Circulating tumor cells in the peripheral blood and bone marrow of patients with ovarian carcinoma do not predict prognosis

BACKGROUND: Ovarian carcinoma is apparently restricted for a long time to the peritoneal cavity. However, about 50% of patients with a surgically documented complete intraabdominal response experience later recurrence. Occult hematogenous micrometastases are common to most epithelial malignancies and have recently been found in 30% of bone marrow samples of ovarian carcinoma patients, as examined by immunocytochemistry. Moreover, these findings were associated with poor progression-free and overall survival. The aim of the current study was to evaluate the possible prognostic significance of tumor cells detected in the peripheral blood and bone marrow of ovarian carcinoma patients by an immunomagnetic method. METHODS: In a total of 90 patients with histologically proven epithelial ovarian carcinoma, blood and (in 73 cases) bone marrow samples were taken. Tumor cells were identified by a microbead coated with the antibody MOC-31, which recognizes an epitope regularly expressed on ovarian carcinoma cells. RESULTS: The authors detected carcinoma cells in the bone marrow in 21% of ovarian carcinoma patients, and in the peripheral blood in 12% of patients. Mean overall survival was 25 and 28 months for patients with or without circulating tumor cells, respectively. CONCLUSIONS: Ovarian carcinoma cells seem to reach peripheral circulation more frequently than expected. However, in contrast to an earlier report, detection of tumor cells in the bone marrow and/or blood was not associated with poor prognosis in ovarian carcinoma patients. This discrepancy remains unexplained, but characterization of circulating ovarian carcinoma cells for their malignant and metastatic capacity is clearly warranted.     

High percentage of false-positive results of cytokeratin 19 RT-PCR in blood: a model for the analysis of illegitimate gene expression

Cytokeratin 19 (CK19) RT-PCR is widely used in order to detect circulating tumor cells in peripheral blood and bone marrow. However, increasing amounts of information support the fact that it is also associated with a high percentage of false-positive results. In our study, we not only managed to demonstrate the significant limitations of this method, but were also able to clarify the reasons behind these limitations. We developed a completely novel RT-PCR for CK19 and sequenced an intron at nucleotide (nt) 980 of the CK19 mRNA to exclude DNA contamination. Tumor dilution experiments were performed in order to analyze the specificity and sensitivity of the method. Control experiments using the blood of healthy donors were performed. Tumor cell dilution experiments gave a detection limit of one tumor cell. If tumor cells were mixed with an equal volume of pure mononuclear cells, the detection limit was 1 tumor cell in 10(5) mononuclear cells. RT-PCR of mononuclear cells from healthy blood donors gave false-positive results in 29% of the cases. We conclude that a significant decrease in the sensitivity of CK19 RT-PCR occurs if it is performed in blood cells and that the illegitimate CK19 gene expression in normal cells can lead to false-positive results. These limitations have to be taken into account if RT-PCR is to be used for the detection of tumor cells either in blood or in bone marrow in clinical practice.     

Flow Cytometric Detection of Proliferative Cells in Leukemias

We studied the proliferative activity of leukemic cells obtained from the peripheral blood and bone marrow of 34 patients; 30 with acute leukemia and 4 with chronic myelogenous leukemia in blastic crisis. Flow cytometry was performed using monoclonal antibody against DNA polymerase α. Since fresh and frozen cells showed virtually identical DNA polymerase α-positive populations and flow cytometric histograms, 52 cryopreserved samples (25 from peripheral blood and 27 from bone marrow) were used in this study. The DNA polymerase α-positive population ranged from 20.4% to 84.7% in peripheral blood, and from 6.5% to 92.1% in bone marrow. A positive correlation ( r =0.76, P ≤0.01) was found between DNA polymerase α-positive populations in peripheral blood and bone marrow from the same patient. This suggests that the DNA polymerase α-positive population in the bone marrow can be estimated from that in peripheral blood. No relationship was observed between the positive population and the response to chemotherapy. Statistical analyses for all cases showed no relationship between the DNA polymerase α-positive population and either the tumor cell count or time to reach a nadir. However, a negative correlation was observed between the positive population in bone marrow samples and the time to reach a nadir ( r =−0.64, P ≤0.05) in those patients who achieved a complete response. In addition, in the cases of acute non-lymphocytic leukemia who did not respond to chemotherapy, a positive correlation was observed between the tumor cell count in bone marrow and the DNA polymerase α-positive population ( r =0.93, P ≤0.01). Thus, the method described here provides a simple and time-efficient means of detecting the proliferative activity of leukemic cells, which is a useful parameter in the treatment of leukemia.     

Minimal residual disease and circulating tumor cells in breast cancer

Tumor cell dissemination in bone marrow or other organs is thought to represent an important step in the metastatic process. The detection of bone marrow disseminated tumor cells is associated with worse outcome in early breast cancer. Moreover, the detection of peripheral blood circulating tumor cells is an adverse prognostic factor in metastatic breast cancer, and emerging data suggest that this is also true for early disease. Beyond enumeration, the characterization of these cells has the potential to improve risk assessment, treatment selection and monitoring, and the development of novel therapeutic agents, and to advance our understanding of the biology of metastasis.     

Immunocytochemical detection of breast cancer cells in marrow and peripheral blood of patients undergoing high dose chemotherapy with autologous stem cell support

Summary Detection of small numbers of breast cancer cells is important in staging the disease and can be helpful in assessing the efficacy of purging regimens prior to autologous stem cell infusion. Immunohistochemical methods are potentially useful and broadly applicable for this purpose since they are simple to perform, sensitive, and may be quite specific. We have used a combination of four monoclonal antibodies [260F9, 520C9, 317G5 (Baxter Corp); BrE-3 (Dr. R. Ceriani)] against tumor cell surface glycoproteins in a sensitive immunocytochemical assay to identify breast tumor cells in bone marrow and peripheral blood. Immunostained cytospin preparations were fixed prior to staining to preserve cytological details of immunopositive cells. After immunostaining, slides were counterstained with hematoxylin to confirm the identity of labeled cells. In cytocentrifuge experiments in which small numbers of CAMA human breast tumor cells were added to bone marrow mononuclear cells, a linear relationship between the number of tumor cells added and the number of tumor cells detected was obtained over a broad range of tumor cell concentrations. The probability of detecting tumor cells was dependent on the number of cytocentrifuge slides examined. When ten slides (5 million cells) were examined, the probability of detecting tumor at a concentration of 4 tumor cells per million bone marrow mononuclear cells was 98%. In clinical specimens, tumor cells were detected in marrow aspirates from 73 of 240 (30%) patients undergoing autologous transplantation, including 70 (37%) of 190 patients with clinical stage IV disease, 0 of 7 patients with clinical stage III disease, and 3 of 43 (7%) patients with clinical stage II disease. Seventy-three of 657 peripheral blood specimens from 26 of 155 patients (17%) contained breast cancer cells with counts ranging from 1 to 97 tumor cells per million leukocytes. Tumor cells were most frequently found in the blood of patients with stage IV disease [21 of 107 (20%)] but were also found in a substantial number [5 of 44 (11%)] of patients with stage II disease. Positive selection of CD34-positive hematopoietic progenitor cells as well as negative purging methods such as incubation with 4-hydroxyperoxy-cyclophosphamide (4-HC) were evaluated with respect to tumor cell depletion. Selection of CD34-positive progenitor cells from bone marrow or peripheral blood resulted in log reduction of 1 to > 4 tumor cells reinfused at autologous transplantation. A lesser log reduction (up to 1) was demonstrated following 4-HC purging. We conclude that properly performed and controlled immunocytochemical staining of bone marrow and peripheral blood cytospins is a sensitive and simple way to detect and quantitate breast cancer cells in hematopoietic specimens harvested for autotransplantation and that CD34-positive progenitor cell selection results in significant reduction in the number of breast cancer cells reinfused with marrow or peripheral blood stem cells.     

High frequencies of functional tumor-reactive T cells in bone marrow and blood of pancreatic cancer patients

Pancreatic cancer is characterized by aggressive growth and treatment resistance. New approaches include immunotherapeutic strategies but the type and extent of spontaneous immune responses against tumor antigens remains unclear. A dominance of TH2 cytokines in patients' sera reported previously suggests systemic tumor-induced immunosuppression, potentially inhibiting the induction of tumor-reactive T cells. We characterized the localization, frequencies, and functional potential of spontaneously induced memory T cells specific for individual tumor antigens or the tumor-associated antigen mucin-1 in the peripheral blood and bone marrow of 41 pancreatic cancer patients. We found high numbers of tumor-reactive T cells in all bone marrow samples and in 50% of the blood samples. These cells secreted the TH1 cytokine IFN-gamma rather than TH2 cytokines upon stimulation with tumor antigens. Although consistently induced during pancreatic cancer, T cells specific for pancreatic antigens were not detected during chronic pancreatitis, suggesting that their evaluation may be of diagnostic use in both diseases. Freshly isolated T cells from cancer patients recognized autologous tumor cells and rejected them in vitro and in a xenotransplant model in vivo, suggesting their therapeutic potential. Thus, tumor antigen-specific T cell responses occur regularly during pancreatic cancer disease and lead to enrichment of tumor cell-reactive memory T cells in the bone marrow. The bone marrow can therefore be considered an important organ for antitumor immune responses in pancreatic cancer.     

Clinical outcome of peripheral blood stem cell support

Two clinical results of peripheral blood stem cell support are commonly considered: (1) the effect on hematopoietic recovery and (2) the effect on the underlying malignancy. The dynamics of hematopoietic recovery after autoiogous bone marrow transplantation and after autologous peripheral blood stem cell transplantation in a clinical setting are similar if no exogenous cytokines are administered and the peripheral stem ceils are collected while their numbers are not deliberately increased (mobilised). If mobilized peripheral stem cells are transplanted, hematopoietic recovery is accelerated. In some circumstances, patients who receive peripheral stem cell transplantation may experience an improved progression-free survival after high-dose therapy when compared with similar patients who receive autologous bone marrow transplantation. Explanations for such a survival advantage might include (1) a lower likelihood of occult tumor cells capable of restoring disease in peripheral stem cell autograft products than in bone marrow harvests, (2) a greater number of cytotoxic effector cells capable of destroying occult tumor ceils in the peripheral stem cell collections than in bone marrow harvests, and (3) a different and advantageous pattern of immunologic recovery following autologous peripheral stem cell transplant compared to autologous bone marrow transplant.     

The happy destiny of frozen haematopoietic stem cells: from immature stem cells to mature applications

Forty years ago, van Putten described in the European Journal of Cancer (see this issue) quantitative studies on the optimal storage techniques of mouse and monkey bone marrow suspensions. Survival of the animals after irradiation following injection with stored bone marrow cell suspensions was the endpoint. He observed some species differences, but based on the data obtained considered a careful trial of the glycerol-polyvinylpyrrolide (PVP) combination for storage of marrow in man was indicated. In spite of this, dimethyl sulphoxide has become the 'standard' cryopreservant for human marrow stem cells. Over the last 40 years, there has been a tremendous increase in knowledge about haematopoietic stem cells and their use in the clinic. Haematopoietic stem cells are now known to travel between the bone marrow and peripheral blood and are the best-characterised adult stem cells. These cells are currently widely used for transplantations in the clinic and are obtained from a wide variety of sources. These include the bone marrow, peripheral blood, cord blood, autologous as well as allogeneic stem cells from related or unrelated donors. Increasingly, data has become available that adult haematopoietic stem cells can generate differentiated cells belonging to other cell types, a process called "developmental plasticity". Thus, they may contribute to non-haematopoietic tissue repair in multiple organ systems. This has created a whole new potential therapeutic armamentarium for the application of haematopoietic stem cells outside of the area of malignancies and haematopoietic disorders.     

Potential use of T cell receptor genes to modify hematopoietic stem cells for the gene therapy of cancer

The purpose of this review is to illustrate some of the technical and biological hurdles that need to be addressed when developing new gene therapy based clinical trials. Gene transfer approaches can be used to “mark” cells to monitor their persistence in vivo in patients, to protect cells from toxic chemotherapeutic agents, correct a genetic defect within the target cell, or to confer a novel function on the target cell. Selection of the most suitable vector for gene transfer depends upon a number of factors such as the target cell itself and whether gene expression needs to be sustained or transient. The TCR gene transfer approach described here represents one innovative strategy being pursued as a potential therapy for metastatic melanoma. Tumor reactive T cells can be isolated from the tumor infiltrating lymphocytes (TIL) of melanoma patients. A retroviral vector has been constructed containing the T cell receptor (TCR) α and β chain genes from a MART-1_(27-35)-specific T cell clone (TIL 5). Jurkat cells transduced with this virus specifically release cytokine in response to MART-1_(27-35) peptide pulsed T2 cells, showing that the virus can mediate expression of a functional TCR. HLA-A2 transgenic mice are being used to examine whether transduced bone marrow progenitor cells will differentiatein vivo into mature CD8⁺ T cells expressing the MART-1_(27-35)-specific TCR. Expression of the human TCR α and β chain genes has been detected by RT-PCR in the peripheral blood of HLA-A2 transgenic mice reconstituted with transduced mouse bone marrow. Expression of the TIL 5 TCR genes in the peripheral blood of these mice was maintained for greater than 40 weeks after bone marrow reconstitution. TIL 5 TCR gene expression was also maintained following transfer of bone marrow from mice previously reconstituted with transduced bone marrow to secondary mouse recipients, suggesting that a pluripotent progenitor or lymphocyte progenitor cell has been transduced.     

Circulating tumor cells and bone marrow micrometastasis

Sensitive immunocytochemical and molecular assays allow the detection of single circulating tumor cells (CTC) in the peripheral blood and disseminated tumor cells (DTC) in the bone marrow as a common and easily accessible homing organ for cells released by epithelial tumors of various origins. The results obtained thus far have provided direct evidence that tumor cell dissemination starts already early during tumor development and progression. Tumor cells are frequently detected in the blood and bone marrow of cancer patients without clinical or even histopathologic signs of metastasis. The detection of DTC and CTC yields important prognostic information and might help to tailor systemic therapies to the individual needs of a cancer patient. In the present review, we provide a critical review of (a) the current methods used for detection of CTC/DTC and (b) data on the molecular characterization of CTC/DTC with a particular emphasis on tumor dormancy, cancer stem cell theory, and novel targets for biological therapies; and we pinpoint to (c) critical issues that need to be addressed to establish CTC/DTC measurements in clinical practice.     

Quantitative tumor cell content of bone marrow and blood as a predictor of outcome in stage IV neuroblastoma: a Children's Cancer Group Study.

PURPOSE: This study investigated the prognostic value of quantifying tumor cells in bone marrow and blood by immunocytology in children with high-risk, metastatic neuroblastoma. PATIENTS AND METHODS: Patients with stage IV neuroblastoma (N = 466) registered on Children's Cancer Group study 3891 received five cycles of induction chemotherapy and were randomized either to myeloablative chemoradiotherapy with autologous purged bone marrow rescue or to nonmyeloablative chemotherapy. Subsequently, they were randomized to 13-cis-retinoic acid or no further treatment. Immunocytologic analyses of bone marrow and blood were performed at diagnosis, week 4, week 12, bone marrow collection, and end induction and were correlated with tumor biology, clinical variables, treatment regimen, and event-free survival (EFS). RESULTS: Immunocytology identified neuroblastoma cells in bone marrow of 81% at diagnosis, 55% at 4 weeks, 27% at 12 weeks, 19% at bone marrow collection, and 14% at end induction. Tumor cells were detected in blood of 58% at diagnosis and 5% at collection. There was an adverse effect on EFS of increasing tumor cell concentration in bone marrow at diagnosis (P =.04), at 12 weeks (P =.006), at bone marrow collection (P <.001), and at end induction (P =.07). Positive blood immunocytology at diagnosis was associated with decreased EFS (P: =.003). The prognostic impact of immunocytology was independent of morphologically detected bone marrow disease, MYCN status, and serum ferritin level in bivariate Cox analyses. CONCLUSION: Immunocytologic quantification of neuroblastoma cells in bone marrow and blood at diagnosis and in bone marrow during induction chemotherapy provides prognostic information that can identify patients with very high-risk disease who should be considered for experimental therapy that might improve outcome.     

Myeloid cell trafficking and tumor angiogenesis

Tumor growth and metastasis depend on neovascularization, the growth of new blood vessels. Recent findings have revealed that tumor neovascularization is regulated in part by monocytes, which are myeloid lineage cells from the bone marrow. Tumors exhibit significant monocyte infiltrates, which are actively recruited to the tumor microenvironment. Upon tumor infiltration, monocytes can participate in tumor neovascularization. Monocytes can either differentiate into macrophages, which express proangiogenic growth factors, or into endothelial-like cells, which may directly participate in neovascularization. Preliminary studies in animals suggest that modulation of bone marrow-derived cell trafficking into tumors will provide a useful new approach in cancer therapy.     

Detection and Relevance of Minimal Disease in Lymphomas

The detection and clinical relevance of minimal disease in non-Hodgkin's lymphoma (NHL) and Hodgkin's disease (HD) is reviewed. Relevant aspects of the basic biology of these diseases are introduced, including the interactions of NHL with bone marrow stromal cells and the consequences of suppressed apoptosis and induced chemoresistance which might explain why minimal lymphoma in bone marrow is a surrogate predictor of a poor clinical outcome. In contrast, NHL cells isolated from stroma, for example mobilized into blood by cytokine, may be more susceptible to apoptosis and clinically less significant. The possible role of angiogenesis in facilitating early metastasis to the bone marrow is considered. Methods of detecting minimal NHL are reviewed and differences in predictive reliability of tumor detected by culture methods versus molecular techniques which identify clonal bcl-2 or antigen receptor rearrangements are discussed. The role of detection of HD by analysis of unique rearrangements of the immunoglobulin heavy and light chain genes is discussed as is the possibility that Reed–Sternberg (RS) cells can be detected molecularly as well as grown in culture from blood and apheresis harvests of patients. It appears that patients with cells resembling RS cells in their harvest do less well following high dose therapy and transplantation and additional studies of this topic are warranted. Future developments including quantitative monitoring of disease burden by real-time automated PCR and the application of genetic profiling to identify genetic markers specific to the tumor and which, potentially can predict prognosis is suggested. Also, the problems which may arise in attempts to monitor the impact of newer therapies such as anti-lymphoma antibodies and vaccines which may preferentially deplete tumor cells from blood and marrow are considered. The past 10 years has witnessed dramatic progress in the application of techniques to monitor minimal lymphoma. This technology has helped demonstrate the success of some new therapeutic approaches e.g. antibody and vaccine therapies, and served to emphasize the failure of others, for example, stem cell selection to purge lymphoma from patient harvests. Technologically, the field is not yet mature and further evolution may be expected.     

Blastic CD4 NK cell leukemia/lymphoma: a distinct clinical entity

We report the findings of three new cases of a distinct clinicopathologic natural killer (NK) cell malignancy characterized by cutaneous, nodal and bone marrow infiltration by CD3-CD4+CD56+ NK blastic cells. Tumor cells were detected in bone marrow and in peripheral blood smears and showed finely distributed nuclear chromatin with nucleoli and a moderate amount of cytoplasm. Epstein-Barr virus (EBV) DNA was negative in the two tested cases. The immunophenotypes determined by flow cytometry were identical concerning mCD3-cytCD3-CD4+weakCD56+ HLA-DR+. The TCR was in germline configuration in the two cases tested. NK cell activity was demonstrated only in one out of the two cases tested. The negative reactions with alpha-naphthyl-acetate-esterase (ANAE), CD11b and CD14 strongly suggested that the tumor cells were not of the monocytic lineage.     

Neuroblastoma cells isolated from bone marrow metastases contain a naturally enriched tumor-initiating cell

Neuroblastoma is a heterogeneous pediatric tumor thought to arise from the embryonic neural crest. Identification of the cell responsible for propagating neuroblastomas is essential to understanding this often recurrent, rapidly progressing disease. We have isolated and characterized putative tumor-initiating cells from 16 tumors and bone marrow metastases from patients in all neuroblastoma risk groups. Dissociated cells from tumors or bone marrow grew as spheres in conditions used to culture neural crest stem cells, were capable of self-renewal, and exhibited chromosomal aberrations typical of neuroblastoma. Primary spheres from all tumor risk groups differentiated under neurogenic conditions to form neurons. Tumor spheres from low-risk tumors frequently formed large neuronal networks, whereas those from high-risk tumors rarely did. As few as 10 passaged tumor sphere cells from aggressive neuroblastoma injected orthotopically into severe combined immunodeficient/Beige mice formed large neuroblastoma tumors that metastasized to liver, spleen, contralateral adrenal and kidney, and lung. Furthermore, highly tumorigenic tumor spheres were isolated from the bone marrow of patients in clinical remission, suggesting that this population of cells may predict clinical behavior and serve as a biomarker for minimal residual disease in high-risk patients. Our data indicate that high-risk neuroblastoma contains a cell with cancer stem cell properties that is enriched in tumor-initiating capacity. These cells may serve as a model system to identify the molecular determinants of neuroblastoma and to develop new therapeutic strategies for this tumor.     

Hairy cell leukemia. An immunologic and ultrastructural study.

A case of hairy cell leukemia in a 39-year-old man is reported. Hairy cells from the peripheral blood, spleen, and bone marrow had lambda-type immunoglobulin on their surfaces; those from the peripheral blood and bone marrow also had IgD on their cell membranes. Frozen sections of spleen reacted with IgGEA, but not IgMEA or IgMEAC markers. Transmission electron microscopy revealed ribosome-lamella complexes in cells from the spleen, but not the peripheral blood. Scanning electron microscopy demonstrated a spectrum of cell surface morphology with many cells characterized by ridges and ruffles. The significance of these findings is considered and it is suggested that the hairy cell is a B lymphocyte.     

Bone marrow micrometastases and circulating tumor cells: current aspects and future perspectives

Early tumor cell dissemination at the single-cell level can be revealed in patients with breast cancer by using sensitive immunocytochemical and molecular assays. Recent clinical studies involving more than 4000 breast cancer patients demonstrated that the presence of disseminated tumor cells in bone marrow at primary diagnosis is an independent prognostic factor. In addition, various assays for the detection of circulating tumor cells in the peripheral blood have recently been developed and some studies also suggest a potential clinical relevance of this measure. These findings provide the basis for the potential use of disseminated tumor cells in bone marrow or blood as markers for the early assessment of therapeutic response in prospective clinical trials.