Cytotoxicity of 4-hydroperoxycyclophosphamide for the blast progenitors of acute myeloblastic leukemia.

The effects of 4-hydroperoxycyclophosphamide (4-HC), an analogue of cyclophosphamide, on the blast progenitors from eight acute myeloblastic leukemia patients were studied in methylcellulose and suspension cultures. Leukemic blast progenitors undergo terminal divisions in methylcellulose culture, making blast colonies. Cells in primary colonies can make secondary colonies after replating in fresh methylcellulose medium. Leukemic blast progenitors grow exponentially in suspension culture for periods of weeks. The ability to form secondary colonies and the exponential growth in suspension culture are considered to reflect the self-renewal of blast progenitors. 4-HC suppressed primary blast colonies in a dose-responsive manner. Secondary colonies were not significantly affected by 4-HC. Although the clonogenic cell recovery was suppressed by 4-HC, leukemic blast progenitors were less sensitive to 4-HC in suspension culture than in methylcellulose culture. The results suggest that 4-HC is effective in suppressing the terminal divisions of blast progenitors but not as effective against the self-renewal of blast progenitors.

Effects on leukemic clonogenic cells in murine myeloid leukemia of 1-beta-D-arabinofuranosylcytosine and the anthracyclines adriamycin, daunomycin, aclacinomycin A, and 4'-epidoxorubicin.

M-3 murine myeloid leukemic cells undergo terminal divisions making colonies in methylcellulose culture and also renew themselves in methylcellulose and suspension; leukemic clonogenic cells are characteristic as stem cells. The effects of 1-beta-D-arabinofuranosylcytosine and four anthracyclines (Adriamycin, daunomycin, aclacinomycin A, and 4'-epidoxorubicin) on M-3 leukemic clonogenic cells were studied. 1-beta-D-Arabinofuranosylcytosine was effective in reducing primary and secondary colonies in methylcellulose and the growth of clonogenic cells in suspension. In contrast, the anthracyclines were not so effective in reducing secondary colonies in methylcellulose or clonogenic cells in suspension as to suppress primary colonies in methylcellulose. The results suggest that 1-beta-D-arabinofuranosylcytosine but not the anthracyclines is effective for not only terminal divisions but also self-renewal of leukemic clonogenic cells. The study will be used as a practical screening test to examine the effects of antitumor agents on leukemic blast progenitors.

Production of growth potentiating factor(s) for autologous blast cells by acute myeloblastic leukemia cells.

The effects of media conditioned by leukemic cells from 11 acute myeloblastic leukemia patients on the growth of autologous blast progenitors were studied. First, it was shown that T-cell-depleted leukemic cells from some patients release high levels of colony-stimulating activity into the culture medium, whereas following further depletion of phagocytic cells, the levels of colony-stimulating activity become undetectable. Second, media conditioned by purified blast cell fraction depleted of both T-cells and phagocytic cells potentiated autologous blast progenitor growth both in methylcellulose and suspension cultures stimulated by optimal concentration of media conditioned by human bladder carcinoma line 5637. Third, media conditioned by these purified blast cells generally did not contain measurable colony-stimulating activity or interleukin 1, whereas substantial levels of granulocyte colony-stimulating factor, granulocyte-macrophage colony-stimulating factor, and interleukin 1 were observed in media conditioned by human bladder carcinoma line 5637 using bioassays and specific immunological assays. Therefore, purified blast cell fraction from acute myeloblastic leukemia patients appears to produce factor(s) other than granulocyte colony-stimulating factor, granulocyte-macrophage colony-stimulating factor, or interleukin 1, which potentiate the growth of autologous blast progenitors both in methylcellulose and suspension cultures.

Expression of interleukin 1 beta receptors on blast cells in acute myeloblastic leukemia: comparison with interleukin 1 beta proliferative activity.

We describe here the presence of a single class of interleukin 1 beta (IL-1 beta) receptors on the surface of blast cells freshly obtained from eight acute myeloblastic leukemia patients and one chronic myelocytic leukemia patient in blast crisis. Blast cells possessed a low number of high-affinity receptors (range, less than 10-173 receptors/cell) with a Kd of 1.8-12.8 x 10(-10) M. At the same time, we have investigated the effects of IL-1 on the growth of leukemic blast progenitors, and a significant heterogeneity of responsiveness was observed. IL-1 beta (1 ng/ml) enhanced blast colony formation in six patients. No significant effect was observed by addition of up to 100 ng/ml of IL-1 beta in the remaining three patients. No significant correlation was observed between the receptor number, receptor affinity, and the cellular responsiveness to IL-1; in some acute myeloblastic leukemia cases with apparent IL-1 receptors, no proliferation response to added IL-1 was observed. Our data show that IL-1 alone can enhance blast colony formation and that lack of responsiveness to IL-1 in some acute myeloblastic leukemia patients is not related to the absence of IL-1 receptors on blast cells.

Fusion of ETV6 to fibroblast growth factor receptor 3 in peripheral T-cell lymphoma with a t(4;12)(p16;p13) chromosomal translocation.

Fusions of the ETV6/TEL gene to receptor or protein tyrosine kinases (TKs), such as PDGFRbeta, JAK2, ABL, ABL2, TRKC, and Syk, have been reported in various hematological malignancies. Expression of the resultant chimeric proteins is believed to lead to constitutive TK activity through activation by the helix-loop-helix (HLH) domain of ETV6. We identified a novel ETV6 partner gene, fibroblast growth factor receptor 3 (FGFR3), in a patient with peripheral T-cell lymphoma (PTCL) with a t(4;12)(p16;p13) translocation. The ETV6-FGFR3 transcript showed a fusion of exon 5 of ETV6 to exon 10 of FGFR3, resulting in an open reading frame for a chimeric protein consisting of the HLH domain of ETV6 and the TK domains of FGFR3. This is the first report of ETV6 and FGFR3 involvement in PTCL.

Effect of recombinant human M-CSF on the proliferation of leukemic blast progenitors in AML patients.

We studied the effects of recombinant human macrophage colony-stimulating factor (M-CSF) on the leukemic blast progenitors from 10 acute myeloblastic leukemia patients. Recombinant human (rh)M-CSF stimulated leukemic blast progenitors in methylcellulose in four patients, but the colonies by rhM-CSF were smaller in size and number than those by rh-granulocyte-CSF or human bladder carcinoma cell line 5637 conditioned medium. rhM-CSF did not increase the number of clonogenic cells in long-term suspension culture. The blast colony formation in methylcellulose and the exponential growth of clonogenic cells in long-term suspension culture are considered to reflect the terminal divisions and the self-renewal of blast progenitors, respectively. The results show that M-CSF stimulates terminal divisions weakly but does not stimulate self-renewal of leukemic blast progenitors. M-CSF did not induce differentiation of blasts either in methylcellulose or in suspension culture.

Suppression of the development of murine myeloid leukemia with granulocyte colony-stimulating factor by inducing apoptosis of leukemic cells.

We studied the antileukemic effect of recombinant human granulocyte colony-stimulating factor (rhG-CSF) by using a radiation-induced murine myeloid leukemia cell line C2M-A5. Intravenous inoculation of C2M-A5 cells into C3H/He mice resulted in the development of myeloid leukemia. However, the leukemic death of the mice was completely suppressed by the subcutaneous injections of rhG-CSF. In order to clarify the mechanism of the suppression, effects of rhG-CSF on C2M-A5 cells were studied in vitro. While C2M-A5 cells grew exponentially in the absence of rhG-CSF, the viability, the growth, and the self-renewal capacity of C2M-A5 cells were all suppressed in cultures in the presence of rhG-CSF. Preincubation with rhG-CSF for 48 h deprived C2M-A5 cells of the ability to induce leukemia in syngeneic mice. Morphological examination revealed the appearance of apoptotic changes of C2M-A5 cells in cultures containing rhG-CSF over the 2-day incubation period. In gel electrophoresis, the DNA from C2M-A5 cells incubated with rhG-CSF for 48 h showed a ladder of degradated DNA bands compatible with apoptosis. From these results, we concluded that the apoptosis of C2M-A5 cells played a key role in the antileukemic effect of rhG-CSF.

Granulocyte-colony stimulating factor induced apoptosis in radiation-induced murine leukemia cell line.

Cytokines regulate proliferation and differentiation of hematopoietic progenitor cells. Recently it has been clarified that physiological cell death, apoptosis plays important role of hematopoiesis. So we evaluated the effects of granulocyte-colony stimulating factor (G-CSF) on leukemic cells, especially focused on apoptosis. Intravenous inoculation of radiation-induced murine leukemia cell line, C2M-A5 into the parent C3H mice resulted in the development of myeloid leukemia. However, the leukemic death of the mice was completely suppressed by the daily subcutaneous injection of recombinant human (rh)G-CSF from the next day (Bessho M. et al., Leuk Res 1989:13:1001-1007). In the in vitro study using C2M-A5 cells, we found that apoptosis appears on the cells at 48 hours after addition of G-CSF in culture. The cells in this stage lost the leukemogenicity to C3H mice (Bessho M. et al., Leukemia 8:1185-1190:1994). To clarify the mechanism of the induction of apoptosis by G-CSF we studied cell cycle and molecular changes in C2M-A5 cells cultured in medium with or without rhG-CSF by means of using the flowcytometry and Northern and Western blot analyses. After addition of rh G-CSF to culture, C2M-A5 cells removed to S phase, next arrested at G0/G1 phase on and after 24 hours, and 48 hours later, apoptosis was observed. Overexpression of mRNAs for c-myc (3-24 hours later) and for p53 (6-24 hours later), were observed in the cell cultured in rhG-CSF administered medium with a concomitant down-expression of bcl-2 mRNA (from 6 hours later). Tyrosine-phosphorylated protein (17 kd) appeared at 48 hours after administration of rhG-CSF to cell culture. This protein was suggested for specific apoptosis induction by rhG-CSF. These results are summarized as follows. (1) rhG-CSF induced apoptosis to C2M-A5 and deprived its leukemogenicity to mice. (2) Induction of apoptosis was associated with cell cycle and correlated to the changes of the expression rates of c-myc,p53, and bcl-2. (3) Tyrosine kinase may play an important role in apoptosis induction to C2M-A5 by rhG-CSF.

Incidence and characteristics of clonal hematopoiesis in remission of acute myeloid leukemia in relation to morphological dysplasia.

We studied 34 patients in remission of acute myeloid leukemia (AML) by performing clonal analysis of peripheral blood polymorphonuclear (PMN) cells and mononuclear (MN) cells, using X-linked DNA polymorphisms, in conjunction with the assessment of morphological myelodysplastic changes, performed by a scoring method. Nine patients demonstrated a non-random or skewed X-chromosome inactivation pattern in PMN cells. Three of these nine patients had an apparently random pattern in MN cells (group A), whereas the remaining six patients demonstrated no difference between the inactivation patterns of PMN and MN cells (group B). The PMN cells of the other 25 patients showed a random X-chromosome inactivation pattern, and the patterns of the PMN cells did not differ from those of the MN cells (group C). The scores for myelodysplasia were high (> or = 4) in all three patients in group A, intermediate (2-3) in two patients and low (score < 2) in four patients in group B, and intermediate in five patients and low in 20 patients in group C. The duration of remission in patients with a myelodysplasia score of > or = 2 was significantly shorter than that of patients with a score of < 2 (P < 0.01). We conclude that clonal remission actually occurs with myelodysplastic features in some patients with AML (around 10%, group A). It is possible that this clonal analysis may not be sensitive enough to detect the preleukemic clone with myelodysplastic features when this clone constitutes only a minor population of remission hematopoiesis. To further elucidate the biology of such preleukemic clones it is essential to develop more sensitive molecular methods for the detection of genetic abnormalities specific to preleukemic hematopoiesis.

A clonal study of hematopoiesis using the M27 beta probe: aberrant band patterns caused by incomplete digestion of a methyl-sensitive enzyme in the the inactive X-chromosome.

The M27 beta probe has been used to determine the clonality of human tumors, based upon X-chromosome inactivation. However, it occasionally gives rise to aberrant results. In this study, the M27 beta probe was used for clonal analysis in Japanese women with clonal stem cell disorders and in those with normal hematopoiesis. Restriction digestion with PstI indicated heterozygosity for the DXS255 locus in 41 out of 50 individuals (82%). Further digestion with HpaII in heterozygous women led to four distinct band patterns: I, both fragments were partially digested; II, either one of the two fragments was completely digested; III, a three-band pattern; and IV, neither fragment was digested. Of 21 hematologically normal females, 17 (81%) and four (19%) had patterns I and III, respectively. In some subjects with pattern I, imbalanced HpaII digestion in the two alleles was seen. Fifteen (65%) of the 23 patients with clonal stem cell disorders had pattern II, while the remainder (35%) had pattern IV. The normal tissues of three acute myeloid leukemia patients with pattern IV all revealed pattern I. It is possible that the aberrant band patterns could be caused by incomplete HpaII digestion in inactive X-chromosomes. In this study, we propose a hypothesis whereby, in normal tissues, aberrant cells, the DXS255 locus of which is not digested with HpaII despite their inactive status, would be mixed with cells demonstrating the usual methylation pattern. In normal tissues, complex of proportion of aberrant cells and skewed Lyonization could produce a variety of band patterns. If a cell with the usual methylation pattern proliferated monoclonally, pattern II would be seen: whereas if an aberrant cell proliferated, pattern IV would be demonstrated.

Refractory anemia with severe dysplasia: clinical significance of morphological features in refractory anemia.

Refractory anemia (RA) in myelodysplastic syndromes (MDS) are very heterogeneous diseases regarding their morphology, clinical features and survival. We proposed the new designations 'RA with severe dysplasia (RASD)' and 'RA with minimal dysplasia (RAminiD)'. In our criteria, RASD is considered present if a bone marrow (BM) examination shows Pseudo-Pelger-Huet anomalies of mature neutrophils > or =3% and/or micromegakaryocytes (mMgk) of megakaryocytes > or =10% in RA patients. RAminiD is defined as RA cases other than RASD. After the reclassification of 58 primary RA patients, the group was composed of 45 RAminiD and 13 RASD patients. The blast percentage in the BM and the frequency of cytogenetic abnormalities observed in the RASD patients were intermediate between those in the RAminiD and RAEB patients. The analysis of survival curves revealed differences among the three groups; the RASD patients had lower survival probabilities than those of the RAminiD group, and significantly higher probabilities than those of the RAEB group. (RAminiD vs RASD, P=0.06; RASD vs RAEB, P=0.004.) Our data indicate that in RA patients, RASD is a distinct subset of RA with an unfavorable clinical outcome.

New system for assessing the prognosis of refractory anemia patients.

Refractory anemia (RA) is a very heterogeneous disease regarding biological and clinical features. The International Prognostic Scoring System (IPSS) was useful for assessing the prognosis in the whole group of 219 myelodysplastic syndrome (MDS) patients. However, the IPSS was not sufficient in 132 RA patients. To predict survival and freedom from acute myeloid leukemia (AML) evolution, we investigated individual prognostic factors based on the clinical parameters (age, gender, morphologic features, cytopenias and cytogenetics) of 132 RA patients using univariate and multivariate analyses. Based on the results, we devised a new system for assessing the prognosis of RA patients. In our system, RA patients with pseudo-Pelger-Huët anomalies >/=3% were classified as high risk (12 patients); of patients without pseudo-Pelger-Huët anomalies >/=3%, those with intermediate/poor karyotype according to IPSS, Hb /=10% were classified as intermediate risk (57 patients); and those without high or intermediate risk were classified as low risk (67 patients). In our system, the analyses of both survival times and leukemia-free survival times revealed significant differences among the three groups (P < 0.0001).

Growth potentiating activity of endogenous production of interleukin-1 and tumor necrosis factor alpha in blast cells of acute myeloblastic leukemia.

For the optimal growth of clonogenic cells in acute myeloblastic leukemia (AML), several cytokines such as tumor necrosis factor alpha (TNF-alpha), interleukin-1 (IL-1) and IL-6 are required in addition to colony-stimulating factor (CSF), which may be produced by blast cells themselves. In the present study, we addressed the potential role of endogenous production of TNF-alpha and/or IL-1 in the in vitro growth of AML clonogenic cells supported by IL-3. Addition of a specific neutralizing antibody against TNF-alpha (anti-TNF-alpha) to the culture significantly reduced the growth-stimulating effect of IL-3 on the cells in 11 of 14 patients. Simultaneous addition of anti-IL-1 alpha and anti-IL-1 beta also partly affected the growth, although to a much lesser extent when compared to the effect observed with anti-TNF-alpha. In 3 patients, the growth-stimulating effect of IL-3 was completely abrogated by anti-TNF-alpha or a combination of all three antibodies. Constitutive TNF-alpha transcript was observed in 5 patients and TNF-alpha protein was present in culture supernatant. Following in vitro culture, a transient but profound increase in c-fos, c-jun, TNF-alpha and IL-1 beta mRNA levels was observed. Anti-TNF-alpha inhibited the accumulation of TNF-alpha transcript, suggesting that membrane-integrated TNF-alpha may be partly responsible for the induction of TNF-alpha mRNA. It seems likely that the accumulation of these genes occurs through a protein kinase C-independent signaling pathway.

Mechanism of action of interleukin 1 on the progenitors of blast cells in acute myeloblastic leukemia.

We tested the effect of interleukin 1 (IL-1) on the growth of leukemic blast progenitors from patients with acute myeloblastic leukemia (AML). A purified blast cell fraction depleted of both T cells and phagocytic cells was tested at different cell densities. Addition of 1 ng/ml of IL-1 alpha alone enhanced blast colony formation in 10 of 13 cases tested, and the enhancement was prominent when plated cell densities were lowered. The conditioned media (CM) from AML patients contained varied levels of IL-1 activity, and following depletion of phagocytic cells, the levels decreased markedly in all cases tested. Addition of either antiserum against IL-1 alpha or IL-1 beta reduced the IL-1 activity in CM, suggesting that AML blasts produce both IL-1 alpha and IL-1 beta. Addition of IL-1 alpha or IL-1 beta antiserum inhibited blast colony formation in a dose-dependent manner, and a combination of both antisera showed the most marked inhibition. However, the augmentation of blast colony formation was almost completely inhibited by addition of anti-granulocyte-macrophage colony-stimulating factor (GM-CSF) serum in all three cases tested. IL-1 is also devoid of this activity when tested in the presence of a combination of granulocyte CSF (G-CSF), GM-CSF, and interleukin 3 (IL-3) at an optimal concentration. These results suggest that blast cells could produce and secrete CSF(s) and/or IL-1, and that the growth-enhancing effect of IL-1 on AML blasts is indirect, via production of CSFs by leukemic cells.

Differential regulation by hematopoietic growth factors of apoptosis and mitosis in acute myeloblastic leukemia cells.

We evaluated the effects of various hematopoietic growth factors (HGFs) on the prevention of apoptosis in blasts from 19 patients with acute myeloblastic leukemia (AML) by assessing DNA ladder formation. After incubation without HGF, apoptosis was noted in all but two patients. HGFs prevented, did not affect, or enhanced apoptosis in 39 (60%), 14 (22%), or 12 (18%) of 65 suspension cultures, respectively. HGFs that prevented apoptosis also stimulated and/or synergized blast colony formation in 35 of 39 corresponding methylcellulose cultures. HGFs that alone stimulated colony formation also prevented apoptosis in all but two of 28 corresponding suspension cultures. In contrast, HGFs that did not prevent apoptosis also failed to stimulate growth in 17 of 26 corresponding methylcellulose cultures. HGFs that enhanced apoptosis alone never stimulated colony formation. After incubation, we noted enhanced c-fos and cjun genes as well as induction of p21 protein. An appropriate dose of HGF elevated c-fos, reduced c-jun and p21, induced G1/S transition, and inhibited apoptosis. In two patients, apoptosis was not induced after incubation. Cells not treated with HGF expressed no c-fos, c-jun, or c-myc, and remained in G0/G1. Taken together, our results support the conclusion that not only c-fos, cjun, and c-myc, but also p53 and p21 are required for blast apoptosis. HGF differentially prevents apoptosis and induces mitosis, and both events seem to be integral to the self-renewal of AML clonogenic cells.

Differential effects of TGF-beta 1 on normal and leukemic human hematopoietic cell proliferation.

We evaluated the effects of transforming growth factor-beta 1 (TGF-beta 1) on the growth of hematopoietic progenitors in normal donors and in patients with hematologic malignancies now designed as clonal disorders of multipotential stem cells. TGF-beta 1 at 80 pM exhibited differential effects on the normal hematopoietic progenitors when cells were stimulated with different growth factors, such as G-CSF, GM-CSF, interleukin-3 (IL-3), or stem cell factor (SCF). The suppressive effect by TGF-beta 1 was increased for growth with GM-CSF, IL-3, and SCF, and growth with G-CSF was unaffected in hematologic malignancies, TGF-beta 1 suppression for growth with G-CSF was increased for essential thrombocythemia (ET) and polycythemia vera; chronic myelogenous leukemia (CML) in chronic phase; CML in accelerated phase; CML in myeloid crisis; myelodysplastic syndrome (MDS) in refractory anemia; MDS in refractory anemia with an excess of blasts; and acute myeloblastic leukemia (AML). In CML-myeloid crisis and AML, TGF-beta 1 almost completely abolished the growth, with some patient-to-patient variation. The mean ED50s for the growth of leukemic blast progenitors were 1.6, 1.2, 0.7, and 0.2 pM in the presence of G-CSF, GM-CSF, IL-3, and SCF, respectively, c-myc and c-myb antisense oligonucleotides significantly suppressed the growth of leukemic blast progenitors, but not that of clonogenic cells from normal donors and patients with ET. We also demonstrated that TGF-beta 1 inhibits mRNA expression by AML blasts for c-myc and/or c-myb. When the data are taken together, growth suppression by TGF-beta 1 appears to increase with the progression of clonal evolution in hematologic malignancies.

The in vitro growth patterns and drug sensitivities of leukemic blast progenitors among the subtypes of acute myelocytic leukemia.

The in vitro growth activities and drug sensitivities of leukemic blast progenitors were compared among the subgroups of acute myelocytic leukemia (AML) classified according to the French-American-British (FAB) cooperative group. Leukemic cells separated from the peripheral bloods of AML patients were cultured in methylcellulose media, and the plating efficiencies of primary colonies (PE1) and secondary colonies after replating (PE2) were determined. PE1 and PE2 have been considered to reflect the capacities of terminal divisions and self-renewal of leukemic blast progenitors, respectively. PE1 and PE2 were variable among AML patients; these findings suggest that AML is a heterogeneous disease in terms of the proliferative activities of leukemic cells. No significant correlation was noted between PE1 or PE2 and the AML subtype. The sensitivities to cytosine arabinoside (Ara-C) of leukemic blast progenitors were studied in methylcellulose and suspension cultures. Ara-C sensitivity was not significantly correlated with the AML subtype, either. In contrast, there was statistically significant correlation between PE2 and the remission outcome of the patients, whereas PE1 was not significantly associated with the clinical outcome. The results in the present study indicate that the proliferative activity, especially self-renewal capacity, of leukemic blast progenitors is highly predictive of the prognosis of AML patients but is not significantly correlated with the AML subtype classified by the blast morphology.

Roles of stem cell factor in the in vitro growth of blast clonogenic cells from patients with acute myeloblastic leukemia.

We investigated the effects of stem cell factor (SCF) on the growth of blast clonogenic cells from 27 patients with acute myeloblastic leukemia (AML) and 3 patients with chronic myelocytic leukemia in myeloid crisis. SCF alone showed a significant stimulatory activity in 15 of 30 patients (50%). A marked reduction in the number of blast cell colonies supported by SCF alone was noted by the addition of neutralizing antibody (Ab) against granulocyte-macrophage colony-stimulating factor (GM-CSF). Ab against interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-alpha) also moderately reduced the number of colonies, whereas Ab against granulocyte CSF (G-CSF) failed to do so. All four Ab together completely abolished the growth in 5 of 6 patients tested. c-kit antisense oligonucleotides reduced the colony formation supported by IL-3 or G-CSF or, in the absence of growth factor, in only 2 of 10 patients tested. SCF caused stimulation by acting synergistically with G-CSF, GM-CSF, IL-3, IL-6, IL-9, IL-11, and IL-12 in 20 of 27 (74%), 17 of 27 (63%), 14 of 28 (50%), 9 of 28 (32%), 1 of 15 (7%), 3 of 28 (11%), and 2 of 15 (13%) patients, respectively. Thus, SCF alone or in combination with some other factor stimulated the growth in 27 of 30 (90%) patients. Of 3 nonresponders, 2 were AML, M3 at presentation. G-CSF at the optimal concentration increased the sensitivity of blasts to SCF. Taken together, SCF acting in combination with other factors, but not alone, stimulates the growth of blast clonogenic cells. GM-CSF, IL-6, and TNF-alpha may be produced endogenously, whereas G-CSF and SCF may be supplied exogenously. Autocrine regulation of the growth of blasts seems to increase the responsiveness of the cells to any of these factors, allowing them to achieve a highly active growth state.

Leukemia in patients following radiotherapy for malignant neoplasms in the pelvic region.

A prospective study of 1572 women treated with radiotherapy for cervical (1478 women) and ovarian cancer (94 women) was done. Patients had been followed clinically and especially by blood tests between 1961 and 1981, comprising 8990 woman-years (WY). Following radiotherapy, 5 patients developed non-lymphocytic leukemia [2 acute myeloblastic leukemia (AML), 1 acute monocytic leukemia (AMoL), and 2 chronic myeloid leukemia (CML)]. Based on rates for the general population, 0.45 case would be expected and, therefore, the relative risk was 11.2. The average mean marrow dose for all our subjects was calculated to be 1177 rad, the risk of radiation-induced leukemia was 0.43 excess case per year per one million women exposed to 1 rad of radiation to the bone marrow. Four patients with cervical cancer who developed leukemia were in a high-dose-rate group treated with both a linear accelerator (Linac) and remote afterloading system (RALS), and 1 patient with ovarian cancer who developed leukemia was treated with a Linac alone. This is the first report of a statistically significant increased risk of leukemia for patients treated with large doses of radiation for malignant neoplasms in the pelvic region.

Effects of recombinant G-CSF and GM-CSF on the growth in methylcellulose and suspension of the blast cells in acute myeloblastic leukemia.

The effects of two recombinant human CSFs (G-CSF and GM-CSF) on the growth of blast progenitors from 36 acute myeloblastic leukemia patients were studied in methylcellulose and suspension cultures. Blast colony formation in methylcellulose and the growth of blast progenitors in suspension were stimulated by G-CSF or GM-CSF. Their responses to CSFs were different from those of normal myeloid progenitors. First, the sensitivity of blasts to 0.01 ng/ml of G-CSF and 0.001 ng/ml of GM-CSF was significantly increased compared with normal. Second, in more than 70% of patients, the pattern of the responsiveness to the two CSFs was aberrant compared with ordered response in normal subjects. Third, in about half of the patients, combination of G-CSF and GM-CSF showed synergism for the growth of blast progenitors in both culture methods, whereas negligible or no synergism was observed in normal subjects. Finally, when stimulated by G-CSF, GM-CSF, or both, a significant relationship was noted between blast colony formation in methylcellulose and blast progenitor growth in suspension, suggesting that CSFs do not affect the balance between self-renewal and terminal divisions of blast stem cells.