Alfredo Velasco

Molecular Alterations in Primary Prostate Cancer after Androgen Ablation Therapy

Purpose: After an initial response to androgen ablation, most prostate tumors recur, ultimately progressing to highly aggressive androgen-independent cancer. The molecular mechanisms underlying progression are not well known in part due to the rarity of androgen-independent samples from primary and metastatic sites. Experimental Design: We compared the gene expression profiles of 10 androgen-independent primary prostate tumor biopsies with 10 primary, untreated androgen-dependent tumors. Samples were laser capture microdissected, the RNA was amplified, and gene expression was assessed using Affymetrix Human Genome U133A GeneChip. Differential expression was examined with principal component analysis, hierarchical clustering, and Students t testing. Analysis of gene ontology was done with Expression Analysis Systematic Explorer and gene expression data were integrated with genomic alterations with Differential Gene Locus Mapping. Results: Unsupervised principal component analysis showed that the androgen-dependent and androgen-independent tumors segregated from one another. After filtering the data, 239 differentially expressed genes were identified. Two main gene ontologies were found discordant between androgen-independent and androgen-dependent tumors: macromolecule biosynthesis was down-regulated and cell adhesion was up-regulated in androgen-independent tumors. Other differentially expressed genes were related to interleukin-6 signaling as well as angiogenesis, cell adhesion, apoptosis, oxidative stress, and hormone response. The Differential Gene Locus Mapping analysis identified nine regions of potential chromosomal deletion in the androgen-independent tumors, including 1p36, 3p21, 6p21, 8p21, 11p15, 11q12, 12q23, 16q12, and 16q21. Conclusions: Taken together, these data identify several unique characteristics of androgen-independent prostate cancer that may hold potential for the development of targeted therapeutic intervention.

Mismatch repair gene expression and genetic instability in testicular germ cell tumor

Human mismatch repair (MMR) genes encode highly conserved interacting proteins that correct replication errors predisposing to hereditary gastrointestinal and genitourinary malignancies. A subset of sporadic genitourinary tumors also exhibits MMR deficiency and can be identified by measuring the frequency of microsatellite instability (MSI) in cancer cell DNA. We investigated the expression of the two most commonly mutated MMR genes, MSH2 and MLH1 in sporadic testicular germ cell tumor (GCT) in order to: 1) determine the expression pattern of MSH2 and MLH1 proteins in normal seminiferous tubules and histologically distinct GCT subtypes, 2) correlate MMR gene expression with genetic instability in GCT and 3) develop a panel of molecular markers that can identify genetically distinct subsets of GCT for prognostic assessment. MSH2 and MLH1 had differential staining patterns in normal seminiferous tubules and malignant tissues. MSH2 was expressed in all stages of spermatogenesis up to but excluding mature sperm whereas MLH1 was predominantly expressed in premeiotic germ cells. All histological GCT subtypes showed differential immunostaining for MSH2 and MLH1 however pure seminoma had statistically significant fewer low MSH2 staining tumors than other subtypes (p=0.046). Twenty-five percent of GCT exhibited increased frequency of MSI (MSI+ tumors) with 73, 70 and 43% of MSI+ tumors exhibiting low MSH2, low MLH1 or low MSH2 and low MLH1 staining respectively. Fifteen percent of testicular GCT exhibited loss of heterozygosity (LOH) but no MSI (LOH only tumors). Only 28, 17 or 6% of LOH only tumors exhibited low MSH2, low MLH1 or low MSH2 and low MLH1 staining respectively.

Mismatch repair expression in testicular cancer predicts recurrence and survival.

We investigated mismatch repair (MMR) gene expression in testicular cancer as a molecular marker for clinical outcome (recurrence, response to chemotherapy and death) using protein expression and specific genetic alterations associated with the presence or absence of MMR activity. One hundred sixty‐two cases of paraffin‐embedded testis cancer specimens were subjected to immunohistochemical analysis using monoclonal antibody for MLH1 and MSH2 MMR proteins and genetic analysis using specific polymorphic markers. The degree of MMR immunoreactivity and genetic instability in the form of loss of heterozygosity (LOH) and/or microsatellite instability (MSI) were determined by comparing matched normal and tumor tissue. The degree of immunohistochemical staining for MMR expression was associated with a shorter time to tumor recurrence, resistance to chemotherapy and death. Furthermore, clinical relapse and cancer specific death was also associated with tumors exhibiting a high degree of MSI, p = 0.01 and 0.04, respectively. In contrast, LOH was not associated with recurrence, resistance to chemotherapy or death. Therefore, MMR expression defines testis cancers with distinct molecular properties and clinical behavior, such that tumors with decreased MMR immunostaining and/or increased frequency of MSI have a shorter time to recurrence and death despite chemotherapy.

Global Expression Analysis of Prostate Cancer-associated Stroma and Epithelia

Characterization of gene expression profiles in tumor cells and the tumor microenvironment is an important step in understanding neoplastic progression. To date, there are limited data available on expression changes that occur in the tumor-associated stroma as either a cause or consequence of cancer. In the present study, we employed a 54,000 target oligonucleotide microarray to compare expression profiles in the 4 major components of the microenvironment: tumor epithelium, tumor-associated stroma, normal epithelium, and normal stroma. Cells from 5 human, whole-mount prostatectomy specimens were microdissected and the extracted and amplified mRNA was hybridized to an Affymetrix Human Genome U133 Plus 2.0 GeneChip. Using the intersection of 2 analysis methods, we identified sets of differentially expressed genes among the 4 components. Forty-four genes were found to be consistently differentially expressed in the tumor-associated stroma; 35 were found in the tumor epithelium. Interestingly, the tumor-associated stroma showed a predominant up-regulation of transcripts compared with normal stroma, in sharp contrast to the overall down-regulation seen in the tumor epithelium relative to normal epithelium. These data provide insight into the molecular changes occurring in tumor-associated stromal cells and suggest new potential targets for future diagnostic, imaging, or therapeutic intervention.

Assessment of normalization strategies for quantitative RT-PCR using microdissected tissue samples.

Gene expression measurement techniques such as quantitative reverse transcriptase (qRT)-PCR require a normalization strategy to allow meaningful comparisons across biological samples. Typically, this is accomplished through the use of an endogenous housekeeping gene that is presumed to show stable expression levels in the samples under study. There is concern regarding how precisely specific genes can be measured in limited amounts of mRNA such as those from microdissected (MD) tissues. To address this issue, we evaluated three different approaches for qRT-PCR normalization of dissected samples; cell count during microdissection, total RNA measurement, and endogenous control genes. The data indicate that both cell count and total RNA are useful in calibrating input amounts at the outset of a study, but do not provide enough precision to serve as normalization standards. However, endogenous control genes can accurately determine the relative abundance of a target gene relative to the entire cellular transcriptome. Taken together, these results suggest that precise gene expression measurements can be made from MD samples if the appropriate normalization strategy is employed.

Microsatellite instability and loss of heterozygosity have distinct prognostic value for testicular germ cell tumor recurrence.

Germ cell tumor (GCT) is the most common genitourinary malignancy of men between the ages of 18 and 35. Therapy is ultimately successful in over 90% of patients, however significant morbidity and mortality can be associated with adjuvant treatment and relapse. Molecular markers that predict treatment response and/or poor outcome would have immediate clinical benefit since adjuvant treatment could be selectively reserved for patients at higher risk for relapse and those patients most likely to respond to treatment. In order to identify potential prognostic molecular markers, we evaluated 118 GCT for microsatellite instability (MSI), loss of heterozygosity (LOH) and MSH2 immunostaining to identify tumors associated with relapse and/or poor outcome following initial surgical, medical and/or radiation therapy. MSI in 3 or more markers and/or low MSH2 staining were associated with relapse while LOH in the absence of MSI and/or high MSH2 staining were not. Twenty-five percent of GCT exhibited genetic instability in 3 or more microsatellite markers (MSI+ tumors), 15% exhibited LOH in the absence of MSI (LOH only tumors) and 44% exhibited decreased or absent MSH2 immunostaining (low MSH2 staining tumors). Thirty-six patients (30%) relapsed and 27 of these patients (75%) had MSI+ and/or low MSH2 staining tumors. Only 1 patient (3%) with an LOH only tumor and no patients with high MSH2 staining and LOH only tumors relapsed. Therefore distinct GCT subpopulations identified by detection of MSI, LOH and MMR expression are associated with different clinical outcomes. MMR deficient testicular GCT with increased frequency of MSI had an increased association with tumor recurrence compared to GCT with an intact MMR system and LOH in the absence of MSI.

La melatonina reduce la respuesta de cortisol al ACTH en humanos

Adrenal glandswere obtained from 4 patients undergoing unilateral nephrectomy-adrenalectomy for renalcancer. Expression of mRNA MT1 and MT2 melatonin receptors was measured by ReverseTranscriptase Polymerase Chain Reaction (RT-PCR). The effect of melatonin on the response tointravenous (i.v.) ACTH was tested (randomized cross-over, double-blind, placebo-controlledtrial) in eight young healthy males pretreated with dexamethasone (1 mg) at 23:00 h. On thenext day, at 08:00 h, an i.v. line was inserted, at 08:30 h, and after a blood sample, subjectsingested 6 mg melatonin or placebo. At 09:00 h, 1-24 ACTH (Cortrosyn, 1

Association of adrenal medullar and cortical nodular hyperplasia: a report of two cases with clinical and morpho-functional considerations.

Arterial hypertension of adrenal etiology is mainly attributed to primary hyperaldosteronism. However, subtle expressions of hyperadrenergic or glucocorticoid excess can also generate arterial hypertension. The present report describes two hypertensive patients cataloged as resistant essential hypertensives, in whom adrenal masses were found incidentally, who highlight the need to recognize these tenuous clinical or laboratory presentations. Case 1 was a 50-yr-old female with hyperadrenergic hypertension associated to a left adrenal node, normal cortisol and aldosterone:renin ratio, marginally increased urinary normetanephrine, and a positive 131I MIBG radioisotope scan. Adrenalectomy normalized blood pressure and urinary metanephrines. Pathology showed a hyperplastic adrenal medulla associated to a multinodular cortical hyperplasia. Case 2 was a 62-yr-old female with progressive hypertension, a slight Cushing phenotype, non-suppressible hypercortisolism, normal urinary metanephrines, and bilateral adrenal nodes. Bilateral adrenalectomy and subsequent replacement normalized blood pressure and phenotypic stigmata. Pathology demonstrated bilateral cortical multinodular hyperplasia and medullary hyperplasia. The clinical study in both patients was negative for MEN. The apparently rare association of cortical and medullary lesions presented by both patients is probably overlooked in routine pathology exams, but should be meticulously searched since the crosstalk between the adrenal cortex and medulla may prompt dual abnormalities.

Cirugía conservadora de riñón para tumores renales pequeños: papel de la radiofrecuencia

Resumen es: Objetivo: La radiofrecuencia con asistencia laparoscopica es una opcion de tratamiento minimamente invasivo para la conservacion de parenquima renal, esp...

El tamaño de los tumores suprarrenales ¿está en relación al tiempo de evolución o expresa una diferencia biológica?

BACKGROUND Adrenal tumor (AT) malignancy has been related to tumor size. Since laparoscopic surgery is being used, smaller adrenal tumors are being excised. AIM To evaluate eventual clinical and histological differences between adrenal tumors smaller than 4 cm. and those larger than 6 cm. PATIENTS AND METHODS Retrospective review of pathological reports and clinical records of patients operated for adrenal tumors, dividing them in two groups. Group 1 had 29 patients aged 52 +/- 13 years with AT < 4 cm operated during the period 2000-2005, and Group 2 was formed by 52 patients aged 46 +/-18 years with AT >6 cm operated between 1984-2005- Tumors between 4 and 6 cm were not included in the study to establish clear cut differences between groups. RESULTS Tumors were functional in 40 and 41% of cases in groups 1 and 2 respectively. Fifty percent of functional tumors of group 1 were pheochromocytomas and the rest secreted aldosterone. In group 2, 66% of tumors were pheochromocytomas and no aldosterone secreting tumors were found. Fifty two and eight percent of tumors in Groups 1 and 2 were adenomas, respectively (p <0.001). Nineteen tumors of group 2 were malignant, compared with one of group 1 (p <0.001). CONCLUSIONS The tumor size of adrenal cortical tumors may represent biological differences, suggesting two different tumor populations. At time of diagnosis adrenal carcinomas are almost always larger than 6 cm.