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de Melo Maia B.,Hospital AC Camargo | Lavorato-Rocha A.M.,Hospital AC Camargo | Rodrigues I.S.,Hospital AC Camargo | Baiocchi G.,Hospital AC Camargo | And 6 more authors.
Journal of Translational Medicine | Year: 2012

Background: Vulvar carcinomas are rare tumors, and there is limited data regarding molecular alterations. To our knowledge there are no published studies on c-KIT and squamous cell carcinomas of the vulva (VSCC). Although there are a significant number of other tumor types which express c-KIT, there remains controversy as to its relationship to patient outcome. Thus, we wished to investigate such controversial findings to determine the prognostic importance of c-KIT by evaluating its protein and mRNA expression in VSCCs, correlating these findings with clinicopathological features and Human Papillomavirus (HPV) infection.Methods: c-KIT expression was scored by immunohistochemistry (IHC) as positive or negative in 139 formalin-fixed paraffin-embedded (FFPE) cases of vulvar carcinomas arrayed in a tissue microarray (TMA) using the DAKO® A4502 rabbit polyclonal c-KIT antibody (diluted 1:100). c-KIT mRNA was evaluated by qRT-PCR in 34 frozen samples from AC Camargo Hospital Biobank (17 tumoral and 17 non-tumoral samples) using TaqMan probes-Applied Biosystems [Hs00174029_m1]. HPV genotyping was assessed in 103 samples using Linear Array® HPV Genotyping Test kit (Roche Molecular Diagnostics, Basel, Switzerland). All results obtained were correlated with clinical and pathological data of the patients.Results: c-KIT protein was positive by immunohistochemistry in 70.5% of the cases and this was associated with a higher global survival (p = 0.007), a higher recurrence-free survival (p < 0.0001), an absence of associated lesions (p = 0.001), lymph node metastasis (p = 0.0053), and HPV infection (p = 0.034). Furthermore, c-KIT mRNA quantitation revealed higher levels of transcripts in normal samples compared to tumor samples (p = 0,0009).Conclusions: Our findings indicate that those vulvar tumors staining positively for c-KIT present better prognosis. Thus, positivity of c-KIT as evaluated by IHC may be a good predictor for use of more conservative surgery techniques and lymph node dissection in vulvar cancer. So part of the essence of our study is to see the possibility of translating our current results from the bench to the bedside. This will help provide patients a more appropriate, less mutilating treatment, in order to keep the maximum physical and psychic quality as possible to these women. © 2012 Maia et al.; licensee BioMed Central Ltd.


De Melo Maia B.,Camargo Cancer Center | Fontes A.M.,Camargo Cancer Center | Lavorato-Rocha A.M.,Camargo Cancer Center | Rodrigues I.S.,Camargo Cancer Center | And 5 more authors.
Human Pathology | Year: 2014

Epidermal growth factor receptor (EGFR) protein expression was assessed by immunohistochemistry (IHC) in 150 cases of invasive vulvar squamous cell carcinoma. In addition, gene copy number status by fluorescence in situ hybridization was performed in a smaller set of samples. Results were correlated with patient's clinical data and prognostic factors. EGFR overexpression (2+ and 3+) was observed on the membrane in 24.66% and 21.33% of all cases, respectively. Higher EGFR expression was associated with depth of invasion (P =.0409) and disease recurrence (P =.0401). Cytoplasm staining was found in 21.33% of the cases and was associated with absence of nodal metastasis (P =.0061) and better survival (P =.0199). Intratumor heterogeneity of EGFR IHC staining was frequently observed (55.33%) and was associated with the presence of nodal metastasis (P =.0207) and tumor invasion (P =.0161). Worse survival outcomes have been demonstrated in tumors with EGFR heterogeneity (P =.0434). EGFR gene status evaluated by fluorescence in situ hybridization did not correlate with protein expression evaluated by IHC. In conclusion, EGFR cytoplasm staining has no link with poorer outcome; still, this pattern of staining is even more related to better prognosis. EGFR heterogeneity of staining correlated with more aggressive tumors, and presented to be an important marker of poor prognosis in vulvar squamous cell carcinoma. The usage of small biopsies or even tissue microarrays for vulvar cancer evaluation should be carefully reconsidered for the assessment of EGFR as the results may be misleading. Protein overexpression may be independent on gene amplification, showing that other molecular mechanisms than copy number variation may regulate protein expression of EGFR in vulvar cancer. © 2014 Elsevier Inc. All rights reserved.


Rodrigues I.S.,Camargo Cancer Center | Lavorato-Rocha A.M.,Camargo Cancer Center | De M Maia B.,Camargo Cancer Center | Stiepcich M.M.A.,Fleury Institute | And 4 more authors.
British Journal of Cancer | Year: 2013

Background:Epithelial-to-mesenchymal transition (EMT) still remains an obscure event in vulvar squamous cell carcinoma (VSCC).Methods: Immunohistochemistry (IHC) expression of E-cadherin, β-catenin, Snail, Slug, Twist and Vimentin was analysed in 87 VSCC, controlled for human papillomavirus (HPV) positivity, considering tumour front and central tumour as different morphological categories from the same tumour.Results:Lower β-catenin and higher Vimentin expression was associated with invasive front when compared with the central tumour (P=0.013 and P≤0.001, respectively). Higher expression of E-cadherin in central tumour was significantly related to absence of vascular and perineural invasion, lower invasion depth and ≤2 lymph node involvement. Loss of β-catenin and high Slug, Snail and Twist expression was associated with HPV-negative tumours. Moreover, β-catenin lower expression associated with gain in Slug expression predicts a subgroup with worst outcome (P=0.001). Lower expression of β-catenin in both central tumour and invasive front correlated with lower overall survival (P=0.021 and P=0.011, respectively). Also, multivariate analysis showed that lower β-catenin expression was independently associated with poorer outcome (P=0.044).Conclusion:Human papillomavirus-related tumours show better prognosis and outcome; besides, they do not progress through EMT phenomenon. Immunohistochemical analysis of β-catenin in invasive tumour front is a key issue for establishing prognosis of vulva cancer. © 2013 Cancer Research UK. All rights reserved.


Correa-Silva S.R.,University of Sao Paulo | Nascif S.O.,University of Sao Paulo | Molica P.,University of Sao Paulo | Sa L.B.P.C.,University of Sao Paulo | And 3 more authors.
Clinical Endocrinology | Year: 2010

Background In Cushing's disease (CD), adrenocorticotrophic hormone (ACTH)/cortisol responses to growth hormone secretagogues (GHS), such as ghrelin and GHRP-6, are exaggerated. The effect of clinical treatment of hypercortisolism with ketoconazole on ACTH secretion in CD is controversial. There are no studies evaluating ACTH/cortisol responses to GHS after prolonged ketoconazole use in these patients. Objective To compare ghrelin- and GHRP-6-induced ACTH/cortisol release before and after ketoconazole treatment in patients with CD. Design/patients Eight untreated patients with CD (BMI: 28·5 ± 0·8 kg/m2) were evaluated before and after 3 and 6 months of ketoconazole treatment and compared with 11 controls (BMI: 25·0 ± 0·8). Results After ketoconazole use, mean urinary free cortisol values decreased significantly (before: 613·6 ± 95·2 nmol/24 h; 3rd month: 170·0 ± 27·9; 6th month: 107·9 ± 30·1). The same was observed with basal serum cortisol (before: 612·5 ± 69·0 nmol/l; 3rd month: 463·5 ± 44·1; 6th month: 402·8 ± 44·1) and ghrelin- and GHRP-6-stimulated peak cortisol levels (before: 1183·6 ± 137·9 and 1045·7 ± 132·4; 3rd month: 637·3 ± 69·0 and 767·0 ± 91·0; 6th month: 689·8 ± 74·5 and 571·1 ± 71·7 respectively). An increase in basal ACTH (before: 11·2 ± 1·6 pmol/l; 6th month: 19·4 ± 2·7) and in ghrelin-stimulated peak ACTH values occurred after 6 months (before: 59·8 ± 15·4; 6th month: 112·0 ± 11·2). GHRP-6-induced ACTH release also increased (before: 60·7 ± 17·2; 6th month: 78·5 ± 12·1), although not significantly. Conclusions The rise in basal ACTH levels during ketoconazole treatment in CD could be because of the activation of normal corticotrophs, which were earlier suppressed by hypercortisolism. The enhanced ACTH responses to ghrelin after ketoconazole in CD could also be due to activation of the hypothalamic-pituitary-adrenal axis and/or to an increase in GHS-receptors expression in the corticotroph adenoma, consequent to reductions in circulating glucocorticoids. © 2010 Blackwell Publishing Ltd.


Lavorato-Rocha A.M.,Cancer Hospital A.C. Camargo | De Melo Maia B.,Cancer Hospital A.C. Camargo | Rodrigues I.S.,Cancer Hospital A.C. Camargo | Stiepcich M.M.A.,Fleury Institute | And 5 more authors.
Annals of Surgical Oncology | Year: 2013

Purpose: This study was designed to determine the prognostic role of p14ARF in vulvar squamous cell carcinoma (VSCC). Methods: Immunohistochemistry for p14ARF and p53 and fluorescent in situ hybridization (FISH) for TP53 were performed in 139 cases of VSCC. Human papillomavirus (HPV) genotyping by hybridization was employed in 100 cases. qRT-PCR for p14ARF and p53 transcript assessment was performed in 16 cases. All results were correlated with clinicopathological variables. Results: Immunohistochemistry analysis showed p14ARF and p53 positivity in 16.4 % and 53 % cases respectively. Positive p14ARF expression was significantly associated with the following variables: shorter cancer-specific survival (P = 0.04) and shorter disease-free survival (P = 0.02), presence of perineural invasion (P = 0.037), vascular invasion (P = 0.047), and node metastasis (P = 0.031). Also, p14ARF-positive HPV-negative cases had the shortest cancer-specific survival (P = 0.03) and disease-free survival (P = 0.04). HPV infection was detected in 32.8 % of the cases; HPV16 was the most prevalent type. Viral infection was more common in poorly differentiated tumors (P = 0.032). qRT-PCR demonstrated that CDKN2A (p14ARF) had higher expression in tumor samples compared with paired noncancerous samples (P < 0.001). The opposite relationship was seen in TP53 expression evaluation (P < 0.001). FISH demonstrated 4 cases with deleted TP53 (6.3 %). Conclusions: p14ARF represents an important marker of poor prognosis in VSCC. p53 and HPV infection did not show any prognostic importance. Further clinical trials concerning p14ARF positivity may result in important contributions due to its relationship with poor outcome. Mainly due to the relationship of p14ARF with lymph node metastasis, the immunohistochemistry evaluation of this marker may help to identify a subset of patients more suitable to less radical procedures. © 2012 Society of Surgical Oncology.

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