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One hundred and twenty-seven patients with acute leukemia were treated with high-dose cytoreductive programs in conjunction with autologous marrow. Transplantation in relapse resulted in a complete remission rate ranging from 33% to 72%, depending on the time of transplantation, the conditioning regimen used and the performance status of the patient. Remission duration was short, ranging from three to nine months. Transplantation in second or subsequent remission did not change the natural history of the disease. In 14% of the cases, transplantation remissions were obtained exceeding the duration of the preceding remission, results equivalent to those obtained by normal dose chemotherapy. Transplantation in first remission (CR1) resulted in a projected two-year disease-free survival of 72% (13% SE) in acute myelogenous leukemia and of 45% (17% SE) in acute lymphocytic leukemia. We conclude from these data that the results look promising in CR1 and that in second and subsequent remissions a realistic study design is possible by which the influence on the natural history of disease by changing the bone marrow transplantation program, such as purging in vitro, can be studied.

We have compared the in vitro differential killing efficacy of doxorubicin, 5-fluorouracil, cis-platinum, etoposide, and bleomycin on human tumor cells in a new adhesive tumor cell culture system (ATCCS), and on normal bone marrow granulocyte-macrophage colony-forming units (GM-CFUC) in culture. All of the above chemotherapy agents were tested with continuous exposure against tumor cells and GM-CFUC. In addition, bleomycin was also tested with a short (60 min) exposure against GM-CFUC. In order to determine chemosensitivity against all five drugs, 48 tumor specimens from patients with heterogeneous tumor types were tested in the ATCCS. Each drug was tested at three different concentrations corresponding to their lethal doses against GM-CFUC. Our results show that all five drugs exhibited a dose-response relationship against tumor cells and GM-CFUC. Bleomycin also showed a pronounced GM-CFUC-sparing quality with 60-min exposure even at very high concentrations (86% survival of GM-CFUC at 10 micrograms/ml and 48% at 50 micrograms/ml). In addition, it has a high tumor response rate with continuous exposure at low concentrations (67% at GM-CFUC LD5 and 91% at LD40). These data suggest that the comparison of differential cytotoxicity of chemotherapy drugs between tumor cells and bone marrow cells may serve as a model to select agents suitable for in vitro chemotherapy of bone marrow for the purpose of autologous bone marrow transplantation. In particular, bleomycin appears very attractive and deserves further investigation.

To determine whether myeloid leukaemic cells could inhibit normal myeloid colony formation, leukaemic cells at concentrations ranging from 0.5 to 8 X 10(6)/ml were co-cultured in agar but separated by a 1 ml underlayer from 10(5) low-density (less than 1.077 g/ml) nonadherent normal marrow cells. Inhibition of normal-marrow myeloid colony formation occurred regularly at high cell concentrations (8 X 10(6)) at a leukaemic:normal cell ratio of 80:1. This suppression persisted with addition of indomethacin (10(-6)M). On the other hand, both low leukaemic cell numbers and irradiated leukaemic cells frequently stimulated normal colony growth. No inhibitor of colony growth could be detected in leukaemic-conditioned media, and absorption of colony-stimulating activity (CSA) with leukaemic cells improved CSA activity. These experiments point to the difficulty in unravelling the effect of leukaemic cells on normal haemopoiesis (both inhibitory and stimulatory) by in vitro agar culture.

T cells from the peripheral blood of patients with chronic myeloid leukemia (CML) were cultured with phytohemagglutinin and T-cell growth factor (TCGF) in agar culture. These T-cell colonies were pooled and expanded further in liquid culture with TCGF and then simultaneously analysed for the E-rosette receptor with the monoclonal antibody OKT11 and for the presence of the Philadelphia (Ph1) chromosome. OKT11 analysis showed these populations to be composed 99.5% or more of T cells. In four of the seven patients the T-cell suspension showed 7/50 (14%), 3/36 (8%), 2/34 (6%), and 4/44 (9%) Ph1 metaphases. Furthermore, Ph1 metaphases were demonstrated in T-cell cultures in two patients when bone marrow metaphases simultaneously showed 90 and 100% Ph1 negative metaphases secondary to human leukocyte interferon therapy or combination chemotherapy. A minority of T cells in benign phase CML have the Ph1 abnormality despite reduced number of Ph1 metaphases in bone marrow from therapy.

Fourteen patients with untreated small cell bronchogenic carcinoma were treated initially with 2 chemotherapy courses of cyclophosphamide (1.5 g/m2 days 1-3), 4-dimethyl epipodophylloxtin (200 mg/m2 days 1-3), vincristine (1.5 mg/m2 days 1 and 3), and with Adriamycin (80 mg/m2 day 1) in 8 patients and without Adriamycin in 6 patients. To modify hematopoietic toxicity from these high doses of chemotherapy, autologous marrow collected and frozen before storage was thawed and infused after each of these high-dose therapies. After this therapy patients received prophylactic brain irradiation (3000 rad), 4 courses of usual doses of these same drugs and then 5000 rad chest irradiation if there was still evidence of disease (PR) or randomized to radiation if in complete remission (CR). Response rate was high, with 54% CR and 46% PR, a total of 100%. However, a median response duration of 41 weeks and median survival of 56 weeks, are similar to other chemotherapy programs. Toxicity was mild except for cardiac arrhythmias when Adriamycin was included. The reasons for no therapeutic increment are discussed.

Patients with solid tumor malignancies who failed extensive front-line treatments were treated with high-dose BCNU followed by autologous bone marrow transplantation. Since 1976, 14 patients were treated in this program with BCNU doses ranging from 600 to 1500 mg/m2 over 2 days. Three patients developed pulmonary toxicity only a few weeks after receiving high-dose BCNU at total doses of 900, 1000, and 1500 mg/m2. Two of the three patients had had prior irradiation to the primary tumor. The third patient did not have prior irradiation to the chest but had received prior nitrosourea therapy. The use of high-dose BCNU in transplantation may be limited to early pulmonary toxicity.

Eight adult patients with refractory Hodgkin's disease (HD) and non-Hodgkin's lymphoma (NHL) were treated with high-dose combination chemotherapy (cyclophosphamide, BCNU and VP-16) or with cyclophosphamide and fractionated whole-body irradiation (TBI), followed by bone marrow transplant (BMT). Six patients received autologous and two patients allogeneic BMT. Five patients achieved complete remissions, and three of them (two with undifferentiated lymphoma, one with lymphoblastic lymphoma) are alive and free of disease 4-18+ months after BMT. The other two complete responders died of opportunistic infections 2 and 5 months, respectively, after BMT. One patient with HD achieved partial remission and is alive 18+ months after BMT. Two patients were considered failures: one developed leptomeningeal disease 24 days after BMT, and the other died of progressive lymphoma 7 months after BMT. Engraftment and prompt hematologic recovery occurred in all patients. The major toxicity included two fatal infections and one case of diffuse idiopathic interstitial pneumonitis. High-dose chemotherapy with or without TBI followed by BMT appears to produce a high response rate and, although associated with toxicity, it demonstrates the potential for salvaging patients with refractory lymphoma who otherwise would have a dismal prognosis.

Eleven patients with Philadelphia chromosome (Ph1)-positive chronic myelogenous leukemia (CML) in blast crisis (ten patients) or accelerated disease (one patient) were treated with piperazinedione (PIP) and fractionated total body irradiation (TBI) followed by autologous bone marrow transplantation (ABMT). Three patients were transplanted with marrow from which the Ph1 clone had been eradicated by prior intensive chemotherapy. All patients responded with disappearance of blasts in bone marrow and peripheral blood. Six patients achieved a second chronic phase lasting 3 to 14 months (median, 6 months). Two patients had incomplete recovery, and three patients failed to engraft and died from infection. Transplantation with Ph1-negative bone marrow did not improve response duration or survival. Recurrence of blast crisis and incomplete engraftment continue to be the two major problems in this patient group, and more active regimens need to be investigated.

Multiple clinical studies have been undertaken to evaluate the role of ABMT in relapse, in second and subsequent remission (CR greater than or equal to 2), and in first remission (CR1). The value of these high dose cytoreductive programs with ABMT is not yet known. No randomized studies have been done yet indicating that ABMT is superior to conventional dose chemotherapy. Besides, the diversity of regimens and patient populations make evaluation of results extremely difficult. When these variables are taken into account, the effect of purging on the clinical results becomes unclear. Multiple methods of purging have been devised, and several clinical trials have been reported. Although the biological role of purging is still unknown, theoretical considerations will be discussed and recommendations of patient subpopulations in which purging might be effective will be given.

The CFU-S potential of mouse bone marrow, the transplantation capacity of autologous and allogeneic monkey bone marrow and the CFU-C potential of human bone marrow can be cryopreserved without loss of viability. It is critical for an optimal stem cell recovery to avoid an osmotic shock after thawing. The intracellular cryoprotectives glycerol and DMSO which provide a high osmotic pressure should be removed from the thawed cells by a slow stepwise dilution procedure.

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 effect of embryo fibroblasts on the growth of erythroid colony-forming cells in vitro (CFU-e) from mouse bone marrow was investigated. First, the maintenance of CFU-e number in suspension culture was assayed. CFU-e recovered from suspension culture fell rapidly to values below 30% of the initial number. When erythropoietin (EP) was added, the initial decline during the first day was followed by a rise to 80%. In cultures supplemented with irradiated fibroblasts, the number of CFU-e did not show an abortive fall, but there was a slight increase during 3 days of culturing. The influence of fibroblasts on the colony-forming ability of CFU-e was studied in a semisolid culture system composed of an agar underlayer and a methylcellulose overlayer. The number of erythroid colonies scored after 5 days of culture in the presence of different levels of EP was proportional to the number of added fibroblasts and the colony size (depending on the number of fibroblasts) increased to macroscopic dimensions. Fibroblasts alone, without EP, induced colony formation by CFU-e if added in concentrations of 1 X 10(5) or higher. EP was not detectable in medium conditioned by the fibroblasts. These data indicate that fibroblasts may stimulate erythroid colony formation (in the absence of EP) and enhance the colony-forming ability of CFU-e in the presence of EP. From these results, it is suggested that fibroblasts exert proliferation activating effects on CFU-e target cells.

We investigated the possibility that the control of leukemic growth by autologous adherent marrow cells may explain the indolent behavior of oligoleukemia. Leukemic cluster growth was measured by in vitro agar culture. The following marrow fractions (Fr) were examined from 13 patients for leukemic growth: Fr I: ficollisopaque interface cells (density less than 1.077 g/ml); Fr II: adherent-cell depleted Fr I (4.8% +/- 2.1 (SE) monocytes); Fr III: adherent and phagocytic cell depleted Fr I (0.3% +/- 0.2 monocytes); and Fr IV: adherent cells removed from plastic surfaces by lidocaine plus EDTA (74% +/- 10.9 monocytes). Two patient groups were recognized: Group A showed cluster growth in Fr IV with no absolute increase in Fr III and no suppression of Fr III cluster growth with addition of Fr IV. In Group B, Fr III showed an absolute increase in cluster growth. Fr IV did not show cluster growth and addition of Fr IV to Fr III suppressed growth. This suggested that in Group B, monocytes may suppress leukemic growth, the failure to observe this in Group A indicated the possible leukemic nature of the adherent cell fraction and the absence of residual normal monocytes. Other possible explanations are discussed.

The effects of coincubation of normal nonadherent bone marrow cells on adherent monolayers created from human peripheral blood mononuclear cells or marrow cells were investigated. Nonadherent marrow cells were coincubated for 4 hours with peripheral blood adherent cells at ratios of adherent cells to marrow cells of 2:1 to 5:1. This coincubation suppressed subsequent neutrophilic agar colony growth but not eosinophilic growth. Further studies suggested that this suppression was a cell-cell-mediated process and not secondary to soluble factors. However, coincubation on marrow adherent cells caused increased neutrophilic colony recovery. The possible in vivo relevance is discussed.

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.

Previous in vitro classification of adult acute leukemia incorporating morphology has been complex and difficult to understand. We have devised a simplified classification based solely on leukemic proliferation in vitro. Forty-four patients with adult acute leukemia previously untreated were included in this study and received identical chemotherapy. Three in vitro groups were recognized. The complete remission rate (CR) was 77% in the 13 patients with no leukemic growth in vitro (Group 1), 81% in 16 patients with leukemic cell growth but aggregated of 20 cells or less (Group 2) and only 27% in the 15 patients with aggregates of greater than 20 (Group 3). There was a highly significant difference in complete remission rates between Group 3 and the other 2 groups (p less than 0.01). Linear logistic regression analysis demonstrated the independence of the in vitro growth from other prognostic variables. The cause of death in failures suggested that this system detects resistance to the chemotherapy. Similar multifactorial analysis including in vitro agar culture may help to predict for chemotherapy response in preleukemia and leukemia with a low blast cell infiltrate when cytotoxic therapy is clinically indicated.

High-dose methylprednisolone (HDMP) was used to treat 18 episodes of severe (grades III and IV) acute graft-versus-host disease (GVHD) that developed after allogeneic bone marrow transplantation in 12 patients with acute leukemia and in 2 with aplastic anemia. Most of the patients showed rapid improvement in GVHD, with complete resolution of the skin and gut manifestations. However, the response of liver disease to the treatment was slow and incomplete. Complications seen were interstitial pneumonia and fungal and viral infections. Seven patients survived for more than two months following the treatment of acute GVHD. Five of these became long-term survivors with a median survival of 22+ months (range 11-38 months); all five long-term survivors developed chronic GVHD and are alive at the time of this report. It appears that HDMP is an effective treatment for severe acute GVHD. However, its true efficacy can only be ascertained in a randomized study comparing high-dose and conventional-dose methylprednisolone.

An in vitro culture technique for colony formation of marrow cells and peripheral blood cells from untreated acute leukemia patients and from patients in relapse is described. The colonies from bone marrow cells of an untreated acute myelogenous leukemia (AML) patient were demonstrated to be of leukemic origin by cytogenetic analysis. Cells obtained from colonies of leukemic origin contained the human malignancy-associated nucleolar antigen (HMNA) as detected by indirect immunofluorescence. This nucleolar antigen was not present in marrow or peripheral blood cells or cells from colonies of marrow from hematologically normal individuals. Colonies could be grown from over 70% of the marrow and peripheral blood samples from untreated acute leukemia patients. The median number of colonies obtained was 75 per 10(5) marrow cells from patients with AML. In 1/3 of the cases an increased number of colonies could be grown from marrow cell suspensions kept in liquid culture for 5 days. This is indicative of the proliferative capacity of the colony forming cell population. This assay may be useful for detection of residual clonogenic leukemic cells in marrow and peripheral blood cell suspensions.

Analysis of myeloid progenitor cells in the peripheral blood (peripheral blood colony-forming unit granulocyte-macrophage; PBCFU-GM) is limited by their low frequency and by the presence of inhibitory cell populations. These factors limit the study of cytokines and cellular influences on PBCFU-GM in semisolid media assays and complicate the interpretation of data. We have developed a limiting dilution assay (LDA) in liquid culture for PBCFU-GM that allows evaluation of inhibitory or accessory effects of other cell populations and estimation of progenitor cell frequency. Using this system we have examined the inhibitory effect of autologous monocytes on in vitro colony growth. After monocyte depletion by counterflow centrifugal elutriation and adherence, colony growth with recombinant human granulocyte-macrophage colony-stimulating factor was linear over a wide range of cell densities, indicating a direct proliferative effect on circulating myeloid progenitor cells. Simultaneous PBCFU-GM assays in agar demonstrated monocyte inhibition but did not afford reliable interpretation of either progenitor frequency or linear growth kinetics in a statistically verifiable fashion. LDA in liquid culture may be a useful tool to study the effects of various cytokines and cell populations on PBCFU-GM in vitro and in vivo.

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.

On radiobiological grounds, a therapeutic advantage should result when total body irradiation (TBI) in preparation for bone-marrow engraftment is given as a fractionated course, rather than as a single exposure at logistically reasonable dose rates. This is because cells of hemopoietic origin in general show less capacity for repair of sublethal radiation injury than do cells of other organs. Dose-limiting lung tolerance, in the context of fractionated TBI, is estimated to be at least 12 Gy (without correction) in increments of 2 Gy regardless of dose rate. A practical method for delivering TBI using a high-energy linear accelerator is described.

Sixty-one patients with relapsed Hodgkin's disease who had failed a mechlorethamine, vincristine, procarbazine, and prednisone (MOPP)- and a doxorubicin, bleomycin, vinblastine, and dacarbazine (ABVD)-like regimen were treated with a high-dose combination chemotherapy containing cyclophosphamide, carmustine, and etoposide (CBV) and autologous bone marrow transplantation (ABMT). Fifty-nine patients were treated in relapse and two were intensified early in third remission. Following therapy, 29 patients (47%) were in complete remission (CR), 18 patients (30%) achieved a partial response (PR), and 14 patients (23%) had progressive disease (PD). Among the partial responders, six patients achieved a CR following addition of local radiation therapy to sites of residual nodal disease. For a minimum follow-up of 2 years, 23 patients (38%) are alive and free of disease. High-dose CBV therapy produced severe myelosuppression, and there were four (7%) treatment-related deaths. A multivariate analysis identified failure of more than two prior chemotherapy treatments and poor performance status as important adverse risk factors for survival. Patients who had no adverse risk factor and/or were intensified with CBV while Hodgkin's disease was still responding to conventional chemotherapy, had a CR rate of 63%, with 77% projected 3-year survival; whereas, all other patients had a CR rate of 31%, and a projected 3-year survival of only 18%. Our results demonstrated that CBV and ABMT can induce remission duration of 2 years or greater in a significant proportion of patients with relapsed Hodgkin's disease.

In 10 cases of CID, acute GVH reaction could be avoided by the use of small numbers of purified stem-cell concentrates. In 3 cases treated with stem cells from nonidentical donors, moderate delayed GVH reaction occurred. Death occurred from complicating infections. Bacteriologic decontamination is recommended as an additional precaution for future cases. This recommendation is supported by findings in germfree, as well as in specifically decontaminated, mice grafted with allogeneic marrow following x-irradiation in which mortality does not occur even after conventionalization after a limited period.

A total of 144 evaluations of cell surface markers and cellular immune functions were carried out in 57 patients undergoing allogeneic bone marrow transplantation for acute leukemia in remission and relapse and for aplastic anemia. The periods tested were pretransplant, and 1-3, 4-6, 7-12 and more than 12 months posttransplant. The determination consisted of lymphocyte counts; lymphocyte surface marking using OKT3, OKT4, and OKT8 antibodies; and determination of adherent cells, lysozymes and antibody dependent cellular cytotoxicity (ADCC) against chicken red blood cells, human red blood cells, and CEM cells. Natural killer cells were determined against K562 target cells. Lymphoblastic responses were tested after stimulation with pokeweed mitogen (PWM), concanavalin-A (Con-A), and phytohemagglutinin (PHA). We found that the progression in the leukemic state (first remission, second remission, and relapse), prior to transplantation was paralleled by a decrease in T4 lymphocytes (976/microliter +/- 462; 411/microliter +/- 222; 372/microliter +/- 419; P = .04). There was a lack of helper cells and an inverted T4:T8 ratio beyond one year posttransplant independent of graft-versus-host disease status. Lymphocyte functions persisted to be depressed for more than one year. We found a direct correlation of T4 helper cells and an inverse correlation of T8 suppressor cells with lymphoblastic responses to mitogens. It is hoped that the longitudinal evaluations of immune functions after allogeneic bone marrow transplantation, and the characterization of the immune defects seen may lead to better immunorestorative treatments.

Experiments were designed to determine which actual differences in the cellular composition between fetal liver and bone marrow account for the distinct types of graft-versus-host (GvH) disease. The assay of reactive lymphocytes (by in vitro mitogenic stimulation) in fetal liver transplants in mice, the purification of hemopoietic stem cells (HSC) of the transplants, and the quantitation of HSC numbers in the grafts traced the basis for the distinctly weak type of GvH disease after fetal liver cell grafts. It was found that transplantation of purified HSC concentrates did not modify the severity of GvH mortality. The moderate character of the delayed GvH disease was shown to depend on the presence of an HSC population in fetal liver with different qualities and not on numerical differences between the HSC in fetal liver and bone marrow. Data collected also demonstrated that when GvH disease occurred in the recipients of transplants of fetal liver, it shared the characteristic histologic features of the bone marrow GvH syndrome. The recovery of mitogen responsiveness of spleen cells may have been delayed in fetal liver allotransplantation as compared to syngeneic grafting. By supportive infusion of lymphoid cells, it was suggested that the immunodeficiency coinciding with GvH disease represented a secondary manifestation of the disease rather than a primary impairment in lymphoid differentiation.

From March, 1976 to February, 1979, 28 cases of adult acute leukemia of which 24 were evaluable were treated in irreversible relapse with high dose chemotherapy (piperazinedione) and supra-lethal total body irradiation (TBI) in conjunction with autologous bone marrow transplantation (ABMT). The marrow cells grafted were collected and stored in liquid nitrogen at the time of remission. In 12 patients the marrow cells were fractionated using discontinuous albumin gradients in an attempt to separate normal cells from residual leukemic cells. Twelve patients achieved complete remission (CR); in 9 additional patients signs of engraftment were evident but death occurred before achievement of CR. Seven of 12 AML patients, which were treated with bone marrow transplantation as first treatment of their relapse, achieved CR. Four of 5 patients with ALL, whose bone marrows were collected during first remission, reached CR. The median CR duration was 4+ months and the median survival of the patients reaching CR was 6+ months. Autologous bone marrow transplantation offers a good chance of CR (66%) when marrow is collected during first remission and used as first treatment for AML in third relapse and ALL in second relapse.

In adult acute lymphocytic leukemia (ALL) cure is rare. The purpose of this study was to try to improve remission duration and survival by administration of two courses of high-dose chemotherapy, each followed by autologous bone marrow rescue, in first remission. Chemotherapy consisted of cyclophosphamide, BCNU and VP-16-213. Rescue bone marrow was fractionated over a discontinuous albumin gradient to minimize contamination with leukemic cells. Fourteen patients entered the study. Median total remission duration was 14 months. Three patients relapsed after one course of treatment. Five patients relapsed after the second course. Four patients died after the second course and two patients remain alive and well in unmaintained remission, with a total remission duration of 42+ and 47+ months. It is concluded that this regimen is toxic but, with careful selection of patients, may lead to long-term unmaintained remissions.

24 cases of adult acute leukaemia, of which 21 were evaluable, were treated in irreversible relapse with high-dose piperazinedione and supralethal total-body irradiation (T.B.I.) in conjunction with autologous marrow transplantation (A.B.M.T.). The grafted marrow cells had been collected and stored in liquid nitrogen at the time of remission. In 12 patients the marrow cells were fractionated on discontinuous albumin gradients in an attempt to separate normal cells from residual leukaemic cells. 11 patients achieved complete remission (C.R.); 7 other patients had signs of engraftment but died before C.R. The median remission duration was 4 months (2-14). 6 of 9 acute myeloblastic leukaemia patients, in whom bone-marrow transplantation was the first treatment of relapse, achieved C.R. 4 of 5 patients with acute lymphoblastic leukaemia, whose bone-marrow cells were collected during first remission, reached C.R. Autologous bone-marrow transplantation is a valuable first treatment for acute myeloblastic leukaemia in relapse and acute lymphoblastic leukaemia in second relapse.

Several proto-oncogenes have been reported to be expressed in normal and malignant hematopoietic cells. Since these studies have been done almost exclusively by Northern and dot-blot analyses using mixed populations of cells, any conclusions concerning quantitative changes in gene expression are difficult to document. We have developed a rapid and sensitive RNA-in situ hybridization technique permitting detection of as few as 5 copies of mRNA per individual cell. Using this technique we have studied the expression levels of several oncogenes including MYC, SIS, FMS, p53, FOS and RAF in both normal hematopoietic cells and bone marrow (BM) cells obtained from acute myelogenous leukemia (AML) patients at presentation, at relapse and in complete remission (CR). Two of these oncogenes, MYC and SIS, are expressed at levels at least 2-5-fold higher in hematopoietic cells obtained from leukemia patients than in any normal hematopoietic cell examined, including cells obtained from regenerating bone marrow. The proportion of abnormal cells correlated well with the percentage of blast cells determined by morphological examination. In 7 out of 10 AML patients in morphological remission, a subpopulation of cells is detectable with abnormally high levels of MYC and/or SIS mRNA. These high levels of MYC expression are similar to those found in BM cells obtained from AML patients at presentation or relapse, but the percentage of cells with this abnormality is generally much lower. Continued follow-up of these patients has shown that 5 of them relapsed within 8 months. At this time, none of the 3 patients which were negative for MYC overexpression has relapsed.(ABSTRACT TRUNCATED AT 250 WORDS)

The role of autologous marrow transplantation has been reviewed in this chapter. The transplantation results in first remission are difficult to interpret due to the prognostically heterogeneous patient population transplanted and the ever-changing natural history of first complete remission due to more aggressive conventional-dose chemotherapy regimen. The biological role of marrow purging, i.e. elimination of leukaemic cells from the graft, is yet unclear. It is the opinion of the authors that studies in second and subsequent remission can resolve burning issues such as the efficacy of various high-dose conditioning regimens and of different methods of elimination of leukaemic cells such as procedures based on immunological methods, monoclonal antibodies and on differences in sensitivity of stem cells and leukaemic cells to in vitro chemotherapy. It may well be that a large number of patients is needed and, therefore, a multi-centre study is indicated.

We have developed an indirect immunofluorescence technique to stain individual colonies in situ in agar to detect the presence of a human malignancy-associated nucleolar antigen. Bone marrow and peripheral blood cells from leukemia patients who were untreated or suffering relapse were used as a source for colony formation. The agar layer containing the colonies was stabilized by overlaying it with an additional agar layer. Cells in the colonies were fixed with methanol, rehydrated, and incubated for prolonged periods in both the primary and the secondary antibodies. The agar was then transferred onto a glass slide, air dried, and examined using a fluorescence microscope. The method is widely applicable for staining colonies in situ in agar with both polyclonal and monoclonal antibodies.

We have evaluated the transplantation potential of bone marrow stem cell concentrates isolated from the 40/60% interface of discontinuous Percoll gradients. This mononuclear fraction is free from platelets and depleted of granulocytes, and contains the majority of granulocyte-macrophage colony-forming cells (GM-CFC), erythroid burst-forming units (BFU-E), and granulocyte, erythroid, macrophage, megakaryocyte colony-forming cells (GEMM-CFC) in less than 10% of the cell number of the original buffy coat. This preparation allows further manipulation without the clumping and cell loss associated with buffy coat cell preparations. Cells isolated by this technique were evaluated for hematopoietic restoration potential in 14 patients who received allogeneic bone marrow transplants as supportive therapy after high dose cytoreduction to treat leukemias or lymphoma. The number of nucleated cells infused varied from 1.6-5.5 X 10(7)/kg, and the number of GM-CFC infused ranged from 0.4 to 3.7 X 10(5)/kg. There was an inverse relationship between the time to recovery of granulocytes and platelets and the number of GM-CFC infused when fewer than 10(5) GM-CFC/kg were transplanted. Above this dose, there was recovery within 10-15 days after transplantation. The stem cell-enriched fraction contained 30-40% of the original number of T lymphocytes, and acute graft-versus-host disease was observed in seven of these patients.

Twenty-nine patients with diffuse large cell lymphoma who failed traditional chemotherapy were treated with high dose chemotherapy with or without total body irradiation followed by infusion of cryopreserved autologous marrow. Complete response was achieved in 11/29 patients (38%), partial response in 13/29 patients (45%) and 5/29 patients (17%) had no response. Six complete responders remain well and free of disease for 5+, 6+, 9+, 10+, 18+ and 25+ months, 3 relapsed at 2, 3 and 8 months after marrow infusion, and 2 died from infectious complications. Complete response was seen more frequently with the absence of bulky tumor (70 vs 21%, P = 0.03), a total body irradiation containing regimen (52 vs 0%, P = 0.03), a history of complete remission with initial chemotherapy (55% vs. 9%, P = 0.03), and a performance status greater than or equal to 80 (56 vs 15%, P = 0.06). High dose therapy had a high response rate (83%) in resistant diffuse large cell lymphoma and yielded durable complete responses in a minority of these patients.

The combination of high-dose cyclophosphamide, BCNU, and VP-16-213 followed by autologous marrow rescue was evaluated in the treatment of relapsed leukemia refractory to normal-dose chemotherapy. Cyclophosphamide was given at a total dose of 4.5 g/m2, BCNU at a dose of 300 mg/m2, and VP-16-213 at a dose of 600 mg/m2. Seven high-dose treatments followed by marrow rescue were administered to six patients. Two patients achieved complete remissions, three had partial remissions, and one achieved a minimal response. The toxicity of this regimen was moderate.

A 62-year-old woman had recurrent fluctuations of neutrophil, monocyte, reticulocyte and platelet counts with a cycling time of 38.5 d. Periodic fluctuations of granulo-, erythro- and megakaryopoiesis were also demonstrated in her bone-marrow. Oxymetholone reduced the magnitude of the profound cyclic fluctuations in all these cell lines. During treatment a reduced number of in vitro CFU-C of the progenitor myeloid series from the bone marrow was observed. Using the patient leucocytes as feeders for normal control marrow cells, indication was found for a fluctuation in colony stimulating activity in vitro, rising above normal at the nadir of the WBC.