Daniela Bonifacio

Cervical cytopathology: An evaluation of its accuracy based on cytohistologic comparison

Background. Although Papanicolaou cytology represents the most effective technique to prevent and detect precancerous conditions of the uterine cervix, its false‐negative yield is still a reason of concern among pathologists and gynecologists.

Identification of the novel KI and WU polyomaviruses in human tonsils

BACKGROUND Three novel polyomaviruses have been recently discovered: KI, WU and MC polyomaviruses. Their role in human pathology is debated while tissue tropism and site of latency remain unknown. OBJECTIVE To test the hypothesis that KI, WU and MC polyomaviruses can infect human tonsils. STUDY DESIGN Archival paraffin-embedded tonsils from 91 patients affected by different tonsil diseases were screened by polymerase chain reaction to detect viral DNA of KIV, WUV, MCV, BKV and JCV. Phylogenetic and evolutionary analysis of the identified polyomaviruses was carried out. RESULTS Of the 91 tested specimens, 11 contained KIV DNA (12%), 4 WUV DNA (4.4%), 5 BKV DNA (5.5%). MCV and JCV were not detected. Phylogenetic analysis showed that KIVs identified in tonsils fall into a clade distinct from that containing KIVs isolated from respiratory secretions, respiratory tissue and feces. Moreover, four positively selected sites (4.5% of t-Ag sites) were found under strong positive selection (omega=11.4), with posterior probabilities above 0.99. All the sites were located in the N-terminal region of the small t antigen. CONCLUSIONS The results suggest that the novel KI and WU polyomaviruses can infect human tonsils. Future studies are needed to define their role in tonsil diseases.

Co-expression of HSV2 and Chlamydia trachomatis in HPV-positive cervical cancer and cervical intraepithelial neoplasia lesions is associated with aberrations in key intracellular pathways.

Objective: Oncogenic human papillomaviruses (HPVs) are the etiological agents of cervical cancer. Different cofactors might be needed for malignant transformation, but they still remain elusive. Methods: To delineate the role of Chlamydia trachomatis (CT) and herpes simplex virus type 2 (HSV2) in HPV-positive cervical intraepithelial neoplasia (CIN) lesions and cervical carcinoma a series of 149 cervical cancer and CIN biopsies were analyzed for CT and HSV2 DNA by PCR, and HPV genotyped by InnoLipa. Monitoring of aberrations in key intracellular pathways due to CT/HSV2 and HPV co-expression were analyzed with 13 biomarkers. Results: Of the 149 samples tested, 136 were HPV DNA positive; 32/136 contained also CT DNA and 29 HSV2 DNA. Detection of CT was significantly (p = 0.0001) related to multiple-type HPV infections, while HSV2 was of borderline significance (p = 0.053). Of the 13 biomarkers tested, cytoplasmic and nuclear NF-ĸB and VEGF-C were significantly increased in CT+/HPV+ lesions; p = 0.023, p = 0.045, and p = 0.020 as well as survivin, p = 0.026. Survivin was the only marker that was overexpressed also in HSV2+/HPV+ lesions, p = 0.027. Conclusions: CT infection favors the entry and persistence of multiple HR-HPV types, which leads to viral integration, inhibition of apoptosis, overexpression of E6/E7 oncogenes and cell transformation.

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 II&agr; and II&bgr; 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 II&agr; (topo II&agr;), 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 II&agr; 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 II&agr; 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 p16INK4a (9) and Survivin (33) in the multivariate regression model. Topo II&agr; 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 II&agr; 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 II&agr; by degrading the wild-type Rb.

Immunohistochemical MIB-1 and p27kip1 as Prognostic Factors in Pleural Mesothelioma

Malignant pleural mesothelioma (MPM) is a tumour that usually presents a short survival time from diagnosis, but in a small number of patients a longer survival time has been observed. We have studied a series of ten long-term and ten short-term survivors with MPM to assess whether the MIB-1 proliferation index and the p27kip1 expression correlated with survival. Our results show significant difference in MIB-1 and in p27kip1 expression between the group of short and longer survival patients. It suggests that the difference in tumour growth fractions may express different biological behaviour and therefore indicate important factors in MPM prognosis.

High Prevalence of BK Polyomavirus Sequences in Human Papillomavirus-16-Positive Precancerous Cervical Lesions

High‐ and low‐grade cervical lesions were analyzed for the presence of polyomavirus (PYV) and human papillomavirus (HPV) sequences. In precancerous cervical lesions, the overall prevalence of PYV sequences was 44% (41/93). Specifically, among the PYV‐positive samples, 83% (34/41) tested positive for BK polyomavirus (BKV) sequences, whereas 17% (7/41) were positive for JC‐virus. None of the samples were positive for simian virus 40. The presence of BKV DNA in high‐grade squamous intraepithelial lesions was confirmed by in situ PCR. BKV sequences were detected more frequently in high‐grade squamous intraepithelial lesions, together with the genotype HPV‐16. The association of BKV with precancerous cervical lesions suggests that this polyomavirus participates with HPV‐16 in the cell transformation process. Alternatively, BKV might multiply better in HPV‐16‐positive cells from precancerous cervical lesions than in HPV‐16‐negative cells. J. Med. Virol. 83:1770–1776, 2011.

Identification and Validation of a New Set of Five Genes for Prediction of Risk in Early Breast Cancer

Molecular tests predicting the outcome of breast cancer patients based on gene expression levels can be used to assist in making treatment decisions after consideration of conventional markers. In this study we identified a subset of 20 mRNA differentially regulated in breast cancer analyzing several publicly available array gene expression data using R/Bioconductor package. Using RTqPCR we evaluate 261 consecutive invasive breast cancer cases not selected for age, adjuvant treatment, nodal and estrogen receptor status from paraffin embedded sections. The biological samples dataset was split into a training (137 cases) and a validation set (124 cases). The gene signature was developed on the training set and a multivariate stepwise Cox analysis selected five genes independently associated with DFS: FGF18 (HR = 1.13, p = 0.05), BCL2 (HR = 0.57, p = 0.001), PRC1 (HR = 1.51, p = 0.001), MMP9 (HR = 1.11, p = 0.08), SERF1a (HR = 0.83, p = 0.007). These five genes were combined into a linear score (signature) weighted according to the coefficients of the Cox model, as: 0.125FGF18 − 0.560BCL2 + 0.409PRC1 + 0.104MMP9 − 0.188SERF1A (HR = 2.7, 95% CI = 1.9–4.0, p < 0.001). The signature was then evaluated on the validation set assessing the discrimination ability by a Kaplan Meier analysis, using the same cut offs classifying patients at low, intermediate or high risk of disease relapse as defined on the training set (p < 0.001). Our signature, after a further clinical validation, could be proposed as prognostic signature for disease free survival in breast cancer patients where the indication for adjuvant chemotherapy added to endocrine treatment is uncertain.

Role of fine-needle aspiration cytology in nonpalpable mammary lesions: A comparative cytohistologic study based on 308 cases

We retrospectively evaluated the accuracy of fine‐needle aspiration cytology (FNAC) in nonpalpable breast lesions detected by imaging techniques between 1995–1997. A total number of 308 lesions was investigated: 273 had been studied by means of either FNAC obtained under ultrasound (175 cases) or stereotactic guidance (98 cases). The overall sensitivity rate was 87.8%; specificity was 95.3%; the positive predictive value was 76.6%; the negative predictive value was 97.8%. Our results confirm that FNAC is quite effective in the approach to patients with nonpalpable breast lesions. It is particularly accurate in diagnosing malignancy, although a lower yield may be encountered in tumor types producing a desmoplastic stroma (tubular carcinoma, infiltrating lobular carcinoma) or in noncomedonic in situ ductal carcinoma. Discrepancy between a suspicious cytology and a negative histology is more frequent with benign lesions usually because of sampling mistake or technically inadequate smears. In particular, when smears are adequate, FNAC safely assists in ruling out the malignant lesions. Diagn. Cytopathol. 23:87–91, 2000.