Beatriz de Melo Maia

Prognostic value of the number and laterality of metastatic inguinal lymph nodes in vulvar cancer: Revisiting the FIGO staging system

OBJECTIVE Inguinal lymph node (LN) metastasis is an important prognostic factor in vulvar cancer. Our aims were to analyze the prognostic value of LN metastasis with regard to the number of LNs that were involved and their laterality and compare these results with the current FIGO staging system. METHODS A retrospective analysis was performed in a series of 234 individuals who underwent inguinal lymphadenectomy for vulvar squamous cell carcinoma from January 1980 to February 2010. RESULTS The mean age was 68 years. One hundred seven (45.7%) patients had LN metastasis. Despite the FIGO staging, we did not observe any significant difference in the risk of recurrence or death between patients with 1 positive LN and ≥ 2 positive LNs. Moreover, there was no difference in outcome between the presence of 1 and 2 positive LNs. On categorizing patients into 3 groups-absence of LN involvement, 1-2 positive LNs, and ≥ 3 positive LNs-we achieved a significantly better prognostic correlation for progression-free survival, disease-specific survival, and overall survival. Extracapsular spread retained a prognostic role for the risk of recurrence in multivariate analysis. Further, for patients with ≥ 2 positive LNs, the presence of bilateral positive LNs did not negatively impact the risk of recurrence or death compared with those with unilateral positive LNs. CONCLUSIONS Our data suggest that the prognostic effect of bilateral LNs reflects the worse prognosis of multiple positive LNs. Regarding prognosis, LN involvement should be categorized into 2 groups-1-2 positive LNs and ≥ 3 positive LNs.

Clinical significance of the interaction between non-coding RNAs and the epigenetics machinery: Challenges and opportunities in oncology

Non-coding RNAs and epigenetics are remarkable mechanisms of cellular control. In this review we underline the processes by which non-coding RNAs (ncRNAs), shown to be involved in various diseases, are capable of modifying and being modified by the epigenetic machinery, emphasizing the clinical importance of this network in cancer. Many ncRNAs have been described that play important roles in the establishment and maintenance of the epigenome. However, only a few studies deeply take into account the role of ncRNAs from a clinicopathological standpoint. The wide range of interactions between the non-coding RNome and the epigenome, and the roles of these networks in the pathogenesis, prognosis and early diagnosis of many diseases, present new challenges and opportunities for future studies regarding therapeutic strategies in oncology.

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

BackgroundVulvar 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.Methodsc-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.Resultsc-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).ConclusionsOur 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.

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 patients 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.

Characterization of sociodemographic and clinicopathological features in Brazilian patients with vulvar squamous cell carcinoma.

Aim: To investigate sociodemographic and clinical-epidemiological profiles of patients with vulvar carcinoma in São Paulo, the largest city of Brazil, to establish a more consistent profile of these features once the incidence of vulvar carcinoma has risen considerably. Data regarding the epidemiological aspects of this tumor are scarce. Methods: A retrospective study was performed using 300 medical records from patients diagnosed with squamous cell carcinoma of the vulva and surgically treated at A.C. Camargo Hospital in São Paulo, Brazil, from 1978 to 2009. Results: The median age of onset was 70 years, ranging from 15 to 98 years, and most women were white (88.51%). Most patients (83.54%) had little or no schooling and had the lowest survival curve. Many patients were diagnosed in the early stages of the disease (57.09% FIGO IB), 59% had complications due to surgery and 43.71% had disease recurrence, of which about 70% died. Conclusions: Our study adds 300 Brazilian cases of vulvar carcinoma to the world literature. Given the high rate of disease recurrence and mortality in Brazil, we conclude that regular gynecologic evaluation and educational policies should be reinforced in order to raise awareness for vulvar cancer.

Design of a miRNA sponge for the miR-17 miRNA family as a therapeutic strategy against vulvar carcinoma

Dysregulation of microRNAs has been studied thoroughly, and has been observed in a variety of tumors including vulvar carcinomas, a rare type of gynecological tumor with increasing incidence. However, very few therapeutic alternatives have reached the clinical setting, and there is an urgent unmet need to develop novel strategies for patients with this tumor type. Thus, a microRNA (miRNA) sponge for the miR-17 miRNA family was designed, synthesized and validated in vitro in order to explore a new therapeutic strategy based on inhibiting this oncogenic miRNA family in vulvar cancer. Members of the miR-17 family were evaluated for expression in a vulvar tumor cell line (SW954) and 20 HPV negative formalin-fixed paraffin-embedded (FFPE) samples by quantitative real-time PCR (qRT-PCR). Six in tandem, bulged sequences that were complementary to these miRNAs were designed, synthesized, cloned, and transfected into SW954 cells. A luciferase reporter assay with a psiCheck2 vector was used to test the specificity of the sponge sequences for miR-17 family miRNA binding. Taqman qRT-PCR was used to test how the sponges affected miRNA expression. In FFPE samples, higher expression of miR-20a and miR-106a correlated with deeper tumor invasion (P = 0.0187 and P = 0.0404, respectively). The luciferase reporter assay validated the specificity of the sponge for miR-17 family members. Using qRT-PCR, we confirmed this specificity with decreased expression in 5 (out of six) miRNAs of the miR-17 family in SW954 cells. Although our results are preliminary, these results demonstrate that these miRNA sponges are potent inhibitors of the miR-17 family of miRNAs in SW954. Therefore, this miRNA-specific sponge may be developed into a novel therapeutic treatment for patients with vulvar cancer.

Does the count after inguinofemoral lymphadenectomy in vulvar cancer correlate with outcome

BACKGROUND Inguinal lymph node (LN) metastasis is an important prognostic factor in vulvar cancer. Our aim was to determine the prognostic value of the number of resected LNs in inguinofemoral lymphadenectomy. METHODS A retrospective analysis was performed in a series of 158 individuals who underwent bilateral inguinofemoral lymphadenectomy for vulvar squamous cell carcinoma from January 1980 to February 2010. RESULTS The mean age was 67 years (range: 15-90). Median tumor size was 5 cm (range: 1-18). A median of 22.5 inguinal LNs (range: 2-57) was resected. Thirteen (8.2%) patients had <12 LNs resected, and 145 (91.8%) had ≥ 12 LNs resected. Eighty (50.6%) patients had LN metastasis, with a median of 2 positive LNs (range: 1-16). Of those with positive LNs, 19 (23.8%), 23 (28.8%), and 38 (47.5%) patients had 1, 2, and 3 or more positive LNs, respectively. Thirty-three (41.2%) patients had bilateral LN metastasis. For patients without LN involvement, we failed to observe any significant difference between patients with <12 LNs and ≥ 12 LNs that were resected with regard to risk of recurrence (p=0.97) and death from cancer (p=0.43) in 5 years. However, resection of <12 LNs in patients with positive LNs negatively impacted the risk of recurrence (p=0.003) and death from cancer (p=0.043). CONCLUSIONS Resection of fewer than 12 LNs in vulvar cancer has a negative impact on outcome for patients with positive inguinal LNs.

Noncoding RNA Profiles in Tobacco- and Alcohol-Associated Diseases

Tobacco and alcohol are the leading environmental risk factors in the development of human diseases, such as cancer, cardiovascular disease, and liver injury. Despite the copious amount of research on this topic, by 2030, 8.3 million deaths are projected to occur worldwide due to tobacco use. The expression of noncoding RNAs, primarily microRNAs (miRNAs) and long noncoding RNAs (lncRNAs), is modulated by tobacco and alcohol consumption. Drinking alcohol and smoking cigarettes can modulate the expression of miRNAs and lncRNAs through various signaling pathways, such as apoptosis, angiogenesis, and inflammatory pathways—primarily interleukin 6 (IL-6)/signal transducer and activator of transcription 3 (STAT3), which seems to play a major role in the development of diseases associated with these risk factors. Since they may be predictive and prognostic biomarkers, they can be used both as predictors of the response to therapy and as a targeted therapy. Further, circulating miRNAs might be valuable noninvasive tools that can be used to examine diseases that are related to the use of tobacco and alcohol. This review discusses the function of noncoding RNAs in cancer and other human tobacco- and alcohol-associated diseases.

Best practice for PTEN gene and protein assessment in anatomic pathology.

There is a lack of standardization of a best practice protocol for Phosphatase and Tensin Homolog (PTEN) assessment by immunohistochemistry in anatomic pathology routine practice. We performed immunohistochemistry for 19 antibodies against PTEN, eleven of which were excluded during the standardization step. Immunohistochemistry of the remaining eight antibodies was performed on a Tissue Microarray containing 55 prostate and 40 renal carcinoma samples. Fluorescent in situ hybridization (FISH) was used as reference standard for immunohistochemistry specificity evaluation. Concerning nuclear staining, polyclonal (Cat#22034-1-AP); 6H2.1 mMAb (Cat#ABM-2052), Y184 RabMAb (Cat#NB110-57441) and 217702 mMAb antibodies presented the highest agreement with fluorescent in situ hybridization (p<0.001 for all) and with regard to cytoplasmic staining, Y184 RabMAb (Cat#NB110-57441); polyclonal (Cat#22034-1-AP) and 217702 mMAb presented the highest agreement (p<0.001 for all). Our results indicate that several commercially available antibodies do not show reliability of sensitivity and specificity for PTEN evaluation and we propose 6H2.1 mMAb (Cat#ABM-2052) as the antibody of choice for laboratory standardization and best practice in clinical routine, which demonstrated excellent sensitivity for both nuclear and cytoplasmic staining, specificity for PTEN by Western blot and good correlation with PTEN status by FISH with regard to nuclear staining.

microRNA Portraits in Human Vulvar Carcinoma

Unregulated expression of microRNAs is well known and has already been demonstrated in many tumor types. However, in vulvar carcinoma this field has been unknown territory. Our study characterizes microRNA in vulvar tumors through an expression profile of 754 miRNAs, relating this with clinical and anatomopathologic data, and presence of HPV infection. Twenty HPV-negative and 20 HPV-positive samples, genotyped for high-risk HPVs (HPV16, 18, 31, 33) and a pool of seven normal vulvar skin samples were used for the identification of differentially expressed miRNAs by TLDA Quantitative Real Time PCR (qRT-PCR). Twenty-five differentially expressed microRNAs between HPV-positive and HPV-negative groups and 79 differentially expressed on the tumor compared with normal samples were obtained. A network between microRNA expression profiles and putative target mRNAs predicted by target prediction algorithms and previously demonstrated as relevant in vulvar carcinomas, such as TP53, RB, PTEN, and EGFR was constructed. Downregulation of both miR-223-5p and miR-19-b1-5p were correlated with the presence of lymph node metastasis; downregulation of miR-100-3p and miR-19-b1-5p were correlated with presence of vascular invasion; overexpression of miR-519b and miR-133a were associated with advanced FIGO staging. In conclusion, our study demonstrates that microRNAs may be clinically important in vulvar carcinomas and our findings may help for further studies on functional implications of miRNA deregulation in this type of cancer. Cancer Prev Res; 6(11); 1231–41. ©2013 AACR.