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We report amplification of the MLL gene region (11q23→11qter) in a 72-year-old woman with myelodysplastic syndrome progressing to acute myelomonocytic leukemia and in a 51-year-old man with a history of hairy cell leukemia and secondary myelodysplasia progressing to acute myelogenous leukemia. The amplicons containing MLL were shown by molecular cytogenetics to extend from chromosomal region 11q23 to the distal long arm of chromosome 11 and to be present in the first patient in five copies on a large ring chromosome and present in the second patient also in five copies on two derived chromosomes. Other karyotypic findings in the first patient included del(5q), +8, and der(21)t(17;21), resulting in the loss of a copy of 17p, whereas deletion 7q was observed in the second patient. Southern-blot analysis for the second patient was consistent with MLL amplification but did not demonstrate rearrangement of the germ-line MLL band. Amplification of MLL and the 11q23 region has been documented in only a few cases and appears to be yet another mechanism by which MLL contributes to the leukemia phenotype.

To show that macrophages can be effectively targeted against malignant B cells, bispecific antibodies (BsAb) were constructed from two antibodies having specificity for the high-affinity Fc receptor for IgG (Fc gamma RI/CD64) and the B-cell differentiation antigens CD19 and CD37. Using a flow cytometry-based assay and confocal imaging, we show that these constructs mediated significant phagocytosis of B lymphocytes by macrophages that could be enhanced with interferon gamma (IFN gamma) and IFN gamma in combination with macrophage colony- stimulating factor. BsAb-dependent phagocytosis was triggered through Fc gamma RI and could be blocked only by using F(ab')2 fragments from the parent molecule or by cross-linking Fc gamma RI. BsAb-dependent phagocytosis was not blocked by antibodies to the other Fc receptors, Fc gamma RII and Fc gamma RIII. Because these antibody constructs bind to an epitope outside the Fc gamma RI ligand binding site, we show that autologous serum, polyclonal IgG, and monomeric IgG1 did not block BsAb- dependent phagocytosis, whereas autologous serum and the IgG fractions blocked parent molecule monoclonal antibody-dependent phagocytosis due to the avid binding of monomeric IgG to Fc gamma RI. Finally, BsAb- mediated phagocytosis was effective against the malignant B cells of patients with mantle cell lymphoma, prolymphocytic leukemia, and chronic lymphocytic leukemia. Based on these studies, we propose that BsAbs may provide an effective means of immunomodulation for patients with B-cell malignancies.

Tissue macrophages derive from monocytes of bone marrow origin. Because monocytes from patients with chronic myelogenous leukemia (CML) contain the Philadelphia chromosome (Ph), it seemed probable that tissue macrophages in CML would originate from the malignant clone. Using powerful molecular techniques, we studied pulmonary alveolar macrophages (PAM) from two patients with CML. PAM from Patient 1, a patient in chronic phase studied before bone marrow transplantation (BMT), contained the Ph by Southern blot analysis. Patient 2, an accelerated phase patient, was studied after post-BMT relapse. PAM from this patient not only contained the Ph, but also expressed the BCR/ABL message documented by a new splice junction in situ hybridization technique. This new technique allows detection of BCR/ABL mRNA and determination of splice useage in individual cells. These data confirm the continued replenishing of PAM from peripheral blood monocytes in non-BMT settings and represent the first direct evidence that tissue macrophages are derived from the malignant clone in patients with CML.

We have utilized the polymerase chain reaction (PCR) to sensitively detect persistence of the chronic myelogenous leukemia (CML) malignant clone and to study bcr/abl mRNA splicing patterns following bone marrow transplantation. Thirteen of sixteen patients displayed persistent malignant cells during post-BMT clinical remission. In two patients bcr/abl mRNA was detected 4 and 9 months prior to clinical relapse. In eleven of fourteen patients in continued clinical remission malignant cells were detected post-BMT. Ten of these eleven patients were also cytogenetically normal. Seven patients have lost all evidence of bcr/abl transcript, but only at 1-2 years posttransplant, while four have shown persistence of the bcr/abl transcript from 28 days to 3 years post-BMT and one has converted from an initially negative result at 1 year post-BMT to detectable levels of chimeric mRNA at 2 years. Thus, 8/9 patients tested at or before 6 months, 7/12 at 1 year, and 3/10 at 2 years showed persistent detectable CML cells. Intriguingly, mRNA splicing patterns changed in 5 patients following BMT, with complete loss of mRNA containing bcr exon 3 (n = 2) or new appearance of mRNA not containing bcr exon 3 (n = 2). A single patient transiently lost evidence of bcr exon 3 expression while persistently expressing the bcr exon 2/abl exon 2 splice. Our data suggest that the majority of patients harbor small numbers of malignant cells following transplantation, and that such persistence may not inevitably predict clinical relapse. Complete elimination of the malignant CML clone post-BMT may rely on immunological mechanisms (e.g., graft-vs-leukemia).

Purpose Because the secretory diarrhea of acute graft-versus-host disease (GvHD) of the gut induces serious metabolic and nutritional disturbances, this study was initiated to assess the use of a somatostatin analogue, octreotide acetate, as adjunctive therapy for severe GvHD of the gut with massive diarrhea. Patients and methods In a pilot study, six patients with biopsy-confirmed acute gut GvHD after allogeneic bone marrow transplantation received octreotide 50 to 250 μg three times a day subcutaneously. Results Three of the six treated patients had a prompt and dramatic reduction in stool volume within 1 to 3 days of initiation of octreotide therapy. Conclusions Somatostatin and its analogues have been used successfully in diarrheal states by antagonism of neuropeptide overproduction, although other potential therapeutic mechanisms include inhibition of fluid secretion, enhanced salt absorption, and inhibition of gut motility. Somatostatin and its analogues may be promising adjunctive agents in the treatment of gastrointestinal GvHD, although assessment in a controlled trial will be required to confirm their therapeutic efficacy.

A phase I/II trial evaluated early administration and dose escalation of interleukin (IL)-2 with granulocyte macrophage colony stimulating factor (GM-CSF) post-transplant. Following melphalan (200 mg/m(2)) and an autologous transplant, IL-2 was initiated (day 0) and continued for 4 weeks. GM-CSF (250 mcg/m(2)/day) began on day 5. Fifteen of 19 patients completed therapy. No treatment-related deaths occurred. IL-2 (1 x 10(6) IU/m(2)/day) was not tolerated in two of six patients due toor =grade 3 fatigue/diarrhea (n=1) or supraventricular tachycardia (n=1). The maximum tolerated dose of IL-2 was 6 x 10(5) IU/m(2)/day; this dose was well tolerated by 11 of 13 patients. Neutrophil and platelet engraftment occurred on day 13 (median; range 10-17 days) and day 13 (median; range 0-74 days), respectively. When compared to control patients, there was a marked increase in the number of CD3+ T cells (P=0.005), CD4+ T cells (P=0.01), CD8+ T cells (P=0.001) and CD4+CD25+Treg cells (P=0.015) post-transplant. Cytotoxicity directed against myeloma cells was markedly increased when compared to control patients (P=0.017). This unique trial design using early administration of IL-2 with GM-CSF during the period of lymphodepletion, demonstrated a marked increase in the number and function of early cytotoxic effector T cells, without suppression of engraftment.

BACKGROUND: Analysis of the peripheral blood (PB) C34 value may determine the optimal time to initiate leukapheresis. STUDY DESIGN AND METHODS: After selecting a threshold PB CD34 value of five CD34 + cells per µL to initiate leukapheresis procedure, a prospective analysis of 50 consecutive patients was initiated to identify the optimal time to initiate leukapheresis and its impact on costs and resource utilization. Clinical decisions were made to commence or to postpone leukapheresis with this PB CD34 threshold number. Based on PB CD34 values for each patient, the number of leukapheresis procedures, postponed or canceled, the number of CD34+ cells per kg, and the total number of cells collected were identified. Costs of mobilization were obtained from the hospital cost accounting system. RESULTS: In 13 months, 50 patients with a hematologic disorder underwent mobilization. There were 34 cancellations or postponements of collections due to a low PB CD34 value in 13 patients. By use of our identified costs per initial collection, this resulted in a savings of $67,660. CONCLUSIONS: This prospective study defines how the implementation of the PB CD34 value results in costs savings. A low PB CD34 value canceled or postponed a significant number of leukapheresis procedures, resulting in a substantial cost savings. Use of the PB CD34 value should be the standard of care during mobilization and peripheral blood progenitor cell collection.

Abstract 2866 Introduction: Diffuse large B cell lymphoma (DLBCL) is the most common lymphoid malignancy and is generally responsive to anthracycline-containing chemotherapy. However, 60% of patients (pts) will relapse after their first line treatment. At the time of relapse the only curative approach includes the use of a stem cell transplant (SCT). The incidence of DLBCL increases with age which creates a subset of pts who are not candidates for first line anthracycline-based chemotherapy, and a large subset of pts who are not candidates for SCT due to advanced age and/or co-morbidities. Thus, there is a significant unmet need for therapies with a low toxicity profile in elderly or medically unfit pts with DLBCL. 90Y-ibritumomab tiuxetan (90Y-IT) is an anti-CD20 murine antibody linked with a beta-emitting isotope approved for use in indolent lymphoma. Maintenance rituximab (R) has been reported to increase response rates and prolong remission duration in some lymphomas. We performed a phase II multicenter clinical trial to examine the efficacy of 90Y-IT induction followed by maintenance R in pts with DLBCL. Patients and Methods: Eligible pts were either intolerant of anthracycline-based chemotherapy or had relapsed or refractory CD20+ DLBCL with measurable disease. Pts had to be ineligible for SCT for reasons other than failure to harvest stem cells. Bone marrow involvement by lymphoma of less than 25% based on bilateral bone marrow aspirate and biopsy was required. R 250 mg/m2 was administered IV immediately followed by 111In-ibritumomab tiuxetan. Nuclear scans were performed at 24 and 48 hours to insure there was no altered biodistribution. On day 8 a second infusion of R 250 mg/m2 followed by 0.4 mCi/kg (for pts with a baseline platelet count >150,000/mm3) or 0.3 mCi/kg 90Y-IT (for pts with a baseline platelet count 100,000-149,000/mm3) was given. Pts with multiple extranodal sites or prior bone marrow involvement received CNS prophylaxis with intrathecal methotrexate or cytarabine. Maintenance R 375 mg/m2 was given on weeks 3–6, then weekly × 4 every 6 months × 4 cycles or until progression. Results: Between 10/2003 and 9/2009, 25 pts have been treated. During the course of the study, the ownership of the therapeutic agent changed three times and therefore enrollment was interrupted on two occasions. The median age of pts was 79 (range 45–91), 36% pts had a sIPI score 3 or more. The median number of prior regimens is 2 [0-5]. The 90Y-IT treatment regimen produced an overall response rate of 36% [9 pts] with 28% CR [7 pts]. To date, the mean OS is 18 months (median 8.1 months) with a median follow-up of 11.2 months. Among responding pts, the median OS has not been reached with a median follow-up of over 26.2 [0.1-71.4] months. Thirteen pts died within the first year, 6 patients (24%) continue to be in remission greater than 18 months, and 4 patients (16%) remain in long-term remission [39.9-71.4 months]. The most frequently observed toxicity was hematologic. Eleven percent of pts had grade 4 neutropenia with only one patient experiencing febrile neutropenia, and 16% of pts experienced grade 4 thrombocytopenia. There were no unexpected non-hematologic toxicities except for 1 patient that experienced extravasation. One late-occurring case of MDS/AML was reported that is possibly related to the study regimen, and one case of adenocarcinoma of the GI tract that is likely unrelated. Of note, none of the pts that progressed on the chemotherapy preceding this study achieved a response to the study regimen. Conclusions: The 90Y-IT treatment regimen has an acceptable toxicity profile in elderly or heavily pretreated pts with DLBCL. The two week outpatient 90Y-IT infusion produces response rates and durations similar to that of more prolonged cytotoxic chemotherapy regimens. Progression on previous chemotherapy predicts for poor response to 90Y-IT. Treatment with 90Y-IT can provide durable remission to a select subset of pts who are not candidates for SCT, or intensive anthracycline based chemotherapy. Disclosures: Off Label Use: We are describing a phase II study of the use of 90Y-Ibritumomab Tiuxetan for treatment of diffuse large B-cell lymphoma. Current FDA approved use of 90Y-Ibritumomab Tiuxetan includes relapsed or refractory, low-grade or follicular B-cell non-Hodgkin's lymphoma (NHL) or previously untreated follicular NHL who achieve a partial or complete response to first-line chemotherapy. Joyce:Spectrum Pharmaceuticals, Inc.: Research Funding; Cell Therapeutics Inc: Research Funding; Biogen Idec: Research Funding.

Approximately 60% of adult patients (pts) with diffuse large B cell lymphoma (DLBCL) will relapse after first line therapy. Second line chemotherapy has response rates of 45–66% with median response duration of 3 to 5 months.90Y ibritumomab tiuxetan (90Y-IT) is a murine IgG1 kappa antibody against CD20 linked with a beta-emitting isotope approved for use in relapsed indolent lymphoma. Maintenance rituximab (R) has been reported to increase response rates and prolong remission duration in some lymphomas. We performed a phase II multicenter clinical trial to examine the efficacy of 90Y-IT induction followed by maintenance R in pts with DLBCL. Eligible pts had histologically confirmed CD20+ DLBCL according to WHO criteria. Pts had disease that was either relapsed or refractory to anthracycline-based chemotherapy or pts were intolerant of anthracycline-based chemotherapy. Adequate baseline hematologic and organ function was required. Pts were required to have 150,000/mm3 or 0.3 mCi/kg 90Y-IT if baseline platelet count 100,000 to 149,000/mm3, total dose not to exceed 32 mCi. Pts received R 375 mg/m2 on weeks 3–6. Pts with multiple extranodal sites or prior bone marrow involvement received CNS prophylaxis with intrathecal methotrexate or cytarabine. Non-progressing pts received maintenance R 375 mg/m2 weekly x 4 every 6 months x 4 or until progression. Toxicity analysis was planned after 15 pts were enrolled. Stopping rules were not met. As of 7/12/2006, 16 pts have been treated of a planned 40. Pts ranged in age from 45 to 95 with a median age of 77. Forty-four percent of patients had a secondary IPI score ≥ 3. The median number of prior regimens is 2 ranging from 1–5. The 90Y–IT treatment regimen produced an overall response rate of 37.5% with 25% CR at 12 weeks. All pts with a CR had a secondary IPI of 1. All pts with PD had a secondary IPI ≥ 2. To date, the median EFS is 2.5 months with a median follow-up of 6.3 months. Among responding patients, the median EFS is not reached with a median follow-up of over 11 [5.7- 22.2] months. Five patients (31%) continue to be in remission greater than 6 months, and three patients (19%) remain in remission for 1 year. The most frequently observed grade 3 or 4 toxicity was hematologic. 55.5% of pts experienced grade 3 or 4 neutropenia. 66.7% of pts experienced grade 3 or 4 thrombocytopenia. The most frequent non-hematologic adverse event was hypotension experienced during the R infusion. The 90Y-IT treatment regimen has an acceptable toxicity profile in pts with relapsed/refractory DLBCL. The two week outpatient 90Y-IT infusion produces response rates and durations similar to that of more prolonged cytotoxic chemotherapy regimens.

Elderly patients with AML (>60) have a poor prognosis due to high risk cytogenetics and a high incidence of secondary AML. Comorbid conditions make induction therapy more risky and less effective. There is a need for less toxic and more effective treatment for these patients. The combination of bortezomib (B) and melphalan (M) demonstrate synergistic cytotoxicity in vitro against AML cell lines providing a rationale for this combination in patients with AML. We report the initial results of a pilot study of low-dose M and B for the treatment of patients with AML and high-risk myelodysplastic syndromes. Eligibility requirements included a diagnosis of AML or high-risk MDS and subjects were not candidates for standard induction therapy or had failed therapy. Strata 1 included patients who had received no previous therapy and strata 2 patients with refractory or relapsed disease. Treatment consisted of M (2mg) oral daily dose and B (1.0mg/M2) on day 1, 4, 8 &11 of a 28 day cycle. Ten patients have been enrolled. Median age was 69 (range 54–79), 8 patients w/ AML and 2 w/ refractory MDS. Five patients were previously treated (strata 2); regimens included induction (7(3), auto SCT (1) and azacytadine (2). Cytogenetic abnormalities were present in 70% of patient specimens. To date 25 cycles of treatment have been given (range 1–6). Six patients are evaluable for response. Two patients (both with MDS) were withdrawn after 1 cycle for cytopenias. Two patients with AML achieved CR by bone marrow morphology. One patient, a 78 year old man with multiple cytogenetic abnormalities and no previous treatment achieved remission with normalization of cytogentetics after 4 cycles. A 74 year old man who had failed previous therapy for his AML achieved a morphologic remission with persistent cytogenetic abnormalities after 6 cycles. Three patients required admission for management of febrile episodes in association with neutropenia that were disease related. There were no treatment related admissions or grade 3/4 non hematologic toxicity events. The 74 year old man who achieved a CR with no “in hospital” days while on study had previously failed to respond to two induction attempts which required a 44 day hospitalization. Accrual continues. In summary, the combination of low dose oral melphalan with low dose bortezomib appears to be a novel, active and well tolerated outpatient treatment for elderly patients with high risk MDS and AML.

Radioimmunotherapy (RIT) is a useful addition to the armamentarium against NHL. Due to concerns about chronic myelosuppression and the possible inability to mobilize stem cells or tolerate further therapy after RIT, this modality has not been commonly utilized in potential transplant candidates. We report the successful mobilization, collection, transplantation and engraftment of 5 patients with NHL following yttrium-90 ibritumomab tiuxetan. Patients had follicular lymphoma, grade I or II (n=3), transformed follicular lymphoma (n=1) or blastic variant mantle cell lymphoma (n=1). Time from RIT to mobilization was 10 months (median; range 7–27 months). The median number of regimens prior to RIT was 2.5 (range 1–4) including prior autologous transplant (n=1). The median number of regimens following RIT and prior to transplant was 1.5 (range 1–2). All patients exhibited chemosensitive disease. Patients were mobilized with R-ICE/G-CSF (n=4) or G-CSF alone (n=1) and required 2.8 leukaphereses (mean; range 2–5). The CD34 yield was 3.8x106 CD34+ cells/kg (median; range 3.4–5.1x106 CD34+ cells/kg). All patients received busulfan, VP-16, ARA-C and cyclophosphamide (BVAC) as the preparatory regimen. One patient received IL-2 from day 0 to day 11. Time to engraftment was 13 days (median; range 9–19 days) for an absolute neutrophil count (ANC) >500 cells/ml and 23 days (median; range 15–44 days) for a platelet count>20x103/ml. The patient on the IL-2 study showed delayed engraftment of both ANC (day 19) and platelets (day 27). The patient with mantle cell lymphoma, who was undergoing his second autologous transplant, exhibited delayed platelet engraftment (day 44). Time to engraftment did not differ significantly for either ANC (p=0.16) or platelets (p=0.10) in 18 RIT-naïve NHL patients receiving BVAC and an autologous peripheral blood stem cell transplant (PBSC) during the same time period. There were no treatment-related deaths. With a median follow up of 12 months, one patient has died of disease recurrence. Two patients remain in complete remission. Two patients are alive with disease (relapse at 12 months and 14 months) and are currently receiving therapy. No patient has demonstrated laboratory or cytogenetic evidence of a myelodysplastic syndrome. In conclusion, heavily pretreated patients who have received radioimmunotherapy with yttrium-90 ibritumomab tiuxetan and subsequently relapse can successfully undergo mobilization, collection, and autologous PBSC transplant. These patients demonstrate full engraftment, durable remissions and tolerate additional therapy should relapse occur following transplant.

Autologous peripheral blood stem cell transplantation (APBSCT) is effective for the treatment of multiple myeloma (MM), however the majority of patients relapse. Recent evidence shows an improved outcome if absolute lymphocyte count is increased immediatedly following APBSCT in patients with hematologic malignancy. We designed a phase II trial evaluating a dose escalation of SQ IL-2 and standard dose GM-CSF post-transplant for myeloma patients. Patients (n=18) received melphalan 200mg/m2 with GM-CSF 250mg/m2/day beginning on day 5. IL-2 began on the day of transplant and continued 5 days per week for 4 weeks. Peripheral blood samples were obtained at baseline (pre-transplant) and every week for 4 weeks post-transplant and evaluated using flow cytometry for cell subsets using antibodies directed against CD3, CD4, CD8, CD25 and CD56. Control patients (n=11) consisted of MM patients who received melphalan 200mg/m2 with either G-CSF or GM-CSF without IL-2. IL-2 at a dose of 1 X 106IU/m2/d was not tolerated in 2 of 6 patients due to grade 4 fatique and diarrhea (n=1) and grade 4 supraventricular arrhythmia (n=1). A dose of 6 X 105 IU/m2/d was well tolerated by 12 patients. Level 3 or greater toxicities included nausea (n=5), diarrhea (n=3), anorexia (n=11), mucositis (n=9). Engraftment of neutrophils occurred on day 12 and platelets on day 18. The LOS was 21 days in IL-2 treated cohort and 18 days in control group. Absolute lymphocyte counts on days 10–15 were increased an average of 152% in the IL-2 cohort (range 25–390%) over the control group. At day 21, there was a marked increase in the number of CD3+CD8+ cytotoxic T cells (43 %, +/− 7.5%) , CD56 +NK cells ( 78.3 %, +/− 9.1), and CD8+CD56+ NKT cells (36.8 %, +/− 9.8%) compared to patients’ baseline levels. Cytotoxicity of Day #21 PBMNC in the IL-2 cohort was strikingly increased at 28.1% (18.5 – 29.2%) compared to 5.5 % (3.59 – 6.52%) at baseline when tested against a human myeloma cell line (RPMI 8226) using chromium release assays (E: T ratio = 100:1). These results demonstrate a tolerable regimen of immediate post-transplant immunotherapy with marked increase in the number and function of early cytotoxic effector cells. The enhanced immune recovery may translate into an improved outcome.