Immunohistochemical WWOX expression and association with angiogenesis, p53 expression, cell proliferation and clinicopathological parameters in cervical cancer / Avaliação da expressão do gene WWOX por avaliação imunohistoquímica, sua associação com marcador de angiogênese, expressão do p53, proliferação celular e parâmetros clinicopatológicos no câncer de colo uterino

Abstract Objective The current study evaluated the expression of WW domain-containing oxidoreductase (WWOX), its association with clinicopathological features and with p53, Ki-67 (cell proliferation) and CD31 (angiogenesis) expression in patients with invasive cervical squamous cell carcinoma (ICSCC). To the best of our knowledge, no other study has evaluated this association. Methods Women with IB stage-ICSCC (n = 20) and women with uterine leiomyoma (n = 20) were prospectively evaluated. Patients with ICSCC were submitted to type BC1 radical hysterectomy and pelvic lymphadenectomy. Patients in the control group underwent vaginal hysterectomy. Tissue samples were stained with hematoxylin and eosin for histological evaluation and protein expression was detected by immunohistochemistry studies. Results The WWOX expression was significantly lower in the tumor compared with the expression in thebenign cervix (p = 0.019). TheWWOXexpressionwas inversely associated with the CD31 expression in the tumor samples (p = 0.018). There was no association betweentheWWOXexpression with the p53 expression (p = 0.464)or the Ki-67expression (p = 0.360) in the samples of invasive carcinoma of the cervix. There was no association between the WWOX expression and tumor size (p = 0.156), grade of differentiation (p = 0.914), presence of lymphatic vascular invasion (p = 0.155), parametrium involvement (p = 0.421) or pelvic lymph node metastasis (p = 0.310) in ICSCC tissue samples. Conclusion The results suggested that WWOX may be involved in ICSCC carcinogenesis, and this marker was associated with tumor angiogenesis.

Resumo Objetivo O presente estudo avaliou a expressão do WWOX, sua associação com características clinicopatológicas e com a expressão do p53, ki-67 (proliferação celular) e CD31 (angiogênese) em pacientes com carcinoma invasivo de células escamosas do colo uterino, ou simplesmente câncer do colo uterino (CCE). Métodos Foram avaliadas prospectivamente pacientes com CCE no estágio IB (n = 20) e mulheres com mioma uterino, no grupo controle (n = 20). As pacientes com CCE foram submetidas à histerectomia radical e à linfadenectomia pélvica do tipo B-C1. As mulheres no grupo-controle foram submetidas à histerectomia vaginal. As amostras de tecido foramcoradas comhematoxilina e eosina para avaliação histológica e a expressão das proteínas foi detectada por imuno-histoquímico. Resultados A expressão do WWOX foi significativamente menor no tumor quando comparada com sua expressão no colo do útero benigno (p = 0,019). A expressão tumoral de CD31 foi inversamente associada à expressão de WWOX (p = 0,018). Sua expressão não foi associada à expressão tumoral de p53 e Ki-67 em pacientes com CCE (p = 0,464 e p = 0,360, respectivamente). Não houve associação entre a expressão de WWOX e o tamanho do tumor (p = 0,156), grau de diferenciação (p = 0,914), presença de invasão vascular linfática (p = 0,155), comprometimento do paramétrio (p = 0,421) ou metástase dos linfonodos pélvicos (p = 0,310) em pacientes com CCE. Conclusão Os resultados sugeriram que o WWOX pode estar envolvido na carcinogênese do CICECU e esse marcador foi associado à angiogênese tumoral.

Evaluation of the p16 and Ki-67 Biomarkers as Predictors of the Recurrence of Premalignant Cervical Cancer Lesions after LEEP Conization / Avaliação dos biomarcadores p16 e Ki-67 como preditores de recidivas de lesões pré-cancerígenas do colo do útero após conização por cirurgia de alta frequência

Abstract Objective To evaluate the expressions of biomarkers p16 and Ki-67 in low-grade (LG) or high-grade (HG) lesions, and to relate them to risk factors and the recurrence of these lesions. Methods A retrospective case-control study of 86 patients with LG and HG lesions who underwent a loop electrosurgical excision procedure (LEEP) between 1999 and 2004. The control group was composed of 69 women with no recurrence, and the study group, of 17 patients with recurrence. All patients were followed-up over a two-year period after surgery, and screened every six months, including cytology and colposcopy. Biopsy samples collected from LEEP were submitted to immunohistochemical analysis for p16 and Ki-67. The statistical analysis was performed using the Statistical Package for the Social Sciences software (SPSS, IBM-SPSS, Inc., Chicago, IL, US), with a significant p < 0.05. Results The biomarkers p16 and Ki-67, separately or combined, showed no relation to recurrence on the total analysis. However, evaluating specifically HG lesions, the positive expression (2+ and 3 + ) of p16/Ki-67 was associated with recurrence (0.010). In addition, p16 isolated was also more expressive in HG lesions (2+ and 3 + , p= 0.018), but it was unrelated to recurrence. Conclusion Proteins p16 and Ki-67, both isolated and combined, are not reliable primary markers for the recurrence of cervical lesions in the majority of LG lesions. However, analyzing only the group with prior diagnosis of HG lesions, the expressions of p16 and of p16/Ki-67 were associated with recurrence, and they may be useful in monitoring these cases.

Resumo Objetivo Avaliar as positividades dos biomarcadores p16 e Ki-67 em lesões de baixo grau (BG) ou de alto grau (AG), e relacioná-las com os fatores de risco e com a recidiva dessas lesões. Métodos Estudo retrospectivo caso-controle, com 86 pacientes com lesões de BG e AG, submetidas à conização por cirurgia de alta frequência entre 1999 e 2004. O grupo de controle foi constituído de 69 mulheres sem recidivas, e o grupo de estudo, de 17 pacientes que recidivaram. Todas as pacientes foram acompanhadas durante dois anos após a cirurgia, com controle a cada seis meses, incluindo citologia e colposcopia. As peças provenientes de cirurgia de alta frequência (CAF) foram submetidas a imunohistoquímica para p16 e Ki-67. A análise estatística foi realizada com o programa Statistical Package for the Social Sciences (SPSS, IBM-SPSS, Inc., Chicago, IL, EUA), com p significante quando < 0,05. Resultados Isoladamente ou em conjunto, p16 e Ki-67 não se relacionaram com as recidivas quando analisados na totalidade dos casos. Entretanto, avaliando especificamente as lesões de AG, a positividade (2+ e 3 + ) do conjunto p16/Ki-67 foi relacionada com recidiva (0,010). No mais, p16, isoladamente, foi também mais expresso nas lesões de AG (2+ e 3 + , p= 0,018), mas sem relação com recidiva. Conclusão Quando testadas na totalidade dos casos, as proteínas p16 e Ki-67, separadas ou em conjunto, se mostraram ineficientes como marcadores primários de recidiva de lesões precursoras. Entretanto, quando avaliadas somente no grupo diagnóstico prévio de lesão de AG, as expressões das proteínas p16 e p16/Ki-67 têm relação com a recidiva, e podem ser úteis no acompanhamento desses casos.

Immunohistochemical Expression of the Tumor Suppressor Protein p16 INK4a in Cervical Adenocarcinoma / Expressão imunhistoquímica da proteína de supressão tumoral p16 INK4a em adenocarcinoma cervical

ABSTRACT Objective: To evaluate the diagnostic utility of the p16ink4a protein expression as a marker for adenocarcinoma of the cervix. Methods: In a cross-sectional study, p16ink4a expression was evaluated in 30 cervical biopsies from patients diagnosed with invasive adenocarcinoma from 2 reference clinics in Brazil, and compared with 18 biopsies of endocervical polyps (control cases). The performance of the tests for p16ink4a was evaluated using a conventional contingency table, and the Kappa (k) index was used to evaluate the agreement of the marker with the tissue diagnosis. Results: In total, 66% of the invasive adenocarcinoma cases were positive for p16ink4a. All of the adenomatous polyps cases used as negative controls were shown to be negative for p16ink4a. The marker showed a high sensitivity and a high negative predictive value. The Kappa index was good for p16ink4a (k 1/4 0.6). Conclusion: Considering the strong association between the p16ink4a marker and the cervical adenocarcinoma, its use represents an important tool for reducing incorrect diagnoses of adenocarcinoma and thereby avoiding overtreatment.

RESUMO Objetivo: Avaliar a utilidade diagnóstica da expressão da proteína p16ink4a como marcador de adenocarcinoma do colo. Métodos: Em estudo transversal, a expressão de p16ink4a foi avaliada em 30 biópsias cervicais de pacientes diagnosticadas com adenocarcinoma invasivo de colo uterino provenientes de dois serviços de referência no Brasil, comparando com achados em 18 biópsias de pólipos endocervicais (grupo de controle). Para avaliar a performance do teste, foi utilizada tabela de contingência convencional, e para avaliar a concordância com o diagnóstico, foi aplicado o índice de Kappa (k). Resultados: No total, 66% dos casos de adenocarcinoma invasivo foram positivos para p16ink4a. Todos os pólipos adenomatosos foram negativos para p16ink4a. O marcador mostrou uma alta sensibilidade e alto valor preditivo negativo. O índice de Kappa foi bom para p16ink4a (k 1/4 0.6). Conclusion: Considerando a forte associação entre o marcador p16ink4a e o adenocarcinoma cervical, seu uso representa uma ferramenta importante para reduzir o risco de diagnóstico incorreto de adenocarcinoma e, por conseguinte, evitar o excesso de tratamentos.

The role of P16ink4a and P53 immunostaining in predicting recurrence of HG-CIN after conization treatment / O papel da expressão imunoistoquímica do P16ink4a e do P53 na predição da recorrência da nic-ag após tratamento por conização

Objective: Io evaluate the expression of p16INK4a and p53 biomarkers in conization specimens from patients with high grade cervical intraepithelial neoplasia (HG-CIN), correlating them with the ability to predict the recurrence. Methods : we conducted a retrospective study of patients with HG-CIN in cervical biopsy treated with conization between January 1999 and January 2006 who had a minimum follow-up of 18 months. The expression of the p16 and p53 was assessed by tissue microarrays and correlated with disease recurrence. For analysis, we used the test of proportions (chi-square), considering value p<0.05, 95% CI and calculations of sensitivity, specificity and accuracy of these immunomarkers in predicting recurrence. Results : the series comprised 83 patients aged between 16 and 86 years (35±11.7), divided into two groups: 30 with HG-CIN recurrence (study group) and 53 without recurrence (control group). Mean age, parity, smoking and conization technique were similar in both groups. The p53 expression was present in 43% of the study group and 57% of the control group, and the p16 was present in 43% of the study group and in 57% of the control group (p>0.05). p53 had a positive predictive value (PPV) of 42% and negative predictive value (NPV) of 73%, sensitivity 70%, specificity of 47% and accuracy of 59%. The p16, PPV 42%, NPV 72%, sensitivity 66%, specificity of 49% and accuracy of 56%. Conclusion : immunohistochemistry expression of p53 and p16 showed low sensitivity and low specificity as predictors of HG-CIN recurrence after conization treatment.

Objetivo : avaliar a expressão dos biomarcadores p16INK4a e p53, nas peças de conização de pacientes com neoplasia intraepitelial cervical de alto grau (NIC-AG), correlacionando com a capacidade de predizer o risco de recorrência. Métodos : estudo retrospectivo de pacientes com NIC-AG em biópsia de colo uterino, tratadas por conização, entre janeiro de 1999 e janeiro de 2006 e seguimento mínimo de 18 meses. A expressão dos biomarcadores p16 e p53 foi avaliada através de técnica de microarranjos teciduais e correlacionada com a recorrência da doença. Para análise utilizou-se o teste das proporções (qui-quadrado), considerando valor p<0,05, IC95% e cálculos de sensibilidade, especificidade e acurácia destes imunomarcadores na predição de recorrência. Resultados : oitenta e três pacientes, idade entre 16 e 86 anos (35±11,7), divididas em dois grupos: 30 com recorrência da NIC-AG (grupo estudo) e 53 sem recorrência (grupo controle). A média de idade, paridade, hábito de fumar e técnica de conização foram semelhantes nos dois grupos. A expressão do p53 esteve presente em 43% do grupo estudo e 57% do grupo controle e para o p16 esteve presente em 43% do grupo estudo e 57% do grupo controle (p>0,05). O p53 apresentou valor preditivo positivo (VPP) de 42% e valor preditivo negativo (VPN) de 73%, sensibilidade de 70%, especificidade de 47% e acurácia de 59%. O p16, VPP de 42% e VPN de 72%, sensibilidade de 66%, especificidade de 49% e acurácia de 56%. Conclusão : a expressão imunoistoquiímica do p53 e do p16 apresentaram baixa sensibilidade e baixa especificidade como marcadores capazes de predizer a recorrência da NIC-AG tratada por conização.

Surface activity, lipid profiles and their implications in cervical cancer.

BACKGROUND: The profiles of lipids in normal and cancerous tissues may differ revealing information about cancer development and progression. Lipids being surface active, changes in lipid profiles can manifest as altered surface activity profiles. Langmuir monolayers offer a convenient model for evaluating surface activity of biological membranes. AIMS: The aims of this study were to quantify phospholipids and their effects on surface activity of normal and cancerous human cervical tissues as well as to evaluate the role of phosphatidylcholine (PC) and sphingomyelin (SM) in cervical cancer using Langmuir monolayers. METHODS AND MATERIALS: Lipid quantification was done using thin layer chromatography and phosphorus assay. Surface activity was evaluated using Langmuir monolayers. Monolayers were formed on the surface of deionized water by spreading tissue organic phase corresponding to 1 mg of tissue and studying their surface pressure-area isotherms at body temperature. The PC and SM contents of cancerous human cervical tissues were higher than those of the normal human cervical tissues. Role of PC and SM were evaluated by adding varying amounts of these lipids to normal cervical pooled organic phase. Statistical analysis: Student's t-test (p < 0.05) and one-way analysis of variance (ANOVA) was used. RESULTS: Our results reveals that the phosphatidylglycerol level in cancerous cervical tissue was nearly five folds higher than that in normal cervical tissue. Also PC and sphingomyelin SM were found to be the major phospholipid components in cancerous and normal cervical tissues respectively. The addition of either 1.5 microg DPPC or 0.5 microg SM /mg of tissue to the normal organic phase changed its surface activity profile to that of the cancerous tissues. Statistically significant surface activity parameters showed that PC and SM have remarkable roles in shifting the normal cervical lipophilic surface activity towards that of cancerous lipophilic component. CONCLUSION: The Langmuir monolayer technique was sensitive to detect changes in tensiometric profiles of cervical cancers and these could be modulated by alterations in phosphatidylcholine and sphingomyelin levels. Therapeutic strategies may be designed to modulate these tensiometric profiles and lipid constituents of cancerous tissues.

Oncoprotein c-erbB-2 in squamous cell carcinoma of the uterine cervix and evaluation of its significance in response of disease to treatment.

Tissues from 50 cases of squamous cell carcinoma of the uterine cervix were analysed for immunohistochemical expression of c-erbB-2 oncoprotein and the patients were followed-up for 2 years. Immunopositivity of c-erbB-2 was studied with reference to clinical stage, histopathological differentiation and response to the cancer therapy. Expression of c-erbB-2 protein was found to be higher (37.5%) in cases with stage II disease, whereas more expressions were noticed in poorly differentiated squamous cell carcinoma (33.3%). Among cases who showed complete response to the treatment, 20.8% were positive for c-erbB-2 oncoprotein. On the contrary, 36.8% of prognostically unfavourable cases revealed positivity for c-erbB-2 immunostaining. However, the difference between c-erbB-2 expressions of these two said groups of patients, which were divided in accordance with the response to treatment, did not attain to statistical significance. Study on c-erbB-2 among larger number of patients with cervical carcinoma may prove to be an important factor in response to cancer therapy.

Significance of mucin stain in uterine cervical cancers.

Histopathological examination of 140 cases of cervical cancer was supplemented with staining for the presence of mucin. Secretion of mucin was detected in 27 cases. Fourteen cases of large cell non-keratinizing (LCNK) squamous cell carcinoma were reclassified, 12 as squamous cell carcinoma with mucin secretion and two as adeno-squamous carcinoma. One case of undifferentiated carcinoma was labelled as adenocarcinoma on the basis of mucin stain. None of the keratinizing squamous cell carcinomas were positive for mucin. The results indicate that mucin expression should be looked for even in those carcinomas where no obvious gland formation is seen.

Detection of HSV-2 antigen in carcinoma cervix and premalignant conditions by immuno-cytochemistry.

One hundred and twelve cases of cervical pathology (58 paraffin sections and 54 cervical smears) were assessed by PAP technique and 30 cases by indirect immunofluorescent technique. Forty-two normal cervical smears from the age matched controls were stained by indirect immuno-fluorescent technique. HSV-2 antigen was detected by PAP method in 86 out of 112 cases (78.57%) i.e. 50/55 squamous cell carcinoma, 13/13 carcinoma in situ, 11/15 severe dysplasia, 3/4 moderate dysplasia and 9/16 mild dysplasia. The amount of antigen was maximum in squamous cell carcinoma and decreased in carcinoma in situ, severe, moderate and mild dysplasia in descending order. Three cases of adenocarcinoma cervix were negative. Only one case out of 42 controls was positive. HSV-2 antigen was detected by an indirect IF technique in 8/9 squamous cell carcinoma, 2/3 carcinoma in situ, 3/7 dysplasia, O/1 adenocarcinoma and 4/10 inflammatory cases. The above findings support the association between HSV-2 and squamous cell carcinoma cervix, as well as carcinoma in situ which is statistically significant.

Serum protein-bound hexosamine in tumours of the genital tract

Serum protein-bound hexosamine was estimated in one hundred and four women. In patients with fibroid uterus there was an increase in its level. In the malignancy group of patients there was a highly statistically significant increase in its mean than that of controls. Its level could be of value in early detection of cancer body