Investigation of EGFR/pi3k/Akt signaling pathway in seminomas.

Activation of the receptor for epidermal growth factor (EGFR) in some testicular tumors activates several signaling pathways. Some components of these pathways are phosphorylated or mutated in testicular germ tumors (TCGT), including EGFR, Kirstein ras oncogen (KRAS) and cell surface protein of the germ cell (KIT). The latter two activate RAF /MEK/ERK and PI3 K/AKT, and interconnect with the EGFR/pI3 k/Akt pathway. We investigated the expression of EGFR/pI3 k/Akt pathway proteins in seminomas and in their precursor lesion, germinal cell neoplasia in situ (GCNIS) and related genetic mutations. We used immunohistochemistry for pEGFR, pI3 k and pAkt expression with a scoring system for 46 seminoma surgical specimens: 36 classical and 10 GCNIS. In 17 samples, the mutations of EGFR (exons 19 - 21), KIT (exons 11, 17) and KRAS (exons 2, 3) were investigated using qPCR and sequencing. Of the 36 seminomas studied, 22 (61%) expressed pEGFR. Ten samples exhibited high scores for pEGFR, pI3 k and pAkt. In 5 of 17 cases (33%) some mutation was exhibited in the exons studied: 21 of EGFR (2), 17 of EGFR (1), 3 of KRAS (1) and 11 of KIT (1). Six cases exhibited nuclear translocation of EGFR; of these, four exhibited mutations of EGFR, KRAS and KIT. Eight of ten of the GCNIS expressed a high pEGFR score (80%). In 2 of 6 cases (33%), mutation was detected in exon 21 of EGFR and one smear showed EGFR translocation to the nucleus. The translocation represents a subpopulation with worse prognosis for TCGT. The EGFR/pI3 k/Akt signaling pathway is linked to TDRG1, which regulates chemosensitivity to cisplatin; this is a mechanism of resistance to treatment. TDRG1 and the EGFR/pI3 k/pAkt pathway could be therapeutic targets for seminomas resistant to cisplatin.

Epididymal mitochondrial status of hypothyroid rats examined by transmission electron microscopy.

Hypothyroid rats show alterations in the mobility of sperm recovered from their epididymides. The AgNOR technique, immunohistochemistry and transmission electron microscopy were used to investigate changes in epithelial cells from epididymides of rats treated with (131)I. Counting of NORs did not permit detection of changes in the proliferative capacity of epididymides of hypothyroid animals. Transmission electron microscopy revealed changes in the mitochondria of hypothyroid rats that probably are associated with incipient apoptosis.

AgNOR, p16 and human papillomavirus in low-grade squamous intra-epithelial lesions of the uterine cervix. Preliminary report.

It has been shown that infection with high-risk human papillomaviruses (HR-HPV) is related to the development of cervical cancer. The persistence of the virus in intra-epithelial lesions of cervix uteri (SILs) is the basis for the application of HPV testing for screening and management of patients. Most infections by HR-HPVs resolve spontaneously, however, and do not progress to dysplasia or cancer. p16INK4a is a useful biomarker of cervical intra-epithelial neoplasia and could be a marker for the progression of low-grade squamous intra-epithelial lesions (LSILs) to high-grade squamous intra-epithelial lesions (HSILs), because it correlates independently with increasing SIL grade. We conducted a preliminary histological study of 28 patients diagnosed with LSIL, HSIL or nondysplastic epithelium (NDE) from whom 28 biopsies of uterine cervix and 28 endocervical brushed biopsies were taken. Argyrophilic nucleolar organizer region (AgNOR) and p16INK4a assays were performed on the biopsies, and endocervical brushings were used for HPV typing. The high risk HPV group showed that the number of patients with AgNOR areas greater than 3.3 µm(2) and with expression of p16INK4a were statistically greater than the number of lower risk patients. None of the biopsies of LR-HPV carriers expressed p16 and AgNOR areas> 3.3 µm(2) simultaneously. Four LSILs and the NDE of this group expressed neither of the two markers. If the correlation between AgNOR areas and p16INK4a is good, we may be able to develop a low cost simple technology for studying patients infected with HR-HPV and diagnosed with LSIL of uncertain behavior.

Sensitivity and specificity of cytodiagnosis of body fluids in a laboratory of urgencies.

The main purpose for studying cytological body fluids is confirmation of a benign or malignant effusion. Our cytology laboratory analyzes body fluids and results are requested urgently. The samples are stained by the Giemsa and Papanicolaou methods to give a preliminary report, then they are examined by other complementary techniques. Three hundred thirty samples of pleural and peritoneal fluids were studied to compare the sensitivity of Papanicolaou and Giemsa stains. AgNOR assay, immunocytochemistry and assessment of ploidy were used to improve the sensitivity of the cytodiagnosis. Two hundred one samples were positive, 84 negative and 45 inconclusive using the Papanicolaou stain, while 135 samples were positive, 72 negative and 123 inconclusive using Giemsa stain. The sensitivity was 79%, 53% and 83% for Papanicolaou, Giemsa, and both techniques together, respectively. Using complementary techniques, the sensitivity reached 95% for AgNOR, 87% for tumor markers (panel), and 92% for Ploidy. There were no false positive in our series; therefore specificity was 100%. The use of both Papanicolaou and Giemsa in conjunction increased the sensitivity of the cytodiagnosis in body fluids. The complementary methods, especially AgNOR assay and assessment of ploidy, diminished the number of inconclusive cases.

Prognostic value of germ cells in the ejaculate: a case study.

The first approach to assessing male fertility is to study a spermiogram, where special attention is given to sperm count, motility and morphology, while less attention is given to other cells in the ejaculate. Normal spermatogenesis requires a balance between cell death and proliferation; therefore, the number of germ cells (GC) in the ejaculate is less than the number of sperm. We propose a new index for altered spermatogenesis, i.e., the rate GC/sperm. We investigated a patient with oligozoospermia and a GC/sperm ratio greater than one, which indicated that spermatogenesis had been damaged. Complementary cytological tests were employed to characterized GC status: Papanicolaou stain, transmission electronic microscopy (TEM), vitality test, AgNOR and TUNEL assay. We also correlated cell morphology with ultrastructure studies that showed apoptosis. Nuclear apoptosis is characterized by vacuolization, misshapen nuclei, and "half moon," dispersed, uncondensed, disrupted and smudged chromatin. Cytoplasmic apoptosis is characterized by vacuolization, cytoplasmic protrusions, lamellar bodies, and swollen endoplasmic reticulum and mitochondria. To date, only testicular biopsy has been used to diagnose complete or incomplete testicular arrest. Our investigation is the first to determine a cytological feature in semen samples that could be used as a biological marker for abnormal spermatogenesis and for predicting the transition from oligospermia to azoospermia.

Assessment of human sperm protein tyrosine phosphorylation by immunocytochemistry in a clinical andrology laboratory. Preliminary data.

We propose that evaluation of protein tyrosine phosphorylation (TP) status in ejaculated spermatozoa under capacitating conditions in an experiment that mimics "in vitro" the physiology of sperm from ejaculation through the female genital tract could potentially be used as a prognostic test for functional competence of sperm in fertilization. Our purpose was to elucidate whether there is a relation between conventional sperm parameters, occurrence of TP and pregnancy outcome obtained from intrauterine insemination (IUI). Semen samples were analyzed according to WHO criteria. TP levels were determined by immunocytochemistry under four different conditions: 1) ejaculated sperm, 2) postselection sperm, 3) postselection sperm incubated 5 h at 37 degrees C and 5% CO(2), and 4) postselection sperm incubated overnight at 37 degrees C and 5% CO(2). Data on sperm tyrosine phosphorylated proteins did not correlate with sperm concentration, progressive motility or normal sperm morphology. TP increased under capacitating conditions and showed a time dependent pattern except for five outlier cases. IUI was performed in 12 selected couples who had neither female nor male infertility factors. The three pregnancies had a time dependent pattern for TP. Of the unsuccessful cases, one had an outlier TP pattern. It appears that a TP time dependent pattern is necessary for fertilization.

Usefulness of ploidy, AgNOR and immunocytochemistry for differentiating benign and malignant cells in serous effusions.

OBJECTIVE: The objective of this study was to establish the value of different markers in differentiating reactive mesothelial cells from metastatic adenocarcinomatous cells in serous effusions (SE). METHODS: Forty-five SE were processed for morphological examination (Papanicolaou stain), assessment of ploidy, AgNOR counting and immunocytochemical assay of carcinoembryonic antigen (CEA), epithelial membrane antigens (EMA), Ber-EP4 and Leu-M1. Ploidy was established in an image-analyser in smears stained by the Feulgen stain method. AgNOR dots were counted in the smears stained with the silver nitrate assay for non-histone proteins present in the nucleolar organizer region. CEA, EMA, Ber-EP4 and Leu-M1 were evaluated by immunocytochemistry using the streptavidin-biotin complex method. RESULTS: All the smears with positive cytology were aneuploid. Three false negatives by morphological studies were aneuploid, with AgNOR values in two of them corresponding to the neoplastic group. CEA and Leu-M1 showed a low specificity; EMA and Ber-EP4 showed moderate sensitivity. CONCLUSIONS: The assessment of ploidy and the study of AgNOR were better methods than immunocytochemistry for distinguishing between reactive mesothelial cells and adenocarcinomatous cells in serous fluid.

[Usefulness of AgNOR technique in the interpretation of serous effusions]. / Utilidad de la técnica de AgNOR en la interpretación de los derrames de cavidades serosas.

BACKGROUND: AgNOR technique detects, using silver salts, argyrophylic proteins of the nucleolar organizer region (NOR). The number and size of NOR reflect cell activity, proliferation and transformation and may help to differentiate benign from malignant cells. AIM: To assess the value of AgNOR assay to differentiate reactive mesothelial cells from malignant cells in serous effusions. MATERIAL AND METHODS: Thirty one fluids obtained from 16 pleural, 14 peritoneal and one pericardial effusion, were studied. The fluids were processed with Giemsa and Papanicolau stains and with the AgNOR technique. The number of AgNOR dots were counted (only when it was possible to distinguish each individual dot) and the mean value per nucleus was calculated for each smear. RESULTS: Mesothelial cells had a mean of 4.88 +/- 1.5 dots compared with 13.78 +/- 3.88 dots in the malignant cells (p < 0.001). CONCLUSIONS: AgNOR assay can be useful for the differentiation of benign and malignant cells in serous effusions.

Epididymal scanning electron microscopy study in hypothyroid rats.

The purpose of this research was to investigate, using scanning electron microscopy, the effect of hypothyroidism on the ultrastructure of the rat cauda epididymis. Thyroidectomy was obtained by ip injection of 270 muCi of 131I per rat. One month later, several portions of cauda epididymis were examined. Morphological and physiological differences were detected in the cauda epididymis of the hypothyroid animals when compared to the control normal rats. The hypothyroid condition was associated with important changes in the luminal surface of the cauda epididymis epithelium. Broken, oblique, and loss of stereocilia, denuded epithelial cells, and flattening of the tubule were observed. The results confirm that hypothyroidism causes marked structural changes in the cauda ductus epididymis and could be adversely affect sperm maturation motility.

Epididymal cytology changes in hypothyroid rats.

The purpose of this research was to investigate, using light, transmission, and scanning electron microscope, the effect of hypothyroidism on the ultrastructure of the rat epididymis. Thyroidectomy was obtained by ip injection of 270 microCi of (131)I per rat. One month later, several portions of cauda epididymis were examined. Morphological differences were detected in the epididymis of the hypothyroid animals when compared to the control normal rats. The hypothyroid conditions were associated with important changes in the epididymis. The light observations showed cells with clearing of the chromatin and increased density and thickness of the chromatic rim, chromatinic net, and disappearance of the segment of the chromatin rim. In the scanning electron microscope broken, oblique, denuded epithelial cells with loss of stereocilia were observed, as well as flattening of the tubule. The hypothyroid condition under transmission electron microscope was associated with a decrease in the height of the cells, diminution of the internal lumen and number of mitoses, and decreased chromatin decondensation. Results obtained confirmed that hypothyroidism causes marked structural changes in the ductus epididymis and could adversely affect the maturation and motility of sperm.