Lidia Virginia Alonio

Human papillomavirus (HPV) DNA in penile carcinomas in Argentina: Analysis of primary tumors and lymph nodes

Among sexually transmitted diseases, infection by human papillomavirus (HPV) has become one of the most important. On the other hand, though epidemiological data show that some HPV types are closely associated with cervical cancer, few reports have been found with reference to penile carcinoma because of its rare occurrence. The aim of this study was to investigate the relationship between HPV infection and penile cancer in Argentina. A retrospective study was carried out on 38 white men with penile squamous‐cell carcinoma. Sixty‐five archival fixed biopsies taken from 34 primary penile tumors, 25 nodal metastases, 1 skin “satellite” metastasis and 5 histologically normal lymph nodes were used as specimens. HPV detection and typing were carried out by the polymerase chain reaction (PCR) using generic primers, combined with single‐stranded conformational polymorphism (SSCP) analysis. HPV DNA was found in 71% patients, corresponding 81% of them to “high risk” types, with predominance of HPV 18. Both primary tumors and metastases showed concordance of HPV occurrence and type in both lesions. In 3 patients, HPV 16 was detected not only in primary tumors and metastases, but also in histologically normal lymph nodes. Our data indicate that most penile carcinomas in Argentine patients are etiologically related to HPV, especially to “high risk” genital types. The agreement in HPV detection between primary tumors and metastases suggests a potential viral role in tumor progression. HPV detection in otherwise histologically normal lymph nodes might be useful as early marker of a metastatic process. J. Med. Virol. 61:65–69, 2000.

Physical status of the E2 human papilloma virus 16 viral gene in cervical preneoplastic and neoplastic lesions

BACKGROUND Integration of human papilloma virus (HPV) 16 DNA is considered an important genetic change in cervical lesion progression towards ICC. The viral E2 gene is often disrupted by this process, releasing suppression of viral E6/E7 oncogenes, a key factor for oncogenic progression. OBJECTIVES To evaluate the physical status of HPV 16 E2 gene in cervical preneoplastic and neoplastic lesions and its relation with lesion severity. STUDY DESIGN A sensitive PCR approach for the detection of an intact E2 HPV 16 gene in infected epithelial cells from the cervix with low grade squamous intraepithelial lesion (LGSIL), high grade squamous intraepithelial lesion (HGSIL) and invasive cervical carcinoma (ICC) diagnosis was applied. The correlation between gene disruption and lesion stage was examined. RESULTS Sixty-two LGSIL, 39 HGSIL and 24 ICC samples were analyzed. Fifty-seven LGSIL [92%], 13 HGSIL [33%] and 4 ICC [17%] showed results compatible with an intact E2 gene, while 5 LGSIL [8%], 26 HGSIL [67%] and 20 ICC [83%] samples gave no signal. CONCLUSIONS HPV 16 E2 gene disruption showed a positive correlation with cervical lesion progression, particularly from LGSIL to HGSIL. Although additional genetic events are very likely to be needed for HGSIL to ICC progression, the E2 gene disruption is a putative early marker to consider in the prognostic analysis of HPV 16 chronically infected women.

Ha-ras oncogene mutation associated to progression of papillomavirus induced lesions of uterine cervix

BACKGROUND Epidemiological and virological surveys suggest that the HPV presence is not enough condition to generate anogenital cancer, others factors (genetic, environmental, hormonal, etc) may have an important role. Mutations of ras genes were observed in several human neoplasias, including cervical cancer. OBJECTIVE The aim of the study was to assess the frequency of Ha-ras oncogene mutations in cervical intraepithelial neoplasia (CIN) grade III and invasive squamous cell carcinomas and to examine this genetic factor in relation to HPV infection and the clinical evolution of cervical lesions. STUDY DESIGN They were selected for (a) evaluation of the frequency of Ha-ras mutations: 39 cases of invasive carcinomas (InCa), 47 CIN III and 12 normal tissues taken from areas adjacent to the tumor (NT). (b) Retrospective follow-up: 18 cases of lesion progression; 9 cases of persistence and 12 of regression to mature or immature metaplasia after specific treatment. All biopsies obtained from each patient during the follow-up done between 5 and 10 years were included. HPV typing and scanning of possible mutations in Ha-ras were made by single-strand conformation polymorphism analysis/polymerase chain reaction. RESULTS HPV-DNA was detected in 95% of InCa and 84% of CIN III; HPV 16/18 was found in 65% of patients, mainly associated with persistent infection and lesion progression. The undetermined HPV types (18%) could indicate the circulation in our country of types other than those screened (6, 11, 16, 18, 31 and 33). Twenty percent of CIN III and 41% of InCa had patterns compatible with Ha-ras mutations. Mutated Ha-ras was detected in 61 and 44% of progression and persistence cases, respectively, including early stages of progression. CONCLUSIONS Ha-ras mutations were detected in CIN II-III lesions; in mutated cases, the progression took place in under 2 years, then this detection may be an early predictive marker of rapid progression.

A semiquantitative PCR method (SQ-PCR) to measure Epstein/Barr virus (EBV) load: its application in transplant patients

BACKGROUND High Epstein-Barr virus load has been related to an increased risk of Posttransplant Lymphoproliferative Disorders (PTLD) in transplant recipients. OBJECTIVES Development of a method to quantitate EBV DNA levels in peripheral blood mononuclear cells (PBMC) and evaluate its usefulness in transplant patients. STUDY DESIGN We designed a semiquantitative nested PCR based on a limiting dilution analysis to detect high viral loads in PBMC. This method was applied to 25 healthy carriers, and 85 solid organ transplant recipients as follows: (A) 53 asymptomatic patients; (B) 24 symptomatic patients; (C) eight patients with PTLD. RESULTS In healthy carriers the reciprocal of the limiting dilution (RLD) ranged between non-detected (ND) and 1, the median RLD was ND, which is equivalent to a viral load of <1 copy per 10(5) PBMC. In the transplant population the medians RLD (range) were: (A) asymptomatic group: ND (ND-64), median equivalent to a viral load of <1 copy per 10(5) PBMC; (B) symptomatic group: 4 (ND-256), median equivalent to a range of viral load of 4-64 copies per 10(5) PBMC

Human papillomavirus and mutated H-ras oncogene in cervical carcinomas and pathological negative pelvic lymph nodes: A retrospective follow-up

The metastasis status of pelvic lymph nodes (PLNs) seems to be a predictive factor of survival. It was suggested that the presence of HPV DNA and other biological markers in PLN may indicate a sub clinical early metastasis. The aim was to describe the prevalence and distribution patterns of HPV DNA and H‐ras mutations in intra operatively obtained cervical tumors and PLN. Thirty‐seven cervical tumors and 61 lymph node biopsies from 37 patients with cervical cancer were selected. HPV typing and location were performed by PCR/dot blot and in situ hybridization (ISH) respectively. PCR/RFLP was used to scan for mutations in H‐ras. Hundred percent of the cervical cancers and 85% of the PLN were HPV positive; co‐infection with more than one type was 27%. HPV 16 was detected alone or co‐infecting with other types in 84% of tumors and 46% of PLN; the second most frequent viral type was HPV 18 (tumor: 27%; PLN: 20%). In PLN, HPV was located in nuclei or/and cytoplasm of lymphocytes, macrophages, endothelial, and /or stromal cells. H‐ras mutations were identified in 5/24 (21%) of patients with cervical tumors showing poor or moderated differentiation. HPV DNA in histological tumor‐free PLN not necessary indicate metastasis, but it may be associated to an active immune reaction. Mutated H‐ras is probably involved in cervical carcinogenesis and its detection in tumor and metastasis free PLN may be related to early metastasis or recurrence in at least a subset of poorly differentiated cervical tumors. J. Med. Virol. 80:694–701, 2008.

Epstein-Barr virus load in transplant patients: Early detection of post-transplant lymphoproliferative disorders

High levels of circulating EBV load are used as a marker of post-transplant lymphoproliferative disorders (PTLD). There is no consensus regarding the threshold level indicative of an increase in peripheral EBV DNA. The aim of the study was to clinically validate a developed EBV quantification assay for early PTLD detection. Transversal study: paired peripheral blood mononuclear cells (PBMC), plasma and oropharyngeal lymphoid tissue (OLT) from children undergoing a solid organ transplant with (n=58) and without (n=47) PTLD. Retrospective follow-up: 71 paired PBMC and plasma from recipients with (n=6) and without (n=6) PTLD history. EBV load was determined by real-time PCR. The diagnostic ability to detect all PTLD (categories 1-4), advanced PTLD (categories 2-4) or neoplastic PTLD (categories 3 and 4) was estimated by analyzing the test performance at different cut-off values or with a load variation greater than 0.5log units. The higher diagnostic performance for identifying all, advanced or neoplastic PTLD, was achieved with cut-off values of 1.08; 1.60 and 2.47log EBVgEq/10(5) PBMC or 2.30; 2.60; 4.47loggEq/10(5) OLT cells, respectively. EBV DNA detection in plasma showed high specificity but low (all categories) or high (advanced/neoplastic categories) sensitivity for PTLD identification. Diagnostic performance was greater when: (1) a load variation in PBMC or plasma was identified; (2) combining the measure of EBV load in PBMC and plasma. The best diagnostic ability to identify early PTLD stages was achieved by monitoring EBV load in PBMC and plasma simultaneously; an algorithm was proposed.

Cutaneous human papillomavirus genotypes in different kinds of skin lesions in Argentina.

Cutaneous human papillomaviruses (HPVs) comprise a large and highly heterogeneous virus group. Some of the cutaneous HPVs of the genus Beta have been suggested as a co‐factor in the development of non‐melanoma skin cancer (NMSC). The aim of this study was to determine cutaneous HPV prevalence and type‐specific distribution in different kinds of skin lesions from Argentine patients visiting Dermatology Departments of three hospitals from Buenos Aires. A cross‐sectional analysis was performed. HPV DNA was analyzed in (i) 3 patients with Epidermodysplasia verruciformis (EV) harboring benign lesions (BL) (n = 1) and squamous cell carcinoma (SCC) (n = 4); (ii) 240 non‐EV patients harboring: (a) BL (n = 38), (b) Actinic Keratosis (AK) (n = 83), (c) SCC (n = 74), and (d) basal cell carcinoma (BCC) (n = 96). Detection and genotyping of 35 cutaneous HPV DNA was carried out by BGC‐PCR and GP5+/6 + PCR followed by reverse line blot assay. In EV patients, Beta types were found in all lesions (5/5), including the potentially high‐risk HPV types 5 and 8, mostly in multiple infections. In non‐EV patients, cutaneous types were found in 50.0% of BL, 43.4% of AK, 31.1% of SCC, and 16.7% of BCC. Beta HPVs were the most frequently found in all lesions, being present in all AK and SCC cases that were positive for HPV. No type‐specific correlation with lesion severity was found. In our series, a wide spectrum of cutaneous HPV types was detected in different skin lesions. A possible role for these HPVs in skin carcinogenesis deserves further study. J. Med. Virol. 89:352–357, 2017.