Câncer/Oncologia/Tumor

A detecção e caracterização eficiente de células micrometastáticas na medula óssea de pacientes com carcinoma de mama são de importância prognostica e terapêutica. A técnica utilizada deve superar os desafios que resultam do pequeno número de células alvo (uma por um milhão de células hematopoiéticas) e expressão heterogênea de marcadores celulares micrometastáticos. Pesquisadores franceses e noruegueses realizaram um estudo, recentemente publicado na revista Cancer, para avaliar e melhorar os atuais métodos de purificação e caracterização de células de carcinoma disseminadas raras.

Os autores desenvolveram um ensaio que não requer separação por gradiente de densidade. Esse ensaio pode ser realizado diretamente com o aspirado de medula óssea e é baseado no uso de grânulos imunomagnéticos cobertos com um anticorpo que reconhece um epítopo de superfície de célula epitelial, a molécula de adesão de célula epitelial (EpCAM). Para determinar a especificidade do ensaio, os autores avaliaram as amostras de medula óssea de 46 pacientes controles.

O novo método foi altamente reprodutível e foi capaz de detectar apenas 10 células de carcinoma entre 50 milhões de células hematopoiéticas. O rendimento foi perto de 100%, com somente 0,01% de célula não-específica drenada. Os autores encontraram que 68 ± 51 células foram aderidas por 50 milhões de células nos aspirados controles e que a separação por gradiente de densidade aumentou esse número em duas a 29 vezes. Essas células aderidas expressaram EpCAM, representada por 1,4.10^-4% da amostra, e foram caracterizadas como de origem de célula hematopoiética (CD45+) ou origem de célula progenitora (CD34+).

A relevant immunomagnetic assay to detect and characterize epithelial cell adhesion molecule-positive cells in bone marrow from patients with breast carcinoma - Cancer; 2004; 101(4): 693-703

A relevant immunomagnetic assay to detect and characterize epithelial cell adhesion molecule-positive cells in bone marrow from patients with breast carcinoma Immunomagnetic purification of micrometastases

Valérie Choesmel, A.I. 1, Philippe Anract, M.D. 2, Hanne Høifødt, A.I. 3, Jean-Paul Thiery, Ph.D. 1, Nathalie Blin, Ph.D. 1 *

1UMR144 CNRS, Research Division, Institut Curie, Paris, France 2Orthopedic Surgery Department, Hôpital Cochin, Paris, France 3Tumor Biology Department, The Norwegian Radium Hospital, Oslo, Norway

email: Nathalie Blin (nathalie.blin@curie.fr)

^*Correspondence to Nathalie Blin, Institut Curie, CNRS UMR144, 26 rue d'Ulm, 75248 Paris Cedex 05, France

Fax: (011) 33 142346349

Funded by:
 Programme Incitatif et Coopératif on Micrometastases at the Institut Curie

Keywords

breast carcinoma • bone marrow • micrometastases • immunomagnetic selection • epithelial cell adhesion molecule

Abstract

BACKGROUND

The efficient detection and characterization of micrometastatic cells in the bone marrow of patients with breast carcinoma are of prognostic and therapeutic importance. The technique used must overcome the challenges that result from the small number of target cells (1 per 1 million hematopoietic cells) and the heterogeneous expression of micrometastatic cell markers. In this study, the authors assessed and improved the current methods for purifying and characterizing rare disseminated carcinoma cells.

METHODS

The authors developed a single-step assay that does not require density-gradient separation. This assay can be performed directly on crude human bone marrow aspirates and is based on the use of immunomagnetic beads coated with an antibody that recognizes an epithelial cell-surface epitope, the epithelial cell adhesion molecule (EpCAM). To determine the specificity of the assay, the authors evaluated bone marrow specimens from 46 control patients.

RESULTS

The novel method was highly reproducible and was capable of detecting as few as 10 carcinoma cells among 50 million hematopoietic cells. The yield was nearly 100%, with only 0.01% nonspecific cell draining. The authors found that 68 ± 51 cells were trapped per 50 million cells in control crude aspirates and that density-gradient separation increased this number by 2-fold to 29-fold. These trapped cells expressed EpCAM, represented 1.4 × 10-4 % of the sample, and were characterized as of hematopoietic cell origin (CD45 positive) or progenitor cell origin (CD34 positive).

CONCLUSIONS

The authors developed a highly efficient and reproducible, single-step immunomagnetic assay that may be performed directly on crude human bone marrow aspirates. The authors believe the current study is the first to demonstrate that some rare bone marrow cells (CD45-positive or CD34-positive cells) may express EpCAM and, to some extent, may contaminate the purified micrometastatic cell fraction. Thus, a universal marker for micrometastatic cells remains to be discovered. Cancer 2004. © 2004 American Cancer Society.