ALBERT

Description: One advantage of the use of peripheral blood stem cells (PBSCs) over autologous bone marrow would be a reduced risk of tumor cell contamination. However, the level of neoplastic cells in the PB of multiple myeloma (MM) patients after mobilization protocols is poorly investigated. In this study, we evaluated PB samples from 27 pretreated MM patients after the administration of high dose cyclophosphamide (7 g/m2 or 4 g/m2) and granulocyte-colony stimulating factor for the detection of myeloma cells as well as hematopoietic progenitors. Plasma cells containing intracytoplasmic lg were counted by microscope immunofluorescence after incubation with appropriate antisera directed against light- and heavy-chain lg. Moreover, flow cytometry studies were performed to determine the presence of malignant B-lineage elements by using monoclonal antibodies against the CD19 antigen and the monotypic light chain. Before initiation of PBSC mobilization, circulating plasma cells were detected in all MM patients in a percentage ranging from 0.1% to 1.8% of the mononuclear cell fraction (mean value, 0.7% +/- 0.4% SD). In these patients, a higher absolute number of PB neoplastic cells was detected after chemotherapy and granulocyte colony-stimulating factor. Kinetic analysis showed a pattern of tumor cell mobilization similar to that of normal hematopoietic progenitors with a maximum peak falling within the optimal time period for the collection of PBSCs. The absolute number of plasma cells showed a 10 to 50-fold increase as compared with the baseline value. Apheresis products contained 0.7% +/- 0.2% SD of myeloma cells (range, 0.2% to 2.7%). Twenty-three MM patients were submitted to PBSC collection. In 10 patients, circulating hematopoietic CD34+ cells were highly enriched by avidin-biotin immunoabsorption, were cryopreserved, and used to reconstitute bone marrow function after myeloablative therapy. The median purity of the enriched CD34+ cell population was 89.5% (range, 51% to 94%), with a 75-fold increase as compared with the pretreatment samples. The median overall recovery of CD34+ cells and colony-forming unit-granulocyte-macrophage was 58% (range, 33% to 95%) and 45% (range, 7% to 100%), respectively. Positive selection of CD34+ cells resulted in 2.5- to 3-log depletion of plasma cells and CD19+ B-lineage cells as determined by immunofluorescence studies, although DNA analysis of CDR III region of IgH gene showed the persistence of minimal residual disease in 5 of 6 patient samples studied. Myeloma patients were reinfused with enriched CD34+ cells after myeloablative therapy consisting of total body irradiation (1,000 cGy) and highdose melphalan (140 mg/m2). They received a median of 4 x 10(6) CD34+ cells/kg and showed a rapid reconstitution of hematopoiesis; the median time to 0.5 x 10(9) neutrophils and to 20 and 50 x 10(9) platelets per liter of PB was 10, 11, and 12 days, respectively. These results, as well as other clinically significant parameters, did not significantly differ from those of patients (n = 13) receiving unmanipulated PBSCs after the same pretransplant conditioning regimen. In summary, our data show the concomitant mobilization of tumor cells and hematopoietic progenitors in the PB of MM patients. Positive selection of CD34+ cells reduces the contamination of myeloma cells from the apheresis products up to 3-log and provides a cell suspension capable of restoring a normal hematopoiesis after a total body irradiation-containing conditioning regimen.

Description: Human interleukin-9 (IL-9) stimulates the proliferation of primitive hematopoietic erythroid and pluripotent progenitor cells, as well as the growth of selected colony-stimulating factor (CSF)-dependent myeloid cell lines. To further address the role of IL-9 in the development of acute leukemia, we evaluated the proliferative response of three leukemic cell lines and 32 primary samples from acute myeloblastic leukemia (AML) patients to recombinant human (rh)-IL-9 alone and combined with rh-IL-3, granulocyte-macrophage CSF (GM-CSF), and stem cell factor ([SCF] c-kit ligand). The colony-forming ability of HL60, K562, and KG1 cells and fresh AML cell populations upon IL-9 stimulation was assessed by a clonogenic assay in methylcellulose, whereas the cell-cycle characteristics of leukemic samples were determined by the acridine-orange flow cytometric technique and the bromodeoxyuridine (BRDU) incorporation assay. In addition, the terminal deoxynucleotidyl transferase assay (TDTA) and standard analysis of DNA cleavage by gel electrophoresis were used to evaluate induction of prevention of apoptosis by IL-9. Il-9, as a single cytokine, at various concentrations stimulated the colony formation of the three myeloid cell lines under serum-containing and serum-free conditions, and this effect was completely abrogated by anti-IL-9 monoclonal antibodies (MoAbs). When tested on fresh AML samples, optimal concentrations of IL- 9 resulted in an increase of blast colony formation in all the cases studied (mean +/- SEM: 19 +/- 10 colony-forming unit-leukemic [CFU- L]/10(5) cells plated in control cultures v 107 +/- 32 in IL-9- supplemented dishes, P 〈 .02). IL-9 stimulated 36.8% of CFU-L induced by phytohemagglutinin-lymphocyte-conditioned medium (PHA-LCM), and it was the most effective CSF for promoting leukemic cell growth among those tested in this study (i.e., SCF, IL-3, and GM-CSF). The proliferative activity of IL-9 was also observed when T-cell-depleted AML specimens were incubated with increasing concentrations of the cytokine. Addition of SCF to IL-9 had an additive or synergistic effect of the two cytokines in five of eight AML cases tested for CFU-L growth (187 +/- 79 colonies v 107 +/- 32 CFU-L, P = .05). Positive interaction was also observed when IL-9 was combined with IL-3 and GM-CSF. Studies of cell-cycle distribution of AML samples demonstrated that IL-9 alone significantly augmented the number of leukemic cells in S-phase in the majority of cases evaluated. IL-9 and SCF in combination resulted in a remarkable decrease of the G0 cell fraction (38.2% +/- 24% v 58.6% +/- 22% of control cultures, P 〈 .05) and induced an increase of G1- and S- phase cells. Conversely, neither IL-9 alone nor the combination of IL-9 and SCF had any effect on induction or prevention of apoptosis of leukemic cells. In summary, our results indicate that IL-9 may play a role in the development of AML by stimulating leukemic cells to enter the S-phase rather than preventing cell death. Moreover, IL-9 acts synergistically with SCF for recruiting quiescent leukemic cells in cell cycle.

Description: Patients with CML in lymphoid blast crisis (LBC-CML) or advanced Ph+ ALL have an unsatisfactory and only brief response to imatinib mesylate (IM). Moreover, treatment options in pts who failed IM are extremely limited. Dasatinib (BMS-354825) is a novel, oral kinase inhibitor that targets BCR-ABL and SRC kinases, and has shown promising clinical activity in a Phase I dose escalating study in patients with BCR-ABL-positive leukemias. Between January 2005 and June 2005, 77 pts (42 CML-LBC and 35 Ph+ ALL) who had failed IM-based therapy were enrolled in this multinational Phase II study investigating the safety and efficacy of dasatinib. This preliminary analysis summarizes data on the first 28 pts accrued (13 CML-LBC and 15 Ph+ ALL) who were accrued prior to March 20, 2005. Dasatinib was administered orally at 70 mg twice daily (BID) on a continuous daily dosing schedule; dose escalation to 100 mg BID or dose reduction to 50 mg and 40 mg BID were allowed for poor initial response or persistent toxicity, respectively. Complete blood counts were performed weekly and bone marrow evaluation, including cytogenetic analysis, was scheduled every month. Mutation analyses were performed in all pts. 27 pts were IM resistant and 1 was IM intolerant; 17 (61%) pts had received prior IM doses 〉600 mg/day, 13 (46%) pts received IM for

Description: Twenty-six adult patients (pts) aged between 22 and 60 years (median age: 46) with BCR-ABL+ acute lymphoblastic leukemia (ALL) were prospectively monitored by Q-RT-PCR between August 2001 and July 2004. All pts entered the GIMEMA LAL 0201/A protocol, in which Imatinib alone, at the dosage of 400 mg x 2 daily for at least six months, was administered as post-consolidation therapy in responding pts after intensive chemotherapy (CHT). Eighteen pts (69%) were p190+and 8 (31%) p210+ with/without the p190. In these two groups, the mean number of BCR-ABL copies at diagnosis (ie: BCR-ABL/ABL x 104) was 13,052 (range: 1,466–35,449) and 22,487 (range: 7,315–78,000) (p=ns), respectively. All pts were in 1st complete hematologic remission (25 pts after the first induction-consolidation course; 1 pt after a salvage treatment). Before Imatinib, 8 of the 18 p190+ pts (44%) showed a BCR-ABL copy reduction of ≥ 3 log compared to the levels at diagnosis, (mean BCR-ABL copies 3.6; range: 0–10), and they were defined as good responders to CHT. The remaining 10 pts (56%), defined as poor responders, showed a reduction of 〈 3 log (mean copies 2,825.8; range: 12 – 25,245). In the 10 poor responders, BCR-ABL copies constantly increased over time and this was predictive of an hematologic relapse in 8/10 patients. By contrast, 7 of the 8 good responder pts during Imatinib treatment persistently showed levels of BCR-ABL below 10. These 7 pts are in CCR maintained by Imatinib alone at 6, 9, 13, 13, 17, 21 and 23 months, respectively. In 1 pt, at 6 months from the start of Imatinib, a CNS relapse preceded a marked increase in the BCR-ABL copy numbers in BM cells. Therefore, after a median follow-up of 6 months (range 3 – 23), for poor responders, and of 13 months (range 6 – 30), for good responders, the actuarial probability of relapse was 100% and 12.5%, at 24 months (p=.027), respectively. p210+ pts achieved a molecular response rate slightly different respect to the p190+ cases. In fact, before starting Imatinib, only 2 of the 8 cases (25%) analyzed showed a reduction in BCR/ABL copies ≥ 3 log whereas in the remaining 6 pts, 1 relapsed at 6 months, but the other 5 showed a decrease of the BCR-ABL copies that in 2 cases fell below the 3 log reduction after 8 and 12 months, respectively. Altogether, after a median follow-up of 6 months (range: 2–28), 7/8 p210+ pts were in CCR maintained by Imatinib alone without transplant at 2, 4, 5, 6, 19, 24 and 28 months, respectively. In conclusion, Imatinib is a highly effective post-consolidation treatment for adult Ph+ ALL pts being able to maintain and/or to induce a minimum level of BCR-ABL expression, without the high morbidity and mortality relating to allografting procedures. In addition, in the p190+ cases an unsatisfactory molecular response rate after CHT was a powerful predictor of a subsequent clinical resistance to Imatinib.

Description: The 2nd-generation bcr-abl inhibitor nilotinib is more potent than imatinib (IC50 200nM: Y253H, E255K, E255V, F359C) representing 13% (11/85) of all patients assessed for baseline mutation, showed 13% (1/11) HR and 13% (1/11) MCyR compared to 74% (17/28) and 18% (5/28) respectively in the mutant group with IC50 of ≤200 nM. The nilotinib resistant T315I mutation occurred in 5 pts. Only one of these 5 pts who had T315I and G250E dual mutation achieved HR conceivably reflecting the sensitivity of G250E or non-mutant clone to nilotinib. At the time of data analyses, 50% of pts with baseline mutation were free of disease progression versus 62% of pts without baseline mutation. Rate of progression was 64% (7/11) in the group with less sensitive mutations and 60% (3/5) in pts. with T315I. However, the mutants most frequently associated with progression were F359V and M244V both having 4/5 pts (80%) progressed. In summary, BCR-ABL kinase domain mutations were identified at baseline in 59% of all pts in this cohort and in 67% of pts with imatinib resistance. Responses were observed across a broad spectrum of mutant genotypes. The rate of responses and disease progression may be affected by the baseline mutation types, although a larger data set with longer follow up is needed to further establish the correlation.

Description: Presently, there is no standard second-line regimen for patients (pts) with diffuse large B-cell lymphoma (DLBCL) who cannot undergo stem cell transplantation. Since yttrium-90 (90Y) ibritumomab tiuxetan (Zevalin) is highly active in pts with follicular lymphoma, we wished to establish whether it is also effective in pts with relapsed DLBCL. We therefore conducted a prospective, single-arm, open-label, non-randomized, multicenter phase II trial to evaluate the efficacy and safety of 90Y ibritumomab tiuxetan in elderly pts with histologically confirmed first relapsed or primary refractory DLBCL not appropriate for autologous stem cell transplantation. Pts were divided into 2 groups: those previously treated with chemotherapy alone [Group A, n=76], and those previously treated with chemotherapy and rituximab [Group B, n=28]. Pts in Group A were further divided into 3 strata: pts with primary refractory disease (stratum 1, n= 33), pts relapsing within a year from presentation (stratum 2, n=10), and those relapsing more than 1 year from presentation (stratum 3, n=33). All pts were to receive a single dose of 90Y ibritumomab tiuxetan 14.8 MBq/kg (0.4 mCi/kg) up to a maximum dose of 1184 MBq (32 mCi). The primary endpoint was overall response (ORR) assessed by using IWNHL criteria at Weeks 6, 12, and 24. Secondary endpoints included progression-free survival (PFS) and overall survival (OS). 104 pts were included. Three pts progressed after the first rituximab dose and in 1 patient the 111In labeling procedure failed; 103 pts were evaluable for efficacy and 104 for safety. An ORR of 44% was observed in the entire study population. In Group A, the ORR was 52% in stratum 1, 40% in stratum 2, 58% in stratum 3. In Group B, where 37% of pts were refractory to CHOP-rituximab, the ORR was 19%. The median PFS was 5.9, 2.3, and 6.2 months in strata 1, 2, 3 of Group A, respectively; the median PFS for Group B was 1.6 months. Median OS in Group A was 22.4 months in stratum 3 and has not yet been reached at a maximum follow-up time of 32 months in strata 1&2. Median OS was 4.5 months for Group B. Adverse events (AEs) with the exception of hematologic AEs were generally mild or moderate (CTC grade 1 and 2). There were 4 deaths due to SAEs. Three pts died of suspected cerebral hemorrhage, preceded by CTC grade 4 thrombocytopenias; another died of bleeding from a duodenal ulcer 280 days after the start of study medication, but this AE was determined not to be related to the study drug. The incidence of severe infection is low with 7% of pts hospitalized for infection during the study. In conclusion, 90Y ibritumomab tiuxetan has useful activity in the treatment of relapsed/refractory DLBCL, with no unexpected toxicities observed. The results of this study support a further evaluation of 90Y ibritumomab tiuxetan in combination with chemotherapy or immuno-chemotherapy earlier in the time course of DLBCL.

Description: Bosutinib (SKI-606) is an orally bioavailable dual Src/Abl inhibitor. Biochemical assays have shown it to be up to 200-fold more potent than imatinib as an inhibitor of Bcr-Abl phosphorylation. Unlike imatinib, bosutinib does not exhibit significant inhibition of c-kit or platelet-derived growth factor receptor (PDGFR), which may result in a relatively favorable safety profile. This is an ongoing open-label study in patients (pts) with Philadelphia chromosome positive (Ph+) accelerated phase (AP) and blast phase chronic myelogenous leukemia and (Ph+) ALL who failed prior imatinib therapy or other TKIs. Objectives are to assess safety and clinical activity of bosutinib. Pts receive bosutinib 500 mg/day. We report preliminary data for 57 pts, median age 54 yrs (range 22–83 yrs), 54% male. 23 pts (40%) were in AP, 15 (26%) in blast crisis (BC), 14 (25%) had Ph+ALL, and 5 (9%) were unclassified. Prior therapy included interferon (22 pts), imatinib (55 pts; data missing for 2 pts), dasatinib (17 pts), nilotinib (10 pts), stem cell transplant (5 pts). Overall median duration of bosutinib treatment was 2.7 mos (range 0.03–10.8 mos). Complete hematological response (CHR) was obtained in 7/25 evaluable pts (28%), including 4/14 (29%) pts with AP-CML, 2/8 (25%) pts with BC-CML, and 1/3 (33%) pts with Ph+ ALL. Among pts with no other TKI exposure, major cytogenetic responses (MCyR) were observed in 5/14 evaluable pts (36%), including 3/6 (50%) pts with AP-CML, 2/5 (40%) pts with BC-CML. Among pts with prior TKI exposure, 3/10 (30%) had MCyR, including 0/3 AP, 1/4 BP, and 2/3 ALL pts. Median time to MCyR was 8.9 weeks for pts previously exposed and 12 wks for unexposed to other TKIs. Duration of MCyR was 18 wks. 19 previously unexposed patients were evaluable for major molecular response. 4 (21%) had major molecular response, 3 (16%) of which were complete. Of 42 pts with samples tested for mutations, 13 different mutations were found in 20 pts (48%), including 5 cases of T315I. CHR occurred in 2/3 pts with P-loop mutations and 5/17 with non-P-loop mutations; MCyR occurred in 2/2 pts and 4/9 pts, respectively. Treatment was generally well tolerated in this cohort of heavily pretreated patients. The most common adverse events were gastrointestinal (diarrhea [56%], nausea [37%], vomiting [35%]) but these were usually grade 1–2, manageable and transient, reducing in frequency and severity after the first 3–4 weeks of therapy. Grade 3–4 hematologic laboratory abnormalities reported included thrombocytopenia in 31 pts (59%), neutropenia in 20 pts (38%), and anemia in 13 pt (25%). Grade 3–4 non-hematologic toxicities were diarrhea (9%) and vomiting (9%). Fluid retention was reported in 8 pts (14%), including 2 cases (3%) of pleural effusion (grade 2 and 3). Both were considered unrelated to treatment. Bosutinib is effective in imatinib-resistant pts with advanced CML. Responses were observed across a wide range of Bcr/Abl mutations. Bosutinib has a favorable toxicity profile with a small number of pts experiencing hematologic toxicity and fluid retention, possibly due to the lack of c-kit inhibition and PDGFR inhibition, respectively.

Description: In chronic myelogenous leukemia (CML), the Philadelphia (Ph) chromosome translocation results in the formation of BCR/ABL genes, normally transcribed in two types of hybrid transcripts with a b2a2 or b3a2 BCR/ABL junction, which give origin to 210-kD fusion proteins (P210). A third type of BCR/ABL (with e1a2 type of junction) has been identified in approximately 50% of the Ph-positive acute lymphoblastic leukemia (Ph+ALL) cases and results in the production of a BCR/ABL protein of 190 kD (P190). The presence of this transcript has been associated almost exclusively with the presence of an acute leukemia phenotype. By contrast, here we describe that in addition to transcripts with the b2a2 and b3a2 types of junction corresponding to the P210 proteins, virtually all CMLs at diagnosis bear also BCR/ABL transcripts showing the e1a2 type of junction, which correspond to the acute leukemia- associated P190 protein. With a quantitative polymerase chain reaction assay we found that the amount of the e1a2 mRNA present in CMLs in chronic phase, although in absolute amount much lower than that present in Ph+ ALLs, represents in most cases approximately 20% to 30% of the total BCR/ABL transcripts. Moreover, using a novel and very sensitive Western blot technique, we detected relevant amounts of P190 protein in addition to P210 from peripheral cells of two of the patients.