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Background: Proteomics is expected to play a key role in cancer biomarker discovery. Although it has become feasible to rapidly analyze proteins from crude cell extracts using mass spectrometry, complex sample composition hampers this type of measurement. Therefore, for effective proteome analysis, it becomes critical to enrich samples for the analytes of interest. Despite that one-third of the proteins in eukaryotic cells are thought to be phosphorylated at some point in their life cycle, only a low percentage of intracellular proteins is phosphorylated at a given time., Methodology/principal Findings: In this work, we have applied chromatographic phosphopeptide enrichment techniques to reduce the complexity of human clinical samples. A novel method for high-throughput peptide profiling of human tumor samples, using Parallel IMAC and MALDI-TOF MS, is described. We have applied this methodology to analyze human normal and cancer lung samples in the search for new biomarkers. Using a highly reproducible spectral processing algorithm to produce peptide mass profiles with minimal variability across the samples, lineal discriminant-based and decision tree-based classification models were generated. These models can distinguish normal from tumor samples, as well as differentiate the various non-small cell lung cancer histological subtypes., Conclusions/significance: A novel, optimized sample preparation method and a careful data acquisition strategy is described for high-throughput peptide profiling of small amounts of human normal lung and lung cancer samples. We show that the appropriate combination of peptide expression values is able to discriminate normal lung from non-small cell lung cancer samples and among different histological subtypes. Our study does emphasize the great potential of proteomics in the molecular characterization of cancer.

The activating mutation BRAF(V600E) is a frequent genetic event in papillary thyroid carcinomas (PTC) that predicts a poor prognosis, leading to loss of sodium/iodide symporter (NIS) expression and subsequent radioiodide-refractory metastatic disease. The molecular basis of such an aggressive behavior induced by BRAF remains unclear. Here, we show a mechanism through which BRAF induces NIS repression and promotes epithelial to mesenchimal transition and invasion based on the operation of an autocrine transforming growth factor (TGF)beta loop. BRAF induces secretion of functional TGFbeta and blocking TGFbeta/Smad signaling at multiple levels rescues BRAF-induced NIS repression. Although this mechanism is MAP/extracellular signal-regulated kinase (ERK) kinase (MEK)-ERK independent, secreted TGFbeta cooperates with MEK-ERK signaling in BRAF-induced cell migration, Matrigel invasion, and EMT. Consistent with this process, TGFbeta and other key components of TGFbeta signaling, such as TbetaRII and pSmad2, are overexpressed in human PTC, suggesting a widespread activation of this pathway by locally released TGFbeta. Moreover, this high TGFbeta/Smad activity is associated with PTC invasion, nodal metastasis, and BRAF status. Interestingly, TGFbeta is overexpressed in the invasive front, whereas NIS is preferentially expressed in the central regions of the tumors, suggesting that this negative correlation between TGFbeta and NIS occurs locally inside the tumor. Our study describes a novel mechanism of NIS repression in thyroid cancer and provides evidence that TGFbeta may play a key role in promoting radioiodide resistance and tumor invasion during PTC progression.

A total of 1482 adult ticks collected from vegetation and animals in central Spain in 2003-2005 were tested for the presence of Coxiella burnetii by polymerase chain reaction and subsequent reverse line blot hybridization (PCR-RLB). C. burnetii was identified in 7.7% of questing ticks (80/1039) and 3.4% of ticks collected from animals (15/443) belonging to four species: Hyalomma lusitanicum, Dermacentor marginatus, Rhiphicephalus sanguineus, and R. pusillus. These findings show an active role of ticks in maintaining C. burnetii in wild and peridomestic cycles in central Spain.

Objectives: To report the second case of solitary neurofibroma arising from the tunica albuginea in the literature and to show its imaging findings., Methods/results: We present a case of neurofibroma arising from the tunica albuginea in an adult patient not affected by neurofibromatosis. We describe the ultrasonographic and magnetic resonance imaging (MRI) features and the histopathological characteristics along with a brief bibliographic review., Conclusion: MRI may be useful to characterize paratesticular lesions. Neurofibroma should be included in the differential diagnosis when MRI depicts a well-circunscribed tumour with high-signal intensity on T2 and marked enhancement after gadolinium administration.

Systemic venous pressure is elevated in right heart failure, and this elevation may be reflected in pulsatile venous flow when there is significant tricuspid regurgitation. The presence of this systolic reversed flow in the femoral and popliteal veins may result in major difficulties for diagnosis and treatment. We report the case of a patient with signs and symptoms of acute ischemia of the right lower limb with palpable pulse in the groin and popliteal fossa. Tricuspid regurgitation was suspected by clinical examination, and Doppler ultrasonographic examination of the extremity revealed pulsatile flow in the femoral and popliteal veins while the iliac arterial axis was occluded. A preoperative angiogram revealed an underlying iliac artery thrombosis, which was successfully treated.

Estimates of the incidence of aortoenteric fistula as a sequela of surgery of the aorta range 1-2%. This complication is less common in patients who have had an aortic endograft implanted for aortoiliac aneurysm. We present three cases of aortoenteric fistula complicating endovascular treatment of abdominal aortic aneurysm (3/423 patients, 0.7% in our series).

Background: TWIST1 is a basic helix-loop-helix (bHLH) transcription factor that has been involved in tumor progression and metastasis in several cancer types, although no evidence has been provided yet on its implication in colorectal carcinogenesis., Methods: We examined the expression pattern of TWIST1 messenger RNA (mRNA) in 54 colorectal cancer biopsies compared with each respective adjacent normal mucosa by real-time reverse-transcriptase polymerase chain reaction (RT-PCR) methodology., Results: TWIST1 mRNA was found significantly overexpressed in colorectal cancer samples compared to nontumorous colon mucosa (P < 0.0001). Receiver operating characteristic (ROC) curve analysis demonstrated that TWIST1 mRNA levels are significantly increased in patients with nodal invasion and, interestingly, a significant correlation with patient sex was also found., Conclusions: Evidence for upregulation of TWIST1 mRNA in colorectal cancer is provided, suggesting its implication in the onset of malignant progression of this disease. In addition, significant higher levels of TWIST1 mRNA were found in men than in women, suggesting a possible transcriptional regulation of TWIST1 by sexual hormones. The use of TWIST1 as a new prognostic marker of advanced malignancy, and as a potential therapeutic target in colorectal cancer, is proposed.

Pluripotent cells can be isolated from the mammalian inner cell mass (ICM) of the embryo at the blastocyst stage, and maintained in culture as undifferentiated, embryonic stem cells (ES). These cells are an important model of mammalian development in vitro and are the focus of a great deal of research for their use in Cell Therapy. In vivo, shortly after the blastocyst stage, the ICM segregates into two layers: the hypoblast which will give rise to the yolk sac, and the epiblast. Epiblast stem cells, like ES cells, are pluripotent. The epiblast will differentiate very early into germ cell progenitors, the primordial germ cells (PGC). PGCs can give rise to embryonal carcinoma cells, the pluripotent stem cells of testicular tumors. During normal embryo development, PGCs migrate into the aorta-gonad-mesonephros region (AGM). Interestingly, this region also harbors the first wave of embryonic hematopoiesis. Subsequent waves of hematopoiesis involve AGM-hematopoietic stem cell (HSC) colonization of the fetal liver, thymus, spleen and ultimately, for adult hematopoiesis, the bone marrow (BM). The BM is also source of mesenchymal stem cells (MSCs). It is accepted that the AGM region cells give rise to the mesothelial cells which are the embryonic precursors of the HSC and MSC of the BM. Recent identification of a subpopulation of cells with markers typical of PGCs in the adult BM, which are capable of differentiating into HSCs, suggests that HSCs originate from a common precursor of PGCs and HSCs derived from the epiblast. Several groups have described the presence of stem cells with the same markers in epidermis, bronchial epithelium, pancreas, retina, hair follicle, heart and dental pulp among, other organs. This presence supports the hypothesis that during early development, epiblast/germ line-derived cells are deposited in various organs which persist into adulthood. The question remains whether these pluripotent stem cells are only developmental remnants or if they continuously contribute to the renewal of tissues, and thus can be reactivated for tissue regeneration without the need for stem cell transplantation for human cell therapies.

Objective: To report a case of a 46,XX male with an intratubular undifferentiated germ cell neoplasia within an extra-abdominal gonad., Design: Case report., Setting: Molecular, cytogenetic, pathologic, and clinical units of three tertiary hospitals., Patient(s): A male with ambiguous genitalia at birth and descended testes observed in a pediatric endocrinology setting., Intervention(s): Physical examination, hormonal assays, cytogenetic investigation, molecular analysis, surgical intervention for biopsies and bilateral orchiectomy, and pathologic evaluation., Main Outcome Measure(s): Pathologic evaluation with immunostaining for placental alkaline phosphatase and C-kit., Result(s): Conventional chromosome analysis revealed a 46,XXq- karyotype, and fluorescence in situ hybridization experiments with the SRY probe found a signal at the short arm of the deleted X chromosome. Molecular analysis indicated the presence of a portion of the short arm of the Y chromosome including the proto-oncogene TSPY. Pathologic evaluation of the gonads revealed an intratubular undifferentiated germ cell neoplasia., Conclusion(s): This is the first case of a 46,XX male with descended testes in whom an intratubular undifferentiated germ cell neoplasia developed. When proposals of management in this subgroup of disorders of sexual differentiation are formulated, the risk of germ cell malignancy must be taken into account.

Context: Although gonadotropins and testosterone are high in the fetal/early postnatal periods, Sertoli cells remain immature and spermatogenesis does not progress. We hypothesized that Sertoli cells do not respond to testosterone because they do not express the androgen receptor., Objective: The objective of the study was to describe the precise ontogeny of androgen receptor expression in the human testis from fetal life through adulthood., Design: This was an immunohistochemical study on testicular biopsies from fetal, neonatal, prepubertal, pubertal, and adult human testes., Main Outcome Measures: Quantification of androgen receptor expression in Sertoli cells was measured. Evaluation of androgen receptor expression in peritubular and interstitial cells as well as anti-Müllerian hormone and inhibin-alpha was also performed., Results: Androgen receptor expression was first observed in the nuclei of few Sertoli cells at the age of 5 months. Labeling was weak in 2-15% of Sertoli cells until 4 yr of age and progressively increased thereafter. High levels of androgen receptor expression were observed in more than 90% from the age of 8 yr through adulthood. Androgen receptor was positive in peritubular cells and variable in interstitial cells. Anti-Müllerian hormone immunolabeling was strong in all Sertoli cells from fetal life throughout prepuberty and weakened progressively as spermatogenesis developed. Inhibin-alpha expression was detected in all Sertoli cells from fetal life through adulthood., Conclusions: A lack of androgen receptor expression could explain a physiological Sertoli cell androgen insensitivity during fetal and early postnatal life, which may serve to protect the testis from precocious Sertoli cell maturation, resulting in proliferation arrest and spermatogenic development.

South Africa has the highest number of HIV-1-infected individuals in the world, with HIV-1 subtype C prevailing. However, HIV-1 subtype C accessory genes are rarely characterized in the country. These genes are important for establishing viral pathogenesis. The Vif protein has been shown to counteract the antiretroviral activity of APOBEC3G/F cytidine deaminases. In this study an additional 50 HIV-1 vif sequences are characterized. These include 48 HIV-1 subtype C and 2 HIV-1 subtype B sequences. Highly conserved HIV-1 subtype C motifs are outlined. The previously identified RLRR (90-93) motif does not seem to be conserved among our newly analyzed sequences. Conserved motifs can be useful for developing new vaccine strategies or antiretroviral drugs.

Cdc42, a member of Rho GTPases family, is involved in the regulation of several cellular functions, such as rearrangement of actin cytoskeleton, membrane trafficking, cell-cycle progression, and transcriptional regulation. Aberrant expression or activity of Cdc42 has been reported in several tumours. Here, the specific role of Cdc42 in development and progression of colorectal cancer was analyzed through microarrays technology. A comparative analysis of Cdc42 overexpressing cells versus cells with decreased Cdc42 levels through siRNA revealed that Cdc42 overexpression down-regulated the potential tumour suppressor gene ID4. Results were validated by quantitative RT-PCR and the methylation status of the specific promoter, analyzed. Methylation-specific PCR and bisulfite sequencing PCR analysis revealed that Cdc42 induced the methylation of the CpG island of the ID4 promoter. Colorectal adenocarcinoma samples were compared with the corresponding adjacent normal tissue of the same patient in order to determine specific gene expression levels. The downregulation of ID4 by Cdc42 was also found of relevance in colorectal adenocarcinoma biopsies. Cdc42 was found to be overexpressed with high incidence (60%) in colorectal cancer samples, and this expression was associated with silencing of ID4 with statistical significance (p<0.05). Cdc42 may have a role in the development of colon cancer. Furthermore, inhibition of Cdc42 activity may have a direct impact in the management of colorectal cancer.

Background: The classification of pulmonary neuroendocrine neoplasms is particularly controversial. Large cell neuroendocrine carcinoma (LCNEC) has emerged as a separate entity among pulmonary endocrine neoplasms and the criteria for its histologic diagnosis are now well-described. However, cytologic diagnosis presents more difficulties and to the authors' knowledge, few cytologic studies concerning the entity have been published to date. The objective of the current study was to describe the cytologic features of LCNEC in an attempt to distinguish it from other pulmonary carcinomas., Methods: A cytohistologic study of 11 surgical lobectomy specimens classified as LCNEC was performed. In all these cases, preoperative fine-needle aspiration cytology (FNAC) material was available for review., Results: The cytologic features of the cases were rather similar, resulting in a repetitive pattern. The majority of smears were hypercellular with numerous single, medium-to-large cells. Naked nuclei were abundant but a variable subset of cells demonstrated evident cytoplasm. Groups were 3-dimensional and of variable size, some of them large. Nuclear pleomorphism, molding, and mitosis were common findings. A necrotic background was evident in 6 cases. In 6 cases, neoplastic groups demonstrated peripheral nuclear palisading. Rosette-like structures were present in samples from 5 patients. In 4 cases, immunocytochemistry for the detection of synaptophysin was performed, with positive results., Conclusions: The authors' experience with 11 FNAC cases of LCNEC, has led them to believe that the cytologic image of LCNEC is peculiar and recognizable in many cases. Nevertheless, it is a difficult diagnosis to make and immunocytochemistry plays a critical diagnostic role., ((c) 2008 American Cancer Society.)

Lysyl oxidase-like 2 (Loxl2) interacts with and stabilizes Snai1 transcription factor, promoting epithelial-mesenchymal transition. Either Loxl2 or Snai1 knock-down blocks tumor growth and induces differentiation, but the specific role of each factor in tumor progression is still unknown. Comparison of the gene expression profiles of the squamous cell carcinoma cell line HaCa4 after knocking-down Loxl2 or Snai1 revealed that a subset of epidermal differentiation genes was specifically up-regulated in Loxl2-silenced cells. In agreement, although both Loxl2- and Snai1-knockdown cells showed reduced in vivo invasion, only Loxl2-silenced cells exhibited a skin-like epidermal differentiation program. In addition, we show that expression of Loxl2 and Snai1 correlates with malignant progression in a two-stage mouse skin carcinogenesis model. Furthermore, we found that increased expression of both LOXL2 and SNAI1 correlates with local recurrence in a cohort of 256 human laryngeal squamous cell carcinomas. We describe for the first time that high levels of LOXL2 are associated with decreased overall and disease-free survival in laryngeal squamous cell carcinomas, lung squamous cell carcinoma, and lymph node-negative (N(0)) breast adenocarcinomas. Altogether, our results show that LOXL2 can be used as a new poor prognosis indicator in human squamous cell carcinomas promoting malignant transformation by both SNAI1-dependent and SNAI1-independent pathways.

Castleman's disease (CD) is a rare disorder of uncertain aetiology characterised by massive proliferation of lymphoid tissue usually localised as mediastinal masses, although abdominal involvement has been reported. Localised forms are usually associated with a good prognosis, but several more aggressive multifocal variants have been observed. Two different histologic subtypes have been described: the hyaline vascular type, more common in unicentric CD and usually asymptomatic, and the plasma cell form. Unicentric CD may be associated with an increased risk of lymphoma, but there was no reported increased risk of other malignancies. A patient with plasma cell subtype unicentric CD localised in retroperitoneum associated with an adenocarcinoma of ileocaecal valve and liver metastasis is reported.

Frasier syndrome is an uncommon genetic disorder featuring progressive glomerulopathy, male pseudohermaphroditism, and gonadal dysgenesis with increased risk of gonadoblastoma and malignant germ cell tumors. It is caused by mutations in the donor splice site in intron 9 of the WT1 gene. However, because of its rarity there is limited literature available on the precise spectrum and recommended treatment modalities of this syndrome. We present the clinicopathological findings in 4 patients: 3 phenotypically female adolescents presenting with proteinuria and primary amenorrhea and a 6-month-old baby girl presenting with nephrotic syndrome in whom this very unusual case of early onset was confirmed by molecular studies. The significance of early recognition of Frasier syndrome and its differentiation from Denys-Drash syndrome is reviewed and discussed. Our observation of a case presenting with early clinical manifestations, in contrast with the classical presentation in adolescence, justifies the expansion of the clinical spectrum of Frasier syndrome and contributes to the understanding and appropriate clinical management of these patients.

Most corneal diseases affect corneal stroma and include immune or infectious diseases, ecstatic disorders, traumatic scars, and corneal dystrophies. Cell-based therapy is a promising therapeutic approach to overcome the current disadvantages of corneal transplantation. We intended to search for a cell source to repopulate and regenerate corneal stroma. We investigated the ability of human processed lipoaspirate derived (PLA) cells to regenerate corneal stroma in experimental animals. In the first set of experiments, we tested the biosafety and immunogenicity of human PLA stem cells transplanted into the corneal stroma of rabbits. No immune response was elicited even though we used immune-competent animals. PLA cells survived up to 10 weeks post-transplant, maintained their shape, and remained intermingled in the stroma without disrupting its histological pattern. Interestingly, transparency was preserved even 10 weeks after the transplant, when PLA cells formed a discontinuous layer in the stroma. In the second set of experiments, regeneration of the corneal stroma by PLA cells was assessed, creating a niche by partial ablation of the stroma. After 12 weeks, human cells were disposed following a multilayered pattern and differentiated into functional keratocytes, as assessed by the expression of aldehyde-3-dehydrogenase and cornea-specific proteoglycan keratocan. Based on our results, we believe that adipose-derived adult stem cells can be a cell source for stromal regeneration and repopulation in diseased corneas. The low health impact of the surgical procedure performed to obtain the PLA cells provides this cell source with an additional beneficial feature for its possible future autologous use in human patients.

Colorectal adenocarcinoma is the second cause of cancer mortality in developed countries. Rac1 is a member of the family of Rho GTPases that regulates many intracellular signaling pathways, including those involved in tumorigenesis, invasion, and metastasis. We have investigated the role of Rac1 in colorectal tumor progression by genetic modification of the human colorectal adenocarcinoma cell line SW620 to either overexpress Rac1 or lack Rac1 expression. Tumor behavior was studied by orthotopic injection of stably modified cell lines into the cecal wall of athymic nude mice, a model that replicates the histopathological appearance and clinical behavior of human colorectal adenocarcinoma in humans. While overexpression of Rac1 resulted in an accelerated tumorigenic process, inducing a faster mortality rate, inhibition of Rac1 completely suppressed tumor formation. These results suggest that Rac1 plays a major role in colorectal adenocarcinoma progression. Finally, interference with Rac1 function may provide an important tool to block the malignant phenotype of colorectal adenocarcinoma cells.

The gonadal biopsy provides essential information for the identification, classification and early detection of neoplasias in patients with disorders of sex development. Histopathological findings in these cases must be analysed together with clinical, hormonal, genetic and molecular information before deciding a therapeutic option. Sexual differentiation is the result of multiple and complex genetic and endocrinal mechanisms; therefore, we first present the events taking place during gonadal embryonic development, focusing on the genetic mechanisms involved in sexual determination and the differentiation of the testis and the urogenital tract. In second place, we describe the different gonads in the intersexual states -in testicular regression syndrome, fibrous streak, testicular dysgenesis, streak testes, ovotestes and microscopically normal testes and ovaries-, highlighting the histological features and the differential findings that allow the pathologist to distinguish between these entities with the aid of clinical, genetic, hormonal and molecular information that are characteristic for each situation. In third place, we studied the incidence of neoplasias in gonadal dysgenesis, male pseudohermaphroditism and true hermaphroditism. Finally, we discuss the limitations of gonadal biopsy to achieve a correct diagnosis in the disorders of sex development.

Aortoiliac aneurysms are frequent entities that have very important clinical implications, especially in the younger patients. We are asked not only to save lives by preventing the rupture or repairing those that are already ruptured but also to provide an acceptable quality of life in the postoperative period. Endovascular approaches certainly give us such an expectative but are not clearly indicated in our younger patients and cannot be used routinely in those aneurysms with a yuxtarrenal origin. This is the case report of a young man with a yuxtarrenal aortoiliac inflammatory aneurysm that was treated by the interposition of an aortobifemoral bypass with the addition of endovascular devices in the hypogastric vessels to preserve the pelvic blood flow; they contribute to seal the common iliac aneurysms. This approach was useful to avoid complications during a difficult iliac dissection and was permeable within 1 year of the operation. Durability must be assessed.

Background: Adequate prognostic markers to predict outcome of patients with lung cancer are still needed. The aim of this study was to assess whether choline kinase alpha (ChoKalpha) gene expression could identify patients with different prognoses. ChoKalpha is an enzyme involved in cell metabolism and proliferation and has a role in oncogene-mediated transformation in several human tumours, including lung cancer., Methods: 60 patients with non-small-cell lung cancer (NSCLC) who had undergone surgical resection in a single centre were enrolled into the study as the training group. We used real-time reverse-transcriptase PCR (RT-PCR) to measure ChoKalpha gene expression and analyse the association between ChoKalpha expression and survival in evaluable patients. Additionally, a second group of 120 patients with NSCLC from a different hospital were enrolled into the study as the validation group. We did an overall analysis of all 167 patients who had available tissue to confirm the cut-off point for future studies. The primary endpoints were lung-cancer-specific survival and relapse-free survival., Findings: Seven of the 60 patients in the training group were not evaluable due to insufficient tissue. In the 53 evaluable patients, the cut-off for those with ChoKalpha overexpression was defined by receiver operator under the curve (ROC) methodology. 4-year lung-cancer-specific survival was 54.43% (95% CI 28.24-80.61) for 25 patients with ChoKalpha expression above the ROC-defined cut-off compared with 88.27% (75.79-100) for 28 patients with concentrations of the enzyme below this cut-off (hazard ratio [HR] 3.14 [0.83-11.88], p=0.07). In the validation group, six of the 120 enrolled patients were not evaluable due to insufficient tissue. For the other 114 patients, 4-year lung-cancer-specific survival was 46.66% (32.67-59.65) for those with ChoKalpha expression above the ROC-defined cut-off compared with 67.01% (50.92-81.11) for patients with concentrations of ChoKalpha below the cut-off (HR 1.87 [1.01-3.46], p=0.04). A global analysis of all 167 patients further confirmed the association between ChoKalpha overexpression and worse clinical outcome of patients with NSCLC: 4-year lung-cancer-specific survival for ChoKalpha expression above the ROC-defined cut-off was 49.00% (36.61-60.38) compared with 70.52% (59.80-76.75) for those with concentrations of ChoKalpha below the cut-off (HR 1.98 [1.14-3.45], p=0.01). The overall analysis confirmed the cut-off for ChoKalpha expression should be 1.91-times higher than concentrations noted in healthy tissues when ChoKalpha is used as an independent predictive factor of relapse-free and lung-cancer-specific survival in patients with early-stage NSCLC., Interpretation: ChoKalpha expression is a new prognostic factor that could be used to help identify patients with early-stage NSCLC who might be at high risk of recurrence, and to identify patients with favourable prognosis who could receive less aggressive treatment options or avoid adjuvant systemic treatment. New treatments that inhibit ChoKalpha expression or activity in patients with lung cancer should be studied further.

Introduction: Mixed atrophy of the testis (MAT), a frequent finding in biopsies of formerly cryptorchid and/or infertile patients, is defined as the synchronous occurrence of both seminiferous tubules containing germ cells and Sertoli cell only-tubules in variable proportions. In tubules containing germ cells, different types of abnormalities in spermatogenesis may be seen. The presence of adult spermatids in the biopsy, even in small numbers, correlates with successful spermatozoa retrieval for "in vitro" fertilization techniques. Currently, it is unknown whether precursor lesions of MAT can be identified in cryptorchid patients during childhood., Material and Methods: Eighteen formerly cryptorchid adults who had undergone testicular biopsies in childhood had a repeat testicular biopsy to evaluate infertility. In prepubertal biopsies, abnormalities of the testicular parenchyma were classified into types I (slight alterations), II (marked germinal hypoplasia), and III (severe germinal hypoplasia). In postpubertal biopsies, the percentage of tubules containing germ cells and Sertoli cell only-tubules were estimated, as well as the presence of complete spermatogenesis. Abnormalities in spermatogenesis were classified into lesions of the adluminal or basal compartments of seminiferous tubules., Results: Comparison between prepubertal and postpubertal biopsies revealed that most specimens developing from type III lesions presented with incomplete spermatogenesis (P<0.0001) and more severe lesions of the germinal epithelium (P=0.049)., Discussion: Type III lesions correlated with MAT characteristics that confer a worse prognosis for in vitro fertilization. Thus, MAT characteristics may be predicted in prepubertal cryptorchid patients, allowing a fertility prognosis. The pathogenesis of these lesions, and their possible inclusion into the spectrum of the testicular dysgenesis syndrome, are discussed.

We present the case of a newborn with sternal cleft (SC) and presternal enterogenous cyst operated on during the neonatal period. SC is an uncommon congenital malformation of the thoracic wall which can occur as an isolated form or in association with other malformations. To our knowledge, the presence of SC and enterogenous cyst has not been described to date. Early surgical repair of SC gives good aesthetic and functional results and is usually the preferred approach.

Sex-cord formation and organization are important steps in testicular development and depend on adequate interactions between mesenchymal cells, pre-Sertoli cells, and germ cells. These elements form the testicular blastema, the precursor of the testicular parenchyma, morphologically characterized by poorly organized sex cords and mesenchymal components. Here, we study two uncommon testicular lesions, unrelated to other gonadal anomalies. In the first group, we describe the features of persistence of testicular blastema in three fetal autopsy cases, discussing its possible pathogenesis and clinical importance. In the second, we analyze 11 cases of ectopic testicular parenchyma in the tunica albuginea, an uncommon benign condition of uncertain clinical significance, whose main differential diagnosis is gonadal dysgenesis. Based on their similar topography within the testis, and on their possibly shared embryological origin, we propose that both lesions may represent the two extremes of a maldevelopmental spectrum resulting from a focal delay in testicular development.

We describe the case of an 18-year-old female with Cowden syndrome in whom a simple x-ray detected a solitary pulmonary nodule that was identified as a sclerosing hemangioma. Pulmonary sclerosing hemangioma is an unusual lung neoplasm which typically presents as a solitary peripheral nodule in asymptomatic women. Although the histology of this entity is well defined, its origin and treatment is debated. One of the main diagnostic problems is to histologically differentiate a pulmonary sclerosing hemangioma from a papillary lung carcinoma.

Fetal gonadoblastoid testicular dysplasia (FGTD) is an extremely rare lesion, which, in its original description, appeared in association with hydrops fetalis and other malformations. Its phenotype strongly resembles gonadoblastoma, although in contrast with that rare tumor, FGTD is not associated with the intersexual states or gonadal dysgenesis that accompany such neoplasm. Two reports described an association of FGTD and a morphologically similar lesion with Walker-Warburg syndrome. However, we have not confirmed such an observation, although a nonspecific muscle disorder was found in one of the examples we describe in this article. Here we study 2 additional cases, providing a detailed topographical, histomorphological, and immunophenotypical analysis. A review of all 5 previously described cases is conducted. The features of this lesion support the notion that a focal defect in testicular development is its most likely pathogenesis.

We describe the role of Leydig cells in normal, hyperplastic and neoplastic testis. Recent acquisitions on etiology and pathobiology of Leydig cell proliferations, unusual microscopic presentations and clinical and morphologic features predictive of malignancy are reported.

Tumor suppressor genes can be inactivated by various mechanisms, including promoter hypermethylation and loss of heterozygosity. We screened the 10q locus for loss of heterozygosity and the promoter methylation status of PTEN, MGMT, MXI1, and FGFR2 in neuroblastic tumors and neuroblastoma cell lines. Expression of these genes in cell lines was analyzed with reverse transcriptase-polymerase chain reaction. Loss of heterozygosity at 10q was detected in 18% of tumors and microsatellite instability in 14%. Promoter hypermethylation of MGMT appeared in 8% of tumors and 25% of cell lines. Correlation between methylation status and lack of expression was evident for PTEN, FGFR2, and MXI1 and was less clear for MGMT. No associations between these alterations and MYCN amplification, 1p deletion, or aggressive tumor histology could be demonstrated, singly or in combination. These data suggest that 10q alterations might be implicated in the development of a small number of neuroblastomas.

Despite metastasis as an important cause of death in colorectal cancer patients, current animal models of this disease are scarcely metastatic. We evaluated whether direct orthotopic cell microinjection, between the mucosa and the muscularis layers of the cecal wall of nude mice, drives tumor foci to the most relevant metastatic sites observed in humans and/or improves its yield as compared with previous methods. We injected eight animals each tested human colorectal cancer cell line (HCT-116, SW-620, and DLD-1), using a especially designed micropipette under binocular guidance, and evaluated the take rate, local growth, pattern and rate of dissemination, and survival time. Take rates were in the 75 to 88% range. Tumors showed varying degrees of mesenteric and retroperitoneal lymphatic foci (57 to 100%), hematogenous dissemination to liver (29 to 67%) and lung (29 to 100%), and peritoneal carcinomatosis (29 to 100%). Tumor staging closely correlated with animal survival. Therefore, the orthotopic cell microinjection procedure induces tumor foci in the most clinically relevant metastatic sites: colon-draining lymphatics, liver, lung, and peritoneum. The replication of the clinical pattern of dissemination makes it a good model for advanced colorectal cancer. Moreover, this procedure also enhances the rates of hematogenous and lymphatic dissemination at relevant sites, as compared with previously described methods that only partially reproduce this pattern.

Myofibroblastic inflammatory tumor is a controversial entity that shows great variability in clinical presentation, histological findings, evolution, and prognosis. It is a rare cause of primary lung tumor in adults; however, it is the most common cause of lung tumors in children. The diagnosis is fundamentally histological, although histological diagnosis is not easy because myofibroblastic inflammatory tumor is characterized by a polymorphic cellular infiltration of variable cellular composition that could be similar to other diseases such as lymphoma or low-grade sarcoma. We report the case of a 23-year-old woman in whom a solitary pulmonary nodule was discovered incidentally at plain-film chest x-ray.

Background: Epigenetic alterations and loss of heterozygosity are mechanisms of tumor suppressor gene inactivation. A new carcinogenic pathway, targeting the RAS effectors has recently been documented. RASSF1A, on 3p21.3, and NORE1A, on 1q32.1, are among the most important, representative RAS effectors., Methods: We screened the 3p21 locus for the loss of heterozygosity and the hypermethylation status of RASSF1A, NORE1A and BLU (the latter located at 3p21.3) in 41 neuroblastic tumors. The statistical relationship of these data was correlated with CASP8 hypermethylation. The expression levels of these genes, in cell lines, were analyzed by RT-PCR., Results: Loss of heterozygosity and microsatellite instability at 3p21 were detected in 14% of the analyzed tumors. Methylation was different for tumors and cell lines (tumors: 83% in RASSF1A, 3% in NORE1A, 8% in BLU and 60% in CASP8; cell lines: 100% in RASSF1A, 50% in NORE1A, 66% in BLU and 92% in CASP8). In cell lines, a correlation with lack of expression was evident for RASSF1A, but less clear for NORE1A, BLU and CASP8. We could only demonstrate a statistically significant association between hypermethylation of RASSF1A and hypermethylation of CASP8, while no association with MYCN amplification, 1p deletion, and/or aggressive histological pattern of the tumor was demonstrated., Conclusion: 1) LOH at 3p21 appears in a small percentage of neuroblastomas, indicating that a candidate tumor suppressor gene of neuroblastic tumors is not located in this region. 2) Promoter hypermethylation of RASSF1A and CASP8 occurs at a high frequency in neuroblastomas.

Different authors have investigated the immunohistochemical expression of some proteins in the adenocarcinoma of the stomach, including cell cycle regulators proteins like p53 and Bcl-2; growth factors (c-erb-B2 and EPO-R); angiogenesis-related markers such as COX-2 and cellular adhesion molecules (beta-catenin and E-cadherin). While these proteins have been studied in gastric adenocarcinoma, their immunophenotyping in non tumoral gastric mucous membrane remains unexplored. In the present study, we investigated the expression, function and behavior of these proteins in normal gastric mucous membrane to contribute to gain further knowledge on the significance of their loss or overexpression in malignant gastric tumors.

The present study aims to establish the nature and frequency of testicular lesions in the parenchyma adjacent to testicular germ cell tumors (TGCT) to improve understanding of the factors involved in the development of testicular cancer. Fifty-three cases of TGCT that were fixed in both neutral-buffered formalin and Bouin solution, allowing for the nuclear characterization of Sertoli cells (SCs), were included in this study. In each case, at least 3 sections of different areas of preserved parenchyma surrounding the TGCT were studied. We found Leydig cell hyperplasia, microlithiasis, angiopathy, adenomatous hyperplasia of the rete testis, SC nodules, SC dysgenesis and involution, SC-only tubules, tubular atrophy, adluminal compartment lesions, hypospermatogenesis associated with spermatocyte sloughing, spermatogonial maturation arrest, and hypertrophic and multinucleated spermatogonia. These lesions were found in regions both adjacent and far away from the tumoral mass, and abnormal seminiferous tubules were found intermingled with those showing complete spermatogenesis, suggesting that these lesions are primary and existed before the development of the tumor. Our study suggests that SCs might play a more important role in the development of testicular tumors than previously thought. Our data supports the hypothesis that there is an abnormal differentiation of SCs, caused either by genetic anomalies or by environmental agents during fetal life. This abnormal SC differentiation may cause not only primary spermatogenesis failure and spermatogenesis arrest at different levels, but may also contribute to the poor differentiation of gonocytes into spermatogonia. The abnormal gonocyte differentiation might favor the development of dysplastic germ cells that may later transform into intratubular germ cell neoplasia, unclassified type.

This retrospective study reviews a series of teratomas of the neck and mediastinum aiming at defining the features of these particular locations. We recorded prenatal diagnosis, perinatal management, clinical and radiologic features, pathology, surgical strategies and results in cervical and mediastinal teratomas treated over the last 10 years. During this period we treated 66 children with teratoma of which 11 (6 male and 5 female) had cervicomediastinal locations. Five babies had cervical teratomas extended into the anterior mediastinum in two cases. Prenatal diagnosis was made in three (two with polyhydramnios). Four babies were born by C-section and only one had a successful EXIT procedure. The diagnosis was confirmed by imaging and increased AFP. Surgical treatment involved total tumor removal and in one case subsequent removal of lymph node metastases. All children survived except one in whom airway could not be cleared at birth. Two children bear mild hypothyroidism. During the same period six patients aged 0-17 years were treated for mediastinal teratoma. Only one was prenatally diagnosed and only two had some dyspnea. Removal was performed either by median sternotomy, thoracotomy, or thoracoscopy. They all survive and are free of disease. Teratomas of the neck may cause fetal disease and unmanageable neonatal airway obstruction. Prenatal diagnosis and planned multidisciplinary management are mandatory at birth. In contrast, only some mediastinal tumors cause respiratory embarrassment. Although benign, these tumors are sometimes immature and may metastasize to regional lymph nodes. Total surgical removal is curative. Thyroid insufficiency may be present at birth in cervical teratomas and may be aggravated by surgery.

Choline kinase alpha (ChoKalpha) is a metabolic enzyme involved in the synthesis of phosphatidylcholine, recently implicated in cancer onset since it is overexpressed in a variety of human cancers such as mammary, lung, colorectal and prostate adenocarcinomas. Furthermore, overexpression of ChoKalpha in human HEK293T cells confers them oncogenic properties with the induction of tumors after subcutaneous injection in nude mice. ChoKalpha levels in tumor samples have been analyzed using polyclonal antibodies and Western blotting. These techniques have considerable limitations and do not allow for a precise and efficient evaluation of the real significance of ChoK overexpression in human carcinogenesis. We developed a set of monoclonal antibodies with high specificity and sensitivity against ChoKalpha, and characterized their properties. We provide evidence that the newly generated MoAbs against ChoKalpha have potential use in cancer diagnosis by conventional immunohistochemistry techniques.

The aim of this study is to establish an estimation of the global genomic alteration in neuroblastic tumors (ganglioneuromas, ganglioneuroblastomas and neuroblastomas) and correlate them with different clinical parameters (age, sex, diagnosis, Shimada index, proliferation index, tumor location, and 1p and v-myc avian myelocitomatosis viral-related (MYCN) status) in order to find new molecular and/or prognostic markers for neuroblastoma. To assess the genomic damage in neuroblastic tumors, we used an arbitrarily primed PCR approach, a technique based on the reproducibility of band profiles obtained by a PCR with a low annealing temperature in its first cycles. Genomic damage was assessed by comparing band profiles of tumors and normal paired samples. Gains and losses in the intensity of the bands were computerized and referred to the total number of bands analyzed. We found a higher genomic damage fraction (GDF) in the female's group (U-Mann-Whitney, P = 0.025), but we could not find any association between GDF and tumor location, proliferation index, diagnosis or age of the patient. There was no relationship between 1p status and GDF, but tumors with MYCN amplification had a slightly higher GDF. MYCN amplification might in some way contribute to genomic instability of neuroblastomas.

The oncogene BRAF(V600E) is the most frequent genetic event in papillary thyroid carcinoma (PTC) but its prognostic impact still remains to be elucidated. We evaluated a representative series of 67 individuals with PTC who underwent total thyroidectomy. BRAF-positive tumours correlated with early recurrences (32% vs 7.6%; P=0.02) during a median postoperative follow-up period of 3 years. Interestingly, within the recurrences, a significant majority had negative radioiodine ((131)I) total body scans, predicting a poorer outcome as treatment with (131)I is not effective. This last observation led us to investigate the role of BRAF(V600E) and the MEK-ERK pathway in thyroid dedifferentiation, particularly in Na(+)/I(-) symporter (NIS) impairment, as this thyroid-specific plasma membrane glycoprotein mediates active transport of I(-) into the thyroid follicular cells. A subset of 60 PTC samples was evaluated for NIS immunoreactivity and, accordingly, we confirmed a significant low NIS expression and impaired targeting to membranes in BRAF-positive samples (3.5% vs 30%; P=0.005). Furthermore, experiments with differentiated PCCl3 thyroid cells demonstrated that transient expression of BRAF(V600E) sharply impaired both NIS expression and targeting to membrane and, surprisingly, this impairment was not totally dependent on the MEK-ERK pathway. We have concluded that BRAF(V600E) is a new prognostic factor in PTC that correlates with a high risk of recurrences and less differentiated tumours due to the loss of NIS-mediated (131)I uptake.

Testicular germ cell tumors (TGCTs) include various malignancies with distinct pathologies that share a common precursor lesion (intratubular germ cell neoplasia, unclassified, ITGCNU, or carcinoma in situ, CIS). TGCTs, as a whole, represent a highly curable tumor paradigm, with high sensitivity to radiotherapy and, especially, to cisplatin-based chemotherapy. However, a percentage of cases display therapeutic resistance, and the molecular mechanisms underlying such resistant phenotype remain to be elucidated. We put forward the notion that expression of oncogenic forms of the PCPH gene, which are known to confer resistance to radiation and chemotherapeutic drugs, including cisplatin, may be expressed in TGCTs, and thus contribute to the development of therapeutic resistance. To begin testing this concept, we studied PCPH expression in human TGCT cell lines and in 54 solid tumors by RT-PCR, western immunoblot and immunohistochemistry. The results demonstrated that: i) PCPH is expressed in TGCT cell lines and tumors, including CIS; ii) its expression levels vary among different TGCT pathologies, being generally higher in well differentiated regions and lower in areas of predominant proliferation; iii) PCPH expression is substantially increased in tumors relative to matched normal testicular tissue; iv) tumor samples express PCPH polypeptides of low molecular mass, consistent with the known size of the PCPH oncoprotein, that are either absent from, or markedly reduced in, matched normal tissue. Collectively, these results positively identify PCPH as a good early molecular marker for testicular neoplasms, and strongly indicate that immunodetection of truncated PCPH polypeptides may be a useful diagnostic tool for TGCT.

This study aims to characterize the epididymis-like intratesticular structures (ELITSs), a rare lesion found in elderly men. ELITSs were identified in 6 patients from a review of 1442 autopsies and 271 surgical specimens of adult men. Bilateral lesions were seen in 5 cases. The lesion was located in the proximity of the mediastinal rete testis (6 testes) and at the testicular periphery (4 testes), and at both central and peripheral locations in 1 case. The lesion is characterized by a pseudostratified cylindrical epithelium, with a robust pankeratin and 8, 18, and 19 keratin expression, focal vimentin expression, and apical CD 10 expression, similar to what is proper of the normal human epididymidis. The epithelial layer of ELITSs was surrounded by a thin layer of smooth-muscle cells. The adjacent testicular parenchyma was atrophied and the rete testis showed some associated degenerative lesions related to arteriosclerosis. The ELITSs are distinct from atrophic seminiferous tubules with a Sertoli cell-only pattern and from the benign glandular teratomatous component of an involution of a malignant testicular germ cell tumor, the so-called burn-out germ cell tumor. Clinical and histopathological data suggest that this lesion represents a late Wolffian differentiation similar to the initial segment of the epididymal duct, which represents an unusual manifestation of the aging process.

Paratesticular cysts with benign epithelial proliferations (BEPs) are rare. Only 10 cases were found in a series of 431 paratesticular cysts and were classified as follows: cystadenoma, 5; papilloma, 2; and hamartoma, 3. Four cystadenomas showed multiple papillae lined by CD10+ epithelial cells with hyperchromatic nuclei. The remaining lesion showed areas with a microcystic, glandular, cribriform pattern, with small, benign glands without atypia. Urothelial papilloma presented BEPs with cytokeratin (CK) 7+ and CD10+ and CK20- umbrella-like cells. The mural papilloma was lined by proliferative cylindrical cells exhibiting strong CK7 and CD10 expression. The 3 Wolffian hamartomas were characterized by strongly CD10+ epithelium surrounded by smooth muscle cells. The consistent CD10 expression in BEPs of paratesticular cysts suggests a Wolffian origin. The differential diagnosis of paratesticular cysts with BEP vs metastatic prostatic and primary borderline or malignant tumors is discussed.

Objective: To characterize lysosomes and histochemical function of granular Sertoli cells in developmental alterations., Design: Prospective and retrospective study., Setting: University hospital and research centers., Patient(s): Nineteen infantile and pubertal patients undergoing testicular biopsy; four rat testes for lysosomal study., Intervention(s): CD-68, alpha-1-antitrypsin, vimentin, inhibin alpha subunit, and anti-mullerian hormone antibodies were evaluated. Morphometric measures in seminiferous tubules with and without granular Sertoli cells were obtained. Ultrastructural data of lysosomes in human and rat Sertoli cells were compared., Main Outcome Measure(s): Quantification of mean diameter of seminiferous tubules, tubular fertility index, and germ and Sertoli cell indexes were obtained in human testis., Result(s): Granular changes in Sertoli cells are due to the accumulation of large amounts of lysosomes. Vimentin immunoexpression in infantile and pubertal granular Sertoli cells was lower than in adjacent nongranular Sertoli cells. Inhibin was negative in granular cells. Anti-mullerian hormone-positive and -negative granular Sertoli cells were present within the same tubules., Conclusion(s): The presence of early granular changes in Sertoli cells in childhood and pubertal cryptorchidic patients, associated with other developmental alterations, suggests an intense and irreversible dysfunction of phagocytosis in the granular Sertoli cells. These alterations might be considered primary and irreversible anomalies of Sertoli cells, which might be contributing factors in the infertility seen in these patients.

Background: Malignant mixed tumor of the larynx is a very rare neoplasm; only five cases have been reported, three in the English-language literature., Methods: We report the case of a 69-year-old man with a 2-month history of hoarseness and a left laterocervical palpable mass., Results: Total laryngectomy and bilateral radical neck dissection were performed. The tumor involved the glottic and subglottic regions and thyroid cartilage and extended to the anterior side of the larynx. Microscopically, the tumor was composed of three cellular types: epithelial cells, chondrocytes, and spindle cells. The epithelial cells resembled a moderately differentiated adenocarcinoma, the mesenchymal cells resembled a high-grade chondrosarcoma, and the spindle cells had immunohistochemical features of myoepithelial cells. The tumor metastasized to a cervical lymph node, with the three described components. The patient died 11 months after surgery., Conclusions: The lesion in this case was considered to be a malignant mixed tumor. Differences between this tumor and that of laryngeal chondrosarcoma are discussed., (2005 Wiley Periodicals, Inc.)

Cystic adenomatoid malformation is an uncommon embryonic developmental abnormality usually diagnosed in neonates and infants. Its presentation in adulthood is rare, with only 27 cases reported up to now. Due to its rarity, it is seldom suspected and adult physicians are not familiar with its clinical and radiologic features. We report two cases of cystic adenomatoid malformation presenting in adults, one as a recurrent pneumonia, and another as a coincidental finding on a chest roentgenogram. We describe the clinical features, radiologic and computed tomographic findings, and the histopathologic characteristics in this article, along with a review of the literature.

Objectives: To review current theories about etiology of varicocele and pathogenic mechanisms leading to a progressive disorder of spermatogenesis in relation to the subfertility or infertility these patients may present with. To evaluate its current anatomical knowledge of the normal venous drain of the testicle and its variations that may condition relapse or failure of the treatment of varicocele. To systematically review pathologic testicular lesions in patients with varicocele. To establish factors that may have prognostic significance on post-treatment fertility., Methods: We performed a systematic search in the Medline database for each of the proposed etiological and pathogenic theories on human varicocele. The evaluation of pathologic testicular lesions in patients with varicocele was obtained from the study of testicular biopsies performed at the Hospital La Paz in Madrid over the lost 30 years., Results: Regarding the anatomical theories of varicocele, congenital absence or incompetence of the internal spermatic vein valves, difficult venous drain, augmented hydrostatic pressure of the internal spermatic vein, disorder of the fascial-muscular pump mechanism, and compression of the venous drainage system are considered, among others, potential etiological factors. Regarding possible pathogenic theories of varicocele, we evaluate disorders of testicular thermoregulation, hypoxia, toxic effect of renal and adrenal metabolites, certain endocrine disorders, obstruction of the spermatic tract, disorders of blood flow and epididymal vasculature, oxidative stress, gonadotoxins, apoptosis, and lastly the effect of varicocele on the contralateral testicle., Conclusions: Available data support the idea that etiopathogenesis of varicocele is multifactorial. Many classic etiopathogenic factors related to anatomy, embryology, obstruction, and hyperthermia still prevail in addition to new factors related to oxygen reactive species and apoptosis. However, many pathogenic and physiopathologic aspects of varicocele need to be elucidated yet. As a matter of fact, neither of these data alone may clearly explain the variable effect varicocele has on spermatogenesis and male fertility. So, it is necessary to establish histological criteria with proved prognostic significance that allow us to detect possible progression of testicular lesions after treatment.

The goal of this study was to perform 5-alpha-reductase type 2 gene (SRD5A2) analysis in a male pseudohermaphrodite (MPH) patient with normal testosterone (T) production and normal androgen receptor (AR) gene coding sequences. A patient of Chinese origin with ambiguous genitalia at 14 months, a 46,XY karyotype, and normal T secretion under human chorionic gonadotropin (hCG) stimulation underwent a gonadectomy at 20 months. Exons 1-8 of the AR gene and exons 1-5 of the SRD5A2 gene were sequenced from peripheral blood DNA. AR gene coding sequences were normal. SRD5A2 gene analysis revealed 2 consecutive mutations in exon 4, each located in a different allele: 1) a T nucleotide deletion, which predicts a frameshift mutation from codon 219, and 2) a missense mutation at codon 227, where the substitution of guanine (CGA) by adenine (CAA) predicts a glutamine replacement of arginine (R227Q). Testes located in the inguinal canal showed a normal morphology for age. The patient was a compound heterozygote for SRD5A2 mutations, carrying 2 mutations in exon 4. The patient showed an R227Q mutation that has been described in an Asian population and MPH patients, along with a novel frameshift mutation, Tdel219. Testis morphology showed that, during early infancy, the 5-alpha-reductase enzyme deficiency may not have affected interstitial or tubular development.

Neuroblastoma is the most common pediatric solid tumor. Although many allelic imbalances have been described, a bona fide tumor suppressor gene for this disease has not been found yet. In our study, we analyzed 2 genes, PTEN and DMBT1, mapping 10q23.31 and 10q25.3-26.1, respectively, which have been found frequently altered in other kinds of neoplasms. We screened both genes for homozygous deletions in 45 primary neuroblastic tumors and 12 neuroblastoma cell lines. Expression of these genes in cell lines was assessed by RT-PCR analysis. We could detect 2 of 41 (5%) primary tumors harboring PTEN homozygous deletions. Three of 41 (7%) primary tumors and 2 of 12 cell lines presented homozygous losses at the g14 STS on the DMBT1 locus. All cell lines analyzed expressed PTEN, but lack of DMBT1 mRNA expression was detected in 2 of them. We tried to see whether epigenetic mechanisms, such as aberrant promoter hypermethylation, had any role in DMBT1 silencing. The 2 cell lines lacking DMBT1 expression were treated with 5-aza-2'-deoxycytidine; DMBT1 expression was restored in only one of them (MC-IXC). From our work, we can conclude that PTEN and DMBT1 seem to contribute to the development of a small fraction of neuroblastomas, and that promoter hypermethylation might have a role in DMBT1 gene silencing., (Copyright 2004 Wiley-Liss, Inc.)

Testicular microlithiasis is a well-defined clinical and pathologic entity easily diagnosed through testicular echography; however, its association with cancer and infertility is now under debate. Many efforts have been done in recent years to clarify the spectrum of lesions observed in testicular microlithiasis, but no published data as to the existence of a possible microlithiasis of the epididymis and the rete testis have been found. We have observed microlithiasis of the epididymis and the rete testis in surgical (8 epididymis and 6 testis) and autopsy specimens (12 cases). In decreased order of frequency, microliths of the proximal spermatic way were seen in rete testis, epididymal duct, and efferent ducts. Intraluminal, subepithelial, and interstitial microliths were localized along these segments of the spermatic way. Subepithelial microliths were the most frequently found. A granulomatous reaction around the interstitial epididymal microliths, mimicking malacoplakia, was observed in 1 case. The differential diagnosis of microliths includes corpora amilacea, Michaelis-Gutmann bodies, calcium deposits, hyaline globules, and parasites, like the giant kidney worm Dioctophyme renale. In infants and young adults, microlithiasis of the epididymis and the rete testis is frequently associated with alterations in the development of the proximal spermatic way. In elderly adults, it is related to ischemia and obstruction of the spermatic way.

Ghrelin, the endogenous ligand for the GH secretagogue receptor (GHS-R), has been primarily linked to the central neuroendocrine regulation of GH secretion and food intake, although additional peripheral actions of ghrelin have also been reported. In this context, the expression of ghrelin and its cognate receptor has been recently demonstrated in rat testis, suggesting a role for this molecule in the direct control of male gonadal function. However, whether this signaling system is present in human testis remains largely unexplored. In this study we report the expression and cellular location of ghrelin and its functional receptor, the type 1a GHS-R, in adult human testis. In addition, evaluation of ghrelin and GHS-R1a immunoreactivity in testicular tumors and dysgenetic tissue is presented. The expression of the mRNAs encoding ghrelin and GHS-R1a was demonstrated in human testis specimens by RT-PCR, followed by direct sequencing. In normal testis, ghrelin immunostaining was demonstrated in interstitial Leydig cells and, at lower intensity, in Sertoli cells within the seminiferous tubules. In contrast, ghrelin was not detected in germ cells at any stage of spermatogenesis. The cognate ghrelin receptor showed a wider pattern of cellular distribution, with detectable GHS-R1a protein in germ cells, mainly in pachytene spermatocytes, as well as in somatic Sertoli and Leydig cells. Ghrelin immunoreactivity was absent in poorly differentiated Leydig cell tumor, which retained the expression of GHS-R1a peptide. In contrast, highly differentiated Leydig cell tumors expressed both the ligand and the receptor. The expression of ghrelin and GHS-R1a was also detected in dysgenetic Sertoli cell-only seminiferous tubules, whereas germ cell tumors (seminoma and embryonal carcinoma) were negative for ghrelin and were weakly positive for GHS-R1a. In conclusion, our results demonstrate that ghrelin and the type 1a GHS-R are expressed in adult human testis and testicular tumors. Overall, the expression of ghrelin and its functional receptor in human and rat testis, with roughly similar patterns of cellular distribution, is highly suggestive of a conserved role for this newly discovered molecule in the regulation of mammalian testicular function.

Testicular biopsies from 80 azoospermic young men were revised and the average numbers per cross-sectioned tubule of each germ cell type were calculated and compared with those of control normal testes. In 53 patients, azoospermia had an obstructive cause, and in 22 of those 53 patients more adult spermatids were found by testicular biopsy than young spermatids (over 100% in some testes), in one or both testes. However, in normal testes fewer mature spermatids than young spermatids (23.3%) were found. In the 22 patients, the causes of azoospermia were: vasectomy (7 patients), bilateral agenesis of the vas deferens (3 patients), Young syndrome (3 patients), bilateral cysts in the caput epididymidis (1 patient), bilateral inguinal herniorrhaphy (1 patient), left varicocele (1 patient), and unknown causes (6 patients). Biopsies were bilateral except for 3 cases (a vasectomized patient, a patient with Young syndrome, and a patient with obstruction due to an unknown cause). Hormonal levels were normal in the 22 patients. In addition, testicular biopsies of 3 twisted testes from 3 young adult men showing a number of adult spermatids higher than that of young spermatids were also included in the study. All testicular biopsies-including those of the twisted testes-showed an obstructive histologic pattern, consisting of a mosaic distribution of testicular lesions: mainly tubular ectasis and germ cell sloughing into the adluminal compartment of seminiferous tubules. The increase in the number of adult spermatids was bilateral in 1 of the 6 vasectomized men who underwent bilateral biopsy, and in 7 of the 11 bilaterally biopsied patients with obstructive azoospermia due to other causes. The most probable explanation for the increased number of adult spermatids is stagnation of testicular fluid, caused by sperm excretory duct obstruction. The unilateral increase in the number of adult spermatids in vasectomized men might be related to the occurrence of a spermatic granuloma (a frequent finding in vasectomy) in the proximal end of the sectioned ductus deferens ipsilateral to the testis with nonincreased adult spermatid numbers, and the absence of spermatic granuloma in the ductus deferens ipsilateral to the testis with increased adult spermatid numbers. This granuloma would produce, in addition to spermatozoon destruction, reabsorption of the testicular and epididymal fluids. The higher rate of bilateral increase, in the number of young spermatids observed in the patients with congenital lesions of the ductus deferens or the ductus epididymidis, might be related to the absence of spermatic granulomas in congenital obstructions.