Lorenzo Pacenti

The effects of HIV-1 Tat protein on cell cycle during cervical carcinogenesis

The role of HPV in the carcinogenesis of intraepithelial and invasive anogenital lesions is currently well established. E6 and E7 oncoproteins of high risk HPV genotypes are known to inactivate p53 and pRb pathways. Several studies have described an increased prevalence and recurrence of both cervical HPV infection and invasive cervical cancer among HIV-1 positive women compared to HIV-1 negative cases. For these reasons, cervical cancer is considered an AIDS-defining neoplasm. Unlike other AIDS-associated neoplasms, the occurrence of cervical cancer is independent of immune suppression. HIV-1 infection in patients with high grade precancerous lesions and cervical cancers results in a therapy refractory and more aggressive disease phenotype, which is not yet well understood at the molecular level. An upregulation of HPV E6 and E7 gene expressions by HIV-1 proteins such as Tat has been documented by some authors. However, the role of HIV-1 in cervical carcinomas is still unclear. It is already known that HIV-1 Tat protein is able to influence cell cycle progression. Altogether, these facts led us to investigate the effects of Tat on the expression of cell cycle regulator genes. After transfection of HeLa cells with Tat, we analyzed the expression of cell cycle regulators from these cells by IHC and Real-time PCR. A significant reduction in the expression of cell cycle inhibitors of transcription and an increase in the levels of proliferation markers were observed. These results suggest that HIV-1 may enhance cervical carcinogenesis by promoting cell cycle progression. We also found that this HIV-1 Tat-induced cell proliferation was not dependent on the E2F family of transcription factors, and therefore postulate that Sp factors may be involved.

Different effects of interferon-alpha on melanoma cell lines: a study on telomerase reverse transcriptase, telomerase activity and apoptosis

Summary Background Although the antiproliferative and proapoptotic effects of interferon (IFN)‐α are widely recognized, its antitumour mechanisms are not completely known. Recent studies indicate that the derepressed expression of the catalytic subunit of telomerase, human telomerase reverse transcriptase (hTERT), and telomerase activity (TA) are involved in the process of human carcinogenesis. Only a few studies have investigated the effects of IFN‐α on hTERT and TA, with controversial results.

Detection of telomerase activity and correlation with mitotic and apoptotic indices, Ki‐67 and expression of cyclins D1 and A in cutaneous melanoma

Telomerase plays a key role in carcinogenesis. It is activated in most immortal cell lines and human cancers, including cutaneous melanoma (CM). Increased cell proliferation and deregulation of the cell cycle occur in human cancers. Links between telomerase activity (TA), cell proliferation, cell death and expression of cell‐cycle regulators have not been extensively elucidated in CM. In this study, we investigated TA, mitotic index (MI), apoptotic index (AI), Ki‐67 and nuclear positivity of cyclins D1 and A (Ki‐67+N/1,000, cyclin D1+N/1,000, cyclin A+N/1,000) in 42 primary cutaneous melanomas (PCMs). TA was detected in all cases and directly correlated with MI, Ki‐67+N/1,000, cyclin D1+N/1,000 and cyclin A+N/1,000 (p < 0.001); it was not correlated with AI. When subdividing PCMs into radial and vertical growth phase melanomas (RGPMs, VGPMs), a correlation was maintained only with MI (p < 0.005) and cyclin D1+N/1,000 (p < 0.005). Although MI and Ki‐67+N/1,000 were highly correlated with cyclin D1+N/1,000 and cyclin A+N/1,000 (p < 0.001) when considering all cases together, a high correlation was found in the RGPM and VGPM groups between cyclin A+N/1,000 and Ki‐67+N/1,000 only (p < 0.001), thus suggesting that cyclin A is more closely correlated with cell proliferation than cyclin D1. Our results further support the association between TA, tumor cell proliferation and cyclin D1 and A expression in PCM, though it is possible that links between TA and proliferation, on the one hand, and TA and cyclin D1 expression, on the other, might occur following various pathways. Int. J. Cancer 88:411–416, 2000.

Cutaneous leishmaniasis: usefulness of PCR on paraffin-embedded skin biopsies as part of routine investigation

Human cutaneous leishmaniasis (CL) caused by Leishmania infantum is an endemic disease in Italy (Gramiccia, 2003). Its true incidence is probably under-estimated because the clinical pictures are extremely variable, the lesions can self-cure, and the usual method of diagnosis — the microscopical demonstration of amastigotes in skin biopsies — is not very sensitive (Faber et al., 2003). Although PCR-based assays can increase the sensitivity of CL diagnosis (Motazediam et al., 2002; Safaei et al., 2002; Muller et al., 2003; Cordoba Lanus et al., 2005), in Italy such assays are still largely confined to reference centres and research applications. The aim of the present, retrospective study, which was based on archived, paraffin-embedded skin biopsies, was to assess the potential value of PCR in the routine diagnosis of CL.

Telomerase Activity, Ki-67, Cyclin D1 and A Expression, and Apoptosis in Solitary Fibrous Tumors:Additional Features of a Predictable Course?

Solitary fibrous tumors (SFTs) are infrequent soft tissue neoplasms which are usually benign and surgically curable. However, their behavior is not always predictable, although several clinical and pathological criteria of malignancy have been established. In many cancers, including some soft tissue tumors, telomerase activity (TA) has been shown to be a new reliable pathological marker of malignancy. Overexpression of some cyclins is associated with higher degrees of malignancy and predictive of the clinical course. In this study, we evaluated TA, mitotic and apoptotic indices (MI, AI), and the expression of Ki-67, cyclins D1 and A in five typical and two clinicopathologically atypical SFTs, the latter two of which had also recurred. High TA was demonstrated in the two atypical cases, which also showed a higher labeling index to Ki-67, as well as higher cyclin D1 and A expression, and either none or very few apoptoses. We suggest that TA, Ki-67, cyclin expression, and AI be evaluated in SFTs as possible adjunctive pathological criteria of behavior.

Epstein-Barr virus infection in sinonasal non-Hodgkin's lymphomas

Sinonasal non-Hodgkins lymphomas (SNHLs) of B- or T-cell immunophenotype have been associated with Epstein-Barr virus (EBV) infection of neoplastic lymphoid tissue. Nine SNHLs were investigated using immunohistochemistry, the polymerase chain reaction (PCR) for EBV genome and in situ hybridization (ISH) for EBV encoded RNAs (EBER), immunoglobulin (CI-gHR) and clonal T-cell receptor (CTCβR) gene rearrangements. Eight cases were diagnosed as peripheral pleomorphic T-cell lymphomas (pPTCL). PCR showed the presence of EBV genome in eight cases; ISH for EBER led to the detection of positive cells in five cases. Late membrane protein (LMP) immunostaining was observed in three cases. No EBV positivity has been detected in control cases. The frequent association with EBV infection in the cases illustrated confirms the previous suggestions that EBV may have a role in the genesis of lymphomas of the sinonasal region.

Possible diagnostic role of telomerase activity evaluation in the differential diagnosis between spitz naevi and cutaneous malignant melanoma.

q 2000 British Association of Dermatologists, British Journal of Dermatology, 142, 1047±1070 in at the end of a therapy cycle by the physician. Its front page contains the patients name, date of birth and residence. On the following pages, the type of UV treatment, duration, cumulative doses and side-effects are recorded. On the back of the passport, the importance of this document is explained to the patient as well as the reason why this document must be presented to the treating physician before and after a UV therapy cycle. The advantages of this UV passport are the following: 1 Conservation of information about UV therapy can be transferred between treating physicians; at present, the majority of patients cannot precisely indicate previous UV treatments. 2 Facilitation of therapeutic decisions; the choice of the optimal mode of therapy requires exact information about type and cumulative doses of previous UV treatments. For example, when a patient has received PUVA therapy with a high cumulative dose, there is a relative contra-indication to continuing this form of irradiation or introducing another immunosuppressive therapy like cyclosporin because of the mutagenic potential of PUVA treatment. 3 Control of therapy; the regular calculation and recording of cumulative UV dose allows the treating physician to supervise the treatment with regard to treatment efficacy and to adapt the therapeutic strategy. 4 Collecting data about long-term side-effects; very little is known about long-term risks of modern methods like topical PUVA, UVB 311 nm or UVA1 therapy. If the UV passport finds wide application, it will be possible to investigate prospectively the cumulative doses, the risks of long-term treatment and the interval between irradiation and appearance of long-term side-effects. 5 It documents the concern of dermatologists for accurate records of UV therapy. In conclusion, the advantages of a UV passport for continuous documentation of type, dose and side-effects of therapeutically applied UV irradiation in patients with chronic or relapsing dermatoses are apparent. In Germany, we have therefore developed and introduced such a document where significant data regarding UV treatment can be recorded.

Correlation of EGFR, pEGFR and p16INK4 expressions and high risk HPV infection in HIV/AIDS-related squamous cell carcinoma of conjunctiva

BackgroundSquamous cell carcinoma of conjunctiva has increased tenfold in the era of HIV/AIDS. The disease pattern has also changed in Africa, affecting young persons, with peak age-specific incidence of 30-39 years, similar to that of Kaposi sarcoma, a well known HIV/AIDS defining neoplasm. In addition, the disease has assumed more aggressive clinical course. The contributing role of exposure to high risk HPV in the development of SCCC is still emerging.ObjectiveThe present study aimed to investigate if immunohistochemical expressions of EGFR, pEGFR and p16, could predict infection with high risk HPV in HIV-related SCCC.MethodsFFPE tissue blocks of fifty-eight cases diagnosed on hematoxylin and eosin with SCCC between 2005-2011, and subsequently confirmed from medical records to be HIV positive at the department of human pathology, UoN/KNH, were used for the study. Immunohistochemistry was performed to assess the expressions of p16INK4A, EGFR and pEGFR. This was followed with semi-nested PCR based detection and sequencing of HPV genotypes. The sequences were compared with the GenBank database, and data analyzed for significant statistical correlations using SPSS 16.0. Ethical approval to conduct the study was obtained from KNH-ERC.ResultsOut of the fifty-eight cases of SCCC analyzed, twenty-nine (50%) had well differentiated (grade 1), twenty one (36.2%) moderately differentiated (grade 2) while eight (13.8%) had poorly differentiated (grade 3) tumours. Immunohistochemistry assay was done in all the fifty eight studied cases, of which thirty nine cases (67.2%) were positive for p16INK4A staining, forty eight cases (82.8%) for EGFR and fifty one cases (87.9%) showed positivity for p-EGFR. HPV DNA was detected in 4 out of 40 SCCC cases (10%) in which PCR was performed, with HPV16 being the only HPV sub-type detected. Significant statistical association was found between HPV detection and p16INK4 (p=0.000, at 99% C.I) and EGFR (p=0.028, at 95% C.I) expressions, but not pEGFR. In addition, the expressions of these biomarkers did not show any significant association with tumor grades.ConclusionThis study points to an association of high risk HPV with over expressions of p16INK4A and EGFR proteins in AIDS-associated SCCC.

Distinction between diffuse cutaneous malignant follicular center cell lymphoma and lymphoid hyperplasia by computerized nuclear image analysis

The difficult differential diagnosis between the diffuse variants of cutaneous lymphoid hyperplasia (CLH; synonym: pseudolymphoma) and malignant follicular center cell lymphomas (FCCL) often requires a multidisciplinary approach. Eighteen CLH and 11 FCCL, diagnosed by conventional histology and immunophenotyping and subsequently examined with a polymerase chain reaction to show clonal immunoglobulin heavy-chain gene rearrangements, were subjected to a novel type of automated nuclear image analysis. Of all nuclear parameters tested in azure A-stained semithin sections, the mean nuclear profile area (TN) of lymphoid cells was the best criterion to distinguish between CLH and FCCL (p = 9 x 10 6). Additional distinctive features, in the order of decreasing significance, were the SD of TN; all chromatin textural parameters combined; and the light and the dark fractions of the central nuclear profile areas. Parameters related to the chromatin pattern were independent of nuclear profile size in FCCL, but not in CLH. Two lesions registered as CLH displayed the nuclear characteristics favoring this diagnosis, but showed B-cell monoclonality at the DNA level. In conclusion, computerized nuclear image analysis is a helpful additional diagnostic tool in the evaluation of diffuse CLH and cutaneous FCCL.