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The brief administration of G-CSF to previously treated solid tumor patients has a positive impact on the overall cellularity and progenitor cell content of harvested bone marrow. Fifty-seven patients, fully recovered from therapy and growth factor support, had approximately 500 mL of steady-state marrow harvested as outpatients under local anesthesia. Each patient then received 5 micrograms/kg of G-CSF every 12 hours subcutaneously for either 24 hours (21 patients), 36 hours (20 patients), or 48 hours (16 patients) just before harvesting 500 mL of activated bone marrow. Bone marrow cellularity (x 10(6)/mL) increased from a steady-state mean of 10.7 (+/- 0.9) to 25.7 (+/- 2.8) after 24 hours, 9.3 (+/- 0.7) to 29 (+/- 2.5) after 36 hours, and 9.6 (+/- 0.7) to 28.4 (+/- 2.5) after 48 hours. Although the percentage of CD34+ cells did not significantly change in stimulated marrow, the total number of CD34+ cells (x 10(6)) collected increased from 34 (+/- 6.3) to 52 (+/- 6.6) after two injections, 28 (+/- 3.6) to 65 (+/- 8.5) after three injections, and 28 (+/- 5.4) to 75 (+/- 18) after four injections of G-CSF. Further phenotyping demonstrated significant increases in CD34+HLA-DR+ cells with all three schedules relative to steady-state marrow. There were no changes in the total number of CD34+HLA-DR- cells after two and four shots; however, this population increased from 10 x 10(6) in steady-state marrow to 23 x 10(6) (p = 0.012) after three injections. Analysis of peripheral blood indicated a statistically significant increase in the circulating white count, but more interestingly, there were significant increases in the number of CD34+ cells x 10(4)/mL, suggesting the onset of mobilization. Steady-state blood contained a mean of 0.86 x 10(4)/mL CD34+ cells, which increased to 4.37 x 10(4)/mL, 7.43 x 10(4)/mL, and 8.62 x 10(4)/mL after two, three, and four injections, respectively-levels of CD34+ cells that are comparable to steady-state marrow. Reinfusion of a median of 1.6 x 10(6) activated CD34+ cells/kg resulted in the recovery of > 100/mm3 neutrophils and > 20,000 platelets by days 9 and 19, respectively, which was faster than our previous patients who received steady-state marrow, and comparable to our patients who received mobilized peripheral stem cells.

This report details a bone marrow harvest procedure performed outside the hospital setting under local anesthesia, thereby avoiding many of the risks associated with the traditional surgical procedure. In approximately 30 minutes, 450 milliliters of marrow can be collected from eight bone punctures, containing a median of 4.18 x 10(9) cells and 33 x 10(6) progenitor cells as defined by CD34 expression. Reinfusion of a median 1.2 x 10(6) CD34+ cells/kg in 10 breast cancer and lung cancer patients after dose-intensive chemotherapy resulted in the recovery of granulocytes > 100/mm3 by day 14 and platelets > 20,000 by day 21. Without progenitor cell support, such recoveries could take 30 and 40 days, respectively. Collection of marrow using this protocol does not compromise the engraftment capability of the progenitor cells, seldom necessitates blood product support, is safer for the patient, and reduces the cost of harvesting by 75% compared to inpatient or day surgery procedures.

Colony formation in vitro by marrow cells from patients with untreated acute myelogenous leukemia (AML) and from patients in AML relapse is infrequent using the standard Robinson assay. A newly developed culture system has been described in which marrow from AML patients in these disease stages form leukemic cell colonies. In this in vitro system, phytohaemagglutinin is the essential stimulator for colony formation. The leukemic origin of the colonies has been proven by ultrastructural morphology and cytogenetics. It appears that colony formation by leukemic cells in this system is predominantly independent from the leukocyte factor which is the main stimulator in the Robinson assay for growing colonies of marrow cells from haematologically normal individuals.

We and other investigators have previously reported our findings on oncogene expression in human leukemia in an attempt to study the possible involvement of these genes in the leukemic state. An important shortcoming of these studies has been the lack of information on the expression of these genes in normal hematopoietic cells. To address this question we analyzed both the transcript size and level of expression of six oncogenes in fresh hematopoietic cells obtained from hematologically normal individuals and compared the results to those found in fresh samples obtained from patients with various forms of leukemia (acute myelogenous leukemia, acute lymphocytic leukemia, and chronic myelogenous leukemia). We found low level expression of c-myc, c-myb, c-fes, and c-raf in normal bone marrow in sharp contrast to the high levels of expression found in some forms of leukemia. C-fos was highly expressed in both normal bone marrow and certain leukemias. We were unable to detect c-sis expression in our normal samples. With the exception of c-fes, there was no variation in transcript size when comparing normal and leukemic samples. Having defined the transcript sizes and levels of expression for these proto-oncogenes in normal hematopoietic cells, we know that aberrant transcript size for the genes we have studied is not a common event in leukemias. The levels of expression, however, vary widely between normal hematopoietic cells and leukemia as well as between different types of leukemia.

The effects of droperidol and fentanyl on three samples of human bone marrow with regard to their colony forming in culture was studied in more than 256 multiple experiments. There was no evidence of any toxic effect of droperidol and fentanyl on the bone marrow cells.

The effectiveness of ex vivo chemotherapy with drugs, such as vincristine, etoposide, and Adriamycin (doxorubicin, Adria Labs, Columbus, OH) for elimination of residual tumor cells from human bone marrow grafts could be undermined by the presence of multidrug-resistant tumor cells in the bone marrow. Therefore, to supplement chemoseparation, we investigated whether MRK-16, a monoclonal antibody (MoAb) to the surface moiety of multidrug resistance-associated P-glycoprotein antigen, can eliminate drug-resistant tumor cells in the presence of rabbit complement (RC). Two doxorubicin (DOX)-resistant human myeloma tumor cell line, 8226/DOX40 (resistant to 4 x 10(-7) mol/L DOX) and 8226/DOX6 (6 x 10(-8) mol/L DOX) with high and low amounts of cell surface P-glycoprotein, respectively, and the drug-sensitive parent cell line 8226/S were used as tumor models in this study. Using the limiting dilution assay, we have shown that three cycles of treatment with 25 micrograms/mL of MRK-16 MoAb and a 1:4 final dilution of RC eliminated 2.90 +/- 0.10 logs of 8226/DOX40 cells and 1.94 +/- 0.18 logs of 8226/DOX6 cells. One and two cycles of treatment were less effective, eliminating 0.47 +/- 0.40 and 1.94 +/- 0.36 logs of 8226/DOX40 and 0.12 +/- 0.20 and 1.63 +/- 0.58 logs of 8226/DOX6 cells, respectively. The 8226/S cell growth was unaffected by one to three cycles of treatment. The cell kill was not impaired when the antibody plus complement treatment was carried out on a mixture of 8226/DOX40 or 8226/DOX6 cells with a ninefold excess of irradiated bone marrow mononuclear cells (MNCs). The three cycles of treatment with antibody plus complement did not adversely affect granulocyte-macrophage colony-forming unit (GM-CFU) survival in hematologically normal marrows (92.5% to 104% survival) or in myeloma patient marrows (85% to 100%). These results show that it is possible to eliminate drug-resistant myeloma tumor cell lines from the admixed human bone marrow by treatment with MRK-16 MoAb plus RC. This method could prove to be effective for elimination of other drug-resistant tumor cell lines including those of leukemia and solid tumors, and will be further useful for supplementing chemopurging, and immunopurging of bone marrow with other antitumor cell antibodies.

Cytologic and immunofluorescent data demonstrate that cells morphologically resembling megakaryocytes appear in thin layer agar cultures of cellular fractions of mouse bone marrow and increase in number from zero to a maximum of approxiamately 60 per culture on day 6. It would appear that these "candidate" megakaryocytes are derived from a morphologically unidentified precursor(s) since they are not present in the original cell suspension cultured but begin to appear 24 hours later. Similarities between the ultrastructure of "candidate" megakaryocytes and that of femoral marrow megakaryocytes support morphological similarities at the light microscopic level. Pretreatment of mice with either vinblastine and nitrogen mustard or antiplatelet serum has no effect on the number of "candidate" megakaryocytes appearing in cultures of their marrow. Preliminary data suggest that the rate of appearance of these cells in culture is responsive to serum from thrombocytopenic donors.

A technique for cloning myeloblastic leukemic cells in semi-solid agar, after prior PHA stimulation, was studied in all phases of chronic myeloid leukemia. Phytohemagglutinin responsive cells were found in blast crisis and accelerated phase. In a small number of patients in the benign phase of their disease, colony growth with PHA was detected. A group of patients with early acute leukemia was also examined by this technique. The incidence of PHA colonies appeared to correlate with the progression of the disease. The potential application of this assay for early detection of leukemic clones and possible prediction of the rapidity of progression of the leukemic process is discussed.

Relatively nonmyelotoxic drugs and drug combinations were investigated for their ability to eliminate malignant cells from human bone marrow. In vitro 90% inhibitory concentration (IC90) doses were established on granulocyte macrophage colony-forming units (GM-CFU) in culture of bone marrow by using the GM-CFU assay for the following drugs: 4-hydroperoxycyclophosphamide (4-HC), Adriamycin, L-asparaginase, bleomycin, hydrocortisone, VP-16, spirogermanium, Taxol, and vincristine. The leukemic cell kill efficiency of these drugs at IC90 doses was compared with that of 4-HC on acute lymphoid leukemia (ALL) cell lines by using the limiting-dilution assay. Under these conditions, no single drug was superior to 4-HC. To increase the in vitro effect in leukemic cell kill, combinations of vincristine with hydrocortisone, Adriamycin, VP-16, and 4-HC were investigated. Vincristine at 1 to 5 micrograms/mL increased the marrow cytotoxicity of hydrocortisone, Adriamycin, and VP-16, but it was protective (subadditive) with 4-HC. Vincristine and 4-HC in combination was additive to supraadditive on ALL cell lines, increased the leukemic cell kill by one to two logs above 4-HC alone at IC90 doses (P less than .05), and was not affected by the addition of excess marrow cells. The recommended doses for chemopurging in clinical studies are vincristine, 1 to 5 micrograms/mL, plus 4-HC, 5 micrograms/mL.

We investigated whether monoclonal antibodies (MoAbs) reactive against both acidic and basic cytokeratins alone were sufficient to detect minimal numbers of contaminating epithelial tumor cells in the bone marrow of breast cancer patients. Monoclonal anti-cytokeratin antibodies (AE1 and AE3) were used to stain 14 breast carcinomas by the avidin-biotin-peroxidase technique. Nine tumors (64.3%) showed high reactivity and five (35.7%) showed low or moderate reactivity. Nine MoAbs that proved to be unreactive to light density bone marrow cells by immunoalkaline phosphatase histochemistry were screened for reactivity to breast carcinomas having only low or moderate positivity to cytokeratin antibodies. Three of nine MoAbs showed high percentages of positivity and were selected to supplement the anti-cytokeratin antibodies for immunohistochemical detection of minimal marrow disease in breast cancer patients. A MoAb cocktail was prepared, further tested for reactivity to another five breast carcinomas, and compared with cytokeratin staining alone. The cocktail labeled 100% of carcinoma cells in all the examined specimens. To determine the sensitivity of this panel for detecting minimal numbers of contaminating tumor cells in bone marrow, in vitro mixing experiments were performed. T47D breast carcinoma cells were mixed with bone marrow mononuclear cells at ratios from one tumor cell per 10 bone marrow cells up to one tumor cell per 1 x 10(6) marrow cells, and cytospin preparations were subsequently stained with the MoAb cocktail by the immunoalkaline phosphatase method. Our approach could detect one tumor cell in 1 x 10(5) hematopoietic cells.

To understand the possible mechanisms of lithium carbonate-induced neutrophilia, the in vitro effect on human myeloid progenitor cells was examined. A significant increase in spontaneous colony formation (15 of 24 experiments) was observed with the addition of lithium. Increased colony formation seldom occurred when human placental conditioned media as a source of colony-stimulating activity (CSA) was simultaneously added to the cultures. Further data suggest that lithium requires an adherent marrow cell population for this action and that increases in CSA-containing cultures may be due to suboptimal CSA concentrations. Lithium was shown to release CSA from marrow cells and adherent cell population prepared from human bone marrow. Lithium possibly increases spontaneous human myeloid colony development indirectly through CSA release by adherent cells.