Micrometástasis: estrategias para su detección / Micrometastases: strategies for detection

La micrometástasis o enfermedad mínima residual ha adquirido una importancia trascendental en oncología al representar un verdadero problema clínico que debe ser solucionado, ya que aún se desconoce la respuesta de estos focos tumorales a los diferentes tratamientos que se usan para el control del cáncer. Aun cuando este es un problema específico fundamental a ser solucionado, ya existen métodos de ensayo inmunohistoquímicos y de biología molecular, que han permitido la ubicación de microfocos de células tumorales en diferentes órganos y tejidos, existiendo diferentes técnicas para determinar y cuantificar estas lesiones. Dentro de estas técnicas destacan la citometría de flujo y diferentes técnicas moleculares que van desde las ya tradicionales hasta las más nuevas y sofisticadas. El objetivo de la presente revisión está dirigido evaluar los nuevos métodos de diagnóstico que permitan la identificación de esta enfermedad residual, lo cual serviría para establecer tratamientos individualizados que pudieran prevenir la recurrencia de la enfermedad en los pacientes de cáncer bajo tratamiento.

Micrometastasis or minimal residual disease has become critically important in oncology since it represents a true clinical problem that must be solved, as the response of these tumor foci to the different treatments that are used for the control of cancer, is still unknown. Even though this is a fundamental specific problem to be solved, there are already immunohistochemical and molecular biology diagnostic methods that have allowed microfoci location of tumor cells in various organs and tissues, and different techniques are available to determine and quantify these lesions. Within these techniques, flow cytometry and different molecular methods are included, and they range from the traditional to the newest and most sophisticated. The goal of this review was aimed to evaluate new diagnostic methods that permit the identification of this residual disease, which would serve to establish individualized treatments and prevent the recurrence of the disease in cancer patients under treatment.

Transcription analysis of TIMP-1 and NM23-h1 genes in glioma cell invasion / Análise transcricional dos genes TIMP-1 e NM23-H1 na invasão celular em astrocitoma difuso e glioblastoma multiforme

PURPOSE: To evaluate using transcription analysis the presence and importance of two genes: NM23-H1 and TIMP-1 on control of tumor cell invasion in diffuse astrocytomas (WHO II) and glioblastoma multiforme (WHO IV). METHOD: Northern blot analysis of NM23-H1 and TIMP-1 was performed. Eight diffuse astrocytomas and 19 glioblastomas (WHO IV) were analyzed to determine if TIMP-1 and NM23-H1 were candidates to inhibition of tumor cell invasion quantitated RNA levels. The samples were collected directly from operating room. Total cellular RNA was extracted from frozen tissue samples using guanidinium-isothiocyanate and cesium chloride gradients. Total RNA (10 mg per sample) from tumor tissue were size fractionated through 1 percent agarose-formaldehyde gel and transferred to nylon filters and then hybridized to 32P-labeled DNA probes and placed for autoradiography. Levels of specific RNAs were determined by computer-assisted laser densitometry. Blot filters were sequentially hybridized to nm23 and TIMP-1 probes in addition to GAPDH, as a control. Statistical analyses were carried out according to t-test for equality of means. RESULTS: NM23-H1 were detected in each sample, however it did not correlate with malignancy and invasiveness. On the other side TIMP-1 gene expression showed a clear correlation between low expression and invasiveness. CONCLUSION: The data suggest that TIMP-1 is an inhibitor of high grade gliomas invasion. NM23-H1 was present in the entire gliomas sample, but it did not vary in diffuse astrocytomas and glioblastomas.

OBJETIVO: Comparar através da análise da expressão dos níveis de RNA, a presença e a relevância dos genes NM23-H1 e TIMP-1 no controle da invasão celular tumoral dentro do tecido cerebral normal em: astrocitoma difuso (OMS II) e glioblastoma multiforme (OMS:IV). MÉTODO: Análise em "Northern blot" dos genes NM23-H1 e TIMP-1. Oito astrocitomas fibrilares difusos (OMS II) e 19 glioblastomas multiformes foram analisados para determinar se TIMP-1 e NM23-H1 estavam relacinados à inibição da invasão tumoral nas neoplasias do sistema nervoso central, quantificando os níveis de RNA dos respectivos genes extraídos diretamente dos tumores. 10 mg por amostra de RNA total foram fracionados de gel de formaldeído e transferidos para os filmes de hibridação. Níveis específicos de RNAs foram determinados na espectrofotometria. Valores das razões entre NM23-H1/GAPDH e TIMP-1/GAPDH foram submetidos à análise de variabilidade das médias. RESULTADOS: A análise da expressão do gene TIMP-1 mostrou supressão em tumores gliais malignos. CONCLUSÃO: Os resultados indicam que existe relação direta entre níveis baixos de TIMP-1 e malignidade dos gliomas. O gene NM23-H1 foi detectado em todas as amostras, mas não foi possível relacionar sua subexpressão ou superexpressão com algum fenótipo de invasividade.

Putrescine, DNA, RNA and protein contents in human uterine, breast and rectal cancer.

AIMS: To find out the status of DNA, RNA and protein in human uterine, ovarian, breast and rectal carcinoma. MATERIAL AND METHODS: In this prospective study, patients of age group between late thirties and late fifties suffering from uterine, ovarian, breast and rectal cancer were taken as subjects of the present study. The total number of cases studied for each cases was ten. Pieces of human carcinomatous tissues of above mentioned cases were taken along with surrounding normal tissues. From the tissue samples, putrescine is separated by the method of Herbst et al, DNA analysed by Diphenylamine method, RNA by Orcinol method and protein by Biuret method. RESULTS: Tissue content of putrescine rises simultaneously with that of DNA, RNA and protein in carcinomatous growths as above in comparison to their respective adjacent normal tissue, the differences being statistically highly significant. CONCLUSIONS: Increase in DNA, RNA and protein concentration may be a pre-requisite for increased synthesis of putrescine in carcinomatous tissue and thereby the concentration of other di- and poly-amines.

Mutation and expression of the p27KIP1 and p57KIP2 genes in human gastric cancer

Cyclin-dependent kinase inhibitors (CDKI) are negative regulators of cell cycle progression by binding the cyclin-CDK complex and inhibiting the CDK activity. Genetic alteration in the CDKI genes has been implicated for carcinogenesis. To test the genetic alteration in the p27 and p57 genes, KIP family CDKI genes, 30 gastric tumor-normal pairs and 8 gastric cancer cell lines were analyzed for mutations by polymerase chain reaction-single strand conformational polymorphism (PCR-SSCP). No mutation was detected in these genes although length polymorphisms in the proline-alanine repeat of the p57 gene were detected. When the p27 and p57 mRNAs were analyzed in gastric cancer cell lines by RT-PCR, the p27 mRNA was expressed considerably high in tumor cells but expression of the p57 mRNA was much low in gastric cancer cell lines compared to that of normal cells. The result suggests that inactivation of gene expression rather than mutations in the p57 gene accounts possibly for the involvement of this gene in tumorigenesis of gastric cancer. However, expression of the p27 gene seems to be essential for cell survival.

The detection of circulating breast cancer cells in peripheral blood by reverse transcriptase-polymerase chain reaction

Some circulating cancer cells in the blood play a central role in the metastatic process and may have a major influence on patient progress. Their numbers can be very small and techniques for their detection need to be both sensitive and specific. Polymerase chain reaction (PCR) has been successfully used to detect small numbers of tumor cells in cancer. We used a reverse transcriptase-polymerase chain reaction (RT-PCR) to detect circulating breast cancer cells in venous blood samples before operations and assessed cytokeratin-19 (CK-19) and cytokeratin-20 (CK-20) as target mRNA markers in the blood of healthy donors (n=6) and breast cancer patients (n=30) with American Joint Committee on Cancer stages 0 to IIIa. CK-19 mRNA was expressed in all blood samples of healthy donors and patients. But CK-20 was the only mRNA marker not detected in the blood from healthy donors. Seven of 30 (23%) venous blood isolates of breast cancer patients yielded a CK-20 mRNA with positive results. There was no correlating CK-20 mRNA expression with stage and axillary lymph node status. In conclusion, CK-19 showed no diagnostic value as a mRNA marker in the detection of circulating cancer cells by RT-PCR assay because this was expressed in the blood of healthy donors. CK-20 mRNA was an useful marker to detect circulating cancer cells in breast cancers.