Prognostication of vulvar cancer based on p14ARF status: molecular assessment of transcript and protein.

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.

Cell cycle suppressor proteins are not related to HPV status or clinical outcome in patients with vulvar carcinoma.

Interactions between the cyclin-dependent kinase inhibitors (CDKI) and human papillomavirus (HPV) infection in the pathogenesis of vulvar carcinoma are still incomplete. This study aimed to evaluate the prognostic relevance of these proteins in vulvar cancer. One hundred and thirty-nine patient specimens assembled in a tissue microarray were evaluated for p16, p21, p27, and pRb by immunohistochemistry. HPV status was assessed by a linear array HPV genotyping test. In 16 cases with available frozen tumor, quantitative real-time reverse transcriptase-polymerase chain reaction for CDKN2A(p16), CDKN1A, and Rb was performed. Protein expression was considered positive in 40 patients for p16, 35 for p21, 28 for p27, and 19 for pRb. HPV was positive in 43 of the 105 evaluable cases. Expression of CDKIs and pRb, with the exception of p16, seem to be linked to the early phases of vulvar carcinogenesis. Although p16 and p21 protein expression was associated with early stages of disease, no prognostic significance was found when analyzing CDKI proteins or detecting HPV status, limiting their clinical usage. No association was observed between expression of CDKI proteins and HPV status, suggesting that in spite of this association found in cervical cancer, this seems not to be valid for vulvar carcinoma.

Prognostic significance of c-KIT in vulvar cancer: bringing this molecular marker from bench to bedside.

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.

Polymorphisms in the AR and PSA genes as markers of susceptibility and aggressiveness in prostate cancer.

The study of genes involved in androgen pathway can contribute to a better knowledge of prostate cancer. Our aim was to examine if polymorphisms in prostate-specific antigen (PSA) and androgen receptor (AR) genes were involved in prostate cancer risk and aggressiveness. Genotypes were determined by PCR-RFLP (PSA) or using a 377 ABI DNA Sequencer (AR). PSA(G/G) genotype (OR = 1.78, 95% CI = 1.06­2.99) and AR short CAG repeats (OR = 1.89, 95% CI = 1.21­2.96) increased risk for prostate cancer and were related with tumor aggressiveness. About 38.3% of tumors showed microsatellite instability. In conclusion, polymorphisms in these genes may be indicated as potential biomarkers for prostate cancer.

Prognostic value of GRIM-19, NF-κB and IKK2 in patients with high-grade serous ovarian cancer.

AIMS: High grade serous carcinoma (HGSC) is an aggressive tumour, and most patients relapse after treatment, acquiring resistance to platinum-based chemotherapy. One of the resistance mechanisms proposed is apoptosis evasion triggered by drug-related cytotoxic effect in the cell. In this context, this study aims to evaluate the protein expression of GRIM-19, NF-κB and IKK2, their association with chemotherapy response and to determine their prognostic values in HGSC. METHODS: GRIM-19, NF-κB and IKK2 expression was evaluated by immunohistochemistry (IHC) in 71 patients with HGSC selected between 2003 and 2013, whose underwent primary debulking surgery with complete cytoreduction. Protein expression was analyzed in relation to platinum response groups, tumour progression, clinicopathological data and survival. RESULTS: Positive IKK2 expression was related to resistance (p = 0.011), shorter disease-free survival (p = 0.001) and overall survival (p = 0.026) and was also a risk factor for relapse (p = 0.002) and death (p = 0.032). The association between IKK2 and NF-κB positivity predicted a subgroup with shorter overall survival (p = 0.004), disease-free survival (p = 0.003) and resistance to platinum-based chemotherapy (p = 0.036). NF-κB positivity was associated with worse overall survival (p = 0.005) and disease-free survival (p = 0.027) and was a positive predictor for relapse (p = 0.032) and death (p = 0.008). Higher expression of GRIM-19 was associated with higher disease-free survival (p = 0.039) and was a negative predictor for relapse (p = 0.046). CONCLUSIONS: GRIM-19 is a potential predictor of prognosis and disease recurrence in HGSC. IKK2 and NF-κB are related to poor prognosis and are potential predictors of response to platinum-based chemotherapy in HGSC. IHC analyses of GRIM19, IKK2 and NF-κB may be important in the attempt to provide prognostic values for relapse and response to treatment in patients with HGSC.

EGFR expression in vulvar cancer: clinical implications and tumor heterogeneity.

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.

Avaliação do processo de transição epitélio-mesênquima e sua correlação com tipos virais de HPV e terapia em carcinomas vulvares / Evaluation of the epithelial mesenchymal transition process and its correlation with viral HPV types and terapy in vulvar carcinomas

O carcinoma de células escamosas da vulva compreende 92% de todos os tipos de câncer vulvar invasivo e corresponde a cerca de 3% a 5% dos tumores malignos do trato genital feminino. A transição epitelio mesênquima (TEM) é um importante evento durante a progressão do câncer e é induzida via ativação de fatores de transcrição como TWIST, SNAI2 e SNAI1 que modulam a expressão de E-caderina, Vinentina e β-catenina. Entretanto, para o carcinoma vulvar pouco se sabe sobre a relação da TEM com a presença de tipos específicos de papiloma vírus humano (HPV), fatores prognósticos e terapia. Mediante tal contexto, os objetivos do presente estudo foram identificar as alterações de expressão proteica de marcadores de transição epitélio-mesênquima (TEM) comparando centro e fronte do tumor e correlacionar os resultados com a presença de infecção por subtipos específicos de HPV para a determinação de valores prognósticos e preditivos em carcinoma de vulva. Também realizamos ensaios in vitro para observar o efeito de NVP-BEZ235, um composto que age indiretamente em fatores de transcrição como o Slug, em uma linhagem metastática de vulva. Nossos resultados demonstraram que a perda de expressão de E-caderina é observada em estágios avançados de invasão tumoral enquanto que β-catenina representa um biomarcador importante para estabelecer o prognóstico no carcinoma vulvar (p=0.044). Além disso, a perda de expressão de β-catenina associada ao aumento de expressão de Slug no fronte de invasão caracteriza um subgrupo de tumores com fenótipo de TEM relacionados com a negatividade para infecção pelo HPV com o pior prognóstico (p = 0,001). No cenário clínico, a avaliação de imuno-histoquímica comparativa da expressão de β-catenina entre fronte de invasão e centro do tumor, pode representar uma ferramenta adicional para estabelecer o prognóstico de carcinoma vulvar...

Vulvar squamous cell carcinoma comprises 92% of all cases of vulvar cancer and accounts for about 3% to 5% of malignant tumors of the female genital tract. Epithelial-mesenchymal transition (EMT) is an important event during cancer progression and it is induced by activation of transcription factors as Twist, Slug and Snail factors that modulate the expression of E-cadherin, Vimentin and B-catenin. However, considering vulvar carcinoma, little is known about the relationship among EMT, presence of specific types of human papillomavirus (HPV), prognostic factors and therapy. In this context, the aims of this study were to analyze expression of markers of EMT comparing invasive front and central tumor and correlate the results with the presence of specific subtypes of HPV for determination of prognostic and predictive factors in vulvar carcinoma. And also we performed in vitro assays to observe the effect of NVP-BEZ235, a compound that acts indirectly on transcription factors such as Slug on a metastatic vulvar cancer cell line. Our results demonstrated that loss of E-cadherin expression is observed in advanced stages of tumor invasion whereas β-catenin is an important biomarker for establishing the prognosis in vulvar carcinoma (p=0.044). Furthermore, β-catenin lower expression associated with gain in Slug expression at the invasive front characterizes a subgroup of EMT-related HPV-negative tumors with the worst outcome, increased invasiveness and progression (p= 0.001). In the clinical setting, IHC comparative assessmentof β-catenin expression between invasive front and central tumor may represent an additional tool for establishing prognosis of vulvar cancer. We also showed that NVP-BEZ235 decreased the migration, invasion and proliferation of metastatic vulvar cancer cell line that presents an EMT-like that represents a potential therapeutic target for the treatment of aggressive tumors, which often do not respond to conventional treatments...