Bayard Clarkson

Treatment of adult acute leukemia with arabinosylcytosine and thioguanine

Forty unselected adults with acute leukemia were treated with CA (2–3 mg/kg) by daily rapid intravenous injection and TG (2.5 mg/kg) orally. Two patients were judged to have had an inadequate trial, having died after 3 and 4 days. Of 38 adequately treated patients, 2 had acute lymphoblastic and 36 acute myeloblastic, monoblastic, or myelomonoblastic leukemia. Twenty‐eight patients had had previous chemotherapy (19 with either CA or 6‐MP alone) but had failed to respond or had relapsed and were refractory. The 38 adequately treated patients had 1 or more courses of CA and TG of 8–32 days each as limited by individual tolerance. The drugs caused moderate or severe pancytopenia and marrow hypoplasia in all but 3 patients treated. Twenty‐one of 38 patients had remission with M‐1 marrows lasting an average 6 (1.5–13) months with 6 patients still in remission. Seventeen of 21 patients had H‐1 peripheral blood ratings (normal platelet, leukocyte and hemoglobin values) and 4 had H‐2 ratings, all because of low hemoglobin. Twenty patients in remission were given maintenance therapy; the dosage varied but is currently TG 2.5 mg/kg 4–5x/wk + CA 2–3 mg/kg 1‐2x/wk.

The physiologic disposition of 5‐fluorouracil and 5‐fluoro‐2′‐deoxyuridine in man

The plasma concentration and the urinary excretion of 5‐fluorouracil (FU) and 5‐fluoro‐2′deoxyuridine (FUdR), administered to patients with cancer, were determined by microbiologic assay; following regional administration, both local and systemic concentrations were measured. Rapid intravenous injections of usual clinical doses resulted in relatively high initial plasma concentrations (100 to 750 mμM per milliliter) which fell below a detectable level by 3½ hours in all patients. Generally, less than one fifth of the dose was excreted in the urine and almost all of this occurred in the first hour. The renal clearance of both drugs exceeded the glomerular filtration rate, and the clearance of FUdR was greater than FU. Gastrointestinal absorption of FU was unpredictable; FUdR was very poorly absorbed. Both drugs were degraded more completely when given by continuous infUSion, as indicated by reduced urinary excretion. After intravenous injection, the drug concentration in the cerebrospinal fluid and in neoplastic effusions was only one‐fiftieth or less that of the plasma concentration. After direct injection into neoplastic effusions, the concentration in the fluid was over 100 times that reached in the effusion after intravenous injection of the same dose. Long‐term infusion of low doses of FU into small arteries resulted in 100 or more times greater local than systemic plasma concentrations, while regional perfusion of FUdR resulted (briefly) in up to 1,000 or more times the concentration in the perfusion circuit compared to the general circulation.

Studies of cellular proliferation in human leukemia.III. Behavior of leukemic cells in three adults with acute leukemia given continuous infusions of3H-thymidine for 8 or 10 days

Three adults with acute leukemia were given continuous infusions of 3H‐thymidine for 8 or 10 days. At the end of the infusions 88 to 93% of the leukemic cells were labeled. The leukemic cells varied in size and the flash 3H‐thymidine labeling indexes were generally progressively higher in direct relation to increasing nuclear size. Almost none of the smallest leukemic cells were labeled initially, but, at the end of the infusions, most were labeled regardless of size. The few remaining unlabeled were usually small, and it is concluded that these had remained dormant for die duration of the infusions. The mean generation time of the labeled fraction of leukemic cells in one patient was about 90 hours prior to treatment and about 200 hours during prednisone therapy. In the other 2 patients, antimetabolite therapy was started immediately after the infusions and the generation times were about 100 and 200 hours. There was considerable variability in generation time among the proliferating cells in each leukemic population. The minimum could not be determined because of continual interchange between cells of different sizes as a result of cell growth prior to division and halving of volume thereafter. The data is consistent with the hypothesis that the leukemic cells behave as a self‐maintaining population; we found no evidence for influx of leukemic “stem cells” from an unrecognized precursor compartment. In one patient, destruction of many of the leukemic cells by chemotherapy resulted in an increase in the 3H‐thymidine labeling index and the average size of the remaining cells. Similar findings have been described for several experimental tumors in vivo and for various cell types in vitro; i.e., their‐rate of proliferation varies according to their population density. Acute leukemic cells apparently grow less rapidly when their population density exceeds a critical level, but the finer alterations in kinetics which occur and the mechanism of growth inhibition are as yet unknown.

Acute myelocytic leukemia in adults

The proposed reasons why acute myelocytic leukemia (AML) is less responsive to chemotherapy than lymphoblastic leukemia are examined. It is concluded that neither difference in age incidence nor difference in pathogenesis provides adequate explanations and that the major reasons why chemotherapy is less successful in AML are because: 1. None of the available cytotoxic drugs is sufficiently selective in killing leukemic myeloblasts and thus remission induction is very hazardous, and 2. There are relatively few drugs available which are dependably lethal to leukemic myeloblasts, thereby hindering effective remission consolidation. Despite these obstacles, several regimens employing combinations of drugs have recently been reported which give remission rates of 50% or better and significant prolongation of survival. The current status of one of these regimens—the “L‐6” protocol —is reviewed and discussed in relation to anticipated future developments.

Continuous culture of seven new cell lines (SK-L1 to 7) from patients with acute leukemia

Continuous suspension cultures of seven lines of cells derived from the blood of seven patients with acute leukemia who had large numbers of circulating leukemic cells are described. Three cultures have been growing for more than 18 months and the other four for more than six months. Two of the cultures were obtained from children with lymphoblastic leukemia and five from patients with myeloblastic or myelomonocytic leukemia. In one of the myelomonocytic cultures, after several weeks in vitro, some of the blasts originally present underwent partial differentiation into pseudoeosinophils; these later died out, to be replaced by continuously‐growing blasts. In all of the established cultures, the cells resembled primitive blast forms morphologically, were highly motile, had diploid or near‐diploid modal numbers of chromosomes, and had doubling times of between 20 and 70 hours.

Chronic myelogenous leukemia as a paradigm of early cancer and possible curative strategies.

The chronological history of the important discoveries leading to our present understanding of the essential clinical, biological, biochemical, and molecular features of chronic myelogenous leukemia (CML) are first reviewed, focusing in particular on abnormalities that are responsible for the massive myeloid expansion. CML is an excellent target for the development of selective treatment because of its highly consistent genetic abnormality and qualitatively different fusion gene product, p210bcr-abl. It is likely that the multiple signaling pathways dysregulated by p210bcr-abl are sufficient to explain all the initial manifestations of the chronic phase of the disease, although understanding of the circuitry is still very incomplete. Evidence is presented that the signaling pathways that are constitutively activated in CML stem cells and primitive progenitors cooperate with cytokines to increase the proportion of stem cells that are activated and thereby increase recruitment into the committed progenitor cell pool, and that this increased activation is probably the primary cause of the massive myeloid expansion in CML. The cooperative interactions between Bcr-Abl and cytokine-activated pathways interfere with the synergistic interactions between multiple cytokines that are normally required for the activation of stem cells, while at the same time causing numerous subtle biochemical and functional abnormalities in the later progenitors and precursor cells. The committed CML progenitors have discordant maturation and reduced proliferative capacity compared to normal committed progenitors, and like them, are destined to die after a limited number of divisions. Thus, the primary goal of any curative strategy must be to eliminate all Philadelphia positive (Ph+) primitive cells that are capable of symmetric division and thereby able to expand the Ph+ stem cell pool and recreate the disease. Several highly potent and moderately selective inhibitors of Bcr-Abl kinase have recently been discovered that are capable of killing the majority of actively proliferating early CML progenitors with minimal effects on normal progenitors. However, like their normal counterparts, most of the CML primitive stem cells are quiescent at any given time and are relatively invulnerable to the Bcr-Abl kinase inhibitors as well as other drugs. We propose that survival of dormant Ph+ stem cells may be the most important reason for the inability to cure the disease during initial treatment, while resistance to the inhibitors and other drugs becomes increasingly important later. An outline of a possible curative strategy is presented that attempts to take advantage of the subtle differences in the proliferative behavior of normal and Ph+ stem cells and the newly discovered selective inhibitors of Bcr-Abl.

Studies of cellular proliferation in human leukemia. VI. The proliferative activity, generation time, and emergence time of neutrophilic granulocytes in chronic granulocytic leukemia

The proliferative kinetics of neutrophilic granulocytes were studied in 3 patients with typical chronic granulocytic leukemia (CGL) by means of differential counts, mitotic indexes, and 3H‐thymidine labeling using autoradio‐graphic methods. The granulocyte precursors in CGL had lower mitotic and 3H‐thymidine labeling indexes than the corresponding normal cells; their median grain count halving times were longer than normal in 2 patients and approximately normal in the third. The proliferative rates appeared similar in the marrow and spleen but the cells rarely divided in the blood. In one patient given a continuous intravenous 3H‐thymidine infusion for 10 days, 100% of the granulocyte precursors were labeled. The minimum maturation time of CGL neutrophilics was approximately normal, but their average transit times through the myelocyte and later maturation compartments was prolonged. The overproduction of granulocytes appears mainly due to increased formation of myeloblasts; whether this is due to an increased rate of stem cell activation or whether the myeloblasts may themselves function to some extent as selfsustaining cells is unknown.

Survival in chronic myelogenous leukemia: influence of treatment and extent of disease at diagnosis.

A retrospective study was conducted on the survival of 178 patients with chronic myelogenous leukemia from January 1948 through December 1967. Through analysis of survival time, comparison has been made between a group of 50 patients treated with chemotherapy alone vs. a group of 84 patients treated with radiation therapy to the spleen and chemotherapy. No difference could be found in these two major therapeutic groups (median survival 31 months for both). Further analysis has been made to determine whether survival could be correlated to the initial extent of disease. Initial hemoglobin value, WBC, platelet counts, absolute blast count, spleen size, and selected combination of these factors showed no significant correlation with survival. Acute blastic transformation occurred in 60% of 117 patients. Infection as a cause of death was less frequent in patients with progressive disease without acute blastic transformation, while renal failure due to hyperuricemia and thromboembolic phenomena occurred more often in this group as compared with patients dying in acute blastic phase.

Studies of cellular proliferation in human leukemia. IV. Behavior of normal hematopoietic cells in 3 adults with acute leukemia given continuous infusions of3 H-thymidine for 8 or 10 days

Proliferative behavior of residual normal hematopoietic cells was studied in 3 patients with acute leukemia given continuous infusions of 3H‐thymidine for 8 or 10 days. The erythrocyte precursors were reduced in number, but were dividing rapidly at about the same rate as reported in normal subjects (i.e., generation time about 1 day or less); this is significantly faster than the mean generation time of the labeled leukemic cells. The proliferative rate of the red cell precursors in 1 patient was not appreciably affected by thioguanine, although the drug caused a complete remission of the disease. The red cell precursors showed no significant impairment of maturation, but the data suggests that they may have undergone more divisions during passage through the several maturation compartments than occurs in the normal state. The main reason for deficient red cell production in acute leukemia appears to be that the leukemic cells somehow interfere with the activation of stem cells and, therefore, an insufficient number of pronormoblasts are formed; the mechanism of inhibition is unknown. The proliferative behavior of the earliest granulocyte precursors could not be determined and that of the myelocytes varied, being fastest in a patient who had an infection. Prednisone and thioguanine had little effect, but 5‐fluoro‐2′‐deoxyuridine slowed the proliferation of the granulocyte precursors more than that of the leukemic cells. Almost all granulocytes were labeled at the end of the infusions and the emergence times of labeled metamyelocytes and later forms were similar to those reported from normal subjects, indicating that their maturation process is not defective. None of the lymphocytes were labeled on short‐term in vitro incubation with 3H‐thymidine, but appreciable numbers of both large and small lymphocytes were labeled at the end of the infusions. Their order of appearance in all patients suggests that most of the small lymphocytes originated in the marrow; in the one patient (S.W.), in whom large lymphocytes could be identified in both marrow and blood, the large cells apparently originated mostly in extramedullary sites. The cells identified as large or small lymphocytes in marrow and blood probably did not divide in these compartments; the exact proliferative rate of their precursors could not be determined, but it was appreciably slowed by chemotherapy.

Current status of treatment of acute leukemia in adults: An overview of the memorial experience and review of literature

The results of treatment of 629 previously untreated adults with acute leukemia at Memorial Hospital are reviewed. During the past 14 years, 135 adults (greater than 15 years) with acute lymphoblastic leukemia (ALL) have been treated with one of three successive multidrug-intensive treatment protocols (L2, L10/10M, and L17/17M), each calling for 2.5 to 3 years of systemic chemotherapy and prophylactic intrathecal methotrexate without cranial irradiation. The complete remission (CR) rates were L2 (n = 22) = 77%; L10/10M (n = 69) = 86%; L17/17M (n = 44) = 77%. The median durations of survival and remission were, respectively, L2 = 33 and 30 months; L10/10M = 62 months and not reached; and L17/17M = not reached. Almost all relapses occurred within the first 3 years while still continuing treatment, and there were only rate late relapses after stopping treatment. It appears that approximately half of the patients may have been cured with the latest two protocols. During the last 17 years, 494 adults aged 15 to greater than 70 with acute nonlymphoblastic leukemia (ANLL) were treated with one of five successive multiple drug treatment protocols of varying intensity (arabinosylcytosine + 6-thioguanine [n = 36]; L6 [n = 101]; L12 [n = 104]; L14/14M [n = 121]; and L16/16M [n = 132]). Patients with myelodysplastic syndromes generally were not treated until they developed acute leukemia, but were then entered and included in the results. Secondary leukemias following treatment of other neoplastic diseases were not included. The complete remission rates were fairly constant between 47 and 64% and the median durations of remissions were between 9 and 21 months. The intensive treatment L14 and L16 protocols were associated with more early deaths and did not result in a significantly improved remission incidence or duration or survival. With all protocols, the majority of relapses occurred within the first 2 years, but relapses continued to occur at a decreasing rate for 4 years and occasionally even later. Whereas a small fraction (approximately 10 to 15%) of adults with ANLL are now apparently being cured with combination chemotherapy, despite intensive efforts there has been little improvement during the last decade and more selective and effective forms of treatment are urgently needed.