Amir A. Toor

Clinical activity of arsenic trioxide for the treatment of multiple myeloma

Arsenic has been used since ancient times as a therapeutic agent. However, until recently its use in modern medicine has been restricted to the treatment of a limited number of parasitic infections. In the early 1990s, reports from China described impressive results with arsenic trioxide in patients with de novo, relapsed, and refractory acute promyelocytic leukemia (APL). Other investigators subsequently confirmed these results leading to approval of its use for relapsed or refractory APL in the United States. Investigations of this agent have demonstrated that its efficacy in APL and preclinical tumor models is dependent upon a number of mechanisms, including induction of apoptosis, effects on cellular differentiation, cell cycling, and tumor angiogenesis. Subsequent preclinical studies showed significant activity of arsenic trioxide in multiple myeloma (MM). Based on this, in a phase II trial, we have evaluated the activity of arsenic trioxide in 14 patients with relapsed MM, refractory to conventional salvage therapy. With the dose and schedule used, treatment with arsenic trioxide produced responses in three patients and prolonged stable disease in a fourth patient, with the longest response lasting 6 weeks. Although treatment was reasonably well tolerated, in these patients with extensive prior therapy, 11 developed cytopenia, five associated with infectious complications and three developed deep vein thromboses. The results of this small trial support further investigation of this novel drug for the treatment of patients with relapsed or refractory MM.

Myeloma of the central nervous system: association with high-risk chromosomal abnormalities, plasmablastic morphology and extramedullary manifestations.

Summary.  Involvement of the central nervous system (CNS) by multiple myeloma, as defined by the detection of malignant plasma cells in the cerebrospinal fluid in the presence of suggestive symptoms, is considered extremely rare. We report on the characteristics of 18 such patients diagnosed and treated at the University of Arkansas over the last 10 years for an overall incidence of approximately 1%. Their evaluation revealed association of CNS involvement with unfavourable cytogenetic abnormalities (especially translocations and deletion of the chromosome 13), high tumour mass, plasmablastic morphology, additional extramedullary myeloma manifestations and circulating plasma cells. The presence of these features should alert clinicians to the possibility of CNS involvement. The outcome of these patients was extremely poor despite the use of aggressive local and systemic treatment including autologous stem cell transplants. Given this universally poor prognosis, the application of allogeneic transplants should be studied in this clinical setting.

Predicting long-term (≥ 5 years) event-free survival in multiple myeloma patients following planned tandem autotransplants

Summary. Although outcome in multiple myeloma (MM) patients has improved significantly with the introduction of autotransplants (AT), the curability of this approach remained to be demonstrated. Therefore, we analysed outcome and prognostic factors using a logistic regression model in 515 consecutive newly diagnosed and previously treated patients intended to receive melphalan‐based tandem transplants with follow up of ≥ 5 years. One quarter ofpatients had event‐free survivals (EFS) ≥ 5 years with no further relapses seen after 7 years (46 patients on plateau). On multivariate analysis, factors associated with EFS ≥ 5 years were absence of chromosome 11 and 13 abnormalities (odds ratio: 6·1), ≤ 12 months of preceding standard‐dose therapy (SDT) (OR: 2·6) and β‐2 microglobulin (B2M) level ≤ 2·5 mg/l at time of first AT (OR: 1·7). Patients with only favourable variables (25%) had a 7‐year EFS in excess of 35%, compared with 15% and 10%, respectively, with one (43%) or two unfavourable variables (27%), and 0% for 5% of patients with three unfavourable variables (P < 0·0001). Using a 1‐year landmark analysis to allow for guaranteed time and thereby excluding early treatment failures, attaining a complete remission (CR) had no significant effect on long‐term survival. Our data are consistent with cure in MM patients with a CR duration ≥ 7 years and re‐establishment of a monoclonal gammopathy of undetermined significance (MGUS) phase in those with persistent evidence of disease post transplantation, but without disease progression ≥ 7 years.

Analysis of expression of heat shock protein-90 (HSP90) and the effects of HSP90 inhibitor (17-AAG) in multiple myeloma.

Heat shock protein 90 (HSP90) is required for structural folding and maintenance of conformational integrity of various proteins, including several associated with cellular signaling. Recent studies utilizing 17-allylamino-17-demethoxygeldanamycin (17-AAG), an inhibitor of HSP90, demonstrated an antitumor effect in solid tumors. To test whether HSP90 could be targeted in multiple myeloma (MM) patients, we first investigated expression of HSP90 by immunofluorescence and flow cytometric analysis in a myeloma cell line (U266) and primary myeloma cells. Following demonstration of HSP90 expression in myeloma cells, archival samples of 32 MM patients were analysed by immunoperoxidase staining. Myeloma cells in all patients showed strong cytoplasmic expression of HSP90 in all samples and 55% also demonstrated concurrent nuclear immunopositivity. Treatment of U266 and primary MM cells with 17AAG resulted in significantly increased apoptosis compared to untreated control cells. Analysis of anti-apoptotic BCL2 family proteins and akt in MM cells incubated with 17-AAG revealed down-regulation of BCL-2, BCL-XL, MCL-1 and akt. Furthermore, although a low concentration of bortezomib resulted in no cell death, a combination of 17AAG and bortezomib treatment revealed a synergistic apoptotic effect on the U266 cell line. These data suggest that targeted inhibition of HSP90 may prove to be a valid and innovative strategy for the development of future therapeutic options for MM patients.

Fractal Organization of the Human T Cell Repertoire in Health and after Stem Cell Transplantation

T cell repertoire diversity is generated in part by recombination of variable (V), diversity (D), and joining (J) segments in the T cell receptor β (TCR) locus. T cell clonal frequency distribution determined by high-throughput sequencing of TCR β in 10 stem cell transplantation (SCT) donors revealed a fractal, self-similar frequency distribution of unique TCR bearing clones with respect to V, D, and J segment usage in the T cell repertoire of these individuals. Further, ranking of T cell clones by frequency of gene segment usage in the observed sequences revealed an ordered distribution of dominant clones conforming to a power law, with a fractal dimension of 1.6 and 1.8 in TCR β DJ and VDJ containing clones in healthy stem cell donors. This self-similar distribution was perturbed in the recipients after SCT, with patients demonstrating a lower level of complexity in their TCR repertoire at day 100 followed by a modest improvement by 1 year post-SCT. A large shift was observed in the frequency distribution of the dominant T cell clones compared to the donor, with fewer than one third of the VDJ-containing clones shared in the top 4 ranks. In conclusion, the normal T cell repertoire is highly ordered with a TCR gene segment usage that results in a fractal self-similar motif of pattern repetition across levels of organization. Fractal analysis of high-throughput TCR β sequencing data provides a comprehensive measure of immune reconstitution after SCT.

Preceding chemotherapy, tumour load and age influence engraftment in multiple myeloma patients mobilized with granulocyte colony-stimulating factor alone

Haematopoietic growth factors, especially granulocyte colony‐stimulating factor (G‐CSF), are frequently utilized alone for peripheral blood stem cell (PBSC) procurement to avoid the morbidity associated with high‐dose chemotherapy (HDT). Moreover, the cytotoxic agents used may not be the most optimal therapy for the malignancy. It also makes scheduling of apheresis easier. Factors having an impact on PBSC procurement and engraftment after HDT were analysed in 117 multiple myeloma patients mobilized with G‐CSF (10–16 µg/kg, median 12 µg/kg) alone using Cox regression analysis. A median of 6·2 × 106 CD34 cells/kg (range 0·6–34·1) were procured during leukapheresis and a median of 2·5 × 106 CD34 cells was infused after the first HDT (range 0·3–23·9). The only factor significantly affecting optimal PBSC procurement was duration of preceding conventional chemotherapy (P = 0·002). Granulocyte recovery was prompt in almost all patients, 75% of whom attained a granulocyte count of 0·5 × 109/l by day 13 (median 11, range 7–19). However, platelet recovery to both 20 × 109/l (median 12 d, range 8–50+) and 50 × 109/l (median 20 d, range 7–205+) varied widely. On univariate analysis, factors influencing platelet recovery were the number of CD34 cells/kg infused, age, β2‐microglobulin levels, response to preceding therapy, bone marrow plasmacytosis and duration of prior therapy. Factors attaining significance on multivariate analysis included number of CD34 cells/kg infused (P = 0·007), β2‐microglobulin levels (P = 0·0001), most probably representing disease load, and age (P = 0·002). Patients with high tumour burden, i.e. β2‐microglobulin levels > 2·5 mg/l, probably benefit from chemotherapy for mobilization both in terms of cytoreduction and adequate stem cell mobilization resulting in accelerated engraftment.

ABO mismatch may affect engraftment in multiple myeloma patients receiving nonmyeloablative conditioning.

BACKGROUND: Blood group incompatibility does not appear to affect the overall outcome in patients undergoing myeloablative conditioning before allogeneic BMT. Data on ABO‐mismatched transplantation in the nonmyeloablative setting are limited.

Favourable results with a single autologous stem cell transplant following conditioning with busulphan and cyclophosphamide in patients with multiple myeloma

Both single and tandem cycles of high dose therapy and autologous peripheral blood stem cell transplantation (ASCT) have been shown to improve survival in multiple myeloma (MM) patients. We report outcomes in 104 MM patients undergoing a single transplant after conditioning with a conventional myeloablative regimen, busulphan and cyclophosphamide. The patients were either in a first (71%), or subsequent remission (29%). Peripheral blood stem cells were mobilized using cyclophosphamide and granulocyte colony stimulating factor. The conditioning regimen consisted of busulphan 0·85 mg/kg given orally every 6 h (16 doses) and cyclophosphamide 60 mg/kg/d given intravenously for 2 d. The entire conditioning, transplant and post‐transplant course were in the outpatient setting for 45% patients. At a median follow‐up of 26 months (range 2–98 months), the median overall and progression‐free survival were 57 months [95% confidence interval (CI) 47–68] and 26 months (95% CI 20–32) respectively. Younger age and higher CD34+ cell dose infused were independently predictive of improved overall and progression‐free survival. Busulphan and cyclophosphamide is an effective and well‐tolerated preparative regimen for ASCT that can be given to MM patients in the outpatient setting.

Posaconazole salvage therapy allows successful allogeneic hematopoietic stem cell transplantation in patients with refractory invasive mold infections

Abstract: We describe the clinical courses of 3 patients with hematologic malignancies (2 with acute myelogenous leukemia and 1 with multiple myeloma) who developed invasive fungal infections due to uncommon molds (Alternaria spp., Paecilomyces lilacinus, and Zygomycetes). Breakthrough invasive fungal infections of the sinus (n=1), lung (n=3), and pericardium (n=1) developed despite fluconazole prophylaxis and failed to respond to treatment with other licensed antifungal therapies, including amphotericin B (n=3), caspofungin (n=2), and voriconazole (n=3), and surgical intervention (n=2). Salvage therapy with posaconazole oral suspension resulted in successful outcomes in all 3 patients, who subsequently underwent allogeneic hematopoietic stem cell transplantation (HSCT) while on continued posaconazole therapy. The median duration of posaconazole treatment before HSCT was 5 months (range: 1.5–6 months). Posaconazole salvage therapy allowed successful allogeneic HSCT in 3 patients with refractory invasive mold infections.

Epigenetic induction of adaptive immune response in multiple myeloma: sequential azacitidine and lenalidomide generate cancer testis antigen‐specific cellular immunity

Patients with multiple myeloma (MM) undergoing high dose therapy and autologous stem cell transplantation (SCT) remain at risk for disease progression. Induction of the expression of highly immunogenic cancer testis antigens (CTA) in malignant plasma cells in MM patients may trigger a protective immune response following SCT. We initiated a phase II clinical trial of the DNA hypomethylating agent, azacitidine (Aza) administered sequentially with lenalidomide (Rev) in patients with MM. Three cycles of Aza and Rev were administered and autologous lymphocytes were collected following the 2nd and 3rd cycles of Aza‐Rev and cryopreserved. Subsequent stem cell mobilization was followed by high‐dose melphalan and SCT. Autologous lymphocyte infusion (ALI) was performed in the second month following transplantation. Fourteen patients have completed the investigational therapy; autologous lymphocytes were collected from all of the patients. Thirteen patients have successfully completed SCT and 11 have undergone ALI. Six patients tested have demonstrated CTA up‐regulation in either unfractionated bone marrow (n = 4) or CD138+ cells (n = 2). CTA (CTAG1B)‐specific T cell response has been observed in all three patients tested and persists following SCT. Epigenetic induction of an adaptive immune response to cancer testis antigens is safe and feasible in MM patients undergoing SCT.