Predicting high-risk human papillomavirus infection, progression of cervical intraepithelial neoplasia, and prognosis of cervical cancer with a panel of 13 biomarkers tested in multivariate modeling.

Comprehensive multivariate models were used to disclose whether any of our previously analyzed 13 markers would be independent predictors of intermediate end point markers in cervical carcinogenesis. The expression of the following biomarkers, E-cadherin, extracellular signal-regulated kinase 1, 67-kd laminin receptor (LR67), matrix metalloproteinase 2, tissue inhibitor of metalloproteinase 2, nuclear factor-kappaB, nm23-H1, p16, proliferating cell nuclear antigen, survivin, human telomerase reverse transcriptase, topoisomerase 2alpha, and vascular endothelial growth factor (VEGF) C in 150 cervical cancer (CC) and 152 cervical intraepithelial neoplasia (CIN) lesions were determined immunohistochemically. Multivariate models were constructed to test predictive power of the markers for 3 outcomes: (1) high-grade CIN, (2) high-risk human papillomavirus (HR-HPV), and (3) CC survival. Performance indicators were calculated and compared by the areas under receiver operating characteristic (ROC) curve. Three marker panels were identified consisting of 5 independent predictors of CIN2 (E-cadherin, extracellular signal-regulated kinase 1, LR67, topoisomerase 2alpha, and VEGF-C), 3 predictors of HR-HPV (survivin, p16, and human telomerase reverse transcriptase), and 2 predictors of CC survival (nm23-H1 and tissue inhibitor of metalloproteinase 2). In predicting CIN2, the best balance between sensitivity (SE) and specificity (SP) was obtained by combining the 2 most powerful predictors in panel 1 (VEGF-C and LR67), giving the area under ROC curve, 0.897 (95% confidence interval [CI], 0.847-0.947); odds ratio, 86.27 (95% CI, 19.71-377.47); SE, 86.0%; SP, 93.3%; positive predictive value (PPV), 99.1%; and negative predictive value (NPV), 43.1%. In a hypothetical screening setting (10,000 women; CIN2 prevalence, 1%), this marker combination should theoretically detect CIN2 with 86.0% SE, 100% SP, 99.1% PPV, and 99.6% NPV, area under ROC curve of 0.930 (95% CI, 0.909-0.951), and odds ratio, 29998.0 (95% CI, 7,879.0-37,338.0). Combining 2 markers (LR67 and VEGF-C) enables accurate detection of high-grade CIN in a clinical setting. However, testing the performance of this marker combination in a screening setting necessitates their analysis in cytological samples.

Over-expression of topoisomerase IIalpha is related to the grade of cervical intraepithelial neoplasia (CIN) and high-risk human papillomavirus (HPV), but does not predict prognosis in cervical cancer or HPV clearance after cone treatment.

OBJECTIVE: One of the pathways leading to cervical cancer is a loss of normal cell cycle control. Topoisomerase IIalpha and IIbeta are important nuclear proteins controlling the G2/M checkpoint, and shown to be over-expressed in many human cancers. Their links to oncogenic human papillomavirus (HPV) types and their prognostic value in cervical cancer are practically unexplored. MATERIAL AND METHODS: As part of our HPV-PathogenISS study, a series of 150 squamous cell carcinomas (SCC) and 152 CIN lesions were examined using immunohistochemical (IHC) staining for topoisomerase IIalpha (topo IIalpha), and tested for HPV using PCR with three primer sets (MY09/11, GP5/GP6, SPF). Follow-up data were available from all SCC patients, and 67 CIN lesions had been monitored with serial PCR for HPV clearance/persistence after cone treatment. RESULTS: Topo IIalpha expression increased with increasing grade of CIN (p = 0.0001), with the most dramatic up-regulation upon progression from CIN2 to CIN3 and peaking in SCC (OR 16.23; 95%CI 7.89-33.38). Topo IIalpha up-regulation was also significantly associated with HR-HPV detection in univariate analysis (OR = 3.07; 95%CI 1.70-5.52), but was confounded by the histological grade (Mantel-Haenszel common OR = 1.622; 95%CI 0.782-3.365), and by entering both p16(INK4a) (9) and Survivin (33) in the multivariate regression model. Topo IIalpha did not predict clearance/persistence of HR-HPV after treatment of CIN, and it was not a prognostic factor in cervical cancer in either univariate or multivariate analysis. CONCLUSIONS: Over-expression of topo IIalpha is significantly associated with progression from CIN2 to CIN3, being a late marker of cell proliferation. Its close association with HR-HPV is plausibly explained by the fact that E7 oncoproteins of these HR-HPV (but not LR-HPV) block the normal pRb-mediated inhibition of topo IIalpha by degrading the wild-type Rb.

Entire genome of a strain of human immunodeficiency virus type 1 with a deletion of nef that was recovered 20 years after primary infection: large pool of proviruses with deletions of env.

We report the complete sequence analysis of the provirus harbored in a long-term nonprogressor (patient SG1) 20 years after the first infection with a human immunodeficiency virus type 1 strain lacking nef. The sequencing showed large deletions in the nef-nef and nef-U3 regions. Except for vpu, all of the other accessory genes were intact. The gag and pol genes did not show significant alterations. We found large deletions in env, spanning the V1, V2, V3, V4, and V5 regions. We believe that, when down-regulation of the class 1 major histocompatibility complex molecules is inhibited by the lack of nef function, the cells containing Env-defective molecules evade cytotoxic T lymphocyte killing and accumulate progressively.

Detection and expression of human papillomavirus oncogenes in non-small cell lung cancer.

Human papillomavirus (HPV) has been found in lung cancer cases with variable frequency. In the present study, we analysed a series of 38 patients with non-small cell lung cancer (NSCLC) (21 paraffin-embedded archival samples and 17 fresh surgical specimens) for the presence of E6 and E7 oncogenes of HPV16, 18 and 31. Eight of the tumours were positive (21%): six HPV16, one HPV16+18, and one HPV31. The normal tissue surrounding the HPV-positive tumour was negative for the presence of the virus. Sequencing analysis of URR, of HPV16, which was the most frequently found HPV type in our cases, showed an adenosine deletion at nucleotide 7861 (E2-binding site) in four out of six patients. Sequencing of the entire E6 and E7 genes of HPV16 showed a T to G transition at nucleotide position 350 of E6, in all examined cases. This mutation is associated to the European variant of HPV16. Analysis of E6 and E7 transcripts was performed on the six fresh surgical specimens infected by HPV16. Our study showed that all of the tumours investigated, except one, contained E6 and E7 transcripts. Only in one case could we identify an unspliced form of the E6 transcript. Our results strengthen the relationship between HPV and NSCLC and support the hypothesis that HPV infection could play a role in bronchial carcinogenesis.

Relationship of up-regulation of 67-kd laminin receptor to grade of cervical intraepithelial neoplasia and to high-risk HPV types and prognosis in cervical cancer.

OBJECTIVE: To evaluate the 67-kd laminin receptor (67LR) in cervical cancer and its molecular links to oncogenic HPV types. STUDY DESIGN: As part of the HPV-PathogenlSS Study, a series of 150 squamous cell carcinomas (SCCs) and 152 carcinoma in situ (CIN) lesions were examined using immunohistochemical staining for LR67 and tested for HPV using polymerase chain reaction (PCR) with 3 primer sets (MY09/11, GP5+/GP6+, SPF). Followup data were available for all SCC patients, and 67 CIN lesions had been monitored with serial PCR for HPV clearance/persistence after cone treatment. RESULTS: 67LR expression increased in parallel with increasing grade of CIN (p = 0. 0001), with the most dramatic up-regulation upon the transition from CIN 2 to CIN 3 and further to SCC. This increased expression was associated with CIN 3/cancer at OR 17.04 (95% CI 7.28-39.87). The seemingly significant association of 67LR with high-risk HPV (HR-HPV) detection (OR 2.20, 95% CI 1.27-3.80) was due to confounding by the histologic grade (Mantel-Haenszel common OR = 1.118, 95% CI 0.576-2.168). Using performance indicators, 67LR expression was of little value as a marker of HR-HPV type, and it did not predict clearance/persistence of HR-HPV after treatment of CIN. Similarly, 67LR expression was not an independent prognostic factor in cervical cancer. CONCLUSION: In cervical carcinogenesis, both integrin- and nonintegrin-type LRs (67LR) probably have functions complementary to each other, mediating transient early and stable adhesions, respectively. Up-regulated 67LR expression is significantly associated with progression from CIN 2 to CIN 3 as a marker of cell proliferation. 67LR is probably orchestrated by mechanisms independent of HR-HPV oncoproteins, which seem to be more closely associated with integrin-type laminin receptors.

Single or multiple HPV types in cervical cancer and associated metastases.

Almost all cervical cancers are human papillomavirus (HPV)-positive. Some aspects of HPV carcinogenesis, such as factors involved in the transformation process and the mono- or polyclonal origin of the carcinogenic process, need to be defined. The latter aspect is addressed in our study. Cervical samples were collected from 102 patients with squamous cell carcinoma. The HPV positivity was established by PCR analysis performed using consensus and specific primers for the L1 and E6/E7 regions, respectively. Eighty-seven samples were positive for the L1 gene and 5 for the E6/E7 genes. Overall, 92 samples contained segments of HPV-DNA (90.2%). HPV-16 was most frequently found either alone or associated with other genotypes (63%). All genotypes identified as a single infection, except HPV-73, belonged to the high-risk HPV group. Among multiple infections, the HPV-31+54 couple was the most frequent. The presence of two genotypes in a primary tumor raises the question of their distribution in a single tumor cell. We attempted to answer this question by comparing the HPV patterns in primary tumors and metastases, considering that metastases derive from cell clones released from the primary tumor. The HPV patterns of primary tumors and metastases overlapped in most patients, even when primary tumors contained a double genotype, thus suggesting that single tumor cells may contain multiple HPV genotypes.

Survivin as a marker of cervical intraepithelial neoplasia and high-risk human papillomavirus and a predictor of virus clearance and prognosis in cervical cancer.

We analyzed survivin as a marker of cervical intraepithelial neoplasia (CIN) and high-risk human papillomavirus (HR-HPV) and a predictor of HPV clearance and disease outcome in cervical cancer in 302 samples (squamous cell carcinomas [SCCs], 150; CIN lesions, 152) by immunohistochemical staining with survivin antibody and HPV testing using polymerase chain reaction. HR-HPV types were associated closely with CIN and SCC. There was a significant linear relationship between grade and intensity of survivin expression (P = .0001). Survivin overexpression also was associated strongly with HR-HPV type (P = .0001). Multivariate regression analysis revealed survivin and p16(INK4a) as equally strong independent predictors of HR-HPV. Deregulated survivin expression did not predict clearance or persistence of HR-HPV after treatment of CIN or survival in cervical cancer in univariate (P = .417) or multivariate analysis. After adjustment for HR-HPV, stage, age, and tumor grade in the Cox regression model, only stage (P = .0001) and age (P = .0001) remained independent prognostic predictors. Survivin seems to be an early marker of cervical carcinogenesis. Up-regulated survivin expression was an independent predictor of HR-HPV in cervical lesions, most plausibly explained by its normal transcriptional repression by wild-type p53 being eliminated by HR-HPV E6 oncoprotein.

Sepharose-bound, highly sulfated glycosaminoglycans can capture HIV-1 from culture medium.

In the search for new strategies against HIV-1 and on the basis of a number of previous studies reporting on the capacity of certain polyanionic compounds to influence the replication of HIV-1, we prepared a few chemically oversulfated dermatan and chondroitin sulfates. Four of these compounds and two samples of heparin were bound to activated Sepharose through either their carboxylic groups, or their aldehydic groups, or their deacetylated primary amino groups. Some of these so-derivatised resins, packed into columns, proved able to remove HIV-1 IIIB, a laboratory adapted strain, and one clinical primary isolate from an AIDS patient, from infected cell culture medium. The resins bind the virus very tightly and could be useful for capturing the virus from infected fluids.

Activation of the ERK/MAP kinase pathway in cervical intraepithelial neoplasia is related to grade of the lesion but not to high-risk human papillomavirus, virus clearance, or prognosis in cervical cancer.

We subjected 302 archival samples (150 squamous cell carcinomas [SCCs] and 152 cervical intraepithelial neoplasia [CIN] lesions) to immunohistochemical staining with extracellular signal-regulated kinase-1 (ERK1) antibody and human papillomavirus (HPV) testing with 3 primer sets. Follow-up data were available for all SCC cases and 67 CIN cases. High-risk (HR) HPV types were associated with CIN (odds ratio [OR], 19.12; 95% confidence interval [CI], 2.31-157.81) and SCC (OR, 27.25; 95% CI, 3.28226.09). There was a significant linear relationship between lesion grade and ERK1 staining intensity (P = .0001). ERK1 staining was a 100% specific indicator of CIN, with a 100% positive predictive value, but a poor predictor of HR HPV. ERK1 expression did not predict clearance or persistence of HR HPV after CIN treatment. ERK1 staining did not significantly predict survival in cervical cancer in univariate (P = .915) or multivariate analysis. After adjustment for HR HPV, stage, age, and tumor grade in the Cox regression model, only stage (P = .0001) and age (P = .002) remained independent prognostic factors. ERK1 expression seems to be an early marker of cervical carcinogenesis. ERK1 overexpression is not a specific marker of HR-HPV in CIN and cervical cancer, nor does it predict virus clearance after CIN treatment or disease outcome in cervical cancer.

Susceptibility to highly sulphated glycosaminoglycans of human immunodeficiency virus type 1 replication in peripheral blood lymphocytes and monocyte-derived macrophages cell cultures.

In the search for new drugs against human immunodeficiency virus type 1 (HIV-1), the replication of III(B) and BaL strains, and of seven primary isolates from AIDS patients, cultured both in peripheral blood lymphocytes (PBLs) and in monocyte-derived macrophages (MACs), was investigated in the presence of two dermatan sulphate and heparin at 10 microg/ml. The three polysaccharides effectively inhibited the replication of III(B) in PBLs and of BaL in MACs, while producing either a slight inhibition or an unexpected large increase in the replication of the seven primary isolates, especially in MAC cultures. In one case, stimulation was found in PBLs and, at lower doses, also with BaL in MACs. Co-receptor use, adaptation to C8166 T cell line, partial sequence of the gp120 V3 loop, variation in positive charge distribution and number of potential glycosylation sites along the V3 loop were assessed for each strain. No explanation could be found for the different susceptibility of the viruses to the polysaccharides. Their presence probably brings about both inhibitory and stimulatory effects, the final outcome depending on the virus, cells and polysaccharide.