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The in vivo effect of in vitro treatment with doxorubicin plus lonidamine on normal and leukaemic cells was investigated in a mouse model of syngeneic bone marrow transplantation. Different numbers of L5178Y tumour cells or normal bone marrow cells alone, or mixtures of bone marrow and leukaemic cells were incubated with doxorubicin (0.25, 0.5, 0.75, 1 microgram/ml) and/or lonidamine (50 micrograms/ml) and reinfused in DBA/2 mice. Lonidamine potentiated the cytotoxic effect of doxorubicin dependent on doxorubicin dosage and tumour cell concentration. Survival after injection of 10(4) in vitro-treated tumour cells was 42% for doxorubicin 0.75 micrograms/ml alone versus 100% for the combination with lonidamine and 50% for doxorubicin 1 microgram/ml alone versus 100% combination. Reinfusion of normal bone marrow incubated with doxorubicin alone or in combination with lonidamine in lethally irradiated mice did not occur in 12-14% of mice injected, indicating that the repopulating ability of stem cells was spared. These data suggest the potential usefulness of lonidamine in ex vivo purging of bone marrow before autologous bone marrow transplants in haemopoietic malignancies.

Southern blot analyses were performed in sequential DNA samples from 4 patients with Ph' + chronic myeloid leukaemia (CML) who underwent lymphoid or mixed blast crisis (BC). Genomic rearrangements at the breakpoint cluster region (bcr) and immunoglobulin heavy chain (IgH) gene level provided, in these cases, a sensitive and specific evaluation of response to therapy both in terms of blasts and Ph' + cell suppression. Recurrent BC was molecularly characterized in the 4 patients, showing each time identical individual specific DNA rearrangement patterns. Residual blasts were detected in 2 cases during intervening chronic phases by IgH rearrangements. Such findings highlight the specificity of these molecular markers, clearly indicating the failure of ablative therapy in eradicating the neoplastic clone. Finally, molecular and phenotypic identity in individual recurrent BC also suggested, in our cases, a lack of clonal evolution during disease progression.

A monoclonal antibody (S17-12), previously described to recognize subsets of myelomonocytic cells, is demonstrated to bind CD9 antigen, a surface marker of B-cell precursors, platelets and several non-hematopoietic tissues. The proportion of S17-12, CD9-positive leukemic cells was determined in 102 patients with acute non-lymphocytic leukemia. It was found to be above 75% in 14/22 M3 cases (including 3/3 of microgranular variant) and only in 1/80 cases of different FAB subtype. Complete reactivity (greater than 95%) of leukemic clonogenic cells with CD9 MoAB was restricted to 4/18 M3 cases. Therefore, a high proportion of CD9-positive cells seems to be peculiar to M3 cases. This may help in the diagnosis of cases that lack typical morphological features.

In this study, cytogenetic and molecular analyses were employed to assess the response to therapy in 29 chronic myeloid leukemia patients undergoing high dose chemotherapy followed by autologous stem cell infusion. Of these, 11 had previously achieved hematologic remission and cytogenetic improvement after alpha-2b interferon (IFN) treatment, whereas 18 underwent autografting in an early phase of the disease. In each case bone marrow samples were examined pre-treatment and at +2, +6 and +12 months in order to verify the degree of Ph1 suppression. In addition, the position of the breakpoint within the BCR region was mapped with multiple restriction enzymes. In 17 cases (59%) a significant Ph1 reduction was observed at +60 days (0-57% residual Ph1+ cells). In three of these cases, a complete cytogenetic response was confirmed at the DNA level by Southern blotting, but specific amplification of the BCR/ABL junction by the polymerase chain reaction (PCR), performed in two cases, still showed residual disease. The remaining 12 patients (41%) revealed a substantial persistence of Ph1+ metaphases (90-100%). Nine of 17 responding patients (53%) showed an increase of Ph1+ cells at 6 months, and five of 20 evaluated had a further increase at 12 months. With the exception of the results seen by PCR, comparison of molecular and cytogenetic techniques did not show significant differences. The variable degrees of Ph1 suppression observed did not appear to be associated with the position of BCR breakpoints. The factors predicting cytogenetic response to IFN and stem cell autograft and long-term durability of cytoconversion should be elucidated in further studies and with longer follow-up.

Fourteen patients with chronic myelogenous leukemia in chronic phase who had achieved hematological remission after alpha-interferon (IFN) therapy were evaluated for their ability to form hemopoietic colonies in vitro. Patients were studied both before and after discontinuation of IFN to assess the optimal timing for a further therapeutic approach consisting of stem cell collection followed by autologous bone marrow transplantation (ABMT). Before stopping IFN a profound inhibition of CFU-GM and BFU-E growth was observed. The numbers of CFU-GM and BFU-E in the bone marrow recovered to 50 per 2 x 10(5) cells plated at a median of 158 and 122 days, respectively, after stopping IFN. At that time, 12 patients were submitted to stem cell harvest and subsequently to ABMT. All patients showed complete engraftment and hematological reconstitution; cytogenetic improvement was observed in eight (67%) cases; two patients displayed complete disappearance of the Ph1 chromosome, confirmed at the molecular level. Eleven of the 12 patients are still in hematological remission at a median of 18 months from autografting and a median of 48 months from diagnosis.

Thirty-nine patients with acute myeloblastic leukemia (AML) in first complete remission (CR) were treated by autologous bone marrow transplantation. All patients received the same induction and consolidation chemotherapy consisting of a combination of daunorubicin (DNR) and cytarabine (Ara-C) followed by four courses of DNR, Ara-C and 6-thioguanine (6-TG). Two different conditioning regimens were used; 25 patients were submitted to the BAVC regimen (BCNU, amsacrine, VP-16 (etoposide) and Ara-C) and 14 to a cyclophosphamide/total body irradiation (CY + TBI) regimen. Six patients (one treated with BAVC and five treated with CY + TBI) died in aplasia. Twelve of the 25 BAVC treated patients and one of the nine CY + TBI treated patients relapsed; 12 (48%) of the BAVC treated patients are in CR with a median follow-up of 45 months and eight (57%) of the CY + TBI treated patients are in CR with a median follow-up of 50 months. All patients in CR have survived for more than 2 years since transplant.

The efficacy of autologous bone marrow transplantation in leukemia and lymphoma may depend upon the selective elimination of malignant cells from human bone marrow in vivo and in vitro. A cyclophosphamide derivative (ASTA-Z 7654) and etoposide (VP16-213) have been tested on lymphoma and leukemia cell lines in a model that may represent a bone marrow situation in complete remission. The influence of different concentrations of normal mononuclear cells and tumor cells in this model and the activity of the two chemotherapeutic agents in the presence of bone marrow cells or peripheral blood cells were evaluated. A major inhibitory effect was observed using the two agents in combination; low doses of ASTA-Z and VP16 consecutively added to the mixture of malignant cells and normal mononuclear cells resulted in a greater elimination of tumor line cells than with ASTA-Z alone at the current 100 micrograms/ml dose. In contrast, no major toxicity on normal human bone marrow precursors was observed; the effect of treatment on hemopoietic recovery with the two agents either alone or in combination was evaluated on CFU-GM growth after long-term bone marrow cultures. Despite a profound growth inhibition at day 0, a recovery was observed in all cases after 7 or 14 days. The use of multiple chemotherapeutic agents in the treatment of bone marrow in vitro could decrease the possibility of malignant cells surviving while sparing normal bone marrow precursors.

14 patients out of a group of 73 affected by CML and treated with alpha-2-IFN, who had obtained hematological and cytogenetic response, were selected to be submitted to autologous BMT. In all patients the IFN treatment was interrupted to obtain an increase of the peripheral WBC necessary for its collection by means of leukapheresis. After a median time of 20.5 weeks from discontinuance of therapy, most patients are still under hematological disease control. Serially performed cell cultures have shown deeply and persistently reduced growth patterns; cytogenetic analyses have displayed Ph' mosaicism, but with a progressive increase in time of Ph' + cells. Such findings reveal that the effect of alpha-2-IFN in CML patients can be unexpectedly prolonged after discontinuance of treatment. A possible explanation might be found in the complexity of the biological mechanisms of action of IFN, thus giving this drug promising therapeutic prospects in the treatment of CML.

A clonogenic assay has been developed that utilizes Burkitt's lymphoma tumor cell lines to detect elimination of up to 5 logs of tumor cell contamination within human bone marrow. Different Burkitt's lymphoma lines bear one or more of a group of markers, including common acute lymphoblastic leukemia antigen gp26 (glycoprotein with a molecular weight of 26,000), B1, surface membrane immunoglobulin, HLA, beta 2-microglobulin, and Ia. Burkitt's tumor cells of the Namalwa line have been mixed with a 20-fold excess of irradiated human bone marrow cells. After treatment with one or more monoclonal antibodies and rabbit complement (RC), mixtures have been grown on a monolayer of irradiated human bone marrow cells and tumor cells enumerated by limiting dilution. Multiple treatments with antibody and RC were more effective than a single treatment in destroying clonogenic tumor cells which bore relevant determinants. Human serum components inhibited the lytic activity of RC in the presence of murine monoclonal antibodies. The total concentration of bone marrow cells proved critical in determining the complete elimination of tumor. Incubation of the Namalwa tumor cell line with RC and the J2 anti-gp26 eliminated more than 3 logs of malignant cells from a 20-fold excess of human bone marrow. Combinations of two monoclonal antibodies were more effective than any single antibody in eliminating Namalwa cells. A combination of three monoclonal reagents was no more effective than a combination of J2 and B1 or J2 and J5 in eliminating Namalwa cells. Treatment of human bone marrow with three antibodies and RC did not, however, produce a selective loss of nonmalignant GM-CFU-C, CFU-E, or BFU-E.

Selective removal of malignant cells from human bone marrow is one requirement for autologous bone marrow transplantation. In earlier studies treatment with multiple monoclonal antibodies and C' was more effective than treatment with individual reagents in eliminating clonogenic Burkitt's lymphoma cells from a twenty-fold excess of human bone marrow. To explore possible mechanisms underlying the additive or synergistic effects observed with multiple reagents, clones of malignant cells that have survived treatment with antibody and C' have now been isolated at limiting dilution. Marked heterogeneity has been observed in binding of different monoclonal antibodies and in resistance to C'-dependent lysis among these different clones. Phenotypic traits were stable for at least two weeks in culture. Clones that survived treatment with the J5 anti-CALLA antibody and C' exhibited a relative decrease in CALLA expression but did not exhibit an increased susceptibility to lysis by anti-CALLA and C'. No selective decrease in gp-26 or B1 was observed in clones treated with J2 or anti-B1. A correlation between CALLA and gp-26 expression was observed in clones treated with J2 and anti-B1, but no clear correlation was observed between antigen expression and susceptibility to C'-dependent lysis. In this model system, escape from C' dependent cytotoxicity does not appear related to the existence of phenotypically stable C' resistant variants within the tumor cell population.

A mouse monoclonal antibody (S4-7) reacting with human myelomonocytic cells has been previously shown to be suitable for bone marrow purging in selected acute myelogenous leukemia (AML) patients with S4-7 positive leukemic clonogenic cells at diagnosis. The results obtained in seven AML patients who underwent such a treatment, followed by autologous bone marrow transplantation (ABMT), are now reported. Six patients underwent ABMT in first complete remission (CR), one in second CR, after BAVC conditioning regimen. One patient died of infection 1 month after ABMT; in the other six a complete recovery of hemopoiesis was observed. In spite of S4-7 reactivity with normal myelomonocytic cells, a prompt recovery of granulopoiesis was however observed both in in vitro liquid culture and in vivo with a median time of 20 days to reach granulocyte values of 500 x 10(6)/l. The patient transplanted in 2nd CR relapsed 3 months after ABMT. Of the five evaluable patients transplanted in 1st CR, two relapsed 8 and 9 months post-ABMT while three remain in continuous CR at 35, 47, 57 months. Leukemic cells of two of the three patients with recurrent disease were studied at relapse and in both could be detected a significant percentage of S4-7 negative cells, detectable neither at diagnosis nor (one patient) at the time of first relapse after standard chemotherapy.

Thirty-one patients affected by advanced ALL entered this study. Twenty (1 in I CR, 9 in II CR, 6 in III CR and 4 extramedullary relapses) were treated with the BMVC conditioning regimen. Eleven (9 in II CR, 2 in III CR) received the Busulfan plus Cytoxan conditioning regimen. Asta-Z 7654-purged marrow was reinfused at day 0. Both protocols were well tolerated. Two patients treated with the BMVC regimen died in aplasia from sepsis; 1 patient died in CR 5 months after transplantation, 13 relapsed after a median time of 4 months (range 1-31). Four patients are in CCR with a median follow-up of 16 months (range 11-24). In the BU + CY treated group no toxic deaths were observed. Four patients relapsed after a median of 3 months (range 2-7) and 7 are in CCR with a median follow-up of 5 months (range 2-28).

Thirteen patients with AML in first relapse were treated with high dose combination chemotherapy followed by cryopreserved autologous bone marrow transplantation (ABMT). The first four patients received the COATA-Roma regimen, consisting of CTX, VCR, CA, 6-TG and ADM; nine additional patients received the BAVC regimen consisting of BCNU, AMSA, VP-16 and CA. A median of 1.6 X 10(8) fractionated nucleated bone marrow cells/kg body weight were reinfused. The median of GM-CFU-C recovered was 4.7 X 10(4)/kg. Out of 13 patients, 10 (76.9%) achieved CR, 3 had profound aplasia and died from hemorrhagic or infectious complications. Of the 10 patients who achieved CR, 1 died after 1 week from heart failure, 5 relapsed respectively 17, 20, 21, 21, 42, weeks after ABMT, 4 are still in CR after 2+, 14+, 17+, and 120+, weeks. Of the 9 patients treated with BAVC regimen, 8(88.8%) achieved CR. Four patients relapsed after a median of 19.7 weeks and 4 are still in complete remission. Of interest is the fact that the second complete remission of one patient is longer than the first one, despite the fact that marrow was not purified by any in vitro treatment. In conclusion we can say that BAVC regimen is highly effective in obtaining second complete remission in patients with AML and prolonged disease free survival can be achieved at least in a small number of cases.

Two AML patients, whose leukemic clonogenic cells totally reacted to the anti-lactofucopentaose III S4-7 monoclonal antibody (MoAb), underwent autologous bone marrow transplantation, in first complete remission, after in-vitro purging with S4-7 MoAb and complement. After ablative chemotherapy (BAVC regimen) and reinfusion of S4-7 purged cells, regeneration of marrow cells occurred with prompt recovery of granulopoiesis and erythropoiesis. A more delayed platelet recovery was observed. The two patients are in complete remission at 20 and 11 months from ABMT. The results indicate that immunologic purging with S4-7 MoAb is safe and suitable for selected AML patients undergoing ABMT.

Effective autologous bone marrow transplantation for leukemia and lymphoma is likely to depend upon the selective removal in vitro of malignant cells from normal human bone marrow precursors. Highly specific cytotoxic conjugates formed by coupling ricin A chain to monoclonal antibodies might prove useful for the selective elimination of malignant cells. Consequently, ricin A chain conjugates have been prepared with several different murine monoclonal antibodies and tested for their ability to eliminate clonogenic Burkitt's lymphoma cells from an excess of human bone marrow. The most active reagents included an antibody:A chain conjugate which bound to the nonpolymorphic chain of the la molecule and another which reacted with the mu heavy chain of cell surface immunoglobulin. Conjugates formed with anti-common acute lymphoblastic leukemia antigen, anti-Mr 26,000 glycoprotein, and anti-B1 were much less active on these Burkitt's cells, contrasting with results of complement-dependent tumor cell lysis. Tumor cell kill was partially inhibited by the addition of greater than 2 X 10(6) human bone marrow cells/ml but could be potentiated by increasing the concentration of conjugate or by the addition of 10 mM ammonium chloride. In the presence of ammonium chloride, at least 4 logs of clonogenic tumor cells could be eliminated within 24 h from a 20-fold excess of bone marrow using 10(-7) M ricin A chain linked to one or two different antibodies. Similar treatment of normal human bone marrow temporarily inhibited granulocyte-macrophage colony-forming units (cell) formation but did not compromise establishment of continuous bone marrow cultures. The degree of selective elimination of tumor cells with A chain antibody conjugates was comparable to that achieved with 4-hydroperoxycyclophosphamide or with multiple monoclonal antibodies and complement.

The progressive and fatal course of chronic myelogenous leukemia has not been affected significantly by chemotherapeutic agents that control the benign phase of the disease. Combination chemotherapy and aggressive treatments may offer some advantages: however, these approaches do not appear to produce stable suppression of Ph1 chromosome or to prolong chronic phase and survival of these patients. Only allogeneic bone marrow transplantation has been demonstrated to be capable of inducing a stable, complete suppression of Ph1+ cells. Recently alpha Interferons (IFN) have been shown to control myeloid proliferation in patients with CML and to determine a progressive and persistent decline of Ph1+ bone marrow cells in some cases. The hypothesis that in most newly diagnosed patients with CML various amount of Ph1 negative cells must still be present, albeit in suppressed state in the bone marrow (BM) or in the peripheral blood (PB), have led some Authors to treat patients with high-dose chemotherapy followed by reinfusion of stem cells collected at diagnosis or after a response to cytoreductive treatments. We report 34 patients with CML treated in chronic phase with Busulohan and Melphalan conditioning regimen followed by reinfusion of BM or PB stem cells.