Development Of Secondary Leukemia Research Articles

Mechanisms for the development of therapy-related leukaemia caused by topoisomerase inhibitors

Leukemia is a blood cancer that originates in the blood and bone marrow. Therapy-related leukemia is a leukemia associated with prior chemotherapy. Cancer therapy with DNA-topoisomerase II inhibitors is one of the most effective among chemotherapies. However, its side effect can be the development of secondary leukemia, characterized by chromosomal rearrangements involving the AML1 or MLL gene. The set of recurrent translocations in such leukemia differs from the set of chromosomal rearrangements in other neoplasia. We review the factors that drive the translocations upon treatment of cells with DNA-topoisomerase inhibitors. Such factors primarily include the mobility of double-strand DNA ends prior to translocation and the gain of functions of the fusion proteins that are formed in the cell as a result of translocation.

Evaluating the association between clonal hematopoiesis and germline pathogenic and likely pathogenic variants in cancer predisposition genes.

1535 Background: Clonal hematopoiesis (CH) is most commonly associated with mutations in genes with a known or putative role in leukemia and with an increased risk for hematologic malignancies. Nearly 50% of primary breast cancers contain leukocytes with CH mutations and CH mutations can be found in breast cancer (BC) tumor infiltrating leukocytes years prior to the development of secondary leukemia. It is currently not known whether CH is more prevalent among BC patients with both germline pathogenic variants and likely pathogenic variants (collectively referred to as PV) in cancer predisposition genes. Here we evaluated the relationship of CH to PV in 546 BC patients (pts). Methods: 546 BC pts underwent targeted capture sequencing of tumor and peripheral blood (PB) samples using MSK-IMPACT. Sequencing results from PB were used for identifying PV affecting up to 89 cancer predisposition genes and somatic mutations in 15 genes commonly associated with CH using previously validated methods. All pts consented via an IRB-approved protocol. Results: The majority (82.3%) of pts had non-metastatic disease. 59.7% of the 546 pts received chemotherapy and 42.7% received radiation. Of the 546 pts, 90 patients had germline PV in a cancer predisposition gene for a total of 98 germline PV identified (8 patients had 2 germline PV). Mutations in DNMT3A followed by TET2 were the most common CH mutations identified. CHEK2 PV were statistically significantly associated with CH in a multivariate analysis after controlling for age, prior chemotherapy and radiation therapy (OR: 3.94, CI: 1.51-10.26, p-value ≤0.005). Age by decade was significantly associated with the presence of CH in that model (OR: 1.93, CI: 1.53-2.42, p value ≤0.001). Of the 8 patients with CHEK2 germline PV and CH, the most common mutated CH genes were DNMT3A (4 pts), followed by TET2 (2 pts), although our sample size is too small to test for significant enrichment for any specific CH gene, type of CH mutation, or trinucleotide context. Further, CH was not associated with germline PV in other cancer predisposition genes. Conclusions: Our findings provide evidence of an association between CHEK2 germline PV with CH in BC pts, which will be further tested in an expanded cohort. If this association is confirmed, it might have theoretical and practical implications, including cancer prevention strategies.

Characteristics and Therapeutic Outcomes of Acute Promyelocytic Leukemia in Children and Adolescents

P=0.045), but their 5-year OS and EFS were not significantly different. Better survival was observed in ATRA plus chemotherapy group both for 5-year OS (100% vs. 60.0±21.9%, P=0.018), and for 5-year EFS (60.0±21.9% vs. 91.7±8.0%, P=0.080) than ATRA only group. Stem cell transplantations were given to 3 patients and they are alive without disease for 8.3-16.5 years of follow-up. One death after relapse, another death in remission and development of secondary leukemia were encountered during the study period. Conclusion: This study analyzed clinical characteristics of pediatric APL and demonstrated very good outcome with the combination of ATRA and chemotherapy.

Mediastinal germ cell tumors and development of secondary leukemia and solid tumors.

e16043Background: Retrospective studies have shown a correlation between mediastinal germ cell tumors (MGCT) and development of AML. Primary treatment for MGCTs is multiagent chemotherapy, which ca...

The In Vitro and In Vivo Effects Of Fermented Papaya Preparation On Radiation Exposure

BackgroundVarious forms of radiation (radioactive, UV) cause cellular damage, which may lead to premature cell death or accumulation of somatic mutations which may lead to initiation and progression of malignancy. The damage is mediated in part by free radicals, particularly reactive oxygen species (ROS) (Reizenstein, Med Oncol Tumor Pharmacother. 1991;8:229). Fermented papaya preparation (FPP) is a product of yeast fermentation of Carica papaya Linn. FPP has been shown to have anti-oxidative potential by scavenging ROS, as well as by chelating excess iron (Prus & Fibach J Biol Regul Homeost Agents. 2012;26:203). Free iron species, such as the labile iron pool (LIP), participate in chemical reactions that generate ROS (Haber-Weiss and Fenton reactions). We studied the potential effect of FPP in preventing radiation-induced damage to various intracellular components in cultured human fibroblasts and myeloid leukemia (HL60) cells and in mice. MethodsCultured cells, human foreskin fibroblasts and myeloid leukemia (HL-60), were irradiated at various doses (0-18 Gy (Gammacell® 220, MDS Nordion, Excel, Canada). FPP (Osato Research Institute, Gifu, Japan) (10-100 µg/ml, was added either before or after irradiation. The cells were assayed after 1-3 days: Their survival was estimated by CellTiter 96® Aqueous Non-Radioactive Cell Proliferation assay. Apoptosis was determined by staining for phosphatidylserine exposure (by fluorescent Annexin V) and for intracellular uptake of propidium iodide. ROS was measured by staining with dichlorofluoresceine diacetate, and the LIP - with calcein-AM. The effects on the DNA were estimated by measuring 8-oxyguanine (a marker of DNA oxidation), using a fluorescent specific probe, and DNA instability was measured by the “comet assay”, a single-cell gel electrophoresis procedure. Apoptosis, ROS, LIP and 8-oxyguanine were quantified by flow cytometry. Experiments were also carried out on irradiated mice treated with FPP (by adding it to the drinking water) either before or after irradiation. The above mentioned parameters were assayed in their bone marrow cells in addition to determining their survival. ResultsThe results indicated that FPP has significant (P<0.05) ameliorating effects on radiation-induced increase of LIP and generation of ROS, as well as on the generation of 8-oxyguanine and DNA instability. In addition, apoptosis was decreased and consequently the survival of the cells was increased (Fig. 1A). Moreover, about 60% of 14 Gy-irradiated mice that received 100 mg/ml of FPP in their drinking water were still alive after 30 days (Fig. 1B). [Display omitted] ConclusionsFPP was able to protect the short-term effects of radiation on cultured cells by increasing their viability. A similar effect was found in mice. The improvement in DNA instability induced by FPP may influence the long-term effects of radiation, such as the development of secondary leukemia, in patients who receive radiotherapy for their primary tumors. Disclosures:Fibach:Osato Research Institute, Gifu, Japan: Consultancy, Research Funding. Rachmilewitz:Osato Research Institute, Gifu, Japan: Consultancy, Research Funding.

A Novel Topoisomerase Inhibitor, Daurinol, Suppresses Growth of HCT116 Cells with Low Hematological Toxicity Compared to Etoposide

We report that daurinol, a novel arylnaphthalene lignan, is a promising potential anticancer agent with adverse effects that are less severe than those of etoposide, a clinical anticancer agent. Despite its potent antitumor activity, clinical use of etoposide is limited because of its adverse effects, including myelosuppression and the development of secondary leukemia. Here, we comprehensively compared the mechanistic differences between daurinol and etoposide because they have similar chemical structures. Etoposide, a topoisomerase II poison, is known to attenuate cancer cell proliferation through the inhibition of DNA synthesis. Etoposide treatment induces G2/M arrest, severe DNA damage, and the formation of giant nuclei in HCT116 cells. We hypothesized that the induction of DNA damage and nuclear enlargement due to abnormal chromosomal conditions could give rise to genomic instability in both tumor cells and in actively dividing normal cells, resulting in the toxic adverse effects of etoposide. We found that daurinol is a catalytic inhibitor of human topoisomerase IIα, and it induces S-phase arrest through the enhanced expression of cyclins E and A and by activation of the ATM/Chk/Cdc25A pathway in HCT116 cells. However, daurinol treatment did not cause DNA damage or nuclear enlargement in vitro. Finally, we confirmed the in vivo antitumor effects and adverse effects of daurinol and etoposide in nude mice xenograft models. Daurinol displayed potent antitumor effects without any significant loss of body weight or changes in hematological parameters, whereas etoposide treatment led to decreased body weight and white blood cell, red blood cell, and hemoglobin concentration.

Therapy-related myeloid leukemia after treatment for epithelial ovarian carcinoma: An epidemiological analysis

Objective Therapy related acute myeloid leukemia (t-AML) is a potential late complication of cytotoxic therapy, and it is of particular concern in the treatment of patients with epithelial ovarian carcinoma (EOC) exposed to multiple courses of chemotherapy during the course of their disease. This study examines the incidence, characteristics and clinical outcomes of patients who developed secondary myeloid-type leukemia after a diagnosis of EOC. Methods National Cancer Institute's Surveillance, Epidemiology and End Results database was pooled for diagnosis of secondary myeloid leukemia after an initial diagnosis of EOC. This group of patients was compared to patients with de novo AML, and to EOC patients who did not develop secondary myeloid leukemia. Demographic, cytopathological and survival data were recorded. Cox Proportional Hazards model was used to calculate hazard ratios (HR) for developing secondary leukemia and to determine the statistically significant variables impacting survival. Kaplan–Meier estimates of survival were obtained and comparisons between the groups were performed using log-rank test. Results One hundred and nine myeloid leukemia cases were identified among 63,359 patients with a prior diagnosis of EOC for an overall incidence of 0.17%. The median latency to development of leukemia was 4 years (range 0–27 years). Median survival from the time of secondary leukemia diagnosis was 3 months and significantly worse than the 6 month median survival in patients with de novo AML ( p < 0.001). Age at leukemia diagnosis greater than 65 and development of secondary vs. de novo leukemia had a statistically worse prognosis on multivariate analysis (HR of 2.69, 95%CI 2.60–2.78 and 1.81, 95%CI 1.49–2.20 respectively). The development of secondary leukemia was more common with EOC diagnosis made prior to the platinum/taxane era (HR 6.70, 95%CI 3.69–12.18). There was no difference in median survival between EOC patients who developed AML and those who did not. Conclusion Development of t-AML is a rare but lethal event among EOC patients, and its incidence has decreased significantly since the use of platinum/taxane-based chemotherapy became the standard of care.

Presenting features and treatment outcome of acute promyelocytic leukemia arising after multiple sclerosis

We report the clinical features and treatment outcome of 33 patients with multiple sclerosis who developed acute promyelocytic leukemia. Thirty patients were previously exposed to mitoxantrone. The median latency period between treatment initiation and acute promyelocytic leukemia diagnosis was 32 months. The PML-RARA bcr1 iso-form was identified in 87% of cases. Twenty-nine (90%) patients achieved hematologic remission after all-trans retinoic acid and chemotherapy (n = 31) or arsenic trioxide and all-trans retinoic acid. Consolidation included modified chemotherapy or arsenic trioxide. At a median follow up of 26 months, 23 patients are in complete remission, 4 relapsed and one developed secondary leukemia. The 5-year cumulative incidence of relapse and overall survival were 23% and 68%, respectively. Although treatment heterogeneity and suboptimal post-remission therapy must be taken into account, overall results and development of secondary leukemia in one patient suggest that effective and less toxic agents like arsenic trioxide warrants further investigation in this context.

Preferential induction of MLL (Mixed Lineage Leukemia) rearrangements in human lymphocyte cultures treated with etoposide

Topoisomerase II inhibitors are effective chemotherapeutic agents in the treatment of cancer, in spite of being associated with the development of secondary leukemia. Our purpose was to determine the effects of etoposide on different genomic regions, aiming at discovering whether there are preferential sites which can be targeted by this drug in peripheral lymphocytes from healthy individuals. The in vitro treatment with low doses of etoposide (0.25, 0.5, and 1 μg/mL, in 1 hour-pulse or continuous-48 h treatment) induced a significant increase in chromosomal aberrations, detected by conventional staining and FISH with specific probes for chromosomes 8 and 11, compared with untreated controls (p < 0.05). Additionally, the frequencies of alterations at 11q23, detected by MLL specific probes, were significantly higher (p < 0.005) in treated cells than in controls. In contrast, an analysis of rearrangements involving the IGH gene did not disclose differences between treatments. The present results demonstrated the potential of etoposide to interact with preferential chromosome sites in human lymphocytes, even at concentrations below the mean plasma levels measured in cancer patients. This greater susceptibility to etoposide-induced cleavage may explain the more frequent involvement of MLL in treatment-related leukemia.

Multicentre phase II study of the baseline BEACOPP regimen for patients with advanced-stage Hodgkin's lymphoma

A German Hodgkin's lymphoma (HL) study group designed the BEACOPP (bleomycin, etoposide, doxorubicin, cyclophosphamide, vincristine, procarbazine, prednisolone) regimen. In the BEACOPP regimen, treatment intervals were shortened and the dose-intensity was increased compared with those in the ABVD regimen (doxorubicin, bleomycin, vinblastine and darcarbacine), resulting in a long-term disease-free survival rate of approximately 75 – 80%. In the present study, we evaluated the safety and efficacy of the BEACOPP regimen. Between April 2001 and February 2004, 20 patients with HL of stage IIB or higher who had received no previous treatment were enrolled. The patients were aged 17 – 69 years (median 22 years). The histologic types were mixed cellularity in four cases and nodular sclerosis in 16 cases. The stages were stage IIB in four cases, stage III in 12 cases, and stage IV in four cases. Nineteen (95%) of the 20 patients achieved complete remission. The 3-year survival rate was 100% and the 3-year progression-free survival rate was 89.7%. Adverse drug reactions were grade 4 neutropenia in 12 patients, grade 3 – 4 thrombocytopenia in seven patients, and grade 3 or higher non-hematologic toxicities in two patients (stomatitis in one patient and ALT/AST elevation in one patient). The BEACOPP regimen for advanced-stage HL showed an excellent complete remission rate and high efficacy even in stage III/IV patients. However, a long-term risk of the BEACOPP regimen is the development of secondary leukemia or myelodysplastic syndrome. Therefore, long-term follow-up of these patients, including monitoring for toxicities, is necessary.

Analysis of Vector Integration with LM-PCR after Retroviral HSV-Tk/ΔLNGFR Gene Transfer.

The development of secondary leukemia as an unexpected complication after retroviral transfer of potentially therapeutic genes was recently reported. In the pathogenesis of this complication, the upregulation of proto-oncogenes due to random vector integration played a crucial role. Therefore, the analysis of vector integration after retroviral gene transfer could become an interesting tool for risk assessment and safety measurement. In this study, 4 patients diagnosed with chronic myelogenous leukemia (CML) and 2 patients suffering from acute myelogenous leukemia (AML) were transplanted with T-cell-depleted CD34+ enriched stem cells (HSCT) from their HLA-identical siblings without further immunosuppression. After day +60 following HSCT, they received gene-modified donor T-cells infected with the replication-deficient retrovirus SFCMM-3, expressing the herpes simplex thymidine kinase (HSV-Tk) as a suicide gene, and the truncated low affinity nerve growth factor (ΔLNGFR) to control graft versus leukemia (GvL) effects and graft versus host disease (GvHD). In order to assess the viral integration sites in the transfused gene-modified donor T-cells, we performed a ligation-mediated PCR (LM-PCR) to amplify the vector flanking sequences. Final PCR products were visualized by gel electrophoresis and silver staining. Afterwards, sequence analysis (CEQ 8000, Beckman Coulter GmbH, Krefeld, Germany) of the clonal products was carried out. Standard National Center for Biotechnology Information (NCBI) blast searches were done to identify the flanking sequences (http://www.ncbi.nlm.nih.gov/BLAST). Within the investigated gene-modified T-cells of our study population, we found several integration sites. Up to now there has been no evidence for retroviral integration in the vicinity of proto-oncogene promoters after sequence analysis and database search. Furthermore, we were able to monitor the presence of the transduced T-cells at several time points after HSCT by detecting the internal retroviral band. In conclusion, the LM-PCR is a specific method to amplify and identify the flanking sequences after retroviral gene transfer, contributing significantly to the safety of gene therapy.

Pharmacological Intakes of Niacin Increase Bone Marrow Poly(ADP-Ribose) and the Latency of Ethylnitrosourea-Induced Carcinogenesis in Rats

Cancer chemotherapy agents cause short-term leukopenia during treatment and the development of secondary leukemias after recovery from the original disease. We reported that niacin deficiency in rats increases the severity of nitrosourea-induced leukopenia and the subsequent development of cancers. This study was designed to test the effects of supplementing an already high quality diet with pharmacologic levels of niacin. For a period of 4 wk, nontumor-bearing weanling Long-Evans rats were pair-fed AIN-93M diets that were niacin adequate (30 mg/kg diet) or pharmacologically supplemented (4 g/kg diet) with nicotinic acid (NA) or nicotinamide (Nam). One week after the initiation of niacin feeding protocols, ethylnitrosourea (ENU) treatment began (12 doses, 30 mg/kg by gavage, every other day). ENU treatment caused leukopenia, which was not prevented by niacin supplementation. At the end of ENU treatment, all rats were switched to a niacin-adequate diet and monitored. Within 36 wk after the start of treatment, all of the ENU-treated rats either lost 5% of peak body weight or had palpable tumors > 1 cm in diameter, and were necropsied. Supplementation with NA or Nam at 4.0 g/kg diet (combined analysis) increased the latency of the ENU-induced morbidity curve, relative to niacin-adequate controls. Morbidity could be attributed in almost all cases to some form of neoplasm, with leukemias the predominant form. In short-term studies, supplementation with either NA or Nam caused dramatic increases in bone marrow NAD(+) (1- to 1.5-fold), basal poly(ADP-ribose) (3- to 5-fold) and ENU-induced poly(ADP-ribose) levels (1.5-fold). These data show that supplementation of a niacin-adequate, high quality diet with pharmacologic levels of nicotinic acid or nicotinamide increases NAD(+) and poly(ADP-ribose) levels in bone marrow and may be protective against DNA damage.

Acute leukemia following a previous malignancy: do acute lymphoid leukemia and acute myeloid leukemia have common risk factors?

Within the framework of the GIMEMA Study Group, the characteristics of acute lymphoid leukemia and acute myeloid leukemia occurring in patients who have suffered a previous malignancy were studied. Assessment was also made of the clinical course, laboratory features and overall outcome of these conditions. A four-year, multi-center retrospective study was conducted to evaluate the effect of treatment for previous hematological malignancy on the development of secondary leukemia. The study collected in the GIMEMA Archive of Adult Acute Leukemia 3934 new cases of acute leukemia (2964 AML, 901 ALL, 60 acute biphenotypic leukemia). Among these cases, data were evaluated from patients with a personal history of a previous malignancy, and included inquiring into demographic data, history of neoplastic diseases in the 1st degree relatives, type and treatment of the previous malignancy, latency until the development of a secondary acute leukemia diagnosis, laboratory features, treatment and outcome at the onset of secondary acute leukemia. Approximately 200 (5.1%) patients presented a previous malignancy. Twenty-one were affected by ALL and 179 by AML. The proportion of patients with secondary AML was higher than that of patients with secondary ALL (179/2964 vs 21/901, O.R. 2.69-95% C.I. 1.66-4.39, P<0.001). The median latency, from the onset of the previous malignancy to the development of secondary ALL was 27 months and to the development of secondary AML was 52 months (P<0.05). Furthermore, of patients who previously received chemotherapy more developed a second AML (66/127 sAML vs 5/21 sALL; O.R. 3.46-95% C.I. 1.10-11.56, P<0.01). In most cases, chemotherapy treatment for a previous malignancy can play a role in the development of secondary AML. In almost all cases of secondary ALL, the role of previous drugs does not appear to be relevant. On the basis of our analysis, performed systematically for the first time on a large adult series of acute leukemia, we conclude that in these patients a biological predisposition to cancer may be suspected.

Niacin Deficiency in Rats Increases the Severity of Ethylnitrosourea-Induced Anemia and Leukopenia

Many chemotherapeutic agents function by damaging the DNA of rapidly dividing cells, leading to side effects in the bone marrow, including anemia and leukopenia during chemotherapy and the development of secondary leukemias in the years following recovery from the original disease. We have created an animal model of alkylation-based chemotherapy, in nontumor-bearing rats, to investigate the effect of niacin deficiency on the side effects of chemotherapy [2 x 2 design, niacin-deficient (ND) vs. pair-fed (PF) control, and ethylnitrosourea (ENU) vs. vehicle control (C)]. Weanling Long-Evans rats were fed ND diet or PF niacin replete diet for 4 wk. ENU or C treatment started after 1 wk of feeding and consisted of 12 doses delivered by gavage, every other day. At 4 wk postweaning, niacin deficiency and ENU treatment ended, the rats were fed a high-quality control diet (AIN-93M) and the recovery of blood variables was monitored. ND alone decreased growth rate and caused anemia and neutrophilia. ENU treatment alone caused anemia, lymphopenia, neutropenia and an increase in circulating reticulocytes. In combination, ND and ENU treatment synergistically decreased hematocrit. ND prevented the ENU-induced increase in reticulocyte numbers observed in control rats. ND also increased the severity of ENU-induced lymphopenia. A combination of ND and ENU abolished the neutrophilia caused by ND alone. In summary, ND significantly increased the susceptibility of young Long-Evans rats to ENU-induced bone marrow suppression, suggesting that niacin-deficient cancer patients may benefit from supplementation.

Emergence of secondary acute leukemia in a patient treated for osteosarcoma: implications of germline TP53 mutations

Background Secondary leukemia and myelodysplastic syndromes have been reported in patients following treatment for a wide range of neoplastic disorders. However, second malignancies after chemotherapy and/or irradiation for osteosarcoma are unusual. Procedure We report the case of a 15-year-old girl who developed a myelodysplastic syndrome with evolution to acute nonlymphocytic leukemia after treatment for osteosarcoma. Therapy-related acute leukemia karyotype findings such as abnormalities of chromosomes 5,7, and 17 were found in the cytogenetic analysis. Moreover, using denaturing gradient gel electrophoresis and DNA sequencing, we detected the presence of a double germline mutation in exon 7 of the TP53 gene. Conclusion This observation supports the possibility of a causal relationship between germline TP53 mutations and the development of secondary leukemia and myelodysplasia. Med. Pediatr. Oncol. 30:165-169, 1998. © 1998 Wiley-Liss, Inc.

Human CD34+ hematopoietic progenitors have low, cytokine-unresponsive O6-alkylguanine-DNA alkyltransferase and are sensitive to O6- benzylguanine plus BCNU

Human bone marrow (BM) cells contain low levels of the DNA repair protein, O6-alkylguanine-DNA alkyltransferase, which may explain their susceptibility to nitrosourea-induced cytotoxicity and the development of secondary leukemia after nitrosourea treatment. Isolated CD34+ myeloid progenitors were also found to have low levels of alkyltransferase activity. The level of alkyltransferase in CD34+ cells or in mononuclear BM cells did not increase after incubation with granulocyte-macrophage colony-stimulating factor, interleukin-3, stem cell factor, the combination, or 5637 conditioned medium. BCNU sensitivity remained unchanged as well. In addition, O6-benzylguanine depleted alkyltransferase activity in BM cells at concentrations as low as 1.5 mumol/L after a 1-hour exposure. O6-benzylguanine pretreatment markedly sensitized hematopoietic progenitor colony-forming cells to BCNU, resulting in a reduction in the dose of drug (termed the dose- modification factor) required to inhibit 50% of the colony formation (IC50) of threefold to fivefold. Since, unlike many other cell types, proliferating early (CD34+) hematopoietic precursors do not induce alkyltransferase, myelosuppression may be the dose-limiting toxicity of the combination of O6-benzylguanine plus BCNU in clinical trials.

Human CD34+ Hematopoietic Progenitors Have Low, Cytokine-Unresponsive O6-Alkylguanine-DNA Alkyltransferase and Are Sensitive to O6-Benzylguanine Plus BCNU

Human bone marrow (BM) cells contain low levels of the DNA repair protein, O6-alkylguanine-DNA alkyltransferase, which may explain their susceptibility to nitrosourea-induced cytotoxicity and the development of secondary leukemia after nitrosourea treatment. Isolated CD34+ myeloid progenitors were also found to have low levels of alkyltransferase activity. The level of alkyltransferase in CD34+ cells or in mononuclear BM cells did not increase after incubation with granulocyte-macrophage colony-stimulating factor, interleukin-3, stem cell factor, the combination, or 5637 conditioned medium. BCNU sensitivity remained unchanged as well. In addition, O6-benzylguanine depleted alkyltransferase activity in BM cells at concentrations as low as 1.5 mumol/L after a 1-hour exposure. O6-benzylguanine pretreatment markedly sensitized hematopoietic progenitor colony-forming cells to BCNU, resulting in a reduction in the dose of drug (termed the dose-modification factor) required to inhibit 50% of the colony formation (IC50) of threefold to fivefold. Since, unlike many other cell types, proliferating early (CD34+) hematopoietic precursors do not induce alkyltransferase, myelosuppression may be the dose-limiting toxicity of the combination of O6-benzylguanine plus BCNU in clinical trials.