Browsing by Author "Velasco, Alfredo"

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    Assessment of normalization strategies for quantitative RT-PCR using microdissected tissue samples
    (2007) Erickson, Heidi S.; Albert, Paul S.; Gillespie, John W.; Wallis, Benjamin S.; Rodriguez-Canales, Jaime; Linehan, W. Marston; Gonzalez, Sergio; Velasco, Alfredo; Chuaqui, Rodrigo F.; Emmert-Buck, Michael R.
    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.
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    Association of adrenal medullar and cortical nodular hyperplasia - A report of two cases with clinical and morpho-functional considerations
    (HUMANA PRESS INC, 2006) Valdes, Gloria; Roessler, Eric; Salazar, Ivan; Rosenberg, Helmar; Fardella, Carlos; Martinez, Pedro; Velasco, Alfredo; Velasco, Soledad; Orellana, Pilar
    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 I-131 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.
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    Comparison of snap freezing versus ethanol fixation for gene expression profiling of tissue specimens
    (2004) Perlmutter, M. A.; Best, C. J.; Gillespie, John W.; Gathright, Y.; González Bombardiere, Sergio; Velasco, Alfredo; Linehan, W. M.; Emmert Buck, Michael R.; Chuaqui F., Rodrigo
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    Global expression analysis of prostate cancer-associated stroma and epithelia
    (2007) Richardson, Annely M.; Woodson, Karen; Wang, Yonghong; Rodriguez-Canales, Jaime; Erickson, Heidi S.; Tangrea, Michael A.; Novakovic, Kristian; Gonzalez, Sergio; Velasco, Alfredo; Kawasaki, Ernest S.; Emmert-Buck, Michael R.; Chuaqui, Rodrigo F.; Player, Audrey
    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.
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    Mismatch repair expression in testicular cancer predicts recurrence and survival
    (WILEY-LISS, 2008) Velasco, Alfredo; Corvalan, Alejandro; Wistuba, Ignacio I.; Riquelme, Erick; Chuaqui, Rodrigo; Majerson, Alejandro; Leach, Fredrick S.
    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. (c) 2007 Wiley-Liss, Inc.