ALBERT

Description: Fludarabine melphalan conditioning has been used widely for conditioning of pts with hematologic malignancies. When combined with in vivo alemtuzumab, this regimen leads to reliable engraftment, minimal acute and chronic GVHD, and low early transplant-related mortality (TRM). Between 2002 and 2004, we conducted a prospective study in hematologic malignancies. Here we report outcomes in 55 patients with high-risk myeloid malignancies. Median age was 54 (range 17–71); 17 were 60 and older. 28 had HLA-identical related donors, 23 had MUD donors, and 4 had 1 Ag-MM related donors. 32 pts had high leukemia burden at transplant (24 active AML, 6 MDS with 〉5% blasts, 1 CML-BC, 1 myelofibrosis in transformation). 23 pts had low leukemia burden at transplant (4 AML in CR1 with adverse cytogenetics, 2 AML CR1 with WBC〉100K, 1 AML CR1 requiring 2 inductions, 6 AML CR2, 1 AML CR3, 4 MDS 0 in 20 pts. Many had other high risk features including 15 with prior transplant and 10 with secondary leukemia. Conditioning consisted of fludarabine-melphalan-alemtuzumab (as per Smith, Blood 2002, abstract 5292), with tacrolimus for GVHD prophylaxis until day 100. Stringent CMV prophylaxis with high dose valacyclovir was given for 180 days. There were two early graft rejections (both from1 Ag-MM related donors) and both pts died of aplasia. There were also 2 deaths during conditioning. Including these 4 pts, cumulative day 100 TRM is 17% (95% CI 7–27). Median follow-up for survivors is 14 months (range 2–30). Estimated one year survival is 53% (95% CI 39–67) and one year PFS is 44% (95% CI 30–68). Donor type did not affect survival. Age 〉 55 (HR 2; p= 0.04), PS 〉 0 (HR 2.4; p= 0.001) and high leukemia burden (HR 7.7; p=0.0001) were associated with decreased survival in univariate analysis. 20 of 23 pts with low leukemia burden remain alive, 18 in ongoing remission. By contrast, one year survival for pts with high leukemia burden is 35 % (figure). Only 1 case of grade III–IV acute GVHD was observed and extensive chronic GVHD occurred in 3 pts. CMV reactivation was common, but documented CMV pneumonia occurred in only one patient. Fludarabine melphalan alemtuzumab conditioning results in excellent long-term outcome for patients with high-risk myeloid malignancies and low leukemia burden, with a very low incidence of acute and chronic GVHD. In these disorders, GVHD is not a prerequisite for achievement of durable CR. For pts with high leukemia burden, therapy provided mainly palliative responses with a low incidence of GVHD. Further improvements are needed to reduce recurrence rate for pts with high leukemia burden. Figure Figure

Description: The chromosomal translocation (8;21) fuses the AML1 gene on chromosome 21 and the ETO gene on chromosome 8 in human acute myeloid leukemias, resulting in expression of the chimeric transcription factor AML1/ETO. AML1/ETO-mediated dysregulation of target genes critical for hematopoietic differentiation and proliferation is thought to contribute to the leukemic phenotype. Several mechanisms, including recruitment of histone deacetylases (HDACs) to AML1 target genes, may be responsible for altered gene expression. We used an ecdysone-inducible expression system in the human monoblastic U-937 cell line to isolate genes that were differentially expressed upon induction of AML1/ETO expression. By representational difference analysis (cDNA-RDA), we identified 26 genes whose expression levels were significantly modulated following AML1/ETO induction for 48 hours. None of these genes has previously been described as a target of AML1, ETO or AML1/ETO. One gene down-regulated by AML1/ETO in vitro, Williams Beuren Syndrome critical region 5 (WBSCR5), was expressed in primary t(8;21) negative AML blasts but not in primary t(8;21) positive AML blasts, strongly implying a role of this gene in the phenotype of t(8;21) positive AML. WBSCR5 is part of the critical region located on chromosome 7q11.23 that is deleted in the Williams Beuren syndrome (OMIM 194050), an autosomal dominant disorder comprising vascular, neurological, behavioral and skeletal abnormalities. WBSCR5 has recently been shown to have a role in the activation and differentiation of B cells (Brdicka et al., J. Exp. Med. 196:1617, 2002) and thus was also termed Non-T cell activation linker.. WBSCR5 as well as seven other regulated genes were further studied using all-trans-retinoic acid (ATRA), an inducer of differentiation of U-937 cells, and Trichostatin A (TSA), an HDAC inhibitor. WBSCR5 and two other out of these eight genes were regulated during ATRA-induced monocytic differentiation of U-937 cells, however none of them antagonistically, upon both ATRA-treatment and AML1/ETO-induction. Since repression of WBSCR5 might be mediated by recruitment of HDACs through the fusion gene, cells were treated with TSA prior to transgene induction. However, the AML1/ETO-associated dysregulation of WBSCR5 gene expression (as well as that of the other seven genes studied) was not mediated by a TSA-sensitive mechanism. The identified genes provide a useful model to study the mechanism by which the AML1/ETO fusion protein exerts its function in transcriptional dysregulation in acute myeloid leukemia. The role of WBSCR5 in malignant hematopoietic cells warrants further study.

Description: Recruitment of histone deacetylases and DNA hypermethylation of promoter regions of specific genes are two mechanisms of transcriptional repression and gene silencing which have been linked, and are implicated in differentiation block in AML. We hypothesized that the histone deacetylase inhibitor (HDI) depsipeptide could result in transcriptional de-repression, upregulation of specific target genes and differentiation of the leukemic clone in AML. Eighteen patients (pts), median age 60 years (range 25–77) with relapsed or refractory AML were enrolled on a multicenter Phase II study of depsipeptide in AML. Patients were stratified into 2 groups on study entry: Group A (n=14) included patients without specific chromosomal abnormalities known to recruit histone deacetylases. Group B (n=4) included patients with chromosomal aberrations such as the t(8;21), inv 16 and t(15;17) known to recruit histone deacetylases. Depsipeptide was administered intravenously at a dose of 18mg/m2/d on days 1, 8 and 15 of a 28 day cycle. Peripheral blood mononuclear cells were obtained prior to (hour 0), and after 4 (hr 4) and 24 hrs (hr 24), on days 1 and 8 of the first cycle of therapy for evaluation of histone acetylation by flow cytometry, and gene re-expression by REAL-time RT-PCR. Target genes of interest include MDR1, a target of HDI mediated upregulation, and p15INK4B (p15), a target of DNA hypermethylation in AML. MDR1 and p15 copy numbers are expressed as a normalized quotient of MDR1 and p15, respectively, to the housekeeping gene ABL. The drug has been well tolerated. The most common adverse effects noted included grade 1/2 nausea, vomiting and fatigue. No objective evidence of response (CR or PR) or other evidence of antileukemic activity has been seen in group A. In contrast, 2 of 4 pts (50%) in Group B, have had a disappearance of bone marrow blasts (blast percentage 〈 5%) in the setting of a normocellular marrow, with concomitant recovery of near-normal hematopoiesis following 1 and 2 cycles of therapy respectively. This anti-leukemic effect was short-lived, with both pts developing an increase in bone marrow blasts within 30 days of the initial response. Both of these patients also had translocations involving the AML1 gene {1 had t(8;21) and the other had a novel translocation t(4;21)}. Interestingly both of these responding pts and one other pt (75%) in cohort B demonstrated an increase in H3 acetylation at 4 and/or 24 hrs, in contrast to 4 of 14 pts (28%) in cohort A. There was an overall mean increase of 41% in MDR1 expression at hr 4 on days 1 and 8 (p=0.04). p15 expression was also upregulated at hr 4 on days 1 and 8 (91% mean increase, p=0.01). We conclude that the HDI, depsipeptide, may have anti-leukemic activity in specific cytogenetic subsets of AML known to recruit histone deacetylases, and this is associated with a concomitant increase in histone acetylation. In addition, upregulation of specific target genes occurred in patient derived mononuclear cells, following depsipeptide treatment. The study remains open to accrual for pts with specific chromosomal abnormalities known to recruit histone deacetylases.

Description: Recent studies suggest that detection of subclinical, or minimal residual disease (MRD) in apheresis products used for autografting correlates with poor disease-free survival following ASCT for mantle cell lymphoma (MCL). To validate this observation and to gain insights into the kinetics of MRD during treatment, we are performing a prospective analysis of MRD using quantitative real-time PCR (Q-PCR) in patients (pts) undergoing treatment for MCL on a CALGB study (59909). Q-PCR of sequential paired bone marrow (BM) and blood (B) samples and of apheresis products was performed using either a patient-specific immunoglobulin heavy chain (IgH) or BCL-1 gene rearrangement. All samples were analyzed in triplicate using LightCycler technology and reported as a normalized ratio of IgH or BCL-1 copy number to GAPDH copy number. The sensitivity of the assay ranged from 1 X 104–1 X 105. To date, a clonal IgH or BCL-1 gene rearrangement was detected in 36 of 41 (88%) pts entered on study. Patient-specific primers and consensus probes were used for Q-PCR monitoring of MRD following two courses of intensive induction therapy, during stem cell mobilization, and 3 and 12 months after ASCT and post-transplant immunotherapy with Rituximab (R). 27 pts have completed all protocol treatment with a median follow-up of 7 months (range: 0–28). 26 pts were evaluated for MRD following two courses of induction therapy with cyclophosphamide, methotrexate, doxorubicin, vincristine, prednisone, and R. 10 of 26 became MRD negative (−) following induction while 16 remained MRD positive (+). Following mobilization with high-dose cytarabine, etoposide and R, apheresis products from 9 of 10 MRD- pts were evaluated and all products were MRD- (1 pt not evaluable). Of the 16 pts who were MRD+ prior to mobilization therapy, MRD- apheresis products were collected in 8; 5 had MRD+ stem cell collections, and 3 were not evaluable. In total, apheresis products were evaluable in 22 of 26 pts; 17 (77%) had MRD- stem cells collected prior to ASCT. None of these MRD- pts has relapsed to date although 2 pts with MRD- products became weakly MRD+ 12 months following ASCT and R. Of the 5 pts with MRD+ apheresis collections, 4 have remained persistently MRD+ following ASCT and R; 1 has relapsed 19 months after completion of all treatment. Rising levels of MRD in BM and B samples were noted in this patient 3 and 12 months post-ASCT. Statistical comparison of MRD values in paired BM and B samples prior to, and post-ASCT demonstrated good agreement with an intraclass correlation coefficient of.814 and.777, respectively. In conclusion, our results demonstrate that prospective MRD monitoring using Q-PCR provides important insights into the kinetics of response during treatment of MCL. MRD- apheresis products were collected in the majority of pts following intensive induction and mobilization chemo-immunotherapy with R on CALGB 59909. To date, no pts with MRD- apheresis products have relapsed following ASCT. In contrast, it appears that pts with MRD+ apheresis products remain MRD+ following ASCT and R and may be more likely to relapse. Longer clinical follow-up is needed to clarify the significance of the persistence of MRD in apheresis products and following ASCT for MCL.

Description: Patients with relapsed AML over the age of 60 have a poor prognosis. Gemtuzumab ozogamicin (GO) has been approved for older pts in first relapse, although many pts who attain complete remission (CR) do not fully recover normal platelet count (so-called CRp). In vitro studies have shown that oblimersen down-regulates Bcl-2 in AML cells and enhances apoptotic cell death induced by GO. We conducted a Phase 2 study to evaluate the safety and efficacy of GO combined with oblimersen for older pts with AML. Eligibility requirements included: age ≥ 60 yrs; AML in 1st relapse; ≥ 3 mos 1st CR duration; ≥ 25% CD33-positive AML cells. Pts received oblimersen at a dose of 7 mg/kg/d for 7 days by CIV beginning on days 1 and 15; GO was given at a dose of 9 mg/m2 IV over 2 hrs on days 4 and 18. A total of 48 pts were enrolled (ITT population), all of whom received at least 1 dose of oblimersen; 9 pts failed to receive the required 2 doses of GO (per-protocol population, n=39). The median age was 67 (range, 59 to 88 yrs). Duration of 1st CR: 〈 6 mos: 7 pts; (15%); 6 to 12 mos: 29 pts (60%); 〉 12 mos: 12 pts (25%). No. of prior regimens: 1 (17 pts, 35%); 2 or 3 (26 pts, 54%); ≥ 4 (5 pts, 10%). Among treated pts, 79% completed 21 days of protocol therapy. Overall, 12 pts achieved a major response, either CR (n=5) or CRp (n=7), for an ITT response rate of 25% and a per-protocol response rate of 31%. The median time to remission was 52 days. Ten of the 12 responders survived 〉 6 mos, whereas only 6 non-responders survived ≥ 6 mos. Serious adverse events for the oblimersen/GO combination were qualitatively similar to those reported for GO alone and included among other reactions: Grade 3-4 febrile neutropenia (42%) or thrombocytopenia 33%; nausea; fever; rigors, and dyspnea. Treatment-emergent adverse reactions led to discontinuation of protocol therapy in 10 pts (21%). The most common serious adverse event was febrile neutropenia (25%). One pt (2.1%) died during treatment (sepsis) and 16 pts (33%) died within 30 days of last study medication (infection, bleeding, respiratory failure, progressive AML, and other disease-related complications). No episodes of VOD were observed. Oblimersen can be safely combined with GO; however, pts enrolled in this study appear to have had more unfavorable characteristics at entry compared with prior studies using GO alone in pts with relapsed AML. Therefore, assessment of an incremental benefit from the addition of oblimersen will require a randomized trial.

Description: Reduced intensity conditioning regimens and improvements in supportive care have allowed for the expansion of allogeneic stem cell transplantation to those previously ineligble. However, such patients remain at significant risk for regimen related toxicity. We examined whether clinical predictors that influence toxicity after standard chemotherapy, such as age, comorbidity and functional status similarly predict increased transplant related mortality (TRM) and decreased survival after a RIST with in-vivo T-cell depletion. We analyzed 81 consecutive patients on a single protocol with high-risk and refractory hematologic malignancies transplanted at our institution from 2002 to 2004. The conditioning regimen consisted of fludarabine 30 mg/m2/d (D-7 to D-3), Campath-1H 20 mg/d (D-7 to D-3), and melphalan 140 mg/m2 (D-2) with tacrolimus given for post-transplant immunosuppression. Comorbidity was scored from a retrospective chart review using the Charlson Comorbidity Index (CC) and the Kaplan-Feinstein scale (KF). Eastern Cooperative Oncology Group performance status (PS) and age at transplant were tabulated. TRM was defined as any death occurring without disease progression. Median age was 51 years (range 17–68). Fifty-five percent had HLA-identical sibling donors, 5% had mismatched related donors, 35% had matched unrelated donors and 5% had mismatched unrelated donors. KF served as a more sensitive indicator of comorbidity than CC. Fifty-three percent of patients scored at least one point for a comorbid condition by KF, as opposed to 25% by CC (P0 0.53 Comorbidity** CC, no comorbidity 0.76 2.3 0.042 0.089 2.25 0.014 0.03 CC, ≥1 comorbidity 0.52 KF, no comorbidity 0.85 2.7 0.027 0.183 2.74 0.004 0.03 KF, ≥1 comorbidity 0.56 Age Age50 0.63