 and 31/33/35, and 1 case showed mixed typing of HPV 6/11, 16/18 and 31/33/35. In 32 cases of squamous cell carcinoma in situ, 1 case of HPV 6/11; 3 cases of HPV 16/18, 3 cases of HPV 31/33/35 and 1 case of mixed typing of HPV 16/18 and 31/33/35 were present. A case of HPV 6/11, 10 cases of HPV 16/18 and 9 cases of HPV 31/33/35 could be detected among the cases of invasive squamous cell carcinoma. Mixed typing of HPV 6/11 and 16/18; HPV 6/11 and 31/33/35; HPV 16/18 and 31/33/35; HPV 6/11, 16/18 and 31/33/35 were revealed in 2, 1, 3 and 5 cases of invasive squamous cell carcinoma, respectively. In 7 cases of adenocarcinoma, 1, 2 and 1 cases exhibited positivity for HPV 16/18; HPV 6/11 and HPV 16/18; and HPV 16/18 and HPV 31/33/35.(ABSTRACT TRUNCATED AT 250 WORDS)

Detection of estrogen and progesterone receptor bindings in mammary carcinoma cells: a comparative study using immunohistochemical and biochemical methods.

A comparative study of estrogen and progesterone receptor bindings of breast carcinoma tissue was done by immunoperoxidase and dextran-coated charcoal (DCC) methods. Fifteen cases of paraffin embedded formalin fixed tissue of mammary carcinomas which had previously been evaluated by the DCC method were selected. Twelve cases were ductal carcinoma and 3 were of lobular origin. Lymph node tissue showing metastasis was available in 3 cases. By immunoperoxidase technique, 12 and 10 (80 and 66.7%) cases were positive for estrogen and progesterone receptor respectively compared with 8 and 3 (53.3 and 20%) cases by the DCC technique. Corresponding results of both methods to detect estrogen and progesterone bindings were 9 and 8 (60 and 53.3%) of all cases, respectively. Five cases for estrogen and 6 cases for progesterone positive by immunoperoxidase could not be detected by the DCC technique. Only one case of estrogen negative by the immunoperoxidase gave a positive result with the DCC technique. Variability of staining occurred between primary and metastatic lesions, 2 out of 3 cases displayed positive staining in both sites; one remaining case was positive only in the lymph node metastasis. Immunoperoxidase is a relatively simple, swift and inexpensive technique in comparison to the DCC technique. Using fixed embedded tissue makes it possible for retrospective studies and providing a permanent record for reevaluation. Moreover, morphology of the tumor can be determined at the same time as detection of hormonal receptor bindings.