One Step Histological Detection and Staining of the PTEN Tumor Suppressor Protein by a Single Strand DNA.

Antibodies are the most used technological tool in histochemistry. However, even with monoclonal antibodies, their standardization is difficult due to variation of biological systems as well as to variability due to the affinity and amplification of the signal arising from secondary peroxidase detection systems. In this article we combined two synthetic molecules to facilitate the standardization of a detection protocol of protein markers in histological sections. The first molecule was an aptamer, a 50-base single-stranded DNA fragment, which recognizes a PTEN tumor suppressor. The second molecule used was also another single stranded 18-base aptamer DNA fragment, which forms a quadruplex structure guanine box. This G-quadruplex recognizes and attaches a molecule of hemin, increasing the catalytic capacity for the hydrogen peroxide. Our results show how the correct structural design of DNA combining an aptamer together with the peroxidase-like DNAzyme allows to detect proteins in histological sections. This tool offers the standardization of the detection of prognostic markers in cancer, in quality and quantity, due to its synthetic nature and its 1:1 antigen:enzyme ratio. This is the first time that reproducible results have been presented in histological sections staining a cancer marker using a single-stranded DNA molecule with dual function.

Contribution of Human papillomavirus in neuroendocrine tumors from a series of 10,575 invasive cervical cancer cases.

AIMS: Neuroendocrine tumors (NET) of the cervix are rare tumors with a very aggressive course. The human papillomavirus (HPV) has been linked to its etiology. The objective of this study is to describe HPV prevalence and genotype distribution of NET. METHODS AND RESULTS: Forty-nine tumors with histological neuroendocrine features were identified among 10,575 invasive cervical cancer (ICC) cases from an international study. HPV DNA detection was done using SPF10/DEIA /LiPA25 system. Immunohistochemical (IHC) staining for neuroendocrine markers (chromogranin A, synaptophysin, CD56) and for p16INK4a as a surrogate for HPV transforming infection was performed. In 13 samples with negative IHC for all 3 neuroendocrine markers studied, it was possible to conduct electron microscopy (EM). NET represented 0.5% of the total ICC series and HPV was detected in 42 out of 49 samples (85.7%, 95%CI:72.8%,94.1%). HPV16 was the predominant type (54.8%), followed by HPV18 (40.5%). p16INK4a overexpression was observed in 38/44 cases (86.4%). Neuroendocrine IHC markers could be demonstrated in 24/37 (64.9%) cases. EM identified neuroendocrine granules in 8 samples with negative IHC markers. CONCLUSIONS: Our data confirms the association of cervical NET with HPV and p16INK4a overexpression. Specifically, HPV16 and 18 accounted together for over 95% of the HPV positive cases. Current HPV vaccines could largely prevent these aggressive tumors.

Argentophilic nucleolus organizer region as a proliferation marker in cervical intraepithelial neoplasia grade 1 of the uterine cervix.

AIM: p16INK4a and argentophilic nucleolus organizer region (AgNOR) can be used as markers for progression of cervical intraepithelial neoplasia grade 1 (CIN1) of the uterine cervix. Our objective was to study the predictive value of the AgNOR technique as a progression marker of CIN1 and its correlation with p16INK4A. MATERIAL AND METHODS: One uterine cervix biopsy from each of 75 patients with diagnosis of CIN1 was selected. All of these patients underwent a second biopsy, and these were also used for the study. RESULTS: The second biopsies showed: regression (20 patients), persistent CIN1 (38 patients), progression to CIN2 (10 patients) and progression to CIN3 (seven patients). p16INK4A showed reactivity in 67 of the 75 first CIN1 biopsies: 12 of the 20 cases that cleared the lesions and the 55 cases with persistent or progressive lesions were positive for p16INK4a (specificity: 40%; sensitivity: 100%; positive predictive value [PPV]: 82%; negative predictive value [NPV]: 100%). Samples with AgNOR areas less than 3.0 µ(2) returned in all cases, but patients whose lesions persisted or progressed to CIN2/CIN3, showed AgNOR areas greater than 3.0 µ(2) in 50/55 cases (specificity: 100%; sensitivity: 91%; PPV: 100%; NPV: 80%). CONCLUSIONS: p16INK4a is expressed in a high percentage of returning lesions. AgNOR might be a better marker of proliferation of CIN1 than p16INK4a (PPV = 100%), which means that a value greater than 3.0 µ(2) indicates the persistence or progression of the lesion. As its NPV is 80%, a value of AgNOR area less than 3.0 µ(2) in CIN1 leaves a margin of doubt about the future behavior of the lesion.

Prenatal hyperandrogenization induces metabolic and endocrine alterations which depend on the levels of testosterone exposure.

Prenatal hyperandrogenism is able to induce polycystic ovary syndrome (PCOS) in rats. The aim of the present study was to establish if the levels of prenatal testosterone may determine the extent of metabolic and endocrine alterations during the adult life. Pregnant Sprague Dawley rats were prenatally injected with either 2 or 5 mg free testosterone (groups T2 and T5 respectively) from day 16 to day 19 day of gestation. Female offspring from T2 and T5 displayed different phenotype of PCOS during adult life. Offspring from T2 showed hyperandrogenism, ovarian cysts and ovulatory cycles whereas those from T5 displayed hyperandrogenism, ovarian cysts and anovulatory cycles. Both group showed increased circulating glucose levels after the intraperitoneal glucose tolerance test (IPGTT; an evaluation of insulin resistance). IPGTT was higher in T5 rats and directly correlated with body weight at prepubertal age. However, the decrease in the body weight at prepubertal age was compensated during adult life. Although both groups showed enhanced ovarian steroidogenesis, it appears that the molecular mechanisms involved were different. The higher dose of testosterone enhanced the expression of both the protein that regulates cholesterol availability (the steroidogenic acute regulatory protein (StAR)) and the protein expression of the transcriptional factor: peroxisome proliferator-activated receptor gamma (PPAR gamma). Prenatal hyperandrogenization induced an anti-oxidant response that prevented a possible pro-oxidant status. The higher dose of testosterone induced a pro-inflammatory state in ovarian tissue mediated by increased levels of prostaglandin E (PG) and the protein expression of cyclooxygenase 2 (COX2, the limiting enzyme of PGs synthesis). In summary, our data show that the levels of testosterone prenatally injected modulate the uterine environment and that this, in turn, would be responsible for the endocrine and metabolic abnormalities and the phenotype of PCOS during the adult life.

Apoptosis is increased and cell proliferation is decreased in out-of-phase endometria from infertile and recurrent abortion patients.

BACKGROUND: Various endometrial abnormalities have been associated with luteal phase deficiency: a significant dyssynchrony in the maturation of the glandular epithelium and the stroma and a prevalence of out-of-phase endometrial biopsy specimens. Out-of phase endometrium is a controversial disorder related to failed implantation, infertility and early pregnancy loss. Given that the regulation of the apoptotic process in endometrium of luteal phase deficiency is still unknown, the aim of this study was to evaluate cell proliferation, apoptosis and the levels of the main effector caspase, caspase-3 in the luteal in-phase and out-of-phase endometrium. METHODS: Thirty-seven endometrial samples from sterile or recurrent abortion patients were included in this study: 21 in-phase samples (controls) and 16 samples with out-of-phase endometrium. Biopsy specimens of eutopic endometrium were obtained from all subjects during days 21-25 of the menstrual cycle. The endometrium with endometrial maturity of cycle day 25 or less at the time of menstruation was considered out-of phase. Endometrial tissues were fixed in 10% buffered formaldehyde. For apoptosis quantification, sections were processed for in situ immunohistochemical localization of nuclei exhibiting DNA fragmentation, by the terminal deoxynucleotidyl transferase (TdT)-mediated dUTP digoxygenin nick-end labeling (TUNEL) technique. Expressions of Proliferating Cell Nuclear Antigen (PCNA) as a marker of cell proliferation, and of cleaved caspase-3 as a marker of apoptosis, were assessed by immunohistochemistry in the luteal in-phase and out-of-phase endometrium from infertile and recurrent abortion patients. RESULTS: Luteal out-of-phase endometrium had increased apoptosis levels compared to in-phase endometrium (p < 0.05). Caspase-3 evaluation confirmed these results: the luteal out-of-phase endometrium showed augmented cleaved caspase-3 expression (p < 0.005). As well, our data demonstrated that the luteal out-of-phase endometrium expresses decreased PCNA levels (p < 0.05), showing that cell proliferation is diminished in this tissue. CONCLUSIONS: this study represents the first report describing variations at the cell proliferation and cell death levels in the out-of-phase endometrium in comparison with in-phase endometrium from infertile and recurrent abortion patients. Further studies are needed to elucidate a potential role of these alterations in the physiopathology of luteal phase deficiency.

Cytosolic accumulation of HPV16 E7 oligomers supports different transformation routes for the prototypic viral oncoprotein: the amyloid-cancer connection.

E7 is the major transforming activity in human papillomaviruses, a causal agent for cervical cancer. HPV16 E7 is a small protein with a natively unfolded domain for which dozens of specific cellular targets were described, and represents a prototypical oncoprotein among small DNA tumor viruses. The protein can form spherical oligomers with amyloid-like properties and chaperone activity. Conformation specific antibodies locate endogenous oligomeric E7 species in the cytosol of 3 model cell lines, strongly co-localizing with amyloid structures and dimeric E7 localizes to the nucleus. The cytosolic oligomeric E7 appear as the most abundant species in all cell systems tested. We show that nuclear E7 levels are replenished dynamically from the cytosolic pool and do not result from protein synthesis. Our results suggest that long-term events related to de-repression of E7 would cause accumulation of excess E7 into oligomeric species in the cytosol. These, together with the known target promiscuity of E7, may allow interactions with many of the non-pRb dependent targets described. This hypothesis is further supported by the detection of E7 oligomers in the cytosol of cancerous cells from tissue biopsies.

The effects of metformin on uterine tissue of hyperandrogenized BALB/c mice.

The present study investigated the role of the N, N'-dimethylbiguanide metformin (50 mg/kg body weight in 0.05 ml water, given orally with a canulla) in preventing the adverse effects generated by hyperandrogenism on uterine function. Daily injection of dehydroepiandrosterone (DHEA: 6 mg/100 g body weight in 0.1 ml oil) for 20 consecutive days induces polycystic ovaries in BALB/c mice. In this model we found that DHEA produced alterations on uterine histology closely related to the development of pre-cancerous structures concomitantly with increased incidence of uterine apoptosis. The injection of DHEA induced a pro-inflammatory status since uterine prostaglandin (PG) F2 alpha levels and cyclooxygenase 2 were increased although PGE levels were decreased. Furthermore, DHEA promoted a pro-oxidant status since it increased nitric oxide synthase (NOS) activity and decreased superoxide dismutase and catalase activities and the antioxidant metabolite glutathione levels. DHEA also regulated the percentages of CD4+ and CD8+ T lymphocyte that infiltrate uterine tissue. When metformin was administered together with DHEA uterine histology and apoptosis did not differ when compared with controls. Therefore, metformin prevented the pro-inflammatory and pro-oxidative status generated by DHEA and restores the ratios of CD4+ and CD8+ T cells to those observed in controls. We conclude that metformin is able to restore either directly or indirectly uterine function by preventing some inflammatory and oxidative alterations produced by hyperandrogenism.

Detrimental effects of hyperandrogenism on uterine functions.

The aim of the present work was to study some of the adverse effects produced by hyperandrogenism on the uterine function. Daily injection of dehydroepiandrosterone (DHEA: 6 mg/ 100 g body weight, sc) for 20 consecutive days induced polycystic ovaries in BALB/c mice. In this model, we found that DHEA produced alterations on uterine histology closely related to the development of tumour structures. In addition, hyperandrogenism induced a pro-inflammatory and a pro-oxidant condition represented by increased levels of prostaglandin F2 alpha production and uterine nitric oxide synthase (NOS) activity and by a decrease in both superoxide dismutase (SOD) and catalase (CAT) activities together with a decrease in the levels of the antioxidant metabolite glutathione (GSH). DHEA also induced an increase in CD4+ together with a decrease in the CD8+ T lymphocytes that infiltrate the uterine tissue. We conclude that this intricate network of regulators could be responsible for the low rate of implantation observed in women with polycystic ovary syndrome.

Association of DRB1 and DQB1 HLA class II polymorphisms in high-grade and neoplastic cervical lesions of women from Argentina.

OBJECTIVES: To investigate associations between HLA-DRB1 and HLA-DQB1 polymorphisms with low- and high-grade cervical lesions in Argentine population and the role human papillomavirus status in these associations. MATERIALS AND METHODS: Cervical biopsies and peripheral blood samples were taken from 32 patients with cervical intraepithelial neoplasia grade 1 (CIN 1) and 44 patients with cervical CIN 3 or invasive squamous cell carcinomas. Cervical cells and peripheral blood samples from 40 healthy women were included as control group. Human papillomavirus detection and typing were done by polymerase chain reaction (PCR) MY09, 11-restriction fragment length polymorphisms, or PCR 5+, 6+ dot-blot hybridization, and HLA DR/DQ typing by the PCR-sequence-specific oligonucleotide probes method. RESULTS: HLA-DRB1*04 and HLA-DQB1*0302 were found to be positive associated with the CIN 3/invasive squamous cell carcinomas subgroup, whereas HLA-DRB1*13 and HLA-DQB1*02 were negatively associated with the same group, when comparing to the control group. CONCLUSIONS: The data support the hypothesis that HLA-DRB1*04 and HLA-DQB1*0302 may be considered risk factors for malignant progression, whereas HLA-DRB1*13 and HLA-DQB1*02 may have a protective role. Further studies with a larger group are needed to confirm these susceptibility and protective roles in disease progression in Argentine population.

Interlaboratory quality control in gynecologic cytopathology using the novel CONQUISTADOR software. Interobserver reproducibility in the Latin American screening study.

OBJECTIVE: The recently developed software (CONQUISTADOR), capable of computing all intralaboratory and interlaboratory quality control (QC) indicators, was used to evaluate the diagnostic agreement among 4 cytology laboratories participating in the LAMS Study. STUDY DESIGN: The study was an interlaboratory exchange of specially designed 5 slide sets, each comprising 20 (conventional cytology) slides. At the first step, 80 slides (with "clear-cut" cases) were divided into four sets (A, B, C, D) of 20 specimens, each including inadequate and negative cases as well as in different proportions of all diagnostic TBS 2001 categories. In the second round, a fifth set (E) of 20 slides ("difficult cases") was designed, with all diagnostic categories, ASC and AGC included. Common measures of reproducibility (kappa and weighted kappa), accuracy (SE, SP, PPV, NPV) and 3 indices of diagnostic variability were calculated for sets A-D and set E, separately. RESULTS: For the 5 slide sets together, the weighted kappa was 0.8 (95% CI 0.76-0.85), which is the lower limit of the "almost perfect" ranking of kappa statistics, indicating an excellent interlaboratory agreement. The interlaboratory reproducibility was lower only for the difficult set (E). Similarly, the sensitivity for set E (70.0%) was lower than that (92.1%) for sets A-D. The diagnostic variability indices were not substantially different between the difficult (set E) and clearcut (sets A-D) cases. CONCLUSION: High interlaboratory reproducibility was obtained for sets A-D ("clear-cut" cases), while more interlaboratory variation was evident in the difficult samples. The new CONQUISTADOR software is a valuable tool in calculating the indicators needed in this intralaboratory and interlaboratory.

Tratamiento conservador en el carcinoma de endometrio estadio inicial en pacientes estériles / Conservative treatment of endometrial carcinoma in sterile patients

Se presentan 5 casos de mujeres que presentaban un adenocarcinoma de endometrio, estadio inicial y que fueron tratadas con acetato de medroxiprogesterona con el fin adicional de preservar su potencial de fertilidad. Habían consultado todas por esterilidad y, durante su estudio, se les diagnosticó un adenocarcinoma de endometrio por biopsia endometral. Se completó la evaluación del resto del endometrio con histeroscopias en 4 casos y con raspado uterino el restante. Clínicamente eran estadios iniciales: en cuatro casos se trataba de un adenocarcinoma bien diferenciado G1 y en un caso semidiferenciado G2. Cuatro pacientes fueron tratadas con acetato de medroxiprogesterona en dosis de 500 mgs por día y con 200 mgs por día la restante. Se realizaron controles histológicos cada dos meses y el tiempo de duración del tratamiento fue de 3 meses en dos casos, de 4 meses en otros dos y de 9 meses en el restante. En 4 mujeres se obtuvo remisión completa del adenocarcinoma. Tres de éstas continuaron con sus estudios y tratamiento de la esterilidad y lograron embarazos que llegaron a término, una paciente en dos oportunidades. Hasta la actualidad ninguna tuvo una recurrencia y el tiempo de seguimiento mínimo es de 30 meses. La cuarta paciente debió abandonar el tratamiento por esterilidad y a los dos años tuvo una recidiva del adenocarcinoma por lo que se le realizó una anexohisterectomía total, comprobándose en la pieza operatoria que se trataba de un adenocarcinoma moderadamente diferenciado G2 y que infiltraba hasta el tercio medio del miometrio. Mantiene curación clínica luego de 42 meses de seguimiento. El tratamiento conservador con progestágenos como primera línea en pacientes que padecen un adenocarcinoma bien diferenciado G1 estadio inicial ofrece la posibilidad de preservar la fertilidad sin disminuir la gran tasa de curación, de estos tumores, alcanzada con la terapia quirúrgica convencional

El tratamiento hormonal primario del carcinoma de endometrio inicial: consideraciones terapéuticas / Primary hormonal treatment of the carcinoma of initial endometrium, therapeutic considerations

Se presenta 4 casos de carcinomas de endometrio estadios I clínicos, que fueron tratados inicialmente con hormonoterapia. Tres de ellos remitieron por completo y dos han conseguido embarazos posteriores. Se analizan las pautas diagnósticas y terapéuticas en pacientes portadoras de carcinomas de endo-metrio que desean preservar la fertilidad

Reproducibilidad del grado nuclear en el cáncer de mama: una experiencia multicéntrica / Reproducibility of nuclear grade in breast neoplasm: a multicenter experience

El grado nuclear es considerado uno de los elementos de valor pronóstico en el carcinoma de mama. Resulta de interés verificar cuál es el grado de reproducibilidad de los patólogos generales al consignar el grado nuclear considerando que la mayor parte de los estudios anatomopatológicos de los carcinomas de mama los realizan patólogos "no expertos". Para ello se tomó una serie de 15 carcinomas de mama de los cuales 10 fueron seleccionados al azar y 5 por su particular dificultad para clasificar el grado nuclear. Diez patólogos generales trabajando en forma separada examinaron los casos y les asignaron el grado nuclear. Se hizo una primera asignación de acuerdo al criterio que cada observador utilizaba habitualmente para sus informes y una segunda siguiendo a una guía escrita. e estudiaron los datos por medio del análisis de la varianza. Los resultados mostraron que tanto para los casos seleccionados al azar como para la serie total no hubo diferencias significativas entre los observadores. La guía escrita, sorprendentemente, en lugar de disminuir las diferencias las aumentó. Un análisis del comportamiento individual de los observadores permitió detectar que dos de ellos tenían una gran variación entre ambas asignaciones, lo que se consideró como una influencia determinante para los resultados de todo el grupo. Cuando se analizó la diferencia interobservador para discriminar los tumores de alto grado (G3) del resto, se observó una buena reproducibilidad en todo el grupo participante. Estos resultados nos permiten concluir que, en esta serie examinada por patólogos generales, se obtuvo una aceptable reproducibilidad, en especial cuando se trató de identificar a los tumores con mayor riesgo.

Carcinoma epitelial del ovario: un futuro predecible? / Epithelial ovarian neoplasm: to predict future?

236 pacientes con carcinoma ovárico fueron estatificadas y reestatificadas según la clasificación FIGO 1985. Objetivos: establecer si: el estadio tumoral, los factores histopatológicos, clínico quirúrgicos y el dosaje de CA 125 modifican el período libre de enfermedad y la sobrevida y pueden ser utilizados como predictores de evolución futura. Si los títulos de CA 125 están relacionados con la magnitud del volumen tumoral y extensión de la enfermedad. La distribución por estadios fue EI ab n = 70, EI c n = 14, EII n = 24, EIII a n = 6, EIII b n = 15, EIII c y IV n = 118. El seguimiento mínimo fue de 3 años y el máximo de 18 las curvas de sobrevida fueron calculadas con el método de Kaplan y Meier y el nivel de significación con el Wilcoxon test. Resultados; 1) La sobrevida para los estadios Iab Ic, II, IIIa, IIIb, IIIc y IV fue respectivamente 72, 65, 57, 55, 36 y 16 por ciento. 2) Con respecto al grado de diferenciación, se establecieron diferencias estadísticamente significativas entre G1 y G2, p<0,01 y G1 vs. G3, p<0,01. Para el grado nuclear hubo diferencias estadísticamente significativas entre GN1/GN2-GN3 p<0,001. Para el grado mitótico se establecieron diferencias estadísticamente significativas entre GM1/GM2 y GM3, p<0,001. 3) Para los factores clínicos quirúrgicos se establecieron diferencias significativas entre las pacientes que estuvieron extirpación completa y PR < de 2 cm. (p<0,05) y entre aquellas con Lr < de 2 cm. y > de 2 cm. (p<0,01). 4) Para los valores de CA 125 se establecieron diferencias estadísticamente significativas (p<0,001) entre los grupos con títulos menores de 100 y mayores de 500 mui/ml., siempre que estos valores se normalizaran en el postratamiento. Conclusiones: los factores pronósticos histopatológicos (G,N y M), los clinicoquirúrgicos (RN, LR < 0 > de 2 cm.) y los niveles de CA 125 modifican la sobrevida y pueden ser utilizados como predictores de evolución e indicadores de terapéuticas complementarias del tratamiento inicial

Tumores Mullerianos mixtos malignos / Malignant mixed Mullrian tumors

Se estudió en seis tumores müllerianos mixtos malignos, previamente clasificados en tres homólogos y tres heterólogos, su capacidad pluripotencial a través de la determinación de expresión de marcadores biológicos de estirpe y diferenciación. Ellos definieron un patrón homólogo caracterizado por la expresión de citoqueratina, vimentina, desmina y actina. Dos casos de este grupo fueron negativos para mioglobina, miosina esquelética y proteínas S 100, confirmando el carácter homólogo; y otro positivo para mioglobina fue considerado heterólogo. El patrón heterólogo la expresión de los marcadores homólogos y tambíén fue positivo para mioglobina, miosina esquelética o proteínas S 100. No se observó marcación para Factor VIII en las células neoplásicas. La inmunotipificación con marcadores biológicos permitio reconocer áreas sarcomatosas de carcinoma y establecer diferenciación celular, contribuyendo a efectuar diagnósticos de certeza y clasificación correcta

Carcinoma y fibroadenoma de mama / Carcinoma and fibroadenoma of the breast

Se revisaron 8 casos de carcinoma de mama en coexistencia con fibroadenoma del archivo de la División Patología del Hospital de Clínicas, comprendidos entre los años 1957 y 1987. En 3 de los pacientes el carcinoma se originó en el fibroadenoma (grupo I), en los 5 restantes en el tejido mamario adyacente (grupo II: carcinomas sincrónicos). Todos los pacientes eran mujeres. Durante el peródo 1957-1987 se estudiaron 987 fibroadenomas y 2004 carcinomas. De acuerdo a estas cifras el 0,30% de los fibroadenomas desarrolló un carcinoma en el componente epitelial, y el 0,15% de los carcinomas se originó a partir del epitelio de un fibroadenoma. La edad promedio general fue 43,3 años. En los carcinomas in situ el promedio descendió a 39 años y en los infiltrantes alcanzó los 45,5 años. La edad promedio fue 20 aös mayor que la edad pico de aparición de los fibroadenomas. En el grupo I, se encontró un carcinoma lobulillar y dos carcinomas ductales, todos ellos in situ. Dos de los 3 pacientes mostraron la coexistencia de otra neoplasia ginecológica. En el grupo II, se observó un carcinoma lobulillar, tres ductales y uno lobulillar y ductal. Todos ellos fueron infiltrantes. El tejido mamario adyacente mostró en todos los casos patologia significativa consistente en: múltiples fibroadenomas, enfermedad fibroquística, adenosis esclerosante, carcinoma que tratan de explicar la asociación entre carcinoma y fibroadenoma mamarios. La baja probabilidad que tiene un fibroadenoma de dessarrollar un...