Phosphorylated S6 as an immunohistochemical biomarker of vulvar intraepithelial neoplasia.

As life expectancy lengthens, cases of non-viral-associated vulvar squamous cell carcinoma and its precursor lesion, so-called differentiated vulvar intraepithelial neoplasia (VIN), continue to increase in frequency. Differentiated VIN often is difficult to recognize and failure to detect it before invasion results in morbidity and mortality. Thus, identification of a reliable biomarker for this type of lesion would be of great clinical benefit. Our recent studies have identified activation (ser235/236 phosphorylation) of ribosomal protein S6 (p-S6) in basal epithelial cells as an event that precedes and accompanies laminin γ(2) overexpression in most preinvasive oral dysplasias. To test this as a potential biomarker of vulvar dysplasia, we immunostained seven differentiated VINs and nine papillomavirus-related 'classic' VINs, most of which were associated with carcinoma, for p-S6. All carcinomas, all differentiated VINs, and most classic VINs contained regions of p-S6 staining in the basal layer, whereas basal and parabasal cells of normal vulvar epithelium and hyperplastic and inflamed lesions lacking cellular atypia were p-S6 negative. Laminin γ(2) was expressed in a subset of VINs, always occurring within basal p-S6 positive regions, as we had found previously for oral dysplasias. Lichen sclerosus is considered a potential precursor of vulvar carcinoma. Two lichen sclerosus lesions of patients with a concurrent carcinoma and one of six lichen sclerosus lesions without atypia or known concurrent carcinoma were basal p-S6 positive. In summary, there is a distinct difference in p-S6 basal cell layer staining between benign and neoplastic vulvar squamous epithelium, with consistent staining of differentiated VIN and of some lichen sclerosus lesions. These results support further studies to assess the potential of p-S6 as a biomarker to identify vulvar lesions at risk of progressing to invasive cancer.

Relationship of margin status and radiation dose to recurrence in post-operative vulvar carcinoma.

OBJECTIVE: To evaluate the effect of margin status and radiation dose in patients treated with radiation therapy (RT) for vulvar cancer. Clinical outcomes included vulvar recurrence (VR), relapse-free survival (RFS) and overall survival (OS). METHODS: We retrospectively reviewed the records of 300 patients with Stage I-IVA vulvar cancer treated between 1988 and 2009. Slides were reviewed and margin status was scored as negative (≥ 1 cm), close (<1cm) or positive after formalin fixation. Cox proportional hazards models were constructed to determine significant prognostic factors for vulvar relapse. RESULTS: Of 205 eligible patients, 69 (34%) had negative surgical margins, 116 (56%) had close margins and 20 (10%) had positive margins. Median follow-up time was 49 months. The 4-year RFS rate was 53% and OS was 73%. Of 78 recurrences, 62 had the vulva as the first site of recurrence. The 4-year rates of freedom from vulvar recurrence were 82%, 63% and 37% for those with negative, close and positive margins, respectively (p for trend=0.005). On multivariate analysis, close margins (HR=3.03, 95% CI 1.46-6.26) and positive margins (HR=7.02, 95% CI 2.66-18.54) were associated with a significantly increased risk of vulvar relapse. Those who received a dose ≥ 56 Gy had a lower risk of relapse than those who received ≤ 50.4 Gy (p<0.05). Though recurrences were noted with margins up to 9 mm, the highest risk of vulvar recurrence was associated with margins ≤ 5 mm (p=0.002). CONCLUSIONS: Close or positive margins were associated with a significantly increased risk of vulvar recurrence. Radiation with a dose ≥ 56 Gy may decrease the risk of vulvar recurrence.

Immunomarkers in gynecologic cytology: the search for the ideal 'biomolecular Papanicolaou test'.

Harnessing the knowledge we have gained on the cell cycle disruption caused by human papillomaviruses (HPV) will likely lead to improved screening modalities for cervical cancer and its precursors. An easily applied biomarker that has high specificity and sensitivity would represent an attractive alternative or complement to cytology and HPV testing. To date, a number of promising markers have been investigated. These include p16(INK4A), MIB-1, BD-ProEx C, and L1. Newer possibilities involve a variety of gene products associated with aberrations of chromosome 3q, such as telomerase, p63, and PIK3CA, as well the combination of biomarkers such as p16(INK4A) and MIB-1 in the same assay. Although none of them has yet been incorporated into screening algorithms or found its way into routine practice, their performance characteristics remain a focus of current investigations. This review summarizes what we know and where we hope to go in translating basic pathobiology into clinical practice.

Differentiated vulvar intraepithelial neoplasia contains Tp53 mutations and is genetically linked to vulvar squamous cell carcinoma.

Differentiated vulvar intraepithelial neoplasia is a unique precursor to vulvar squamous cell carcinoma that is typically HPV-negative and frequently associated with nuclear p53 staining. These features imply a mode of pathogenesis involving somatic mutations. However, the genetic relationship of differentiated vulvar intraepithelial neoplasm and vulvar squamous cell carcinoma and the role of Tp53 mutations in this process have not been resolved. We analyzed 11 differentiated vulvar intraepithelial neoplasms and 6 associated vulvar squamous cell carcinomas. Sections were stained for p53 and p63 and DNA from multiple epithelial sites, representing normal control tissues (n=10), differentiated vulvar intraepithelial neoplasias (n=18), and vulvar squamous cell carcinomas (n=6), were obtained by laser capture microdissection, and sequenced for exons 2-11 of Tp53. Six of 10 cases contained at least one Tp53 mutation-positive differentiated vulvar intraepithelial neoplasia focus; 4 strongly p53 immuno-positive and 2 negative. Staining for p53 and p63 co-localized, targeting the immature epithelium, but surface epithelium was Tp53 mutation-positive. Four of five vulvar squamous cell carcinomas were Tp53 mutation-positive; two shared identical Tp53 mutation with adjacent differentiated vulvar intraepithelial neoplasia. Disparate foci of differentiated vulvar intraepithelial neoplasia often showed different mutations consistent with multiple neoplastic clones. Differentiated vulvar intraepithelial neoplasia is, with few exceptions, associated with Tp53 mutations and will be p53 immunopositive when missense mutations (versus some nonsense and all deletion mutations) are present. Multiple Tp53 mutations in different sites supports the presence of multiple independent genetic events, but shared Tp53 mutations in both differentiated vulvar intraepithelial neoplasia and vulvar squamous cell carcinoma support a genetic relationship between the two. The confinement of p53 staining to immature cell nuclei is consistent with maturation-dependent degradation of mutant p53 protein.

Biomarker (ProEx C, p16(INK4A), and MiB-1) distinction of high-grade squamous intraepithelial lesion from its mimics.

Topoisomerase IIalpha and minichromosome maintenance protein 2 are proteins associated with aberrant S-phase induction. The current study evaluated the performance of these biomarkers (ProEx C; TriPath Oncology, Burlington, NC) compared with p16(INK4A) and MiB-1 in distinguishing high-grade squamous intraepithelial lesions (HSILs) from HSIL mimics. We collected archival cervical biopsy, cone, and curettage specimens from 96 cases in which the differential diagnosis of HSIL vs reactive epithelial changes was considered. Hematoxylin- and eosin-stained slides were reviewed independently by three pathologists and scored for the presence or absence of SIL. Immunostains for ProEx C, p16, and MiB-1 were available for 95, 96, and 59 samples, respectively, and classified blinded to histological interpretation. Strong nuclear and cytoplasmic staining for p16 and staining for MiB-1 and ProEx C that extended beyond the lower one-third of the epithelium were scored as positive. Chi(2)-tests and receiver operating characteristic analysis were conducted to statistically compare biomarker immunostaining performance against majority histological interpretation of SIL. Agreement between pathologists was also assessed by the kappa-statistic. Inter-observer agreement ranged from fair to moderate (kappa=0.37-0.57). All three biomarkers correlated strongly with the majority diagnosis of SIL (P<0.001). Positive staining for ProEx C, p16, and MiB-1 was observed in 87% (N=52/60), 84% (N=51/61), and 94% (34/36), respectively, of SIL and negative in 71% (N=25/35), 63% (N=22/35), and 52% (N=12/23), respectively, of majority diagnoses of NoSIL. The combination of p16/ProEx C predicted more SIL (92%, N=33/36) and NoSIL (61%, N=14/23) than p16 plus MiB-1 (94%, N=34/36 and 43%, N=10/23), although this difference was not statistically significant. ProEx C appears to provide an equivalent level of sensitivity and a higher level of specificity for HSIL alone or in conjunction with p16. Its principal value may be in providing a lower false positive rate for NoSIL relative to MiB-1.

Repeating cytological preparations on liquid-based cytology samples: A methodological advantage?

This study investigates the rule that repeating cytological preparations on liquid-based cytology improves sample adequacy, diagnosis, microbiological, and hormonal evaluations. We reviewed 156 cases of pap-stained preparations of exfoliated cervical cells in two slides processed by DNA-Cytoliq System. After sample repeat/dilution, limiting factors affecting sample adequacy were removed in nine cases and three unsatisfactory cases were reclassified as satisfactory. Diagnosis was altered in 24 cases. Of these, the original diagnosis in 15 was atypical squamous cells of undetermined significance; after the second slide examination, diagnosis in 5 of the 15 cases changed to low-grade squamous intraepithelial lesion, 3 to high-grade squamous intraepithelial lesion, and 7 to absence of lesion. Microbiological evaluation was altered, with Candida sp. detected in two repeated slides. Repeat slide preparation or dilution of residual samples enhances cytological diagnosis and decreases effects of limiting factors in manually processed DIGENE DCS LBC.

Cervical cancer screening program of Paraná: cytohistological correlation results after five years.

Since its inception in November 1997, the Cervical Cancer Screening Program of Paraná (CCSPP), Brazil, has resulted in the cytological screening of 2,244,158 women, the coverage of the female population increasing from 43% to 86%. One thousand six hundred one cases screened by cytology, submitted to colposcopy, and subjected to treatment were selected. Cytopathological results were compared with those obtained on the basis of histological analyses of the loop electrical excision procedure specimens, and were subjected to statistical analyses. The data obtained were then compared with cytohistological correlation results from the first year of the program. Considering the exact correlation between cytological and histological diagnoses, the correlation index increased from 53.34% in the first year to 67.3% at the end of 5 yr of the program. Variations that occurred in each diagnostic category are discussed. This study demonstrates a significant improvement in the concordance between cytological and histological results for the 5-yr period compared with the first year of the CCSPP.

Nonneoplastic findings in loop electrical excision procedure specimens from patients with persistent atypical squamous cells of uncertain significance in two consecutive pap smears.

The present study aimed to determine the histopathological correspondence of the diagnosis of atypical squamous cells of uncertain significance (ASCUS) in the absence of squamous intraepithelial neoplasia (SIL) in loop electrical excision procedure (LEEP) products. Retrospective histopathological analysis of 70 LEEP products preceded by ASCUS was consistently detected by two consecutive Pap smears. The presence or absence of several histopathological findings in cases with (23/70) and without (47/70) histological diagnosis of SIL were compared. Immature squamous metaplasia, severe cervicitis, and tube-endometrial metaplasia were found with similar frequency on positive and negative neoplastic disease specimens. Reactive squamous atypia, keratosis, atrophy, glandular reactive/inflammatory atypia, tunnel clusters, and microglandular hyperplasia were less frequently found in LEEP specimens presenting with SIL. Reactive squamous atypia and keratosis were consistently associated with SIL absence and appear to be responsible for the cytological diagnosis of ASCUS (P < 0.05).

[Hpv cofactors in cervical carcinogenesis]. / Co-fatores do HPV na oncogênese cervical.

Human papillomavirus (HPV) plays a central rule in uterine cervix carcinogenesis. Other factors direct or indirectly influence the installation of this mechanism in cervical squamous epithelium. Investigations regarding mechanisms of interaction of these factors with viral elements are found in the literature of the last 20 years. The present review article discusses possible co-factors of HPV in the genesis of the squamous carcinoma of uterine cervix, taking into account only the factors whose association with the virus or cervical cancer has been documented by experimental studies, and not based just on clinical or epidemiological data. Among the approached parameters are immunological factors (local and humoral immune response), the association with Acquired Immune Deficiency Syndrome, genetic factors as protein p53 polymorphism, tabagism and the use of oral contraceptives. All these factors interact in variable intensity with oncoproteins and other HPV elements, increasing and facilitating the virus action in host cells, leading to the development of immortalization and carcinogenesis.

MOLECULAR MARKERS OF EARLY CERVICAL NEOPLASIA.

Pure morphological distinction of high-grade squamous intraepithelial lesions (HSILs) from their mimics can be challenging. Diagnosis can be difficult with nonconventional HSILs associated with a metaplastic phenotype, squamous intraepithelial lesions (SILs) that defy precise classification such as "eosinophilic dysplasias", and those that overlap with columnar neoplasms, including stratified variants of adenocarcinoma in situ ("SMILE"). Gene expression and protein profiling have identified biomarkers with the potential to decrease diagnostic variability and increase specificity of histological and cytological analysis. Among the ones clinically useful for HSIL detection are p16(INK4A) and MIB-1 which complement each other, differentiating SIL from normal/atrophic (MIB-1 low) or reactive/immature metaplastic (p16(INK4A) scattered) epithelium. Additional markers, including ProEx(TM) C, have been proposed but their added value is yet to be established. In the final analysis, biomarkers are most helpful for distinguishing benign immature or atrophic proliferations from HSIL. The distinction of LSIL from HSIL must be made on the hematoxylin and eosin-stained section and should be made with care, given the potential consequences of a diagnosis of CIN2 or CIN3.

Prognostic significance of lymph node variables and human papillomavirus DNA in invasive vulvar carcinoma.

OBJECTIVE: The present study investigates the influence of lymph node pathological features and HPV DNA status on the prognosis of vulvar invasive tumors. METHODS: This study includes 184 consecutive cases of primary invasive squamous cell carcinoma of the vulva treated by radical surgery from 1975 to 1992, in São Paulo, Brazil. Clinical follow-up data was collected from patient files and hematoxilin-eosin sections were reviewed. HPV detection and typing was done by polymerase chain reaction (PCR), using specific and generic primers, followed by dot blot hybridization (DBH) with type-specific oligonucleotide probes for 19 HPV types. Age-adjusted Kaplan-Meier survival curves and Cox proportional hazards models were used to analyze the cancer risk associations for all DNA and pathology-related variables. RESULTS: Among 161 cases tested by PCR, 38 (23.6%) were positive for high-risk HPV types. Regional lymph nodes of 43 cases, including all those of HPV-positive tumors and a sample of the ones removed from patients with HPV negative tumors, were evaluated by the same method. HPV DNA was found in the lymph nodes of 10 cases. In every case, at least one lymph node was metastatic and the HPV detected in the lymph nodes were of the same type as those found in the primary tumor in all cases. Multivariate analysis including age, race, pattern of invasion, tumor thickness, inflammatory reaction, surgical margins, number of node metastases, presence of extracapsular growth, depth of invasion, and presence of high-risk HPV DNA was performed. Following automated selections of this model, node variables important for prognosis that remained were number of node metastases and presence of extracapsular growth. CONCLUSIONS: Patients with four or more node metastases associated with extracapsular spread were 5.6 (95%CI: 2.3-13.1) times more likely to die from cancer and 10.0 (95%CI: 4.0-24.9) times more likely to have a recurrence than patients without metastases. The HPV status in the tumor was not important as a prognostic factor.

Cervicites e seus agentes na rotina dos exames colpocitológicos / Cervicitis, and its agents in the Pap smear routine

Introdução: cervicite é um dos assuntos mais controvertidos na patologia cervical e sua definição varia muito de acordo com a análise dos aspectos clínico, citológico, colposcópico e histológico. Objetivo: considerando a capacidade do teste de Papanicolaou na identificação de agentes microbiológicos e a leucorréia como causa mais freqüente de consulta ginecológica, este estudo tem como objetivo analisar a prevalência de cervicite e seus agentes microbiológicos na rotina dos exames colpocitológicos. Métodos: foram analisados 500 esfregaços cérvico-vaginais da rotina da Divisão de Patologia do Instituto Nacional do Câncer (DIPAT-INCA). Foram excluídos 171 (34,2%) casos por não conterem células glandulares endocervicais. Os critérios utilizados para o diagnóstico de cervicite foram: 1) alterações nas células metaplásicas e endocervicais-metacromasia, pseudo-eosinofilia, binucleação ou multinucleação, aumento do volume nuclear e nucléolos proeminentes; 2) intensidade do exsudato inflamatório; e 3) a quantidade de muco cervical presente no esfregaço. Resultados: dos 329 casos estudados, 207 casos (63%) apresentaram cervicite. A idade das pacientes variou de 15 a 74 anos, sendo a idade média de 36 anos. Quanto aos critérios utilizados para o diagnóstico, 98% apresentavam metacromasia, 97,8% pseudo-eosinofilia, 47,5% ativação nuclear e 6,7% binucleação ou multinucleação. Nenhum caso apresentou nucléolo proeminente. Apenas 0,8% dos casos apresentou cervicite linfócita. De acordo com a microbiota bacteriana e os agentes causais de inflamação, 47% apresentaram microbiota de bacilos de Dodërlein, 23,8% microbiota de bacilos curtos, 5,3% microbiota de cocos, 7,7% microbiota mista, 21,8% microbiota sugestiva de Gardnerella vaginalis, em 6,3% microbiota não-visualizada, 0,5% apresentou célula com inclusões sugestivas de clamídia, 4,3% tricomoníase e 2,4% candisíase. Conclusão: o presente estudo demonstrou a eficácia do método de Papanicolaou para o diagnóstico de cervicite.

Variantes de lesões intra-epiteliais escamosas: relato de quatro casos / Variants of intraepithelial squamous lesions: report of four cases

Entre a rotina de biópsias e produtos cirúrgicos provenientes do colo uterino, um número significativo de lesões intra-epiteliais escamosas (LIE) pode causar dificuldade quanto a caracterização e graduação histológica. Tais lesões têm sido identificadas e descritas isoladamente por artigos científicos como variantes histológicas de LIE cervicais. São elas a metaplasia papilar imatura atípica (MPIA) e as variantes de neoplasia intra-epitelial cervical graus II/III: queratinizante, com padrão metaplásico imaturo de crescimento e escamomucinosa. Neste artigo são exemplificados quatro casos representativos das entidades citadas acima, provenientes das rotinas do Programa de Prevenção do Câncer Ginecológico do Estado do Paraná e de um laboratório privado especializado em patologia ginecológica de Curitiba, o Laboratório de Citopatologia e Anatomia Patológica Annalab. Os principais critérios diagnósticos são descritos, assim como a correlação citológica e molecular relacionada à presença e à localização do ácido nucleico viral (papilomavírus humano [HPV]) nas lesões.

Etiopatogenia do câncer vulvar / Etiopathogenesis of vulvar cancer

Existem poucos trabalhos na literatura a respeito dos mecanismos moleculares envolvidos na patogênese do carcinoma vulvar. Estudos que servem como base para a compreensão destes mecanismos são discutidos de forma compreensiva neste artigo de revisão. As alterações genéticas, a associação com o papilomavírus humano (HPV) e outros possíveis fatores envolvidos na carcinogênese dos tumores vulvares são abordados

Co-fatores do HPV na oncogênese cervical / HPV Cofactors in cervical carcinogenesis

O papilomavírus humano (HPV) exerce um papel central na carcinogênese do colo uterino. Em torno a ele orbitam outros fatores que influenciam direta ou indiretamente a instalaçäo deste mecanismo no epitélio escamoso cervical. Investigaçöes a respeito dos mecanismos de atuaçäo e interaçäo desses co-fatores com os elementos virais encontram-se na literatura dos últimos 20 anos. O presente artigo de revisäo explora os possíveis co-fatores do HPV na gênese do carcinoma escamoso do colo uterino, levando em conta apenas os fatores cuja associaçäo com o vírus ou câncer cervical tenha sido documentada experimentalmente, e näo apenas clínica ou epidemiologicamente. Dentre os parâmetros abordados estäo os fatores imunológicos (resposta imune local e humoral), a associaçäo com a Síndrome da Imunodeficiência Adquirida Humana, fatores genéticos como o polimorfismo da proteína p53, o tabagismo e o uso de contraceptivos orais. Todos estes fatores interagem em menor ou maior intensidade com oncoproteínas e outros elementos do HPV, potencializando a açäo do vírus na célula hospedeira e facilitando o desenvolvimento dos processos de imortalizaçäo e carcinogênese

Revisäo das alteraçöes citomorfológicas da infecçäo pelo vírus do papiloma humano em citologia cervicovaginal / Cytological effects of human papillomavirus infection in cervical smears: a review article

Os autores revisam os principais critérios propostos pela literatura para o diagnóstico da infecçäo pelo vírus do papiloma humano (HPV) em esfregaçös cervicovaginais. A evoluçäo do conhecimento sobre as lesöes pré-malignas escamosas do colo uterino, os diferentes tipos de hpv e a interligaçäo entre estas entidades resultaram em diversas mdificaçöes no conceito e na nomenclatura cito-histológica destas lesöes. No decorrer deste artigo de revisäo, estas informaçöes säo equacionadas, e validade da aplicaçäo de critérios para o diagnóstico indireto do HPV pelo citopatologista é discutida

Correlaçäo cito-histológica em 326 pacientes submetidas a cirurgia de alta freqüência (CAF) no Programa de Prevençäo de câncer ginecológico do Estado do Paraná / Cyto-histological correlation in 326 patients submitted to loop electrosurgical excision oricedure (LEEP) in the cervical cancer program of state of Paraná, Brazil

O presente estudo teve como objetivo avaliar a correlaçäo dos laudos citopatológicos com os respectivos diagnósticos histopatológicos obtidos a partir de produtos de CAF. Para tanto, foram selecionados 326 pacientes submetidas a CAF no Programa de Prevençäo de Câncer Ginecológico do Estado do Paraná (PPCGPR), durante o período de um ano. Os diagnósticos citológicos emitidos foram confrontados com os diagnósticos histológicos e analizados pelo teste do qui-quadrado. A idade das pacientes foi, em média de 38 anos. Considerando-se a presença ou ausência de lesäo, a correlaçäo cito-histológica foi de 83 por cento. O maior índice de concordância entre as categorias diagnosticadas foi verificado nas lesöes de alto grau (63 por cento dos casos). Quanto às lesöes diagnosticadas pela citologia como de baixo grau, notou-se que, em 74 por cento das pecientes (17 casos), a avaliaçäo histológica revelou lesäo de alto grau. Dentre os casos dignosticados como lesöes de significado indeterminado (ASCUS e AGUS), verificou-se que 51 por cento (35 casos) dos casos näo apresentaram lesäo ao exame histológico. Dos nove casos diagnosticados citologicamente como carcinoma invasor, dois (22 por cento) foram confirmados, cinco (55 por cento) apresentaram lesäo no exame histológico. Dentre os casos citologicamente negativos, em que a CAF foi indicada a partir de achados colposcópicos, 71 por cento (20 casos) apresentaram lesäo na histologia. O presente estudo demonstrou a eficiência do programa na detecçäo de LIAG e revelou as categorias diagnósticas onde ocorreram as maiores dificukdades no diagnóstico