Prognosis in colorectal cancer beyond TNM / Prognóstico no carcinoma colorretal para além do TNM

ABSTRACT Introduction: Colorectal cancer is one of the neoplasms with the greatest social impact. Given the great molecular heterogeneity and diversity of pathophysiological mechanisms, it is difficult to define prognostic factors that could guide therapy. Objectives: To identify the molecular prognostic factors that may be of interest in clinical practice and to synthesize the existing evidence. Material and methods: The search for the articles was carried out using the PubMed platform and the keywords "sporadic colorectal cancer and prognosis", for articles published between 2014 and 2019. We selected all articles published on studies in humans and written in English or Portuguese. Of the 215 articles found, 35 articles were selected to perform this review. Results: Current evidence supports the use of four molecular markers in clinical practice − KRAS, NRAS and BRAF (EGFR signalling pathway) and the mismatch repair status. Conclusion: The use of molecular biomarkers in clinical practice to define prognosis is still little supported by the existent evidence. The studies are slightly contradictory, so new projects and international collaborations must be carried out in this area to obtain more robust evidence.

RESUMO Introdução: O carcinoma colorretal é uma das neoplasias com maior impacto social. Dada a grande heterogeneidade molecular e diversidade de mecanismos fisiopatológicos, torna-se difícil definir fatores de prognóstico que orientem a terapêutica. Objetivos: Identificar os fatores de prognóstico moleculares que poderão vir a ter interesse na prática clínica e fazer uma síntese da evidência existente. Material e métodos: A pesquisa dos artigos foi realizada recorrendo à plataforma PubMed e utilizou-se as palavras-chave "sporadic colorectal cancer and prognosis", para artigos publicados entre 2014 e 2019. Foram selecionados todos os artigos publicados sobre estudos em humanos e escritos em inglês ou em português. Dos 215 artigos encontrados, foram selecionados 35 artigos para realizar esta revisão. Resultados: A evidência atual apoia a utilização de quatro marcadores moleculares na prática clínica - KRAS, NRAS e BRAF (via de sinalização do EGFR) e o estado mismatch repair. Conclusão: A utilização na prática clínica de biomarcadores moleculares para definir o prognóstico é ainda pouco apoiada pela evidência disponível. Os estudos são algo contraditórios, pelo que novos projetos e colaborações internacionais devem ser realizados neste âmbito para se obter evidência mais robusta.

Citometría de flujo en reticulocitos de sangre periférica como indicador de inestabilidad cromosómica en pacientes con gliomas de alto grado / Flow cytometry in peripheral blood reticulocytes as a marker of chromosome instability in highgrade glioma patients

Resumen Introducción. La cuantificación de la inestabilidad cromosómica es un parámetro importante para evaluar la genotoxicidad y la radiosensibilidad. Las técnicas convencionales requieren cultivos celulares o laboriosos análisis microscópicos de cromosomas o núcleos. La citometría de flujo en reticulocitos ha surgido como una alternativa para los estudios in vivo, ya que reduce los tiempos de análisis e incrementa hasta en 20 veces el número de células analizables. Objetivos. Estandarizar los parámetros de citometría de flujo requeridos para seleccionar y cuantificar reticulocitos micronucleados (RET-MN) a partir de muestras de sangre periférica, y cuantificar la frecuencia de esta subpoblación anormal como medida de inestabilidad citogenética en sendas poblaciones de voluntarios sanos (n=25) y pacientes (n=25) recién diagnosticados con gliomas de alto grado antes de iniciar el tratamiento. Materiales y métodos. Las células sanguíneas se marcaron con anti-CD71-PE para reticulocitos, anti- CD61-FITC para la exclusión de plaquetas y yoduro de propidio para detectar el ADN en reticulocitos. La fracción celular MN-RETCD71+ se seleccionó y se cuantificó con un citómetro de flujo automático. Resultados. Se describió detalladamente la estandarización de los parámetros citométricos, con énfasis en la selección y la cuantificación de la subpoblación celular MN-RETCD71+. Se establecieron los niveles basales de MN-RETCD71+ en la población de control y en los pacientes se encontró un incremento de 5,2 veces antes de iniciar el tratamiento (p<0,05). Conclusión. Los resultados evidenciaron la utilidad de la citometría de flujo acoplada a la marcación de las células RETCD71+ como método eficiente para cuantificar la inestabilidad cromosómica in vivo. Se sugieren posibles razones del incremento de micronúcleos en células RETCD71+ de pacientes con gliomas.

Abstract Introduction: The quantification of chromosomal instability is an important parameter to assess genotoxicity and radiosensitivity. Most conventional techniques require cell cultures or laborious microscopic analyses of chromosomes or nuclei. However, a flow cytometry that selects the reticulocytes has been developed as an alternative for in vivo studies, which expedites the analytical procedures and increases up to 20 times the number of target cells to be analyzed. Objectives: To standardize the flow cytometry parameters for selecting and quantifying the micronucleated reticulocytesCD71+ (MN-RET) from freshly drawn peripheral blood and to quantify the frequency of this abnormal cell subpopulation as a measure of cytogenetic instability in populations of healthy volunteers (n =25), and patients (n=25), recently diagnosed with high-grade gliomas before the onset of treatment. Materials and methods: Blood cells were methanol-fixed and labeled with anti-CD-71-PE for reticulocytes, antiCD-61-FITC for platelet exclusion, and propidium iodide for DNA detection in reticulocytes. The MN-RETCD71+ cell fraction was selected and quantified with an automatic flow cytometer. Results: The standardization of cytometry parameters was described in detail, emphasizing the selection and quantification of the MN-RETCD71+ cellular fraction. The micronuclei basal level was established in healthy controls. In patients, a 5.2-fold increase before the onset of treatment was observed (p <0.05). Conclusion: The data showed the usefulness of flow cytometry coupled with anti-CD-71-PE and anti- CD-61-FITC labeling in circulating reticulocytes as an efficient and high resolution method to quantify chromosome instability in vivo. Finally, possible reasons for the higher average of micronuclei in RETCD71+ cells from untreated high-grade glioma patients were discussed.

Current applications of molecular pathology in colorectal carcinoma

Molecular pathology is playing an increasingly important role in the treatment and overall management of patients with colorectal carcinoma. Three distinct genetic pathways have been identified that play a role in carcinogenesis: the chromosomal instability pathway, the microsatellite instability pathway, and the CpG island methylator phenotype pathway. Certain genetic mutations, some of which overlap with the aforementioned pathways, can also indicate that a carcinoma patient has a genetic predisposition syndrome, such as familial adenomatous polyposis, Lynch syndrome, and hamartomatous polyposis syndromes. A variety of advanced methods, including next-generation sequencing, are available to test for these and other mutations, such as targetable mutations that may allow tailoring of a treatment regimen to a patient's specific cancer (e.g., KRAS and BRAF mutations). The possible future role of testing circulating tumor cells is also addressed. New mutations and syndromes continue to be discovered, ensuring that our knowledge of colorectal carcinoma and our ability to treat it will advance in the future (AU)

Carcinogenesis Colorrectal. Nuevas perspectivas e implicancias clínicas para su detección / Colorectal Carcinogenesis. New perspectives and clinical implications for its detection

El cáncer colorrectal (CCR) es una de las principales causas de morbilidad y mortalidad a nivel mundial. Clásicamente se considera a los adenomas como las lesiones precursoras del CCR y se estipula un tiempo de 10 a 15 años para completar la secuencia adenoma-carcinoma. El CCR evoluciona a través de la acumulación progresiva de alteraciones genéticas y epigenéticas, las que conducen a la transformación de la mucosa colónica normal en cáncer invasivo. La identificación de diferentes vías moleculares de carcinogénesis colorrectal ha demostrado la naturaleza heterogénea del cáncer colónico. De reciente descripción, las lesiones aserradas muestran cambios moleculares y patológicos distintos a los adenomas tradicionales, estimándose que presentan un tiempo más acelerado de evolución hacia la malignidad. El objetivo de esta revisión es actualizar conocimientos sobre la génesis tumoral y sus bases biomoleculares a fin de posibilitar su aplicación a etapas clínicas concretas como la prevención y el tratamiento

Colorectal cancer (CRC) is one of the main causes of morbidity and mortality worldwide. Adenomas are classically regarded as precursor lesions of CRC and between 10 and 15 years is thought to elapse to complete the adenoma-carcinoma sequence. CRC evolves through the progressive accumulation of genetic and epigenetic alterations that lead to invasive cancer through the transformation of normal colonic mucosa. The identification of different molecular pathways of colorectal carcinogenesis has demonstrated the heterogeneous nature of colon cancer. Recent description of serrated lesions shows molecular and pathological changes other than traditional adenomas with an estimated faster time of progression to malignancy. The aim of this review is to update the knowledge about tumorigenesis and its biomolecular basis for clinical application in early stages providing firm ground for prevention and treatment

Genetic evaluation of mesenchymal stem cells by G-banded karyotyping in a Cell Technology Center

OBJECTIVE: To present the initial results of first three years of implementation of a genetic evaluation test for bone marrow-derived mesenchymal stem cells in a Cell Technology Center. METHODS: A retrospective study was carried out of 21 candidates for cell therapy. After the isolation of bone marrow mononuclear cells by density gradient, mesenchymal stem cells were cultivated and expanded at least until the second passage. Cytogenetic analyses were performed before and after cell expansion (62 samples) using G-banded karyotyping. RESULTS: All the samples analyzed, before and after cell expansion, had normal karyotypes, showing no clonal chromosomal changes. Signs of chromosomal instability were observed in 11 out of 21 patients (52%). From a total of 910 analyzed metaphases, five chromatid gaps, six chromatid breaks and 14 tetraploid cells were detected giving as total of 25 metaphases with chromosome damage (2.75%). CONCLUSION: The absence of clonal chromosomal aberrations in our results for G-banded karyotyping shows the maintenance of chromosomal stability of bone marrow-derived mesenchymal stem cells until the second passage; however, signs of chromosomal instability such as chromatid gaps, chromosome breaks and tetraploidy indicate that the long-term cultivation of these cells can provide an intermediate step for tumorigenesis. .

Increased frequency of micronuclei in the lymphocytes of patients chronically infected with hepatitis B or hepatitis C virus

In this study, we analysed the frequency of micronuclei (MN), nucleoplasmic bridges (NPBs) and nuclear buds (NBUDs) and evaluated mutagen-induced sensitivity in the lymphocytes of patients chronically infected with hepatitis B virus (HBV) or hepatitis C virus (HCV). In total, 49 patients with chronic viral hepatitis (28 HBV-infected and 21 HCV-infected patients) and 33 healthy, non-infected blood donor controls were investigated. The frequencies (‰) of MN, NPBs and NBUDs in the controls were 4.41 ± 2.15, 1.15 ± 0.97 and 2.98 ± 1.31, respectively. The frequencies of MN and NPBs were significantly increased (p < 0.0001) in the patient group (7.01 ± 3.23 and 2.76 ± 2.08, respectively) compared with the control group. When considered separately, the HBV-infected patients (7.18 ± 3.57) and HCV-infected patients (3.27 ± 2.40) each had greater numbers of MN than did the controls (p < 0.0001). The HCV-infected patients displayed high numbers of NPBs (2.09 ± 1.33) and NBUDs (4.38 ± 3.28), but only the HBV-infected patients exhibited a significant difference (NPBs = 3.27 ± 2.40, p < 0.0001 and NBUDs = 4.71 ± 2.79, p = 0.03) in comparison with the controls. Similar results were obtained for males, but not for females, when all patients or the HBV-infected group was compared with the controls. The lymphocytes of the infected patients did not exhibit sensitivity to mutagen in comparison with the lymphocytes of the controls (p = 0.06). These results showed that the lymphocytes of patients who were chronically infected with HBV or HCV presented greater chromosomal instability.

Contributions of cytogenetics to cancer research / Contribuições da citogenética em pesquisas sobre o câncer

The two conflicting visions of tumorigenesis that are widely discussed are the gene-mutation hypothesis and the aneuploidy hypothesis. In this review we will summarize the contributions of cytogenetics in the study of cancer cells and propose a hypothetical model to explain the influence of cytogenetic events in carcinogenesis, emphasizing the role of aneuploidy. The gene mutation hypothesis states that gene-specific mutations occur and that they maintain the altered phenotype of the tumor cells, and the aneuploidy hypothesis states that aneuploidy is necessary and sufficient for the initiation and progression of malignant transformation. Aneuploidy is a hallmark of cancer and plays an important role in tumorigenesis and tumor progression. Aneuploid cells might be derived from polyploid cells, which can arise spontaneously or are induced by environmental agents or chemical compounds, and the genetic instability observed in polyploid cells leads to chromosomal losses or rearrangements, resulting in variable aberrant karyotypes. Because of the large amount of evidence indicating that the correct chromosomal balance is crucial to cancer development, cytogenetic techniques are important tools for both basic research, such as elucidating carcinogenesis, and applied research, such as diagnosis, prognosis and selection of treatment. The combination of classic cytogenetics, molecular cytogenetics and molecular genetics is essential and can generate a vast amount of data, enhancing our knowledge of cancer biology and improving treatment of this disease.

As duas visões conflitantes da tumorigênese que são amplamente discutidas são a hipótese da mutação gênica e a hipótese da aneuploidia. Nesta revisão vamos resumir as contribuições da citogenética no estudo das células tumorais e propor um modelo hipotético para explicar a influência dos eventos citogenéticos na carcinogênese, enfatizando o papel da aneuploidia. A teoria da mutação gênica estabelece que mutações específicas ocorrem e mantêm o fenótipo alterado das células de um tumor, enquanto a hipótese da aneuploidia estabelece que a aneuploidia é necessária e suficiente para a iniciação e progressão da transformação maligna. A aneuploidia é considerada um marcador do câncer e esta desempenha um importante papel tanto na tumorigênese, quanto na progressão tumoral. Células aneuplóides podem ser derivadas de células poliplóides, que surgem espontaneamente ou são induzidas por agentes ambientais ou compostos químicos. A instabilidade genética observada em células poliplóides leva a perdas ou rearranjos cromossômicos, resultando em cariótipos variavelmente aberrantes. Devido à grande quantidade de evidências indicando que um balanço cromossômico correto é crucial para o desenvolvimento do câncer, as técnicas citogenéticas são ferramentas importantes tanto para a pesquisa básica, tais como pesquisas para elucidar a carcinogênese, quanto pesquisas aplicadas, como no diagnóstico, prognóstico e escolha do tratamento. A combinação da citogenética clássica, citogenética molecular e genética molecular é essencial e pode gerar uma grande quantidade de dados, aumentando o nosso conhecimento da biologia do câncer, melhorando assim o tratamento desta doença.

Síndrome de Nijmegen: una enfermedad genética a considerar en un paciente con microcefalia, retardo mental y lesiones de la piel / Nijmegen breakage syndrome

Se presenta el caso de un paciente masculino que a los 6 años de edad es derivado a Neurología Infantil para su estudio por presentar microcefalia y retardo mental. Tras ser evaluado por Inmunología y Genética se realiza en el Laboratorio de citogenética humana, programa de genética ICBM, Facultad de Medicina Universidad de Chile, PCR para deleción 657 del5 que confirma el diagnóstico de Nijmegen dando como resultado deleción nucleótido 5, mutación 657 del5, característico del Síndrome de Nijmegen. Actualmente el niño tiene 13 años y es tratado en el Servicio de Oncología infantil por el desarrollo de linfoma difuso de células grandes B, patología frecuente en este sindrome.

A case of a male patient at 6 years old was referred to child neurology for study due to microcephaly and mental retardation. After being evaluated for Immunology and Genetics Laboratory is performed in human cytogenetics, genetic program ICBM, Faculty of Medicine University of Chile, PCR for deletion 657 of the 5 that confirms the diagnosis of Nijmegen nucleotide deletion resulting in 5, 657 mutation del 5 Characteristic of the syndrome of Nijmegen. Currently the child is 13 and is treated at the Children’s Oncology Service in the development of lymphoma diffuse large B cell, common pathology in this syndrome.

Detección de aneuploidías del cromosoma 17 y deleción del gen TP53 en una amplia variedad de tumores sólidos mediantehibridación in situ fluorescente bicolor / Detection of chromosome 17 aneuplody and TP53 gene deletion in a broad variety of solid tumors by dual-color fluorescence in situ hybridization (FISH)

Introducción. El TP53 es un gen supresor de tumores localizado en la región cromosómica 17p13.1; controla el ciclo celular y se encuentra alterado en cerca de 50% de todas las neoplasias.Objetivos. Determinar las aneuploidías del cromosoma 17 y la deleción en el locus 17p13.1 del gen TP53, en diversos tumores sólidos primarios utilizando la técnica FISH-bicolor. Materiales y métodos. Se analizaron 38 muestras de diversos tipos de tumores sólidos primarios. Todas las muestras se disociaron mecánica y enzimáticamente con colagenasa al 0,2%, antes de la obtención de los núcleos interfásicos. La técnica de FISH-bicolor se realizó en núcleos interfásicos, mediante sondas marcadas directamente con fluorocromos para el centrómero del cromosoma 17 (CEP 17; señal verde; VYSIS) y para el locus específico del gen TP53 (LSI 17p13.1; señal naranja; VYSIS). Resultados. Se encontró que 63% (24/38) de las muestras tenían aneuploidías del cromosoma 17. La monosomía fue la aneuploidía más frecuente (75%; 18/24), seguida de la trisomía (17%; 4/24); la nulisomía y la tetrasomía fueron menos frecuentes. El 89,5% (34/38) de los casos presentaron deleción del gen TP53. Sólo cuatro casos fueron normales para el número de copias del cromosoma 17 y del gen TP53. El estudio histopatológico mostró que la mayoría de las muestras eran tumores malignos. Conclusiones. La aneuploidía del cromosoma 17 y la deleción en el locus 17p13.1 del gen TP53 son alteraciones muy frecuentes en los tumores sólidos. La técnica FISH-bicolor permite detectar simultáneamente alteraciones cromosómicas numéricas y estructurales en núcleos interfásicos.

Introduction. TP53 is a tumor suppressor gene located on chromosome 17p13.1. This gene is essential for the control of cell cycle and has been found altered in about 50% of all tumor types.Objective. The presence of aneuploidy of chromosome 17 and TP53 gene deletion at 17p13.1 locus was determined in primary solid tumors using the dual-color FISH (fluorescence in situ hybridization).Materials and methods. Thirty-eight samples consisted of several types of primary solid tumors. All samples were mechanically and enzymatically disaggregated with 0.2% collagenase prior to obtaining interphase nuclei. The dual-color FISH was performed using direct fluorescent labeling probes for the chromosome 17 centromere (green signal) and for the TP53 gene locus-specific (orange signal). Results. Characteristic aneuploidy on chromosome 17 was found in 63% (24/38) of the samples. Monosomy occurred most frequently (75%, 18/24), followed by trisomy (17%, 4/24); nullisomy and tetrasomy were less frequent. TP53 gene deletion was found in 89.5% (34/38) of cases. Only four tumors were normal for copy number of chromosome 17 and TP53 gene. The histopathologic study showed that most of the samples were malignant tumors. Conclusions. Aneuploidy of chromosome 17 and deletion at 17p13.1 locus of TP53 gene were genetic alterations found to be very frequent in solid tumors. The dual-color FISH was able to detect both numerical and structural chromosomal abnormalities in interphase nuclei.

Principales factores que producen fragilidad cromosómica transitoria en los pacientes referidos para estudio citogenético / Main factors that produce transitory chromosomic fragility in the patients referred for cytogenetic study

The fragile sites are specific loci that show fractures during karyotyping perform under specific laboratory conditions. Are present in normal individuals and are classified by their population frequency. These sites have been associated with an increase in chromosome fragility, fractures and other chromosomal abnormalities. In recent years, the fragile sites have taken great importance because they represent regions in the genome that are particularly sensitive to replicative stress and are frequently rearrenged in tumor cells. Multiple risk factors endogenous and exogenous have been involved in the increase in chromosome fragility, including microorganisms, drugs, illegal drugs and toxins. The fragile sites have provided insight into understanding of the effects of replicative stress on DNA damage and genomic instability in cancer cells. In this work we aim to summarize the limited information available about the topic, and the clinical significance of fragile sites in vivo in the laboratory.

Inestabilidad cariológica durante la formación de células madres del polen en Aloe vera (Aloaceae)

Con el objetivo de esclarecer la posible existencia de anomalías citogenéticas que aminoren la fertilidad del polen de Aloe vera, se analizó la etapa de proliferación celular que lleva a la formación de células madres del polen (CMPs). Se recolectaron botones florales (BF) en 25 plantas de una población ubicada a 10°34’15’’ N, 64°12’08’’ W, los cuales fueron fijados en Carnoy I por 24 h y almacenados en etanol (70 % v/v). Las observaciones se realizaron en preparaciones temporales obtenidas por la tinción del contenido de las anteras suspendidas en orceína acética (1.5 % p/v) por 5 minutos. De las 9 411 células analizadas, 17 % mostraron 1-8 puentes entre cromátidas hermanas, 13 % 1-7 micronúcleos de 0.9-4.8 µm, 8.1 % estaban unidas por puentes y 0.1 % no contenían cromatina. El resto de las células (61.8 %) presentó configuraciones aparentemente normales y sin variaciones morfométricas. La proliferación irregular de una fracción de CMPs (39.2 %) sugiere que las condiciones ambientales de la zona árida donde se realizaron los muestreos inducen inestabilidad cromosómica y cambios fisiológicos que afectan el normal desarrollo de la mitosis premeiótica, generando pérdida o adición de fragmentos, asociados a deficiencias y duplicaciones génicas.

Cassava genetic resources and their utilization for breeding of the crop

Wild cassava relatives are perennials and vary in growth pattern from nearly acaulescent subshrubs to small trees. They have been used as a source of useful characters such as high protein content, apomixis, resistance to mealybug and mosaic disease, and tolerance to drought. Indigenous clones are a potential source of beta-carotene and lycopene. Apomixis genes have been transferred to the crop successfully through interspecific hybridization, and apomictic clones arising from these hybrids are now being grown at the Universidade de Brasília. Interspecific hybrids produced earlier were polyploidized and had their fertility restored. Different useful types of chimera were also produced.

Chromosome instability in industrial strains of Saccharomyces cerevisiae batch cultivated under laboratory conditions

Industrial ethanol fermentation is a complex microbiological process to which yeast cells must adapt for survival. One of the mechanisms for adaptation is thought to involve chromosome rearrangements. We found that changes in chromosome banding patterns measured by pulsed-field gel electrophoresis can also be produced in laboratory media under simulated industrial conditions. Based on analysis of their generational variation, we found that these chromosome changes were specific to the genetic backgrounds of the initial strains. We conclude that chromosome rearrangements could be one of the factors involved in yeast cell adaptation to the industrial environment.

Chromosome instability in carcinomas / Inestabilidad Cromosómica en Carcinomas

Los modelos actuales de carcinogénesis consideran la ocurrencia de mutaciones crecientes y mecanismos selectivos, favoreciendo la sobrevivencia y proliferación celular aumentada. Mecanismos epigenéticos también participan en la oncogénesis, destacando la metilación del DNA. La característica de las células tumorales que permite el aumento de la ocurrencia de mutaciones es denominada inestabilidad genética, donde son identificados dos mecanismos: inestabilidad de microsatélites, caracterizada por alteraciones nucleotídicas con errores en los sistemas de reparación del DNA; inestabilidad cromosómica, en la cual las aberraciones suceden en grandes segmentos cromosómicos. Los carcinomas son caracterizados por alteraciones citogenéticas complejas y grandes mezclas génicas. Alteraciones teloméricas, quiebres de DNA reparados inadecuadamente y deficiencia en los sistemas de chequeo del huso mitótico, son eventos capaces de generar inestabilidad cromosómica y aneuploidía que caracterizan estas neoplasias más agresivas. El conocimiento de los mecanismos que provocan la inestabilidad cromosómica puede permitir la utilización clínica de información en el desarrollo de estrategias terapéuticas más adecuadas, dirigidas a puntos específicos involucrados en procesos de malignización.

In the current carcinogenesis models, the occurrence of increasing mutations and selection mechanisms favoring cell survival and higher proliferation rates are taken into account. Epigenetic mechanisms, among which DNA methylation stands out, also take part in oncogenesis. The characteristic of tumor cells that allows the increase of mutations is named genetic instability, encompassing two mechanisms: microsatellite instability, characterized by nucleotide alterations with errors in the DNA repair systems; and chromosomal instability, represented by aberrations occurring in large chromosome segments. Carcinomas are characterized by complex cytogenetic alterations and large gene amalgamations. Telomeric alterations, inadequately repaired DNA breaks, and deficiencies in the mitotic spindle checking systems are events capable of generating the chromosomal instability and aneuploidy which characterize more aggressive neoplasias. A better understanding of the chromosomal instability mechanisms can show the way towards a clinical utilization of such information, like developing more adequate therapeutic strategies, targeted at specific sites involved in the malignization process.

Allelic imbalance studies of chromosome 9 suggest major differences in chromosomal instability among nonmelanoma skin carcinomas

CONTEXTO: Vários estudos de perda de heterozigozidade na região 9p21-p22, que abriga os genes supressores tumorais CDKN2a/p16INK4a, p19ARF e p15INK4b, têm sido realizados em uma ampla série de tumores humanos, incluindo os melanomas familiares. Perdas e ganhos em outras regiões do cromossomo 9 também têm sido observados com freqüência e podem indicar mecanismos adicionais no processo de tumorigênese dos carcinomas basocelulares da pele. OBJETIVO: Investigar o equilíbrio alélico existente na região 9p21-p22 em carcinomas basocelulares. TIPO DE ESTUDO: Análise molecular de marcadores de microssatélites em tumores e controles. LOCAL: Dois serviços de dermatologia de atendimento terciário em universidades públicas de São Paulo e o Laboratório de Genética Molecular do Câncer da Universidade Estadual de Campinas (Unicamp), Brasil. PARTICIPANTES: Examinamos 13 casos benignos, incluindo 4 queratoses solares, 3 queratoacantomas, 3 nevos melanocíticos, 2 doenças de Bowen e 1 neurofibroma cutâneo, além de 58 tumores malignos da pele: 14 de células escamosas, 40 carcinomas basocelulares e 4 melanomas; em pacientes consecutivamente encaminhados à clínica de Dermatologia da Unicamp e que concordaram em participar do estudo. VARIÁVEIS ESTUDADAS: O tumor principal e uma porção normal de pele não-adjacente foram removidos cirurgicamente de pacientes que consecutivamente procuraram os ambulatórios de dermatologia da Universidade Estadual de Campinas (Unicamp) e da Universidade Estadual de São Paulo (Unesp), São Paulo, por causa de lesões cutâneas. Extraímos DNA tanto de tecido tumoral como do correspondente tecido normal de cada paciente. Para amplificar regiões de repetição polimórfica de microssatélites do cromossomo 9, foram utilizados quatro pares de primers, sendo dois deles destinados à região 9p21-p22. RESULTADOS: Identificamos oito casos (20%) de desequilíbrio alélico entre os carcinomas basocelulares, sendo dois casos de perda de heterozigozidade e seis casos de instabilidade de microssatélite na região 9p21-p22. Outros marcadores também mostravam anormalidades em três destes tumores, enquanto nenhuma alteração foi detectada entre os casos benignos e nos outros tumores malignos. CONCLUSÃO: Esta dependência fenotípica sugere que existem diferenças importantes no comportamento das formas mais comuns de tumores cutâneos não-melanocíticos em relação à sua tendência para instabilidade de microssatélite no cromossomo 9...

Alelotipificación en cáncer de testículo / Alelotypification in testicular cancer

Los marcadores tumorales existentes (alfafetoproteína y gonadotrofina coriónica humana) son patrones indirectos y no están expresados en todos los tumores testiculares (TT) por lo que no permiten una confiabilidad absoluta. Por esto, creemos que la identificación de los genes involucrados en la iniciación y progresión de TT es importante. El estudio del genoma humano (GH) ha ayudado a la identificación de la mayoría de los genes supresores (GS) de tumores descubiertos (p53,VHL,APC,CDKN2,RB). Para lograr detectarlos, se identifican las regiones cromosomales con alta frecuencia de deleciones (AFD) y mediante su estudio sistemático se puede identificar si existen GS involucrados. El objetivo de este trabajo es el estudio del GH(alelotipificación) mediante la amplificación por PCR de secuencias de bases repetidas polimorfas, que se encuentran distribuidas a lo largo del GH. Se seleccionaron 50 casos de TT del tipo seminoma puro y se microdisecó DNA de tejido tumoral y normal en TT. Este material fue amplificado mediante PCR, posteriormente se amplificaron mediante primers conocidos, secuencias cromosómicas determinadas de los sitios cromosomales con altas frecuencias de LOH y MSI buscando zonas calientes. Después de identificadas las regiones hot del mapa cromosómico, se acotó el estudio a los cromosomas 5 y 12. Se utilizaron 12 marcadores, para franquear zonas específicas del 5 y 12, donde encontramos previamente la mayor frecuencia de LOH. Un completo estudio de alelotipificación de alta densidad en los diferentes tipos histológicos de TT permitió detectar una AFD y LOH en cromosomas 12 y 5, detectándose que 47 de 50 casos de seminoma presentan algún marcador con inestabilidad genética en estas zonas, un 78 por ciento de los tumores estudiados (38/50) presentan más de 3 marcadores con inestabilidad. Al franquear las regiones específicas, la frecuencia de inestabilidad (FI) aumenta significativamente en el brazo p de ambos cromosomas. A la luz de los resultados concluimos que existe una FI significativamente alta en los cromosomas 5 y 12, lo que sugiere que en alguna región cercana del brazo corto de estos, se puede encontrar algún GS. Este gen se encontraría posiblemente inactivo, dado que la FI aumenta al cercar el área, lo que hace pensar en un rol supresor. Este es el primer estudio que logra identificar las zonas de inestabilidad cromosomal más frecuentes del TT y ahora realizaremos el estudio de baja densidad con la intención de encontrar este gen.