High-risk human papillomavirus (hrHPV) E6/E7 mRNA testing by PreTect HPV-Proofer for detection of cervical high-grade intraepithelial neoplasia and cancer among hrHPV DNA-positive women with normal cytology.

Our aim was to investigate whether high-risk HPV (hrHPV) mRNA detection by PreTect HPV-Proofer can be used to stratify hrHPV DNA-positive women of different cytology classes for risk of high-grade cervical intraepithelial neoplasia or worse (cervical precancer or cancer, i.e., cervical intraepithelial neoplasia grade 2 or higher [≥ CIN2]). A total of 375 women participating in population-based screening, with a GP5+/6+-PCR hrHPV DNA-positive cervical scrape with normal cytology (n = 202), borderline or mild dyskaryosis (BMD) (n = 88), or moderate dyskaryosis or worse (>BMD) (n = 85), were enrolled. Cervical scrapes were additionally subjected to HPV16/18/31/33/45 E6/E7 mRNA analysis by PreTect HPV-Proofer (mRNA test). Referral and follow-up policies were based on cytology, hrHPV DNA, and mRNA testing. The primary study endpoint was the number of ≥CIN2 detected within 3 years of follow-up. The mRNA positivity increased with the severity of cytological abnormality, ranging from 32% (64/202) in hrHPV DNA-positive women with normal cytology to 47% (41/88) in BMD and 68% (58/85) in >BMD groups (P < 0.01). Women with ≥ CIN2 were more likely to test positive by mRNA test (63%) than women without evidence of ≥ CIN2 (32%; P < 0.01). A positive mRNA test result conferred an increased ≥ CIN2 risk in hrHPV DNA-positive women with normal cytology, i.e., 0.55 (95% confidence interval [95% CI], 0.34 to 0.76) in mRNA-positive versus 0.20 (95% CI, 0.07 to 0.33) in mRNA-negative women. In hrHPV DNA-positive women with BMD or >BMD, the result of the mRNA test did not influence the ≥ CIN2 risk. In conclusion, mRNA testing by PreTect HPV-Proofer might be of value to select hrHPV DNA-positive women with normal cytology in need of immediate referral for colposcopy.

A comprehensive evaluation of the accuracy of cervical pre-cancer detection methods in a high-risk area in East Congo.

BACKGROUND: Given the high burden of cervical cancer in low-income settings, there is a need for a convenient and affordable method for detecting and treating pre-cancerous lesions. METHODS: Samples for comparing the accuracy of cytology, virology and histology were collected. Identification of HPV E6/E7 mRNA was performed using PreTect HPV-Proofer. HPV DNA detection was performed by GP5+/6+ PCR, followed by reverse line blot (RLB) for typing. RESULTS: A total of 343 women, aged 25-60 years, attending gynaecological polyclinics in DR Congo were included for sample enrolment. The test positivity rate was conventional and liquid-based cytology (LBC) at cutoff ASCUS+ of 6.9 and 6.6%, respectively; PreTect HPV-Proofer of 7.3%; and consensus DNA PCR for 14 HR types of 18.5%. Sixteen cases of CIN2+ lesions were identified. Of these, conventional cytology identified 66.7% with a specificity of 96.2%, LBC identified 73.3% with a specificity of 96.9%, all at cutoff ASCUS+. HR-HPV DNA detected all CIN2+ cases with a specificity of 85.9%, whereas PreTect HPV-Proofer gave a sensitivity of 81.3% and a specificity of 96.6%. CONCLUSION: Both HPV detection assays showed a higher sensitivity for CIN2+ than did cytological methods. Detecting E6/E7 mRNA from only a subset of HR HPVs, as is the case with PreTect HPV-Proofer, resulted in a similar specificity to cytology and a significantly higher specificity than consensus HR HPV DNA (P<0.0001).

Allelic imbalance at chromosome region 11q23 in cervical carcinomas.

The long arm of chromosome 11 has received much scrutiny as a high frequency of deletions of various sites has been observed in different tumour types, indicating the presence of putative tumour suppressor genes. In the present study, 81 primary cervical carcinomas were examined for allelic imbalance (AI) using nine microsatellite markers, mapping to the chromosomal region 11q23.1 where the ATM gene is located. AI at any locus in the region was found in 34 of 81 (42%) tumours. AI frequencies varied from 12 to 31% for the different markers used, with the highest frequency at marker D11S1294. Based on the findings of 17 cases with restricted areas of deletions, four chromosomal regions of possible importance in cervical carcinomas could be distinguished. The first region is located between the markers D11S1325 and D11S1819, the second region between D11S2179 and D11S1294, the third region between D11S1778 and D11S1818 and the fourth region between D11S1818 and D11S1347. The second region may thus contain part of the ATM gene. No association between AI of any marker and histopathological or clinical parameters was seen. When comparing the AI findings of the different loci with TP53 protein overexpression, the only significant association found was with D11S2179 located within the ATM gene. The results indicate that a tumour suppressor gene (or genes) on chromosome 11q.23.1 may be involved in carcinogenesis of the cervix and the involvement of the ATM gene remains a possibility.

Deregulation of D-type cyclins in uterine cancers. Cyclin D1/D3 is differentially expressed in cervical cancer.

BACKGROUND: We have previously found cyclin D1 to be overexpressed in 45% of corpus cancers, but in only 3% of cervical cancers. To see whether and how D-type cyclins contribute to the neoplastic phenotype in uterine cancers, aberrant expression and association between cyclins D1 and D3 proteins were studied. MATERIALS AND METHODS: Expression of cyclin D3 was investigated by immunohistochemistry in 51 patients with primary cancer of the corpus and 73 cases of primary cervical carcinomas. Amplification of the cyclin D1 and D3 genes was investigated by fluorescence in situ hybridisation (FISH). RESULTS: We found high/moderate levels of cyclin D3 in 53.5% of cervical cancers and 55% of corpus cancers. High/moderate expression of cyclin D1 and D3 was associated in the corpus cancers, but not in cervical cancers. The accumulation of cyclin D3 but not D1 protein could, in some cases, be explained by increased gene dosage since extra copies of chromosome 6 were found. CONCLUSION: This study demonstrates that in cervical cancers cyclin D3 may compensate for low levels of cyclin D1, whereas in corpus cancers both isoforms may contribute to the neoplastic phenotype.

Human papillomavirus E6/E7 mRNA expression in women younger than 30 years of age.

OBJECTIVE: The prevalence of human papillomavirus (HPV) is high in women younger than 30 years of age, most infections being transient. It is not clear, however, to what extent the E6/E7 transcripts are being expressed. This may be of prognostic importance. In this study, we have determined the prevalence of HPV DNA and mRNA in 283 women younger than 30 years of age. METHODS: E6/E7 transcripts from HPV types 16, 18, 31, 33 and 45 were detected using PreTect HPV-Proofer, while the presence of HPV DNA was detected using Gp5+/6+ consensus PCR and type-specific PCR. RESULTS: A total of 92 women (32.5%) were positive by consensus PCR, 59 (20.8%) were positive by type-specific PCR, while 41 (14.5%) were positive by PreTect HPV-Proofer. E6/E7 mRNA expression was detected in 38 (64.4%) of the 59 HPV type-specific DNA positive women. For HPV 16, E6/E7 mRNA expression was observed in 8 (32%) of the 25 DNA positive women. No high-grade lesions were observed in the concomitant cytology. CONCLUSIONS: Among young women having a normal Pap smear, a high HPV prevalence was found. Hence, use of consensus PCR will most probably give a low prognostic value for identifying subsequent severe dysplasia. The five HPV types 16, 18, 31, 33 and 45 accounted for the majority of infections with two out of three having a detectable E6/E7 mRNA expression. Yet, repeated type-specific testing for HPV mRNA may identify young women with a persistent transforming infection being at increased risk for severe dysplasia.

Expression of retinoblastoma tumor suppressor gene protein, epidermal growth factor receptor, and c-erbB-2 oncoprotein in primary vaginal carcinomas.

Forty-six primary vaginal carcinomas were examined immunohistochemically for expression of retinoblastoma (RB) protein, epidermal growth factor receptor (EGFR), and c-erbB-2 oncoprotein. The results demonstrated that RB protein was not lost in any of the cases, suggesting that structural abnormalities of the RB gene may not play an important role in the pathogenesis of vaginal carcinoma. Fifteen and 11% of the cases showed increased expression of EGFR and c-erbB-2 oncoprotein, respectively, indicating that these oncoproteins may be involved in the neoplastic process of a minority of vaginal carcinomas. Overexpression of EGFR and c-erbB-2 oncoprotein had no prognostic significance in vaginal carcinomas.

Arabinogalactan blockade of experimental metastases to liver by murine hepatoma.

Blocking of organ-specific experimental metastasis was investigated in a syngeneic tumor-host system using a new tumor which primarily colonizes the liver upon intravenous injection. The study included systemic treatment with D-galactose and arabinogalactan as well as cell pretreatment with arabinogalactan and two other glycoconjugates. Treatment with arabinogalactan reduced the amount of liver metastases and prolonged the survival times of the animals in both studies. Host treatment was more effective than tumor cell pretreatment. This could be an effect of arabinogalactan blockade of potential liver receptors by covering of galactose-specific binding sites.

Overexpression of p53 protein and c-erbB-2 protein in small cell carcinoma of the cervix uteri.

Twenty-eight primary small cell carcinomas of the cervix uteri were examined immunohistochemically for overexpression of p53 protein and c-erbB-2 protein. Twenty-one per cent of the cases showed positive immunostaining for p53 protein, whereas no staining was observed using the antibody against c-erbB-2. Our results indicate that altered expression of p53 protein may be involved in the development of small cell carcinoma of the cervix uteri.

Human papillomavirus oncogenic expression in the dysplastic portio; an investigation of biopsies from 190 cervical cones.

In this study, we investigated the presence of E6/E7 transcripts of seven common high-risk human papillomavirus (HPV) types in 190 cervical biopsies. The RNA-based real-time nucleic acid sequence-based amplification assay (NASBA) and type-specific PCR, both detecting HPV 16, 18, 31, 33, 45, 52, and 58, as well as consensus PCR, were performed on all 190 biopsies. High accordance between type-specific and consensus PCR confirms that the HPV types included in this study are the most common types present in cervical dysplasia. Furthermore, we see a clear increase in the incidence of HPV, both DNA and RNA, along with the histological severity of dysplasia. HPV RNA was detected in all but two PCR-positive cases, confirming that the virus exerts E6/E7 mRNA expression in cases of high-grade dysplasia. Out of 19 women given a normal or borderline diagnosis at conisation, only four were found HPV positive, which may suggest that unnecessary conisations can possibly be reduced by introducing HPV testing into the preoperative routine assessment.

Immunohistochemical analysis of p53 protein overexpression in normal, premalignant, and malignant tissues of the cervix uteri.

Two hundred and thirty-eight cervical lesions ranging from normal to malignant were examined for overexpression of p53 protein. Whereas p53 protein was identified in 62 per cent of invasive squamous cell carcinomas, 11 per cent of invasive adenocarcinomas, and 7 per cent of squamous cell carcinomas in situ, no staining was found in adenocarcinoma in situ, dysplastic tissue, condyloma, and normal tissue. In 9 per cent of the positive cases of invasive squamous cell carcinomas, 5-50 per cent of the tumour cells were immunoreactive for p53 protein, whereas the other positive specimens were characterized by only rare p53-positive cells. We conclude that in invasive cervical carcinomas widespread overexpression of p53 protein is unusual, but occasional positive nuclei can be found frequently. Furthermore, our results indicate that altered expression of p53 protein may be involved in the progression of cervical carcinomas.

Aberrant expression of the cell cycle associated proteins TP53, MDM2, p21, p27, cdk4, cyclin D1, RB, and EGFR in cervical carcinomas.

OBJECTIVE: The aims of this study were to study aberrant expression and coexpression of the cell cycle associated proteins TP53, p21, p27, cyclin D1, cdk4, RB, EGFR, and MDM2 in cervical carcinomas, to correlate protein alterations with histopathological and clinical parameters, and to evaluate whether these alterations provide prognostic information. METHODS: Seventy-four cervical carcinomas and 10 cases of normal cervical epithelium from patients with benign uterine leiomyomas were investigated immunohistochemically for aberrant expression of the cell cycle associated proteins using the biotin-streptavidin-peroxidase method and the OptiMax Plus automated cell staining system. RESULTS: In normal cervical epithelium p27 immunostaining was identified in more than 50% of the cells, cdk4 in 5-50% of the cells, and EGFR in less than 5% of the cells, whereas no immunostaining for TP53, p21, MDM2, or cyclin D1 was detected. Positive RB protein staining was identified in all cases of normal cervical epithelium. RB protein staining was also identified in all carcinomas of the cervix uteri. Overexpression of p21 was found in 96% of the tumors, MDM2 in 35%, cdk4 in 69%, cyclin D1 in 3%, and EGFR in 20% of the tumors. A low level of p27 was observed in 65% of the cases. In a previous study, the TP53 protein level has been found to be elevated in 41 of the 74 (55%) cases included in this work. Significant coexpression was seen for TP53 and MDM2 (P = 0. 001); concording results were observed in 67% of the cases. There was no difference in aberrant expression or coexpression of any of the cell cycle regulatory proteins related to histological type, grade of differentiation, FIGO stage, or relapse-free survival. CONCLUSION: The high number of cases showing increased levels of p21 and cdk4 and decreased levels of p27 suggests that these proteins may be important in the pathogenesis of cervical carcinoma. Furthermore aberrant expression of MDM2 in a smaller but significant fraction of cases indicates that these proteins could also be involved in the development of these cancers. Finally our results indicate that MDM2 may protect against HPV-induced TP53 protein degradation.

Expression of p53, MDM2, and p21 proteins in high-grade cervical intraepithelial neoplasia and relationship to human papillomavirus infection.

The relationship between human papillomavirus (HPV) infection and cell cycle regulators p53, MDM2, and p21 in cervical intraepithelial neoplasia (CIN) has not been investigated. p53, MDM2, and p21 immunoreactivity were analyzed with respect to HPV DNA status in high-grade CIN (CIN II-III). Formalin-fixed, paraffin-embedded tissue sections from 169 biopsy specimens with high-grade CIN were examined for HPV DNA by polymerase chain reaction with the L1 and E1 consensus primers Gp5+/6+ and CpI/CpIIG. HPV-positive specimens were typed with the E6 type-specific primers for HPV 16 and 18. The biopsy specimens were stained with the monoclonal antibodies p53, MDM2, and p21. The interpretation of nuclear staining was regarded as focal (< 5%), regional (5% to 50%), or diffuse (> 50%). HPV DNA was found in 156 cases (92%); 122 (78%) were positive for HPV 16, 18, or both. Immunohistochemically, p53 was detected in 50 specimens (30%); nuclear staining was mainly focal. Focal nuclear staining for MDM2 was found in 6 specimens (4%), and regional and diffuse nuclear staining for p21 was present in 137 (81%). Significant correlation was found only between p53 and MDM2 immunoreactivity. These results indicate that there is no correlation between HPV status and expression of the cell cycle regulators p53, MDM2, and p21. Inactivation of p21 and p53 protein may be important, and MDM2 abnormalities seem to play a minor role in the development of high-grade CIN.

Neuroendocrine dysdifferentiation and bombesin production in carcinogen-induced hepatocellular rat tumours.

Primary rat hepatocellular tumours, induced by a combination of diethylnitrosamine and 2-acetylaminofluorene, were examined for the presence of neuroendocrine peptides by immunocytochemical methods. Two-thirds of the tumours showed positive immunostaining for either neuron-specific enolase (NSE), protein S-100 or bombesin. NSE was commonly observed both in hepatocarcinomas and in neoplastic nodules, whereas protein S-100 was more frequently seen in carcinomas (49% positive) than in nodules (13% positive). Bombesin, previously shown to function as an autocrine growth factor in small-cell carcinoma of the lung, was present in neurosecretory granules in 13% of the nodules and 29% of the carcinomas. Normal, preneoplastic and peritumorous liver tissue, including the frequent atypical foci present in the latter two categories, was uniformly negative for all neuroendocrine markers. The foci, like the nodules and carcinomas, generally stained positively for the liver tumour marker glutathione S-transferase type P (GSTP). The results suggest that dysdifferentiation of altered hepatocytes in a neuroendocrine direction may be a common, late event in liver carcinogenesis which could possibly contribute to tumour formation, e.g. by establishing autocrine or paracrine circuits.

TP53 and p21WAF1/CIP1 behave differently in euploid versus aneuploid bladder tumours treated with radiotherapy.

The aim of this study was to examine any relation between DNA ploidy and previously detected TP53 (p53) or p21WAF1/CIP1 expression in 94 patients with muscle-invasive transitional cell carcinoma of the urinary bladder and to associate these factors with survival. DNA ploidy was determined by image cytometry. In a subgroup of patients, the mutational status of the TP53 gene was assessed by temporal temperature gradient electrophoresis (TTGE) or perpendicular denaturant gradient gel electrophoresis (DGGE) and subsequent sequencing. Significantly more aneuploid than euploid tumours showed TP53 accumulation (p = 0.003). Patients with aneuploid tumours lived longer than patients with euploid tumours (p = 0.003). In the euploid, but not in the aneuploid group, TP53 and p21WAF1/CIP1 were associated with cancer-specific survival (p = 0.002 and 0.02, respectively). Patients with > 50% TP53 expression had the longest survival time. Mutation analyses showed acceptable concordance with TP53 expression. We conclude that DNA aneuploidy may confer increased radiosensitivity in bladder cancer patients and that TP53 accumulation may confer increased radiosensitivity, but its effect is detectable only in euploid tumours.

Reduced expression of p21WAF1 is an indicator of malignant behaviour in anal carcinomas.

AIMS: p21 and p27 protein expression were examined in a comparatively large series of patients with squamous cell carcinoma of the anal canal and compared with clinical and histopathological data (tumour stage, nodal status and differentiation). METHODS AND RESULTS: We analysed the expression of p21 and p27 protein in 94 anal carcinomas by immunohistochemistry. Nuclear p21 and p27 staining were detected in 71% (67/94) and 75% (71/94) of the cases, respectively. There was no significant association between p27 staining and tumour stage, nodal status or overall survival. We observed that negative p21 immunoreactivity was significantly associated with poorly differentiated anal carcinomas. Furthermore, a shorter overall survival for patients with no p21 protein expression was seen. CONCLUSIONS: Our data indicate that p21 levels, but not p27 expression, may be a useful predictor of survival in patients with anal carcinomas.

TP53 gene mutations and protein accumulation in primary vaginal carcinomas.

Primary carcinomas from 46 patients were screened for TP53 alterations. Immunohistochemistry demonstrated nuclear TP53 protein accumulation in 22 (48%) cases using the polyclonal CM1 antiserum, whereas 15 (33%) cases showed positive nuclear staining with the mononuclear antibody PAb 1801. Constant denaturant gel electrophoresis (CDGE) was used to screen 27 of the vaginal carcinomas for mutations in the conserved regions of the TP53 gene (exons 5-8). Six of these tumours (22%) contained mutations: four were found in exon 5 and two in exon 8. A total of 50% of the primary vaginal carcinomas carried a TP53 alteration. These results indicate that TP53 abnormalities may be involved in the development of these tumours. However, there was no significant association between TP53 abnormalities and survival.

TP53 alterations in relation to the cell cycle-associated proteins p21, cyclin D1, CDK4, RB, MDM2, and EGFR in cancers of the uterine corpus.

In the present study, TP53 alterations have been analysed and compared with the expression of the proteins p21, cyclin D1, cdk4, RB, EGFR, and MDM2 in 53 cancers of the uterine corpus. TP53 gene mutations analysed by CDGE/DGGE and direct sequencing showed a TP53 gene mutation in 18 per cent of the cases. TP53 gene mutations were not significantly related to overexpression or down-regulation of any of the proteins. Immunohistochemically, there was an increased protein level of TP53 in 77 per cent, p21 in 36 per cent, cyclin D1 in 45 per cent, cdk4 in 77 per cent, EGFR in 8 per cent, and MDM2 in 32 per cent of the cases. Expression of RB protein was normal in all cancers. Significant association of protein expression was seen between TP53 and MDM2 (p=0.005) and p21 and MDM2 (p=0.001). Furthermore, there may be an association between TP53 and p21 (p=0. 038) and cyclin D1 and cdk4 (p=0.045). The results revealed increased levels of TP53 protein in all MDM2-positive cases that did not show TP53 mutations, indicating TP53 protein stabilization and inactivation by complex formation with MDM2. In summary, the high number of cases showing an increased level of TP53 and cdk4 proteins suggests that these proteins play an important role in the neoplastic process in cancers of the uterine corpus.

TP53 protein accumulation and gene mutation in relation to overexpression of MDM2 protein in ovarian borderline tumours and stage I carcinomas.

Three hundred and seventy-four early-stage ovarian tumours, including 27 borderline tumours and 347 stage I carcinomas, were investigated immunohistochemically for overexpression of the TP53 and MDM2 proteins. TP53 (p53) and MDM2 alterations were detected in 15 and 4 per cent of borderline tumours, and in 50 and 13 per cent of stage I carcinomas, respectively. Mutations in the TP53 gene (exons 5-8) were demonstrated in 29 of the 50 stage I carcinomas studied, using denaturing gel electrophoresis followed by direct sequencing. TP53 overexpression was seen less often in tumours of mucinous and endometrioid type than in tumours of other histological types and more often in moderately and poorly differentiated than in well differentiated tumours. MDM2 protein overexpression was seen more often in clear cell carcinoma than in tumours of other histological types. These results indicate that TP53 abnormalities play a crucial role, and MDM2 abnormalities a minor role, in the development of early-stage ovarian carcinoma. There was no significant association between TP53 or MDM2 alterations and survival in multivariate analysis.

TP53 mutations in early-stage ovarian carcinoma, relation to long-term survival.

We conducted the present study to evaluate the frequency and prognostic importance on long-term survival of TP53 mutations and TP53 protein accumulation in a cohort of 178 patients with early-stage ovarian carcinomas. TP53 mutations scored as aberrant temporal temperature gradient gel electrophoresis pattern from all exons were observed in 39.9% of the tumours. Full screening of exons 5-8, followed by sequencing, was successful in 135 cases, and 48 mutations altering the protein were detected in 39 cases (28.9%). TP53 mutations were slightly less common in the Federation of Gynecologists and Obstetricians stage IA than in IB/IC (P=0.05). No significant correlations with histological type, grade of differentiation, DNA ploidy status or age at diagnosis were found. TP53 protein accumulation analysed by immunohistochemistry was found in 32.6% of all tumours, and was a poor predictor of TP53 mutations with 56.4% sensitivity, 77.1% specificity, 50% positive predictive value and 81.3% negative predictive value. Neither TP53 mutations nor TP53 protein accumulation influenced the prognosis significantly in this group of patients.