Pretransplant Conditioning Therapy Research Articles

Clofarabine (Clo) +/− Fludarabine (Flu) with IV Busulfan (Bu) and Allogeneic Stem Cell Transplantation (Allo-SCT) for Relapsed, Refractory Myeloid Leukemia (ML) and MDS

Fludarabine (Flu) combined with IV Busulfan (Bu) is a safe, reduced toxicity pretransplant conditioning program. To enhance the cytoreductive effect of the conditioning regimen, we decided to replace Flu with Clofarabine (Clo), a (superior) second generation nucleoside analog/antileukemic agent, whose immunosuppressive capability in support of allogeneic progenitor cell engraftment is unknown. In 2006 we initiated a trial where Clo would gradually replace Flu combined with IV Bu in a myeloablative program, aimed atretaining the safety profile, whileevaluating the engraftment-promoting capacity, and hopefullyimproving the antileukemic activity.METHODS and PATIENTS: Patients were enrolled/randomized as follows: Arm I: Clo:Flu 10:30 mg/m2, Arm II 20:20 mg/m2, Arm III 30:10 mg/m2, and Arm IV single-agent Clo at 40 mg/m2, the nucleoside analog(s) were/was infused over 60 min each once daily for 4 days on each day followed by Bu infused over 3 hours to an average daily AUC of 6,000 mcMol-min +/− 10%. So far, 20 patients have been enrolled, 12 males and 8 females, with a median age of 43 years (range: 6–57). Seven patients had CML (BC: 1, first AP: 3, second AP: 2, and late first CP: 1). Thirteen patients had AML: 5 with induction failure, 3 were in chemotherapy-refractory relapse, 3 in untreated relapse, 1 had a second PR, and, finally 1 was in a high-risk CR1 [cytogenetics of −7 and t(3;12)]. Of the 13 AML patients 1 had a favorable cytogenetic (CG) pattern, 6 were intermediate, and 4 had poor prognosis CG, while 2 had unknown CG data. GVHD-prophylaxis was based on tacrolimus and mini-MTX, and those who had an unrelated or one antig-mism. related donor received rabbit-ATG (Thymoglobulin™).RESULTS: One patient died of pneumonia, there was no other treatment-related death in the first 100 days, the main toxicity was mucositis grade 2–3 in about half the patients. Specifically there was no significant hepatic or neurologic toxicity. All 19 evaluable patients engrafted uneventfully (one had recurrent AML before day +30). Marrow and blood (T-cell) chimerism studies at day +30 revealed that groups I+II (lower Clo doses; n=9) had a median of 87% (range 17–100) donor (T-cell)-derived DNA, groups III+IV (higher Clo-doses, n=11) had a median of 100% range (64–100) donor-DNA. By day +100 both cohorts had a median of 100% donor-derived DNA, maintained beyond 6 months in all evaluable patients. 16 of the 19 patients with active disease at SCT entered CR, and 11 patients remain in clinical CR (August 2008) with a median duration of 8 mos (range, 2–19). One previously TKI-resistant CML patient (SCT in second AP) progressed by CG at 4 mos, but responded to dasatinib, with a maintained CG CR exceeding 22 mos. Median disease-free and overall survival has not been reached with 11 patients in unmaintained CR, and 12/20 being alive. The projected overall and disease-free survival at one year of 70% and 55%, respectively.CONCLUSION: We conclude that a Clo-Bu-based regimen appears safe in (relapsed, chemotherapy-refractory) high-risk patients, 2) there should be little or no concern about the immunosuppressive capability of Clo in support of allo-SCT in myeloid leukemia, and 3) (an) additional study(-ies) is/(are) warranted to fully evaluate the antileukemic efficacy of Clo combined with IV Bu + ATG as pretransplant conditioning therapy for myeloid leukemia and MDS.

Pre-Transplant Rituximab Therapy Is Associated with Improved Progression-Free and Overall Survival in Patients Undergoing Autologous Hematopoietic Stem Cell Transplantation for Diffuse Large B-Cell Lymphoma (DLBCL).

Outcomes following first-line therapy for patients (pts) with DLBCL have improved significantly with the availability of the chimeric anti-CD20 monoclonal antibody rituximab (R). Despite this progress, many pts develop refractory or recurrent DLBCL and are considered candidates for autologous hematopoietic stem cell transplantation (AuHCT). For such pts, it is possible that R given pre-transplant and/or during conditioning therapy affects the natural history of DLBCL, such that traditional methods of risk assessment and patient selection for AuHCT may need revision. We therefore studied the outcomes of 1,006 pts who underwent peripheral blood AuHCT for DLBCL between 1996 and 2003 reported to the CIBMTR, analyzed according to whether R was (n=188, “+R” group) or was not (n=818, “-R” group) administered prior to AuHCT. Using the chi-square statistic for categorical and the Kruskal-Wallis for continuous variables, there were no significant differences between the +R and -R groups with regard to gender, pre-transplant performance status, disease status at transplant, pre-transplant chemosensitivity, second-line aa-IPI score distribution, Ann Arbor stage at transplant, interval from diagnosis to transplant, bulky disease, bone marrow involvement, post-transplant radiation therapy, or post-transplant myeloid growth factor therapy. The +R group had a higher proportion of pts age 61 or older (40% vs. 23%, p<0.001). AuHCT occurred between 1999–2003 in 96% of pts in the +R group, and between 1996–2001 in 93% of the -R pts (p<0.001). For the +R pts, 94% received R only with pretransplant chemotherapy, 3% only with conditioning therapy, and 3% with both pretransplant chemotherapy and conditioning therapy. Conditioning regimens were similar in the +R and -R groups, with the majority receiving the BEAM regimen. In univariate analysis, platelet and neutrophil engraftment were not affected by use of R. Treatment-related mortality (TRM) at 1, 3, or 5 years did not differ significantly between the +R and -R groups. Progression-free survival (PFS) at 1 and 3 years was superior in the +R group (62% vs. 49% at 1 year, p=0.002; 49% vs. 38% at 3 years, p=0.010). Overall survival (OS) was superior in the +R group (68% vs. 60% at 1 year, p=0.032; 57% vs. 45% at 3 years, p=0.003). In multivariate analysis, later year of transplant (2000–2003) and age <55 were associated with lower TRM, but pre-transplant R was not. Conversely, pre-transplant R, age <55, and fewer than 3 lines of chemotherapy were associated with improved PFS, while year of transplant was not. Finally, pre-transplant R, age <55, fewer than 3 lines of chemotherapy, and year of transplant 2000–2003, were all associated with improved OS. We conclude that pre-transplant rituximab is associated with improved PFS and OS following AuHCT for DLBCL, with no evidence of impaired engraftment or increased TRM.

Anti-Apoptotic Proteins, HSP90 and Activated STAT3 Contribute to Busulfan Resistance of Myeloid Leukemia Cells.

Busulfan(Bu)-based chemotherapy is a conditioning treatment prior to hematopoietic stem cell transplantation (HSCT) of patients with acute and chronic myelogenous leukemia (AML, CML). A major obstacle to successful HSCT is Bu resistance, which might be attributed to individual differences in drug pharmacokinetics and metabolism, or inherent resistance of cancer cells. We hypothesize that gene expression profiling of leukemia cells can be used to dissect the factors that contribute to their Bu resistance. Two Bu-resistant leukemia cell lines were established, characterized and analyzed by microarray and real-time RT-PCR techniques to identify differentially expressed genes. The CML B5/Bu2506 cells are 4.5-fold more resistant to Bu than their parental B5 cells. The AML KBM3/Bu2506 cells are 4.0-fold more Bu-resistant than KBM3 parental cells. Both resistant sublines evade Bu-mediated G2-arrest and apoptosis with constitutively up-regulated anti-apoptotic genes (BCL-XL, BCL2, BCL2L10, BAG3 and IAP2/BIRC3) and down-regulated pro-apoptotic genes (BIK, BNIP3, and LTBR).). Bu-induced apoptosis is partly mediated by activation of caspases; use of the inhibitor Z-VAD-FMK completely abrogated PARP1 cleavage and reduced apoptosis by ∼ 50%. Furthermore, Bu resistance in these cells may be attributed in part to up-regulation of HSP90 protein and activation of STAT3. Inhibition of HSP90 with geldanamycin attenuated phosphorylated STAT3 and made B5/Bu2506 and KBM3/Bu2506 more Bu-sensitive. Analysis of cells derived from patients classified as either clinically resistant or sensitive to high-dose Bu-based chemotherapy had alterations in gene expression that were analogous to those observed in the in-vitro model cell lines, confirming the potential clinical relevance of this model for Bu resistance. Our results suggest the important roles of apoptotic signaling mechanism, HSP90 and STAT3 and should be considered in the classification of AML patients who will likely benefit from busulfan-based pretransplant conditioning therapy and those who should be offered alternative treatment modalities.

IV Busulfan (Bu) with Fludarabine (Flu) or Cyclophosphamide (Cy) - Comparing Ablative Conditioning Regimens for Allogeneic Transplantation in AML/MDS.

As part of our evaluation of Flu as immunosuppressive (and DNA damage repair inhibiting) agent, and as an alternative to Cy in combination with IV Bu, preparing AML/MDS patients for allogeneic transplant (AlloSCT), we postulated that the IV Bu-Flu regimen would yield improved safety, yet increased antileukemic efficacy. We now compared two consecutive series of patients receiving pretransplant conditioning therapy with either IV Bu-Flu or IV BuCy2.Methods: Flu 40 mg/m2 IV was given once daily, each dose followed immediately by Bu 130 mg/m2 IV over 3 hr (days −6 till −3) (n=128 patients), and in the IV BuCy2 the Bu was given at 0.8 mg/kg over 2 hr every 6 hr for 16 doses (days −7 till −4), followed by Cy 60 mg/kg on days −3 and −2 (n=73 patients). Patients with an unrelated (MUD) or mismatched donor received ATG on days −3 to −1. Graft-versus-host disease (GVHD) prophylaxis was based on tacrolimus and mini-methotrexate.Patients: Median age was 46 (19–66) (IV Bu-Flu group) and 39 (13–64) (IV BuCy2 group) years, respectively. At transplant 45% and 46% patients were in CR1–3, the remainder had active disease. Cytogenetic risk categories were equivalent for the two groups, with poor or intermediate risk in 88 % in the IV Bu-Flu, and in 82 % of the BuCy2group. Donors were HLA-identical siblings (SIBS) in 53 %, and 73%, MUDs in 39% and 15 %, others in 8 % and 12% respectively. Median follow-up for patients still alive (August 2004) was 15 mos (3–37; n=78) for the IV Bu-Flu group, and 45 mos (24–76; n=27)) for the IV BuCy2 patients.Results: Both regimens were well tolerated; 100-day treatment-related mortality (TRM) was for SIB/MUD 2.7 / 2% after IV Bu-Flu, and 7 / 9% in the IV BuCy2 group, respectively. Overall, one-year TRM was for 7% after IV Bu-Flu and 18% after IV BuCy2. The overall aGvHD rates were 42% and 45%. 2-year overall survival (OS) were 50% for the IV Bu-Flu group, 42% after IV BuCy2. For patients transplanted in CR the 2-yr OS and event-free survival were 69% and 68 % after IV Bu-Flu, and 48% and 41% in the IV BuCy2.We conclude, that IV Bu-Flu is a safe and highly effective, reduced toxicity myeloablative conditioning regimen for AML/MDS. Its antileukemic efficacy appears to be at least equal to what is experienced with the IV BuCy2 regimen. Thus, our data demonstrate the IV Bu-Flu regimen to be a new standard for myeloablative conditioning therapy for alloSCT in AML/MDS. [Display omitted]

Genome-wide analysis of epigenetically silenced genes in prostate cancer

Busulfan (Bu) is a DNA-alkylating drug used in myeloablative pretransplant conditioning therapy for patients with myeloid leukemia (ML). A major obstacle to successful treatment is cellular Bu-resistance. To investigate the possible contribution of DNA hypermethylation to Bu-resistance, we examined the cytotoxic activity of combined 5-aza-2′-deoxycytidine (DAC) and Bu. Exposure of Bu-resistant B5/Bu2506 ML cells to 0.5 μM DAC resulted in G2-arrest and apoptosis. The observed G2-arrest was associated with hypomethylation and subsequent expression of epigenetically controlled genes including p16INK4A, activation of the p53 pathway, and phosphorylation of CDC2. The DAC-mediated apoptosis was partly due to hypomethylation and up-regulation of XAF1, which resulted in down-regulation of the anti-apoptotic proteins XIAP, cIAP1 and cIAP2. The pro-apoptotic PUMA and BNIP3 proteins were up-regulated while pro-survival STAT3 and c-MYC were suppressed. Combination of 0.05 μM DAC and 5 μg/ml Bu resulted in synergistic cytotoxicity, which was associated with PARP1 cleavage and activation of caspases 3 and 8, suggesting induction of an apoptotic response. P53 inhibition in B5/Bu2506 cells using pifithrin-α alleviated these effects, suggesting a role for p53 therein; this observation was supported by the relative resistance of p53-null K562 cells to [DAC + Bu] combinations and by the effects of an anti-p53 shRNA on the OCI-AML3 cell line. We conclude that the synergistic effects of [DAC + Bu] are p53-dependent and involve cell cycle arrest, apoptosis induction and down-regulation of pro-survival genes. Our results suggest that, depending on tumor p53 status, incorporation of DAC might synergistically improve the cytoreductive efficacy of Bu-based pretransplant regimen in patients with ML.

Extending the role of allogeneic stem-cell transplantion

Allogeneic bone-marrow transplantation (allo-BMT) permits the administration of high-dose chemotherapy and radiotherapy for tumour eradication. When followed by infusion of normal donor haemopoietic cells and posttransplant immunosuppression to prevent graft rejection, allo-BMT is curative for a wide variety of haematological cancers, including acute and chronic myeloid leukaemia (CML). The success of allo-BMT is not entirely dependent on the administration of myeloablative pretransplant conditioning therapy to eliminate residual tumour cells and allow engraftment.

Allogeneic stem cell transplantation (BMT) for AML and MDS following i.v. busulfan and cyclophosphamide (i.v. BuCy).

Pretransplant conditioning therapy with i.v. BuCy followed by allogeneic hematopoietic stem cell transplantation (BMT) was investigated in a phase II trial in patients with acute myeloid leukemia (AML) or myelodysplastic syndrome (MDS). We gave i.v. Bu at a dose of 0.8 mg/kg every 6h x 16 doses, followed by Cy 60 mg/kg daily for 2 days. Twenty-six AML patients (18 males/eight females) were treated, only eight of whom were in CR1. The rest were either refractory to induction chemotherapy (four patients) or in a more advanced stage of their disease (14 patients). In addition, nine patients with MDS (1M/8F) were treated. Their median age was 41 years (range 21-64). Engraftment to > or =500 neutrophils/microl was reached at 14 days (range 10-29 days) post BMT, and the median time of neutropenia was only 11 days (range 4-28 days). The most common regimen-related toxicity was grade 2-3 nausea. In the post-BMT period (including BMT day +30), two patients died, one each from pulmonary hemorrhage secondary to CMV pneumonia and hepatic veno-occlusive disease (VOD), for an early treatment-related mortality (TRM) of 5.7%. Three patients developed VOD and two of them died. There was no direct regimen-related pulmonary or neurologic toxicity. Overall, the clinical side-effect spectrum was analogous to what would be expected from a high-dose oral Bu-based regimen; there was no unique toxicity experienced with the used solvent system. The disease-free survival in the high-risk subgroup (all patients not in CR1) at 1 and 2 years post transplant was 44% and 31%, respectively. The 13 patients still alive in CR have been followed for a median of 24 months (range 18-32). Pharmacokinetic analysis showed very good interdose reproducibility, and limited interpatient variability in area under the plasma concentration vs time curve, peak concentration, and clearance of Bu after this i.v. formulation. We conclude, that this new i.v. Bu formulation is well tolerated; it has an impressive safety profile, and we suggest that it should be considered as appropriate replacement for oral busulfan in pretransplant conditioning therapy prior to allogeneic BMT for patients with AML or MDS.

Diagnosis of invasive aspergillosis in bone marrow transplant recipients by polymerase chain reaction.

A nested polymerase chain reaction (PCR) test targeting Aspergillus spp. large ribosomal subunit genes was evaluated retrospectively on 175 serum samples from 37 bone marrow transplant recipients, 70% of whom received grafts from unrelated donors. Six patients had proven infection, seven had probable infection, and three had possible infection, using the revised EORTC case definitions. These 16 patients were all PCR positive (57 out of 93 samples tested). Two additional patients who did not fulfil current diagnostic criteria, but in whom invasive aspergillosis (IA) was thought clinically probable, were also PCR positive (five out of nine samples). Invasive aspergillosis was unlikely in the remaining 19 patients, four of whom were PCR positive on a single occasion (four out of 70 samples). Three samples were inhibitory to PCR. Sensitivity of PCR in diagnosing patients with IA was 100%, specificity was 79% and positive predictive value was 80%, using the criterion of a single positive result. If two positive results were required, these values were 81%, 100% and 100% respectively. The median duration of infection documented by PCR was 36 days (range 3-248 days) in 17 out of 18 patients (94%) who did not survive. Positive PCR results predated the institution of antifungal therapy in two-thirds of patients. Four patients became PCR positive during pretransplant conditioning therapy.

Caries-related salivary microorganisms and salivary flow rate in bone marrow recipients

Cancer treatments often induce oral complications. In this study we investigate longitudinally the salivary gland function, the salivary caries-related microorganisms, and buffer capacity in bone marrow recipients. Stimulated saliva samples were taken midmorning. The salivary factors were studied in 42 patients from before transplant until 4 months after transplant. A dramatic reduction (66%) of salivary flow rate is noticed in all patients at 1 month after transplant, and only a partial recovery (42% reduction) is seen after 4 months. A clear shift toward a lower buffer capacity and a higher amount of cariogenic microorganisms is seen posttransplant. This shift is more pronounced when total body irradiation was included in the pretransplant conditioning therapy. These findings indicate that the studied parameters in transplant recipients can contribute to a higher caries risk and oral complications during the early posttransplant period.