Efficient enrichment and detection of disseminated tumor cells

#The field of this invention is the detection of tumor cells.

#One of the most serious problems that faces a practicing oncologist is the metastasis of malignant tumor cells from the primary site to multiple, distant sites. In most cases of cancer, it is the metastatic lesions that kill the patient. While surgery is often effective against a primary tumor, it cannot excise all malignant tissue if the cancer has metastasized. For example, one-third of patients with operable breast cancer develop metastases after primary therapy. Adjuvant therapy can improve this prognosis, but requires identification of high-risk patients. Staging primary tumor by size and axillary nodes status is insufficient for this purpose.

#Bone and bone marrow are frequent sites of metastasis, but radiologic and scintographic techniques can detect bone involvement only when destruction of bone matrix has occurred. The presence of epithelial tumor cells in bone marrow generally correlates with conventional risk factors, such as size and histological grade of the primary carcinoma, distant metastasis and lymph node involvement. Clinical follow-up studies have demonstrated a significantly increased relapse rate in patients that presented with epithelial tumor cells in bone marrow at the time of primary surgery.

#FIGS. 1A to 1D show the characterization of human epithelial antigen (HEA) and cytokeratin 8/18 expression during the magnetic enrichment of experimentally added breast carcinoma cells from a sample of leucocyte-rich buffy coat cells.

#FIGS. 2A to 2C show the characterization of CD45 and cytokeratin 8/18 expression during the magnetic enrichment of tumor cells from a leucocyte-rich buffy coat fraction of peripheral blood from a breast cancer patient with distant metastases, or from control samples.

#Methods are provided that utilize magnetic sorting to identify the presence of rare disseminated tumor cells from samples of hematopoietic cells. The tumor cells are generally distant from the site of the primary tumor, and their presence in samples of patient hematopoietic cells may be indicative of the metastatic potential of the tumor. The cell sample may be treated with a permeabilizing agent and fixative in order to detect cytoplasmic proteins. Antibodies directed to tumor antigens or lineage specific antigens are used to magnetically label the tumor cells. The labeled cells are separated from unlabeled hematopoietic cells by magnetic separation. The fraction of cells enriched for tumor cells is useful for quantitating the tumor cells, and as a source of tumor cells for further characterization.

#Antigens that are markers for particular cell types may be proteins, carbohydrates, lipids, or other complex biomolecules. In a few types of tumors, specific antigens have been identified that are expressed at elevated levels on the malignant cells. However, it is more common to find tumor cells that do not have distinctive antigens. In many cases, the tumor cells share many phenotypic markers with the cell type from which they originated. For example, carcinomas express antigens that are typical of epithelial cells; lymphomas of lymphocytes, etc. Such antigens may also be expressed on cells of other lineages, or may be found only at detectable levels with cells of a particular lineage, e.g. common leukocyte antigen, epithelial membrane antigen, etc. The latter are termed “tissue-specific” antigens. When tumor cells have disseminated from their tissue of origin, the tumor cells may be the only cells of that lineage present in a population of cells from a different lineage. For example, carcinomas, of epithelial origin, metastasize to the bone marrow, which is comprised of hematopoietic cells. This expression of tissue specific antigens can then be a basis for separation and detection of the tumor cells.

#The dissemination of tumor cells through the hematopoietic system is medically important as an early indication of metastasis. Hematopoietic cells are developmentally derived from a common embryonic source, and share a common antigenic profile for tissue specific antigens. Sources for samples of hematopoietic cells include the blood and fractions thereof, particularly buffy coat preparations, aphoresis or leukophoresis samples; lymph and lymph nodes; and bone marrow. The term “hematopoietic cells” shall be intended to mean the normal cell populations found in blood, bone marrow, lymph, etc., and will include lymphocytes, e.g. B-cells, T-cells and natural killer cells; myeloid cells, e.g. macrophages, monocytes, polymorphonuclear cells; megakaryocytes, basophils, eosinophils, neutrophils; etc.; erythroid cells, e.g, reticulocytes, platelets and red blood cells; dendritic cells; and the progenitors thereof. Stromal and fibroblast cells found in the bone marrow are also included. The term “hematopoietic cell sample”, particularly with reference to patient samples, shall be intended to include small numbers of disseminated tumor cells that may be present.