Cells capable of colony formation in the peripheral blood of man.

Colony-forming cells have been found in the peripheral blood of man and have been grown in vitro by use of a soft agar gel technique. It has been possible to collect these cells with a blood-cell separator in numbers similar to those found in the peripheral circulation. Repeat leukapheresis of the same donor does not reduce the number of circulating colony-forming cells.

Antigen solubilized from human leukemia: lymphocyte stimulation.

Soluble antigen was extracted with hypertonic (3 molar) potassium chloride from the malignant cells of seven patients with acute leukemia. The antigen and leukemia cells were used to stimulate autologous patients' and allogeneic normal donors' lymphocytes in mixed lymphocyte cultures. The lymphocytes of six patients showed significant blastogenic responses to autologous antigen. In contrast, the lymphocytes of only one of seven normal donors responded to the soluble antigens. Both patients' and normal subjects' lymphocytes responded to the intact leukemia cells. The use of these antigens should facilitate the study of specific tumor immunity in human leukemia.

Serial studies of immunocompetence of patients undergoing chemotherapy for acute leukemia.

Immunocompetence was followed serially for 1 yr from the onset of treatment in 55 adult patients with acute leukemia. The tests used were delayed hypersensitivity responses to a battery of five recall antigens (dermatophytin, dermatophytin 0, candida, streptokinase-streptodornase, and mumps) and in vitro lymphocyte blastogenic responses to phytohemagglutinin and streptolysin 0. There was a strong correlation between immunocompetence at the start of treatment and a good prognosis; 32/39 patients who subsequently entered remission were initially immunocompetent compared to 4/15 who failed to enter remission. In the complete remission group there was a decline in competence starting from 2 to 5 mo after the onset of treatment. In those who remained in remission for 1 yr, competence recovered at 6 mo and remained vigorous thereafter. In those who relapsed before 1 yr, the decline in competence occurred 1 mo before relapse and competence continued to decline progressively during the 1 yr follow-up period. These studies suggest that therapeutic approaches which restore immunocompetence or prevent its decline will improve both the remission rate and the remission duration of patients with acute leukemia.

Pretreatment of cytokinetics in acute myelogenous leukemia. Age-related prognostic implications.

To determine the clinical and biologic relevance of cellular kinetics in leukemia, DNA flow cytometric analysis was performed on bone marrow biopsy specimens from 148 previously untreated adult patients with acute myelogenous leukemia. The proportion of cells in synthesis, second growth, and mitosis (S + G2M) ranged from 4% to 33% with a median of 14%. The overall incidence of complete remission was not affected by the pretreatment cell cycle distribution. As in earlier studies, there was a marked decline in remission rate with advancing age from 73% for patients age less than or equal to 50 yr to 50% for those greater than 50 (P less than 0.01). Although not affecting remission induction overall, an increasing proportion of cells in S + G2M phase was favorable in patients under the age of 50 yr, but was associated with a progressive decline in remission rate in older patients (P = 0.01). This age-related divergent effect of cell cycle kinetics on initial response to therapy was confined to the less favorable subgroup of patients with karyotypic abnormalities, whereas patients with normal diploid cytogenetics had a consistently higher response rate regardless of proliferative activity. A positive correlation was also observed between percent of S + G2M cells and the proportion of diploid metaphases in young patients, contrasting with a negative correlation in the older age group. Our observations strongly suggest that the well-recognized prognostic effect of age on remission induction is not entirely host-mediated, but is at least partly an expression of disease-intrinsic differences between young and older patients.

Human adherent cell contact-mediated modulation of normal myeloid colony formation.

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.

The effects of acetaldophosphamide, a novel stable aldophosphamide analogue, on normal human and leukemic progenitor cells in vitro: implications for use in bone marrow purging.

Acetaldophosphamide (A-ALD), a novel in vitro active and stable derivative of aldophosphamide, kills human bone marrow-derived granulocyte-macrophage colony-forming cells (GM-CFC) independent of the cell cycle. The surviving fraction of GM-CFC is an exponential function of the drug concentration and time of exposure. Variation of marrow light-density cell concentration between 2 x 10(6) and 10 x 10(6)/ml does not significantly influence its GM-CFC toxicity. Marrow depleted of GM-CFC by A-ALD subsequently generates GM-CFC when grown in suspension cultures. During the early period after treatment with A-ALD the number of surviving GM-CFC (size of surviving GM-CFC compartment) does influence the speed of the GM-CFC repopulation in suspension cultures. The importance of the number of surviving GM-CFCs for the growth and maintenance of GM-CFC population in such suspension cultures diminishes with time. No significant differences are observed after 2 wk, indicating that the ancestor stem cell population and its regenerative potential responsible for in vitro hematopoiesis have not been significantly affected by the drug treatment. A-ALD-treated progenitor cells retain their ability to integrate with the previously established marrow stromal cell layer and generate GM-CFC within this layer to an extent comparable to that of untreated marrow cells. The effect of A-ALD on human hematopoiesis is comparable to that of 4-hydroperoxycyclophosphamide. Its advantage over 4-hydroperoxycyclophosphamide is a greater stability in vitro. It has sparing effect on GM-CFC ancestor cells. Its toxicity to myeloid leukemia cell line (KBM-3)-derived clonogeneic cells is higher than to the GM-CFC. It is similar in doxorubicin-sensitive (KBM-3) and -resistant (KBM-3/DOX) leukemic cells. Thus, A-ALD appears to be a promising drug for in vitro purging of bone marrow cells.

Drug sensitivity and cross-resistance of the 4'-(9-acridinylamino)methanesulfon-m-anisidide-resistant subline of HL-60 human leukemia.

A subline of the HL-60 leukemia resistant to 4'-(9-acridinylamino)methanesulfon-m-anisidide (HL-60/AMSA) was developed by intermittent long-term in vitro treatment. Resistance to 4'-(9-acridinylamino)methanesulfon-m-anisidide remained unchanged after 180 doublings in the absence of the drug, suggesting a stable phenotypic alteration. The pattern of cross-resistance of HL-60/AMSA was evaluated for a spectrum of antileukemic agents using the clonogenic assay. Modest cross-resistance to doxorubicin (Adriamycin) was observed in the resistant subline on continuous exposure to the drug for 8 to 9 days; however, HL-60/AMSA cells retained their sensitivity to doxorubicin following short-term exposure for 60 min. HL-60/AMSA was also sensitive to the anthracycline aclacinomycin, Vinca alkaloids, and alkylating agents. Furthermore, enhanced sensitivity to 1-beta-D-arabinofuranosylcytosine was observed. The subline was cross-resistant to etoposide.

Association of circulating immune complexes with cytogenetic abnormalities but not with prognosis in acute nonlymphocytic leukemia.

Sera from 91 adult acute nonlymphocytic leukemia patients were tested for circulating immune complexes by the C1q binding test. In 42 patients tested prior to the initiation of therapy, C1q binding activity was inversely correlated with leukemic infiltrate of the bone marrow (rs = -0.34, p less than 0.02), but it was not related to peripheral white blood cell count or presence of infection. Patients with abnormal C1q binding activities (greater than 6%) were more likely to have cytogenetic abnormalities in their bone marrow (p = 0.02); the most frequent abnormality was an extra No. 8 chromosome. The median survival of 8 patients with abnormal values was 1.5 months as compared to 7.8 months in 34 patients with normal values. However, the C1q binding test did not identify patients likely to achieve remission; and after remission was achieved, it did not identify those likely to relapse. Differences between these findings and those recently published by other investigators are discussed.

In vivo sensitivity and resistance of chronic myelogenous leukemia cells to alpha-interferon: correlation with receptor binding and induction of 2',5'-oligoadenylate synthetase.

Fourteen patients with chronic myelogenous leukemia were treated with partially pure leukocyte interferon (HuIFN alpha). The binding of recombinant leukocyte clone A IFN and the induction of 2',5'-oligoadenylate synthetase (2,5A) in peripheral blood cells were studied to determine whether they correlate with clinical response to IFN therapy. The mean pretherapy binding of radiolabeled recombinant leukocyte clone A IFN to peripheral blood cells was 0.053 +/- 0.02 (SE) fmol (53 +/- 20 amol)/10(6) cells and 0.049 +/- 0.015 fmol/10(6) cells in sensitive and resistant patients, respectively. Twenty-four h after the first HuIFN alpha dose, the binding of recombinant leukocyte clone A IFN decreased 3- to 8-fold in both sensitive and resistant patients. The activity of 2,5A synthetase was induced approximately 100-fold in sensitive patients from a pretherapy mean of 3 +/- 2 nmol/mg to a maximum of 317 +/- 184 nmol/mg during therapy. In contrast, 2,5A synthetase was induced from a pretherapy mean of 0.9 +/- 0.9 nmol/mg to only 6.7 +/- 4.9 nmol/mg in resistant patients. In two patients originally sensitive to HuIFN alpha who developed resistance to therapy, receptors were present in both sensitive and resistant disease stages and appeared to down regulate with therapy regardless of response. In these two patients, 2,5A synthetase was significantly induced with therapy in the sensitive stage but not in the resistant stage. This study shows that lack of clinical response to interferon therapy may coincide with failure to induce 2,5A synthetase activity. This suggests that resistance to alpha-interferon therapy may be mediated by events beyond receptor binding resulting in a failure to induce enzymes responsible for mediation of interferon antiproliferative effects.

Reduction of ifosfamide toxicity using dose fractionation.

Ifosfamide was given in i.v. doses of 600 to 1200 mg/sq m/day for 5 days to 32 cancer patients, refractory to prior therapy, in an attempt to investigate the possibility of reducing toxicity by dose fractionation. Microscopic hematuria occurred in 14% and gross hematuria in only 10% of the patient trials. Azotemia did not occur in any patient on this study. Reversible myelosuppression was comparable to that found by other investigators. Other side effects such as nausea and mental confusion occurred infrequently. Ifosfamide produced antitumor effect in 7 of 27 evaluable patients. This study indicates that the renal and bladder toxicity of ifosfamide can be substantially reduced if the drug is administered in i.v. infusions of 1 to 2 hr daily for 5 days.

Penetration of 3-deazauridine into human brain, intracerebral tumor, and cerebrospinal fluid.

The antitumor agent 3-deazauridine (DAU) was administered rapidly to four patients before surgical removal of intracerebral tumor. Tumor, adjacent brain tissue, and temporalis muscle were assayed for DAU by high-pressure liquid chromatography. DAU penetrated comparably into tumor, brain, and muscle; in one patient, tissue concentrations were higher than concurrent plasma concentrations. The active metabolite 3-deazauridine 5'-triphosphate was quantitated in one tumor sample and greatly exceeded its Ki for cytidine 5'-triphosphate synthetase. DAU was also present in autopsy brain specimens from two patients treated shortly antemortem. Cerebrospinal fluid concentrations were 22.1 and 59.0%, respectively, of concurrent plasma concentrations during continuous infusion of DAU in two patients. Cerebrospinal fluid concentration was 3.1 microgram/ml 2 hr after a 30-min infusion of 1.5 g of drug per sq m and fell to 1.9 microgram/ml at 16 hr. Thus, DAU is capable of penetrating into intracerebral tumor, brain, and cerebrospinal fluid and is worthy of investigation in the treatment of intracerebral and meningeal neoplasms.

The characteristics and outcome of patients with late relapse acute myelogenous leukemia.

The characteristics and outcome of 58 patients with acute myelogenous leukemia (AML) who experienced relapse after a first remission duration longer than 18 months (late-relapse AML) were analyzed and compared with those of 278 patients with earlier relapses. Late-relapse AML was associated with a lower incidence of antecedent hematologic disorder, leukocytosis, and elevated creatinine and lactic acid dehydrogenase (LDH) levels. A favorable karyotype (inversion of chromosome 16; translocations between chromosomes 8 and 21, or 15 and 17) was more frequent in patients whose first remission was 12 months or longer compared with less than 12 months (30% v 10%; P less than .0001). An unfavorable karyotype (chromosome 5 and 7 abnormalities, trisomy 8, other changes) was more frequent in the latter category (16% v 42%; P less than .0001). Thirty-seven of the 58 patients (64%) with late-relapse AML achieved complete remission (CR). The incidence of CR increased significantly with an increased first remission duration from less than 12, 12 to 18, and greater than 18 months (17% v 41% v 64%; P less than .0001), while the incidence of resistant disease was significantly lower (59% v 36% v 19%; P less than .0001). When effective antileukemic regimens were considered, remission rates were also significantly increased by the duration of first remission (24% v 48% v 72%; P less than .001). Compared with patients with earlier relapse, those with late-relapse AML had a longer median survival from salvage therapy (3.5 v 12 months; P less than .01), and longer median second remission durations (3.5 v 11 months; P less than .01). We conclude that late-relapse AML has unique clinical, cytogenetic, and prognostic characteristics, and remains extremely sensitive to chemotherapy with a potential cure fraction. The duration of first remission is an important prognostic parameter in AML relapse and may be useful in the design and analysis of future salvage programs.

Association of granulocytosis with poor prognosis in patients with acute myelogenous leukemia and translocation of chromosomes 8 and 21.

Forty-three patients with acute myelogenous leukemia (AML) and a translocation 8;21 were reviewed. The patients' median age was 30 years, and 62% were men. Twenty-three patients (53%) had loss of a sex chromosome and ten (23%) had other chromosomal abnormalities in addition to the 8;21 translocation. Complete remission (CR) with induction chemotherapy was achieved in 40 patients (93%). The median CR duration was 18 months (range, 1 to 137+ months). Median survival time was 17 months (range, 0.5 to 138+ months) with a 3-year survival rate of 31%. Twenty-three patients (53%) relapsed between 1 and 58 months after entering remission with reinduction therapy resulting in 8 CRs (35%). Thirteen characteristics were examined for an effect on survival. The most striking finding was a significant association between elevated absolute granulocyte count and poor survival (P = .002). WBC count greater than 10,000/microL was also associated with shorter survival (P = .05). Patient age, albumin level, and platelet count showed trends for survival association. Although patients with AML and t(8;21) are regarded as a favorable group with respect to survival, we found a subset of patients who do very poorly. Intensive or more investigational approaches in first remission should be considered for these patients.

A strategy for evaluation of new treatments in untreated patients: application to a clinical trial of AMSA for acute leukemia.

This clinical trial (DT7995) was designed to evaluate amsacrine (AMSA) plus cytosine arabinoside (ara-C), vincristine, and prednisone (OAP) therapy in previously untreated patients with adult acute leukemia and to investigate a new strategy for assignment of patients to treatment using estimated probabilities of complete remission (PPR) based on six prognostic factors. In the first stage of the trial, patients with unfavorable prognosis (PPR less than .40) received AMSA-OAP for remission induction and patients with favorable prognosis (PPR greater than or equal to .40) received Adriamycin [Adria Laboratories, Columbus, OH] plus OAP (Ad-OAP). As AMSA-OAP was found to be promising in patients with unfavorable prognosis, it was administered to relatively more favorable patients (PPR less than .60) in the second stage of the trial and to all patients in the third stage. There were 242 patients entered into study; 134 received AMSA-OAP and 108 received Ad-OAP. Outcomes were compared with 242 paired patients who received Ad-OAP therapy from 1973 to 1977. The estimated complete remission rate in previously untreated adults with acute leukemia is 61% for patients receiving Ad-OAP (95% confidence interval, 59% to 64%). Overall, the survival experience for the 242 patients on DT7995 was significantly better than that in the control series (P = .03), but there was no strong statistical evidence (P = .10) that the 134 patients receiving AMSA-OAP had better survival than control patients receiving Ad-OAP, with a median of 32 v 21 weeks, respectively. It is concluded that AMSA-OAP is equivalent to Ad-OAP in the induction of complete remissions (estimated complete remission rate, 61%) and that assignment of patients to treatment based on predicted prognosis is an ethical and efficient strategy for the evaluation of new therapies in previously untreated patients with acute leukemia.

Piperazinedione, total body irradiation, and autologous bone marrow transplantation in chronic myelogenous leukemia.

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.

Therapy-related leukemia and myelodysplastic syndrome: clinical, cytogenetic, and prognostic features.

One hundred twelve patients who developed acute leukemia or a myelodysplastic syndrome (MDS) after chemotherapy or irradiation for another malignancy were reviewed. The median time from initial therapy to development of secondary leukemia or MDS was 71 months (range, 7 to 331 months). The initial malignancy was hematologic in 43%. An MDS presentation occurred in 57 patients (51%), 55% of whom subsequently transformed to acute leukemia. Chromosomal abnormalities were documented in marrow specimens from 70 of 89 patients with analyzable metaphases (79%; 69% of the total group). Compared with 34 patients with metachronous secondary leukemia without prior chemotherapy or irradiation, those with therapy-related leukemia exhibited a significantly higher frequency of abnormalities of chromosomes 5 and/or 7 (43% v 18%), and lower incidence of diploid karyotypes (18% v 50%). Chromosome 5 and/or 7 abnormalities were also significantly higher in patients previously treated with alkylating agents, procarbazine, and nitrosoureas (72% to 83%), compared with those who had received cyclophosphamide-based regimens (29%), other chemotherapies (14%), or irradiation alone (29%). The median overall survival from diagnosis of the secondary leukemia or MDS was 30 weeks. Survival was significantly shorter for patients with acute leukemia compared with MDS presentation (21 v 45 weeks); in the latter category, it was similar whether evolution to acute leukemia occurred or not. Of 72 patients treated with antileukemia therapy, 29% achieved complete remission (CR). A multivariate analysis of prognostic factors demonstrated the cytogenetic pattern to be the most important characteristic determining remission rate and survival. Other important prognostic features were the morphologic presentation (MDS v acute leukemia) for probability of achieving remission, and patient age and marrow blasts percentage for survival.

High-dose cytosine arabinoside: treatment and cellular pharmacology of chronic myelogenous leukemia blast crisis.

Twenty-one patients with chronic myelogenous leukemia (CML) in blastic transformation underwent 22 remission induction attempts with high-dose cytosine arabinoside (ara-C), administered as a two-hour infusion of 3 g/m2 for six to 12 doses. Ara-C doses were administered every 12 hours in 15 patients and every six to ten hours in six patients. Median patient age was 35 years (range, 20 to 62). The median duration of benign phase was 25 months (range, 0 to 167). Morphology of blast crisis blast cells was myeloid in 15 patients and lymphoid in six. Five patients achieved complete remission (CR), three had partial remission (PR), and one had hematologic improvement, for an overall response rate of 41%. Median remission duration was 2.5 months (range, 0.5 to 6 months). Survival duration was 6 months for responding patients and 1.5 months for those with resistant disease. The response rate was similar for patients with myeloid and lymphoid blast crisis (31% v 50%, respectively). The response rate was significantly higher for patients whose benign phase was less than 1 year (75% v 21%, P = .05) and who had prolonged marrow aplasia after ara-C (86% v 27%, P = .05). Myelosuppression was the major dose-limiting toxicity, and cerebellar toxicity occurred in two patients. Intracellular ara-C 5'-triphosphate (ara-CTP) levels were similar in blood and bone marrow leukemic cells and were slightly greater in the cells of responding patients compared to those with resistant disease. We conclude that high-dose ara-C is an effective regimen for CML blast crisis, resulting in a substantial response rate but modest remission duration. Its combination with other agents may further improve the prognosis of patients with this resistant disease.

Response to salvage therapy and survival after relapse in acute myelogenous leukemia.

The response to and survival following first salvage therapy regimens for 243 patients with acute myelogenous leukemia (AML) treated between 1974 and 1985 were evaluated. Eighty (33%) patients obtained a complete remission (CR), 24% died prior to achieving a response, and 43% were resistant on their first salvage regimen. The median survival was 18 weeks. Five percent overall and 16% of the CR patients are predicted to survive for more than 5 years. The factor most strongly associated with response and survival was the duration of the initial remission with 49 of 82 (60%) patients whose initial CR duration was at least 1 year in duration obtaining a second CR v 31 of 161 (19%) for patients with a shorter remission (P less than .01). Age, liver function, serum lactic dehydrogenase (LDH), karyotype, and the proportion of blasts plus promyelocytes present at the time of starting salvage therapy were strongly associated with probability of response and survival. Multivariate analysis was used to develop logistic regression and proportional hazard models to predict probability of response and survival, respectively. The major regimens used were conventional-dose cytarabine (ara-C) (combined with anthracyclines or amsacrine), high-dose ara-C, rubidazone, amsacrine (AMSA), other anthracyclines, and autologous or allogeneic transplant programs. After allowing for the prognostic factors in the models, specific treatment regimens were not strongly associated with prognosis.

Prediction of complete remission in patients with refractory acute leukemia treated with AMSA.

The relation between characteristics known at start of therapy and response in 102 adults with refractory acute leukemia who received 4'-(9 acridinylamino)-methane-sulfon-m-anisidide (AMSA) was examined. Twenty-three (23%) of these patients attained complete remission (CR). Univariate analysis showed that the following characteristics were associated with CR: a fewer number of prior induction and maintenance regimens, a shorter time between latest relapse and AMSA therapy, the presence of Auer rods, a circulating blast cell count of less than 25,000/mm3, a marrow cellularity less than 90%, a ratio of marrow blasts and promyelocytes to more mature myeloid cells (differentiation ratio) less than 15, and a first-course AMSA dose of greater than or equal to 375 mg/m2. Some of these factors were interrelated. Multivariate analysis using logistic regression techniques was carried out to determine which of the above factors added independent prognostic information. This analysis produced a statistical model that related probability of response to the following in order of selection: Auer rod status, first-course dose, differentiation ratio, and absolute circulating blast cell count. Such a model could be useful in identifying patients with high, intermediate, or low probability of response to AMSA. AMSA could then be prescribed only for patients likely to respond while affording other patients alternate salvage programs at an earlier time.

Fludarabine: a new agent with marked cytoreductive activity in untreated chronic lymphocytic leukemia.

Thirty-three patients with chronic lymphocytic leukemia (CLL) with advanced Rai stage (III-IV) or progressive Rai stage (0-II) disease were treated with fludarabine as a single agent. Eleven patients (33%) obtained a complete remission (CR), 13 (39%) a clinical CR with residual nodules as the only evidence of disease (nodular partial remission [PR]), and two patients (6%) achieved a PR for a total response rate of 79%. Response was rapid, usually occurring after three to six courses of treatment. The major morbidity was infection. Febrile episodes occurred in 13% of the courses (pneumonia 6%, minor infection 4%, and transient fever of undocumented cause 3%). Fludarabine appears to be the most cytoreductive single agent so far studied in CLL.