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Objective.—Polymerase chain reaction (PCR) and indirect in situ hybridization were combined to detect human papillomavirus (HPV) DNA on Papanicolaou (PAP)-stained cervical smears. To our knowledge, this is the first report of an experiment using indirect in situ PCR (IS-PCR) on PAP-stained cervical smears. Design.—We collected native cell specimens from cervicovaginal lavage of 162 patients with squamous intraepithelial lesions. Solution-phase PCR (SP-PCR) was performed as the reference method in the detection of HPV DNA. Indirect IS-PCR was carried out for the same patients to detect the HPV DNA types 6/11 and 16/18 after the PAP-stained smears had been decolorized. Low-risk and high-risk HPV DNA types were also detected by both SP-PCR and indirect IS-PCR. Results.—In the evaluation by indirect IS-PCR, 48 of 81 PAP-stained cell smears of low-grade squamous intraepithelial lesions were positive for HPV DNA, as compared to 40 positive cell smears determined by indirect SP-PCR (sensitivity of indirect IS-PCR compared to SP-PCR, 98.1%). Forty-two of 42 high-grade squamous intraepithelial lesion samples were positive for HPV DNA, as determined by both methods (sensitivity of IS-PCR, 100%). Cell lines investigated in this study as positive or negative controls for HPV DNA were confirmed by indirect IS-PCR and SP-PCR. Conclusions.—Our data show that in comparison to SP-PCR, indirect IS-PCR is a highly sensitive method to detect HPV DNA in cell smears from the uterine cervix. The advantages of indirect IS-PCR are (a) low numbers of cells needed, (b) the possibility of using PAP-stained specimens, and (c) cytologic details of smears can be preserved.

The aim of this study was to detect activated c-K-ras by gene point mutation and to find c-erbB-2 gene amplification with p185 expression in association with the c-K-ras gene product p21 in the human endometrium. Specimens obtained from 25 normal, 31 hyperplastic and 72 malignant samples of the human endometrium were examined for point mutation in codons 12, 13 and 61 of the c-K-ras by direct sequencing and c-erbB-2 gene amplification with p185 and p21 expression by differential polymerase chain reaction (DPCR) and immunohistochemistry. Neither the normal endometrium nor endometrial hyperplasias were found to have mutations in the c-K-ras gene, although a double mutation of codons 12 and 13 as a single-point mutation was observed in one case of endometrioid carcinoma (2.8%). In each of two other cases of endometrioid carcinoma (2/72), two single-point mutations of codon 13 (5.6%) were shown. Using DPCR, we found c-erbB-2 to be amplified in 15 premalignant (48%) and 45 malignant (63%) samples. We noticed that nonamplification of the c-erbB-2 gene was associated with the absence of immunoreactivity. Our data indicate that, while c-erbB-2 plays a role in the early development of endometrioid carcinomas, c-K-ras gene activation by point mutation does not.

The presence of hepatitis C virus (HCV) in normal cervical smears (CS) obtained from 22 HCV-seropositive and 50 HCV-seronegative patients was assessed by reverse-transcriptase-polymerase chain reaction (RT-PCR). The presence of HCV in serum was established by use of enzyme-linked immunosorbent assay, Western blot test, and RT-PCR. HCV was detected in 36.4% (n = 8) of CS cells recovered from 22 HCV-seropositive patients, but not in CS samples obtained from 50 HCV-seronegative patients. Furthermore, cells from the CS of 2 seropositive/smear-positive patients and 1 seropositive/smear-negative patient were isolated; HCV RNA was detectable in the cervical lymphocytes of the 2 smear-positive patients, but not in epithelial cells or granulocytes. HCV RNA is detectable in the CS of some HCV-seropositive women. The clinical importance of these data requires further study.

22206 Background: This study combines a summary of proteomics techniques and protein analysis in mammary cancer with a report of expressed and up-regulated proteins in 10 benign mammary tissues and...