Relationship between progression-free survival and overall survival in chronic lymphocytic leukemia: a literature-based analysis.

BACKGROUND: The endpoints of progression-free survival (pfs) and time-to-progression (ttp) are frequently used to evaluate the clinical benefit of anticancer drugs. However, the surrogacy of those endpoints for overall survival (os) is not validated in all cancer settings. In the present study, we used a trial-based approach to assess the relationship between median pfs or ttp and median os in chronic lymphocytic leukemia (cll). METHODS: The pico (population, interventions, comparators, outcomes) method was used to conduct a systematic review of the literature. The population consisted of patients with cll; the interventions and comparators were standard therapies for cll; and the outcomes were median pfs, ttp, and os. Two independent reviewers screened titles, abstracts, and full papers for eligibility and then extracted data from selected studies. Correlation coefficients were calculated to assess the relationship between median pfs or ttp and median os. Subgroup correlation analyses were also conducted according to the characteristics of the selected studies (such as line of treatment and type of treatment under investigation). RESULTS: Of the 1263 potentially relevant articles identified during the literature search, twenty-three were included. On average, median pfs or ttp was 16.0 months (standard deviation: 12.4 months) and median os was 43.5 months (standard deviation: 31.2 months). Results of the correlation analysis indicated that median pfs or ttp is highly correlated with median os (Spearman correlation coefficient: 0.813; p ≤ 0.001). A significant correlation between median pfs or ttp and median os was observed in second- and subsequent-line therapies, but not in the first-line setting. CONCLUSIONS: Our study demonstrates a strong correlation between median pfs or ttp and median os in previously treated cll, which reinforce the hypothesis that pfs and ttp could be adequate surrogate endpoints for os in this cancer setting.

Increased risk of second malignancies in chronic lymphocytic leukaemia patients as compared with follicular lymphoma patients: a Canadian population-based study.

BACKGROUND: Chronic lymphocytic leukaemia (CLL) patients have an increased risk of other malignancies. This may be due to surveillance bias, treatment or immunosuppression. METHODS: Cohort study of 612 consecutively diagnosed CLL patients in a Canadian province, with comparisons to follicular lymphoma (FL) patients. RESULTS: Treated CLL patients had a 1.7-fold increased risk of second cancers compared with untreated CLL patients. As compared with untreated FL patients, untreated CLL patients had a two-fold increased incidence of second malignancies. CONCLUSION: Chronic lymphocytic leukaemia patients have an inherent predisposition to second cancers and the incidence is further increased by treatment.

Rapid improvement in symptoms and physical function following ibrutinib initiation in chronic lymphocytic leukemia and the associated changes in plasma cytokines.

A prospective pilot study was carried out on 34 CLL patients treated with ibrutinib, evaluating the effects on symptoms and physical function with changes in plasma exosomes (EXs), ß2-microglobulin (ß2M) and 26 plasma cytokines. The revised Edmonton Symptom Assessment Scale (ESAS-R) demonstrated moderate fatigue, shortness of breath and a sense of unwellness before treatment, which significantly improved within 2 weeks of starting ibrutinib. These changes were associated with a rapid improvement in sit-to-stand and 4 m walking speeds. The plasma levels of CCL11, IL-7, -8 and -10 dropped initially while the levels of TNF-α/-ß, CCL3, CCL4, CCL17, and IL-16 continued to decline for 12 months. Despite the initial lymphocytosis, plasma ß2M levels fell but no consistent change in plasma EXs occurred. Thus, ibrutinib can produce a rapid and sustained improvement in symptoms and physical function in CLL, associated with a decline in multiple plasma cytokines.

Descriptive analysis of dosing and outcomes for patients with ibrutinib-treated relapsed or refractory chronic lymphocytic leukemia in a Canadian centre.

Background: Ibrutinib is an approved treatment for relapsed or refractory chronic lymphocytic leukemia (cll) and small lymphocytic lymphoma (sll). The effect of ibrutinib dose reduction compared with discontinuation in a population-based setting is unclear. Methods: To examine the patterns of ibrutinib use in a Canadian population-based setting, we analyzed a retrospective cohort of patients with relapsed or refractory cll or sll treated with ibrutinib. Results: The 64 patients diagnosed with cll or sll had a median age of 76.5 years. Most had unmutated ighv (immunoglobulin variable heavy chain). A hematologic response occurred in 39 patients regardless of the ibrutinib dose. The most common toxicities were infection, bruising or bleeding, and musculoskeletal problems, with a median time to first toxicity of 14 days. More than half the cohort experienced a dose reduction, with musculoskeletal problems, cytopenias, and infection being the leading causes; surgery was the most frequent indication for holding treatment. Only 26 of the 64 patients (40.6%) stayed on the recommended maximal dose of ibrutinib. No differences in reported toxicities or hematologic response rates were evident between the patients receiving maximal and submaximal therapy. At the end of the study period, 53 patients from the initial cohort remained on ibrutinib. Conclusions: More than half the study patients received ibrutinib therapy at a submaximal dose without evidence of increased frequency of toxicities or disease progression. The rate of ibrutinib discontinuation was lower in our cohort than has been reported in other settings. Submaximal ibrutinib dosing will have to be further systematically evaluated.

Xenoinfection of nonhuman primates by feline immunodeficiency virus.

New viral infections in humans usually result from viruses that have been transmitted from other species as zoonoses. For example, it is accepted widely that human immunodeficiency virus (HIV) is the result of the propagation and adaptation of a simian immunodeficiency virus (SIV) from nonhuman primates to man [1]. Previously, we reported productive infection of primary human cells in vitro by feline immunodeficiency virus (FIV) [2], a lentivirus that causes an immunodeficiency syndrome in cats similar to HIV in humans [3]. The present study extends these findings by demonstrating that cynomolgus macaques (Macaca fasicularis) infected with FIV exhibited clinical signs, including depletion of CD4+ cells and weight loss, that are consistent with FIV infection. The development of an antibody response to FIV gag-encoded proteins and detection of virus-specific sequences in sera, blood-derived cells, and necropsied tissue accompanied these changes. Moreover, the reactivation of FIV replication from latently infected cells was observed after stimulation in vitro with phorbol esters and in vivo with tetanus toxoid. The proposed use of lentiviruses in human gene therapy [4, 5] and of nonhuman cells and organs in xenotransplantation [6] has raised concerns about zoonoses as potential sources of new human pathogens. Therefore, the study of FIV infection of primate cells may provide insight into the principles underlying retroviral xenoinfections.

Elevated expression of the c-fes proto-oncogene in adult human myeloid leukemia cells in the absence of gene amplification.

Expression of the 93-kd tyrosine kinase encoded by the human c-fes proto-oncogene (also known as FES) is restricted to mature hematopoietic cells of the granulocytic and monocytic lineages, suggestive of a function essential to normal myeloid differentiation. However, recent studies have shown that c-fes can transform fibroblasts if sufficient levels of gene expression are achieved. These findings indicate that strict regulation of the c-fes gene is critical to normal myeloid development, whereas elevated c-fes expression may contribute to malignant transformation. In the present study, we compared the c-fes messenger RNA (mRNA) levels in leukemia blasts from patients with myeloid or lymphoid leukemia with those of peripheral monocytes from a normal donor with the use of a quantitative ribonuclease protection assay. The presence of c-fes mRNA was readily detected in both acute myeloid leukemia (AML) and chronic myeloid leukemia (CML) cells, but c-fes mRNA was present in low levels or was absent in lymphoid leukemia cells. The leukemia cells of two of five AML patients and four of four CML patients expressed more c-fes mRNA than monocytes from a normal donor, with more than a threefold elevation in the cells of one CML patient. No evidence of amplification or rearrangement of the c-fes gene was detectable by Southern blot analysis of myeloid leukemia DNA, suggesting that the variation in c-fes mRNA levels are related to differences in transcriptional activity and/or message stability. These results indicate that elevated c-fes expression is a common feature of myeloid leukemia cells that could potentially contribute to the leukemia phenotype.

Efficacy of 2'-deoxycoformycin in hairy-cell leukemia: a study of the National Cancer Institute of Canada Clinical Trials Group.

Thirty-one patients with hairy-cell leukemia were treated with 2'-deoxycoformycin (DCF) in a National Cancer Institute of Canada multicenter trial. The DCF was administered in a cycle (4 mg/m2 iv weekly X 3), which was repeated every 8 weeks. Following a complete remission, consolidation was done with two further cycles of DCF. Of 28 patients evaluable for response, 25 obtained a complete remission; 3 had a partial response. To date there has been only one relapse; the median time with no therapy was 429.5 days (range 99-743 days). Toxicity was moderate and included nausea and vomiting, lethargy, and skin rash; with the first cycle of treatment, neutropenia and an increased incidence of fever or infection were also observed. We conclude that low-dose DCF is highly effective in treating hairy-cell leukemia.

Glutathione S-transferase activity, sulfhydryl group and glutathione levels, and DNA cross-linking activity with chlorambucil in chronic lymphocytic leukemia.

Glutathione (GSH) levels and glutathione S-transferase (GST) activities were measured in the leukemia cells of 12 patients with chronic lymphocytic leukemia. Both were correlated with prior clinical exposure to alkylating agents and with DNA cross-link formation by chlorambucil in these cells in vitro. No correlation was observed between prior exposure to alkylating agents and GSH level or GST activity. An inverse correlation was observed between GST activity and cross-linking by chlorambucil, which was enhanced if both GST activity and GSH level were related to cross-linking. These findings suggest that the combination of GST and GSH protects the DNA of leukemia cells from chlorambucil, but the role of this combination in clinical resistance remains to be determined.

Activity of 3'-(3-cyano-4-morpholinyl)-3'-deaminoadriamycin in sensitive and resistant L5178Y lymphoblasts in vitro.

An L5178Y murine lymphoblast cell line resistant to 3'-(3-cyano-4-morpholinyl)-3'-deaminoadriamycin (MRA-CN), L5178Y/MRA-CN, was isolated and characterized. L5178Y/MRA-CN cells were 9.6-fold resistant to MRA-CN compared with parental cells. The resistant cell line also displayed 2-fold resistance to 3'-(4-morpholinyl)-3'-deaminoadriamycin but was not cross-resistant to Adriamycin or chlorambucil. Uptake of MRA-CN was slightly reduced in the resistant cells compared to sensitive cells, but the distribution of the drug within the cells was unchanged. DNA interstrand cross-linking by MRA-CN was not significantly different in the sensitive and resistant cell lines, but MRA-CN was slightly less effective in inhibiting both DNA and RNA synthesis in L5178Y/MRA-CN cells compared with parental cells. NADPH cytochrome P-450 reductase activity was increased in L5178Y/MRA-CN cells compared to parental cells, while the activity of DT-diaphorase was decreased in the resistant cells. The levels of glutathione and glutathione S-transferase activity were increased in the resistant cells compared to sensitive cells; however, pretreatment of L5178Y/MRA-CN cells with buthionine sulfoximine to reduce the glutathione level did not reverse the resistance of these cells to MRA-CN. MRA-CN induced DNA fragmentation that was characteristic of apoptosis in both L5178Y and L5178Y/MRA-CN cells at equitoxic drug concentrations. However, apoptosis occurred more rapidly in L5178Y/MRA-CN cells compared with parental cells. Thus, MRA-CN induces apoptosis in L5178Y cells, and this effect may be important for the anti-tumor activity of this agent. In contrast, DNA interstrand cross-linking does not appear to be the primary mechanism responsible for the cytotoxicity of MRA-CN in these cells.

Cellular pharmacology of 3'-(3-cyano-4-morpholinyl)-3'-deaminoadriamycin and structural analogues in human colon carcinoma HT-29 cells in vitro.

The new Adriamycin (ADR) analogue, 3'-(3-cyano-4-morpholinyl)-3'-deaminoadriamycin (CMA), is the most potent anthracycline yet developed. The cellular pharmacology of CMA and 3'-(4-morpholinyl)-3'-deaminoadriamycin (MA), and their 5-imino derivatives, ICMA and IMA, were compared with ADR in a human colon carcinoma (HT-29) cell line in vitro. In a soft agar clonogenic assay, the order of antitumor activity was CMA greater than ICMA greater than ADR greater than MA greater than IMA, for both 2- and 24-h drug exposure periods, indicating a requirement for the cyanide group and an intact quinone ring for the potent antitumor effect of CMA. The cellular uptake of CMA was 2-fold less than that of MA, although, consistent with its greater nuclear binding, the degree of efflux of CMA was less than that of MA. The order of cytotoxicity of the analogues correlated approximately with their effects on cellular DNA synthesis, indicating that this feature may contribute to the antitumor effect. Using isolated nuclei, the order of inhibition of DNA transcription by the analogues was CMA greater than MA greater than ADR, which was similar to their nuclear affinities, suggesting that their effects on cellular nucleic acid synthesis were due to a direct interaction of drug with DNA. However, CMA did not appear to differ from the other drugs in its base specificity as all the analogues preferentially inhibited Escherichia coli RNA polymerase activity directed by poly(dAdT).poly(dAdT) compared to poly(dGdC).poly(dGdC).

Effects of 3'-(3-cyano-4-morpholinyl)-3'-deaminoadriamycin and structural analogues on DNA in HT-29 human colon carcinoma cells.

The potent Adriamycin (ADR) analogue, 3'-(3-cyano-4-morpholinyl)-3'-deaminoadriamycin (CMA), produces DNA-DNA cross-links in human and murine tumor cells. The cellular pharmacology of CMA, its derivative, 5-imino-3'-(3-cyano-4-morpholinyl)-3'-deaminoadriamycin (ICMA), 3'-(4-morpholinyl)-3'-deaminoadriamycin (MA), and ADR was evaluated in HT-29 human colon carcinoma cells to determine the structural requirements for the cross-linking activity of CMA, and the role of this activity in the antitumor effect of this agent. CMA was 50-fold more cytocidal than ICMA to HT-29 cells, 300-fold more toxic than MA, and 150-fold more potent than ADR. Both CMA and ICMA produced DNA-DNA cross-links in HT-29 cells but, consistent with its reduced cytotoxicity, the imino derivative was 30-fold less active than CMA. No DNA-DNA cross-links were observed with MA or ADR. CMA also showed cross-linking activity in isolated HT-29 nuclei, indicating that cytoplasmic activation was not required for this effect. Both CMA and ICMA produced cross-links in isolated lambda-phage DNA with CMA being 40-fold more active than the imino derivative, and this activity was unchanged in the presence or absence of a reducing agent. While MA and ADR produced DNA strand breaks in HT-29 cells, this damage was not observed with CMA and ICMA. This study indicates that the potent antitumor activity of CMA may be related to its ability to induce DNA cross-links, which can occur without the need for metabolic activation. The cyanide group appears to be essential for cross-linking and the quinone group may also be involved, but by a mechanism unrelated to its reduction.

Enhanced cytotoxicity and inhibition of DNA damage repair in irradiated murine L5178Y lymphoblasts and human chronic lymphocytic leukemia cells treated with 2'-deoxycoformycin and deoxyadenosine in vitro.

The effects of irradiation were evaluated in L5178Y lymphoblasts treated with the adenosine deaminase inhibitor, 2'-deoxycoformycin, and deoxyadenosine. A synergistic antitumor effect was observed in resting cells between irradiation and 2'-deoxycoformycin/deoxyadenosine, with the dose required to reduce the surviving cell fraction to 0.1 being 25% lower than predicted for an additive effect. Synergy was enhanced with increasing deoxyadenosine concentration or with increasing radiation dose. When cells were treated with 2'-deoxycoformycin/deoxyadenosine for 1 h prior to irradiation, synergy was increased by prolonging postirradiation drug treatment. With 4-h postirradiation exposure to drug, varying the preirradiation incubation time did not affect synergy. In contrast, only a small enhancement of antitumor activity was observed in irradiated proliferating cells treated with 2'-deoxycoformycin/deoxyadenosine. Incubation of resting cells with 2'-deoxycoformycin/deoxyadenosine resulted in inhibition of the rate and extent of repair of radiation-induced DNA single strand breaks and an increase in dATP, but had no effect on NAD or ATP. With removal of drug, the dATP level fell rapidly and DNA repair resumed. Repair of DNA single strand breaks was more rapid in proliferating cells than in resting cells and was minimally affected by 2'-deoxycoformycin/deoxyadenosine, although the accumulation of dATP in these cells was 2-fold greater than in resting cells. The repair of DNA single strand breaks in chronic lymphocytic leukemia cells was as rapid as for proliferating L5178Y cells, but repair was significantly inhibited by 2'-deoxycoformycin/deoxyadenosine. These results suggest that 2'-deoxycoformycin/deoxyadenosine can function as a radiosensitizer, and this effect is associated with the cellular accumulation of dATP and inhibition of repair of DNA single strand breaks.

Characterization of the DNA-DNA cross-linking activity of 3'-(3-cyano-4-morpholinyl)-3'-deaminoadriamycin.

3'-(3-Cyano-4-morpholinyl)-3'-deaminoadriamycin (CMA) is a highly potent analogue of the antitumor agent, Adriamycin (ADR), being up to 1500 times more cytotoxic both in vivo and in vitro. In contrast to ADR, CMA, and 5-imino-3'-(3-cyano-4-morpholinyl)-3'-deaminoadriamycin (ICMA) have been shown to possess alkylating activity, as seen by their ability to produce DNA-DNA cross-links in human and murine tumor cells and in isolated lambda-phage DNA. We have compared the pharmacological activities of CMA, ICMA, and the alkylating agent, chlorambucil (CHL), in order to determine the roles of intercalation, the quinone ring, and DNA base composition, in cross-linking by CMA. CMA was 27-and 1000-fold more active than ICMA and CHL, respectively, in cross-linking DNA in L5178Y cells. In addition, the maximum level of cross-linking in L5178Y cells was reached more rapidly with CMA than with CHL, and the CMA cross-links were removed faster and more efficiently by these cells. CMA was 26- and 450-fold more active than ICMA and CHL, respectively, in producing DNA cross-links in isolated lambda-phage DNA. In contrast, the alkylating activity of CMA was only 6-fold greater than CHL, as measured by the ability of the drugs to bind to the nucleophile, p-nitrobenzyl pyridine. CMA was a better DNA intercalator than ICMA, whereas CHL did not intercalate. In addition, the intercalating agent, ethidium bromide, inhibited the cross-linking activity of CMA, but not that of CHL, suggesting that intercalation contributed to the cross-linking activity of CMA. CMA produced an increasing level of cross-linking, but showed no difference in intercalation, with isolated DNA of increasing G-C content, suggesting a preference for alkylating G-C bases. Both the cross-linking and intercalating, but not the alkylating, activities of CMA and ICMA were decreased by the reducing agent, sodium borohydride, providing additional evidence that the intercalative interaction of the ADR analogues with DNA contributes to their DNA cross-linking activity. Thus, alterations to the quinone group may effect the intercalating activity of these analogues and may contribute to the difference in cross-linking activity between CMA and ICMA.

Effect of dietary protein concentration on yield of mutagenic metabolites from 1,2-dimethylhydrazine in mice.

The effects of varying dietary protein concentrations on the metabolism of 1,2-dimethylhydrazine (DMH) to mutagenic products by male C57BL/6 X C3H F mice were assayed by in vivo and in vitro methods. DMH and its metabolite, azoxymethane (AOM), did not increase the mutation frequency of Salmonella typhimurium (strain G-46) in vitro alone or in the presence of mouse liver homogenates capable of activating the promutagen dimethylnitrosamine. Methylazoxymethanol (MAM), another metabolite of DMH, was mutagenic in vitro without activation. S.c. administration of DMH, AOM, or MAM at dosages ranging from 0.2 to 0.8 mmol/kg of body weight caused dose-dependent increases in mutations of S. typhimurium in the host-mediated assay, and molar potencies increased progressively from DMH to AOM to MAM. S.c. or i.p. injections of AOM increased host-mediated mutagenesis within 20 min, while increases in mutagenesis by DMH required at least 1 hr. When [14C]DMH was administered, [14C]azomethane was expired immediately, while 14CO2 began to appear 1 hr after DMH administration. The percentage of administered [14C]DMH expired as azomethane varied inversely with dietary protein concentration, while AOM-induced host-mediated mutagenesis was directly proportional to dietary protein (p less than 0.01). The percentage of DMH converted to mutagenic end products was limited by losses of the volatile metabolite azomethane, especially in protein-deficient mice. Greater expiration of azomethane and decreased conversion of AOM to MAM, both seen with restriction of dietary protein, were associated with a smaller body burden of DMH metabolites.

Induction of DNA strand breaks in chronic lymphocytic leukemia following treatment with 2'-deoxycoformycin in vivo and in vitro.

Four patients with refractory chronic lymphocytic leukemia were treated with the adenosine deaminase inhibitor, 2'-deoxycoformycin, and initially received 4 mg/m2 i.v. weekly. Clinical responses to therapy varied: Patient A had a minimal response; whereas Patient D showed an 85% decrease in lymphocyte count at 2 wk; and Patients B and C had intermediate responses. The pretreatment mononuclear cell adenosine deaminase activities, which ranged from 1.6 to 44.6 nmol adenosine/h/10(6) cells, decreased to approximately 1 nmol adenosine /h/10(6) cells 24 h following 2'-deoxycoformycin, and increased to 15 to 50% of the pretreatment activity prior to the second drug treatment. The clinical response to 2'-deoxycoformycin was unrelated to the pre- or posttreatment adenosine deaminase activities or to the rate of return of enzyme activities following treatment. The plasma deoxyadenosine levels and the leukemic cell dATP concentrations rose slightly with therapy, but there was no correlation between the magnitude of increase and clinical response. No significant levels of DNA strand breaks were observed in the leukemic cells following treatment, although the NAD levels decreased slightly in two patients. When peripheral mononuclear cells from the patients and two controls were incubated in vitro for 24 h with 2'-deoxycoformycin and increasing concentrations of deoxyadenosine, a concentration-dependent increase in dATP and decrease in NAD were observed in both the patients and normals. The normal cells, and cells from two patients, developed a significant number of DNA strand breaks. However, there was no relationship between the formation of DNA breaks and the degree of accumulation of dATP or depletion of NAD, or between any of these changes and subsequent clinical responses to 2'-deoxycoformycin. Based on this study, it appears that the antitumor activity of 2'-deoxycoformycin in chronic lymphocytic leukemia is unrelated to the induction of DNA strand breaks or to changes in the levels of dATP or NAD in the leukemic cells.

Biochemical changes induced in hairy-cell leukemia following treatment with the adenosine deaminase inhibitor 2'-deoxycoformycin.

The adenosine deaminase inhibitor 2'-deoxycoformycin and interferon are highly effective in the treatment of hairy-cell leukemia. In this study, a patient with type 2 hairy-cell leukemia was treated with one cycle of 2'-deoxycoformycin (4 mg/m2, i.v. weekly for 3 weeks), which was repeated at 9 wk. No toxicity was observed, and the hairy cell count fell from 72,000/mm3 to 5,000/mm3 in 3 mo, with a concomitant 50% decrease in the spleen size. The erythrocyte deoxyadenosine triphosphate content increased to 13.6 pmol/10(6) cells following the initial three weekly treatments, but there was no decrease in the adenosine triphosphate pool size and no evidence of hemolysis. The hairy cell adenosine deaminase activity was inhibited by greater than 95% 24 h following the first 2'-deoxycoformycin injection and returned to the pretreatment value at Day 8, although there was a linear decline in peripheral hairy cell count (50%) during this period. No ultrastructural changes were observed in the hairy cells following 2'-deoxycoformycin to suggest lymphocytotoxicity or cellular differentiation. The antitumor activity of 2'-deoxycoformycin could not be attributed to alterations in the hairy cell deoxyadenosine triphosphate/adenosine triphosphate levels or to the induction of DNA strand breaks. Additionally, the plasma levels of interferon did not change during therapy, making it unlikely that 2'-deoxycoformycin exerts its activity by inducing endogenous interferon synthesis.

Lysosomotropic agents selectively target chronic lymphocytic leukemia cells due to altered sphingolipid metabolism.

Lysosome membrane permeabilization (LMP) mediates cell death in a variety of cancer cells. However, little is known about lysosomes and LMP in chronic lymphocytic leukemia (CLL). Owing to drug resistance and toxicity in CLL patients, better treatment strategies are required. Our results show that CLL cells were sensitive to the lysosomotropic agent siramesine. Furthermore, this drug was more effective in CLL cells, regardless of prognostic factors, compared with normal B cells. Siramesine caused LMP, lipid peroxidation and transcription factor EB nuclear translocation followed by mitochondrial membrane potential loss and reactive oxygen species release. Siramesine-induced cell death was blocked by lipid antioxidants, but not by soluble antioxidants or protease inhibitors. To determine whether CLL cells had altered lysosomes, we investigated sphingolipid metabolism as the lysosome is a hub for lipid metabolism. We found that CLL cells had more lysosomes, increased sphingosine-1-phosphate phosphatase 1 (SPP1) expression, and increased levels of sphingosine compared with normal B cells. Raising sphingosine levels increased LMP and cell death in CLL cells, but not in normal B cells. Together, these results show that excess sphingosine in CLL cells could contribute to their sensitivity toward LMP. Thus, targeting the lysosome could be a novel therapeutic strategy in CLL.

Chlorambucil induced apoptosis in chronic lymphocytic leukemia (CLL) and its relationship to clinical efficacy.

Chlorambucil induced apoptosis was measured in CLL cells treated with clinically achievable drug doses in vitro. While spontaneous apoptosis occurred in CLL cells incubated in vitro in the absence of drug, the level of apoptosis, as measured by the extent of DNA fragmentation, was greater in cells treated with chlorambucil. In addition, macrophages were shown to engulf drug treated CLL cells in vitro. To determine if chlorambucil can also induce apoptosis in vivo, CLL cells were isolated from patients before treatment and at intervals after clinical therapy with chlorambucil (0.9 mg/kg given over 3 days). Apoptosis was measured in these cells immediately after isolation and following incubation in vitro for 72 hr. No apoptotic changes were detected in cells immediately after isolation either before or after clinical treatment. In contrast, apoptosis was observed in cells that were incubated ex vivo for 72 hr, and the level of apoptosis was greater in cells that were isolated after chlorambucil treatment compared with cells obtained prior to therapy. The increased apoptosis observed in CLL cells ex vivo after therapy was related to the fall in the patient's lymphocyte count. In general, a large increase in apoptosis ex vivo after treatment was followed by a significant decrease in the patient's lymphocyte count. Thus, chlorambucil may produce its antitumor effect in CLL by inducing apoptosis-associated membrane changes that result in rapid clearance of the apoptotic cells by the immune system.

Combination therapy with nucleoside analogs and alkylating agents.

The nucleoside analog, 2'-deoxycoformycin (dCF), and the alkylating agents, chlorambucil (CLB) and cyclophosphamide, are effective agents in the treatment of chronic B cell leukemias and lymphomas. The cyclophosphamide analog, 4-hydroperoxycyclophosphamide (4-HC), generates the same active metabolite as cyclophosphamide in cells and has been used extensively for bone marrow purging in vitro. We have observed that deoxyadenosine (dAdo) plus dCF (dAdo/dCF) inhibit the repair of x-irradiation-induced and bleomycin-induced DNA damage in vitro, and that this results in either synergistic or additive cytotoxicity, respectively. In the present study we examined whether dAdo/dCF, can enhance the antitumor activity of CLB and 4-HC in chronic lymphocytic leukemia (CLL) cells in vitro. CLL cells were treated with CLB for 6 hr and then with dAdo/dCF for 18 hr and cytotoxicity was measured by the MTT assay. Synergy was observed between CLB and dAdo/dCF in CLL cells from 2 patients, with synergy increasing as the CLB dose was raised. In contrast, similar treatment of human bone marrow cells resulted in little or no synergistic cell kill. Treatment of CLL cells from 2 patients with 4-HC for 30 min followed by dAdo/dCF for 18 hr resulted in little synergistic cytotoxicity, although this drug combination did produce an additive cell kill. Thus, combination therapy with nucleoside analogs and alkylating agents may be useful for improving treatment of CLL.

In vitro cytotoxicity of 2-chlorodeoxyadenosine and chlorambucil in chronic lymphocytic leukemia.

Chronic lymphocytic leukemia (CLL) is most commonly treated with the alkylating agent chlorambucil (CLB), although the nucleoside analogs, fludarabine (Flu) and 2-chlorodeoxyadenosine (CdA), are also effective in this disease. In this study, we investigated the in vitro cytotoxicity of CdA and CLB in CLL cells from 12 patients in vitro. Treatment with CLB for 6 h, followed by CdA for 18 h, resulted in 2.3- to 7.5-fold synergistic cytotoxicity in leukemic cells from 10 patients and an additive effect in cells from two patients. In general, synergy was greatest in patients who were sensitive to CLB or CdA, and could be enhanced by increasing the concentrations of CLB or CdA. Synergy was only observed if the cells were treated with CLB prior to CdA. Synergy could not be explained by an increase in the incorporation of CdA into DNA, or by the inhibition of repair of CLB-induced DNA crosslinks by CdA. In contrast to CLL cells, treatment of human marrow in vitro with CLB and CdA resulted in a low level of synergy for CFU-GM cells, and additive cell kill in erythroid progenitors. Thus, treatment with CdA and CLB can produce selective synergistic cell kill in CLL cells, and combination therapy may improve the therapeutic index of these agents in chemosensitive patients.