Mutation status of genes encoding RhoA, Rac1, and Cdc42 GTPases in a panel of invasive human colorectal and breast tumors.

PURPOSE: The constitutive activation of Ras proteins by point mutation is the most frequently observed oncogene activation in human malignancies. The goal of this study was to investigate whether the constitutive activation of RhoA, Rac1, and Cdc42 proteins by point mutations, which can lead to experimental transformation of cultured cells, actually occurred in a panel of invasive colorectal and breast tumors. METHODS: We performed denaturing gradient gel electrophoresis and sequencing of transcripts amplified by reverse transcription and PCR for RhoA; we used direct sequencing of PCR-amplified genomic DNA to search for mutations in coding exons of the Rac1 and Cdc42 genes. RESULTS: Although mutations of the Kras4B and the p53 genes were detected using these methods, no mutation was found in the coding sequences of RhoA, Rac1, and Cdc42 genes, in primary as well as in associated metastasis. CONCLUSIONS: Point mutations in the coding sequences of genes encoding RhoA, Rac1, and Cdc42 GTPases do not occur at high frequency in invasive breast and colorectal tumors.

Human papillomaviruses and DNA ploidy in anal condylomata acuminata.

Previous studies have emphasized the usefulness of DNA ploidy measurement and Human Papillomavirus (HPV) detection as prognostic markers in low grade cervical lesions. We addressed the eventual relationship between HPV type, DNA profile, and p53 tumor suppressor protein expression in anal condylomata acuminata to eventually determine parameters which may be considered as predictive risk factors for the development of cancer. DNA ploidy was assessed by image cytometry after Feulgen staining of contiguous serial sections of 45 anal condylomata acuminata without atypia containing HPV detected by in situ hybridization and Polymerase Chain Reaction (PCR). p53 expression was detected by immunohistochemistry. DNA aneuploidy was found in 53.3% of these lesions, 48.9% containing non oncogenic HPV types 6 and/or 11 and 4.4% harbouring HPV types 11 and 18. The DNA diploid lesions were all associated with non oncogenic HPV types 6 and/or 11 and one case also contained HPV type 33. There was no significant correlation between the detection of DNA aneuploidy and the presence of immuno-detected p53. DNA aneuploidy was not related to the presence of oncogenic HPV in anal condylomata acuminata. The DNA aneuploid profile frequently observed, especially in lesions associated with non oncogenic HPV types, is not yet well explained and cannot be considered as a prognostic factor. In contrast, a more intensive clinical follow-up should be proposed in patients with oncogenic HPV associated to DNA aneuploidy.

DNA-EIA to detect high and low risk HPV genotypes in cervical lesions with E6/E7 primer mediated multiplex PCR.

BACKGROUND: Oncogenicity of human papillomavirus (HPV) DNA in premalignant and malignant uterine cervical diseases is mainly induced by E6/E7 open reading frame (ORF). The presence of an oncogenic HPV DNA may be a diagnostic marker for the detection of cytologically negative smears. AIMS: To evaluate an original polymerase chain reaction enzyme immunoassay (PCR-EIA) for the detection and typing of oncogenic and non-oncogenic HPV types. METHODS: The test was an original multiplex labelled PCR-EIA for the detection and typing of oncogenic and non-oncogenic HPV using three consensus sequence primers within the oncogenic E6/E7 ORF. One primer was dinitrophenyl (DNP) labelled and the DNP labelled amplimers could be further hybridised with specific biotinylated oligoprobes mixed in only two cocktails: oncogenic (16, 18, 31, 33, 35, 52, and 58) and non-oncogenic (6 and 11) HPV types in only two wells; then biotinylated oligoprobes were deposited in streptavidin-coated microplates. The PCR-EIA was validated on HPV plasmids (types 6, 11, 16, 18, 31, 35, 52, and 58) and used to evaluate cervical scrapes from 181 patients (median age 32 years) at high risk for cervical cancer. RESULTS: HPV were detected in the cervical scrapes of 88 of 181 patients (48.6%); nine with non-oncogenic HPV (5.0%) and 79 with oncogenic HPV (43.6%) including 29 coinfections with oncogenic and non-oncogenic HPV. The number of oncogenic HPV infections increased with the presence of high grade lesions: 95.8% of the cervical scrapes from patients with high grade lesions contained oncogenic HPV compared with 32.1% of the specimens from patients without any lesions detectable by colposcopy and/or by cytological examination of the cervical smears. Moreover, 60% of cervical scrapes exhibiting low grade lesions contained oncogenic HPV. CONCLUSIONS: This test is simple, specific, sensitive, safe, fast, reproducible, and easy to use in routine practice. Thus, it is possible to detect simultaneously on a simple cervical scrape, two kinds of HPV--oncogenic and non-oncogenic--in just two microplate wells with non-isotopic oligoprobes.

Comparative analysis of human papillomavirus detection by hybrid capture assay and routine cytologic screening to detect high-grade cervical lesions.

A commercial HPV detection test, Hybrid Capture (HC), designed to detect 14 HPV types divided into high-risk and low-risk groups, has been evaluated. A total of 1064 scrapes from 1028 unselected women attending routine cytologic screening were tested and results were compared with those of classic cytologic screening and cervical biopsies. The reliability of the test was also evaluated on 38 fresh conization samples. HPV DNA was detected in 108 women (10.5%), including 90 infected by a high-risk HPV (8.8%); 25 high-grade lesions were detected histologically, and high-risk HPV was found in 16 of these 25 women (64%), and in 27 (71%) of the 38 conization samples. The overall sensitivity of HC in detecting high-grade SIL on cervical scrapes and conization samples was 71.2%, while its positive predictive value was 17.8%. Classic cytologic screening appeared to be the most sensitive method (84%) for detecting high-grade SIL, with a positive predictive value of 91.3%. The lower sensitivity of HC limits its use for screening high-grade lesions on a large scale, even though it may be useful for reducing cytologically false-negative results. Moreover, the quantitative approach provided by the HC assay for assessment of the viral load cannot clearly distinguish among cases with or without high-grade lesions.

Oncogenic human papillomaviruses and ploidy in cervical lesions.

AIM: To compare ploidy measurements obtained on tissue sections of selected low and high grade squamous intraepithelial lesions containing oncogenic HPV (types 16, 18 or 33) detected by in situ hybridisation (ISH) or PCR. METHODS: DNA ploidy was assessed by image cytometry after Feulgen staining of contiguous serial sections of eight lesions exhibiting atypical squamous cells or squamous atypia and 53 low and 63 high grade squamous intraepithelial lesions in which HPV had been detected by ISH or PCR. RESULTS: Aneuploidy was strongly associated with the presence of oncogenic HPV, being detected in 50% of lesions with squamous atypia and 75.5% of the low and 95.2% of the high grade squamous intraepithelial lesions. The multiploid profile was highly associated with high grade lesions and with the pattern of HPV DNA integration. CONCLUSIONS: The presence of aneuploidy is strongly suggestive of the presence of oncogenic HPV types. Combining the detection of HPV by ISH and PCR with DNA image cytometry may provide the pathologist and the physician with important prognostic information about low grade lesions, especially when these lesions have a multiploid DNA profile and contain oncogenic HPV.