Gian Franco Zannoni

Class III β-Tubulin Overexpression Is a Marker of Poor Clinical Outcome in Advanced Ovarian Cancer Patients

Purpose: Overexpression of β III tubulin has been involved in paclitaxel resistance in several experimental models. We investigated the role of β III tubulin as predictor of clinical outcome in ovarian cancer patients given platinum/paclitaxel treatment. We also investigated whether β III tubulin expression could be modified after the selective pressure represented by chemotherapy in vivo. Experimental Design: The study was designed to include a series of consecutive ovarian cancer patients with unresectable disease at time of first surgery, who underwent interval debulking surgery with pathologic assessment of response to treatment with platinum/paclitaxel chemotherapy. Immunostaining was done on formalin-fixed, paraffin-embedded tissue sections from pretreatment and posttreatment tissue biopsies by using the polyclonal rabbit anti–class III β-tubulin antibody. Results: β III Tubulin immunoreaction was observed in 51 of 62 (82.2%) cases. β III Tubulin positivity was neither associated with clinicopathologic variables nor with pathologic response to chemotherapy. Significantly lower percentages of β III tubulin positivity were observed in posttreatment (range, 5-80%; median, 20%) versus pretreatment (range 10-100%; median, 40%) tissue biopsies (P = 0.0011). Cases with high β III tubulin expression showed a worse overall survival with respect to cases with low β III tubulin expression (median overall survival, 25 versus 46 months; P = 0.002). Multivariate analysis showed that high content of β III tubulin remains independently associated with a worse prognosis. Conclusions: Assessment of β III tubulin could be useful to identify poor prognosis ovarian cancer patients candidates to more aggressive and/or targeted therapy.

Possible involvement of hMLH1, p16(INK4a) and PTEN in the malignant transformation of endometriosis.

Endometriosis is a common gynecologic disease, which generally follows a benign course. Notwithstanding, several clinical and histologic studies as well as molecular data show that endometriosis could be a precursor of sporadic endometrioid and clear cell carcinomas at extrauterine loci. Several reports have implicated alterations of the hMLH1 and p16ink4a (p16) genes, in particular hypermethylation of the promoter region, and of the PTEN gene, principally genetic mutations, in endometrial and ovarian cancers and have indicated that these alterations are already present in precancer conditions. In this report, we analyzed the methylation status of hMLH1 and p16 and the protein expression of PTEN and hMLH1 in 46 cases of endometriosis stages III and IV to better define the possible involvement of these genes in the malignant transformation of endometriosis. We found abnormal methylation of hMLH1 in 4 of the 46 cases (8.6%). In addition, these cases had no detectable hMLH1 protein expression. Regarding patients with hMLH1 alterations, 2 were classified as stage IV and 2 showed coexistent endometriosis and carcinoma. Only 1 case of endometriosis (2.17%), classified as atypical, showed abnormal methylation of p16. Reduced PTEN protein expression was detected in 7 of 46 cases (15.21%): 5 were clinically classified as stage IV, and the other 2 presented both cancer and hypermethylated hMLH1. Our preliminary study suggests that reduced expression of both hMLH1 and PTEN may be involved in the malignant evolution of endometriosis and should be used as markers of neoplastic transformation in aggressive endometriosis with elevated tumor markers.

Cyclooxygenase-2 expression in endometrial carcinoma: correlation with clinicopathologic parameters and clinical outcome.

Cyclooxygenase‐2 (COX‐2) is overexpressed in endometrial hyperplasia and carcinoma, but no data have been reported until now about the expression of COX‐2 and its possible clinical significance in endometrial carcinoma. We investigated by immunohistochemistry the expression of COX‐2 in a single institutional series of primary untreated endometrial carcinoma patients. The relationship between COX‐2 expression and microsatellite instability (MI) status was also analyzed.

Follicular thyroid neoplasms can be classified as low- and high-risk according to HBME-1 and Galectin-3 expression on liquid-based fine-needle cytology

DESIGN Fine-needle aspiration biopsy (FNAB) is the most reliable diagnostic tool in the diagnosis of thyroid nodules. A cytologic diagnosis of follicular neoplasm with atypical cells of undetermined significance (FN/AUS) implies that the selection of patients between surgery and follow-up is difficult. In this setting immunocytochemical stainings might be helpful. The efficacy of a panel made up of HBME-1 and Galectin-3 antibodies is evaluated in cases processed by liquid-based cytology (LBC). METHODS Out of 7091 thyroid FNAB processed by LBC method, 120 cases undergoing surgery successively were selected. These cases were classified as benign lesion (BL, eight cases), FN, including the ACUS category of the Bethesda classification (FN/AUS, 50 cases), suspicious for malignancy (SM, 59 cases), and malignant neoplasm (MN, three cases). Immunostains for HBME-1 and Galectin-3 were carried out on the LBC slides. RESULTS All MN and BL were histologically confirmed. FN/AUS and SM showed a malignancy risk of 24 and 72.9% respectively. The complete immunocytochemical panel was positive in 83.3% of the cases resulting in malignancy and negative in 87.5% of cases resulting in benign histology. Among the FN/AUS, the complete positive immunocytochemical panel was detected in 76.9% of cases resulting as malignant and the complete negative immunocytochemical panel was observed in 96.8% of cases resulting as benign at histology. CONCLUSIONS The expression of HBME-1 and Galectin-3 in cases classified as FN/AUS on LBC-processed FNABs can effectively distinguish lesions, which need immediate surgery (high risk or FNH or Thy 3h) from those which can be followed-up (low risk or FNL or Thy 3l).

BRAF (V600E) mutation analysis on liquid-based cytology-processed aspiration biopsies predicts bilaterality and lymph node involvement in papillary thyroid microcarcinoma

Activating mutations in the valine‐to‐glutamic acid substitution at position 600 of the v‐raf murine sarcoma viral oncogene homolog B1 (BRAF‐1) gene are detected frequently in patients with papillary thyroid carcinoma (PTC). These mutations have been identified in approximately 29% to 69% of PTCs and in >80% of PTCs of the tall cell variant, whereas they have not been detected in benign lesions or in the majority of those (80%) with the follicular variant of PTC. The objective of the current study was to evaluate the role of liquid‐based cytology (LBC) for the detection of BRAF mutations in the outcome of patients who have thyroid PTC measuring ≤1 cm and, hence, in guiding their clinical and surgical management.

P16ink4a and HPV L1 Immunohistochemistry is Helpful for Estimating the Behavior of Low-grade Dysplastic Lesions of the Cervix Uteri

As only a minority of low-grade dysplastic lesions of the cervix uteri will eventually progress to carcinoma, predicting the behavior of these lesions could be of high value in clinical practice. The aim of the study was to evaluate p16ink4a and L1 as immunohistochemical markers of the biologic potentiality of low-grade dysplasia of the uterine cervix. The study included 38 conization specimens with coexisting cervical intraepithelial neoplasia grade 1 (CIN1) and 3 (CIN3) (group A) and 28 punch biopsies from women with CIN1 and proven spontaneous regression in the follow-up (group B). In group A, all CIN3 were p16ink4a positive (p16+) and L1 negative (L1−). The CIN1 of this group were p16+L1− and p16+L1+ in 68.42% and 31.57%, respectively. No other expression pattern was found in this group. In group B, the p16+L1−, p16+L1+, p16−L1+, and p16−L1− patterns were found in 3.57%, 25%, 14.29%, and 57.14%, respectively. Overall, 96.29% p16+L1− CIN1 were found in group A, whereas all the p16−L1+ and p16−L1− CIN1 were found in group B. A significant difference between staining pattern distributions of group A and B was observed (P<0.0001). The results of the study show that p16ink4a and L1 immunohistochemistry can be helpful for estimating the biologic potentiality of low-grade squamous cervical lesions. Particularly in cases in which the grade of the lesion is morphologically difficult to assess, the p16/L1 expression pattern could be useful for planning the clinical management of these women.

Early-stage cervical cancer: Tumor delineation by magnetic resonance imaging and ultrasound - A European multicenter trial

OBJECTIVE To compare the diagnostic accuracy of ultrasound (US) and magnetic resonance imaging (MRI) in the preoperative assessment of early-stage cervical cancer using pathologic findings as the reference standard. PATIENTS AND METHODS Prospective multi-center trial enrolling 209 consecutive women with early-stage cervical cancer (FIGO IA2-IIA) scheduled for surgery. The following parameters were assessed on US and MRI and compared to pathology: remaining tumor, size, tumor stromal invasion<2/3 (superficial) or ≥2/3 (deep), and parametrial invasion. RESULTS Complete data were available for 182 patients. The agreement between US and pathology was excellent for detecting tumors, correctly classifying bulky tumors (>4cm), and detecting deep stromal invasion (kappa values 0.84, 0.82, and 0.81 respectively); and good for classifying small tumors (<2cm) and detecting parametrial invasion (kappa values 0.78 and 0.75, respectively). The agreement between MRI and histology was good for classifying tumors as <2cm, or >4cm, and detecting deep stromal invasion (kappa values 0.71, 0.76, and 0.77, respectively). It was moderately accurate in tumor detection, and in assessing parametrial invasion (kappa values 0.52 and 0.45, respectively). The agreement between histology and US was significantly better in assessing residual tumor (p<0.001) and parametrial invasion (p<0.001) than the results obtained by MRI. Imaging methods were not significantly influenced by previous cone biopsy. CONCLUSION US and MRI are highly accurate for the preoperative assessment of women with early-stage cervical cancer, although US may be more accurate in detecting residual tumors and assessing parametrial invasion.

Class III β-tubulin and the cytoskeletal gateway for drug resistance in ovarian cancer.

The Class III β‐tubulin isotype (βIII‐tubulin) is a predictive biomarker in ovarian cancer and other solid tumor malignancies. We discovered that βIII‐tubulin function is linked to two GTPases: guanylate‐binding protein 1 (GBP1), which activates its function, and GNAI1, which inhibits it. This finding was demonstrated in a panel of ovarian cancer cells resistant to several chemotherapeutic agents. Using a protein microarray, we identified PIM1 as the downstream partner of GBP1, recruited into the cytoskeleton under hypoxic conditions. The clinical value of these observations was tested by performing an archive study of 98 ovarian cancer patients, which demonstrated that the βIII‐tubulin ‐/PIM1‐ cohort responded to treatment, exhibiting long overall survival (OS), while βIII‐tubulin +/PIM+ patients experienced poor outcomes and OS times similar to patients receiving palliation alone. βIII‐tubulin expression is commonly believed responsible for paclitaxel resistance due to its enhancement of the dynamic instability of microtubules, which counteracts the activity of taxanes. In contrast, our research reveals that βIII‐tubulin behaves as a gateway for prosurvival signals, such as PIM1, to move into the cytoskeleton. When cells are exposed to microenvironmental stressors, they activate this pathway by telling the cytoskeleton to incorporate PIM1 through GBP1 and βIII‐tubulin, which ultimately leads to drug resistance. This discovery reveals that βIII‐tubulin does not act alone but requires partners to play its role. The discovery of such protein:protein interactions underlying this prosurvival cascade makes feasible the development of therapeutic approaches using novel compounds that are capable of inhibiting the transmission of prosurvival signals into the cytoskeleton. J. Cell. Physiol. 227: 1034–1041, 2012.

Clinical Performance of Human Papillomavirus E6 and E7 mRNA Testing for High-Grade Lesions of the Cervix

ABSTRACT Infection with high-risk (HR) human papillomavirus (HPV) is the major cause of cervical cancer. However, relatively few infections progress to malignant disease. Progression to malignancy requires the overexpression of the E6 and E7 genes in the integrated HPV genome. It follows that the E6 and E7 transcripts could be useful markers of disease progression. The study presented here tests this possibility, using data from colposcopy and from cytological and histological tests to compare RNA assays for the E6 and E7 genes with DNA testing. A total of 180 women underwent colposcopy, cytology, and biopsy of suspected lesions (143 cases). Cervical brush specimens were analyzed for HPV DNA and for E6 and E7 mRNA. DNA from HR HPV was found in 57.8% of the specimens; E6 and E7 transcripts were found in 45%. The rates of detection of HPV DNA and of E6 and E7 transcripts were 33.3% and 25%, respectively, for specimens with normal findings; 51.4% and 31.9%, respectively, for specimens with cervical intraepithelial neoplasia grade 1 (CIN1); and 61.1% and 44.2% for specimens with CIN2, respectively. All specimens with CIN3 and 95.5% of specimens from patients with squamous cell carcinoma were positive by both assays. Thirty-seven patients with normal colposcopy findings did not undergo biopsy. HPV DNA and mRNA transcripts were found in 32.4% and 18.9% of these cases, respectively. Comparisons with cytological tests produced similar results. Overall, the mRNA tests showed a higher specificity than the DNA tests for high-grade lesions (72.7% and 56.2%, respectively) and a higher positive predictive value (59.3% and 49.0%, respectively). These findings suggest that mRNA assays could be more powerful than DNA testing for predicting the risk of progression and offer a strong potential as a tool for triage and patient follow-up.

Chemoprevention of DMBA-induced mammary cancer in rats by dietary soy

This study was designed to assess the potential chemopreventive effect of the administration of a standardized soy extract, SOYSELECTTM, on 7,12-Dimethylbenz[a]anthracene (DMBA)-induced mammary tumors in rats. Three groups, 24 females each, were used. Animals were fed either a phytoestrogen-free diet alone (control) or the same diet supplemented with 0.35% or 0.7% of soy extract. Treatment started at weaning and continued to the end of the study (24 weeks after DMBA administration). At day 50 of age all animals received via oral gavage 80 mg/kg DMBA. Only tumors subsequently classified as adenocarcinomas were considered for data evaluation. In rats on the soy diet, mammary tumors took a longer period of time to develop as compared to control rats. However, at the end of the study, no relevant difference in tumor incidence and multiplicity was observed among the groups. The most significant changes were seen between control and soy-treated groups when tumor dimension and results from histopathologic examination were considered. The latter, in fact, showed a dose-dependent reduction in the percentage of poorly differentiated tumors in treated animals. This change was statistically significant in animals receiving 0.7% soy. In addition, assessment of estrogen and progesterone receptor (ERα, PR) levels, revealed a significant reduction in the percentage of ERα and PR positive tumors in animals receiving 0.7% dietary soy, when compared to controls. Interestingly, genistein and daidzein plasma levels determined at the end of the study were within the range of those detected in people consuming large amounts of soyfoods.