Genetic manipulation of primitive leukemic and normal hematopoietic cells using a novel method of adenovirus-mediated gene transfer.

Gene transfer into early hematopoietic cells has been problematic due to the quiescent nature of primitive cells and the lack of gene transfer vehicles with high efficiency for hematopoietic cell types. Previously, we have shown that adenoviral vectors can be used for the transduction of normal human progenitors with gene transfer efficiencies of approximately 30%. However, this approach is limited by relatively slow uptake kinetics (24-48 h) and a strong dependence on the presence of exogenous cytokines. Thus, we have modified this approach by combining adenoviral vectors with polycations to generate a virus-polycation complex, or VPC. Vehicles of this nature, when composed of conventional adenoviral vectors and polyamidoamine dendrimers, are a highly efficient means of transducing both normal and acute myelogenous leukemia (AML) cells. Moreover, the kinetics of gene transfer are markedly increased using the VPC strategy, with approximately 70% of transduction complete within 2 h. In this study, using viruses that encode green fluorescence protein (GFP), or the T cell costimulatory molecule B7.1 (CD80), we show that VPC-mediated gene transfer is an effective means of transducing normal and AML cells, including those with a highly primitive phenotype. Our data suggest that transient genetic manipulation of primitive hematopoietic cells can readily be achieved and should therefore permit a variety of research and clinical endeavors.

The interleukin-3 receptor alpha chain is a unique marker for human acute myelogenous leukemia stem cells.

Recent studies suggest that the population of malignant cells found in human acute myelogenous leukemia (AML) arises from a rare population of leukemic stem cells (LSCs). LSCs have been documented for nearly all AML subtypes and have been phenotypically described as CD34+/CD38- or CD34+/HLA-DR-. Given the potentially critical role of these primitive cells in perpetuating leukemic disease, we sought to further investigate their molecular and cellular characteristics. Flow cytometric studies using primary AML tissue showed that the interleukin-3 receptor alpha chain (IL-3Ralpha or CD123) was strongly expressed in CD34+/CD38- cells (98 +/- 2% positive) from 16 of 18 primary specimens. Conversely, normal bone marrow derived CD34+/CD38- cells showed virtually no detectable expression of the CD123 antigen. To assess the functional role of IL-3Ralpha positive cells, purified CD34+/CD123+ leukemia cells were transplanted into immune deficient NOD/SCID mice. These experiments showed that CD123+ cells were competent to establish and maintain leukemic populations in vivo. To begin to elucidate a biological role for CD123 in leukemia, primary AML samples were analyzed with respect to signal transduction activity in the MAPK, Akt, and Stat5 pathways. Phosphorylation was not detected in response to IL-3 stimulation, thereby suggesting CD123 is not active in conventional IL-3-mediated signaling. Collectively, these data indicate that CD123 represents a unique marker for primitive leukemic stem cells. Given the strong expression of this receptor on LSCs, we propose that targeting of CD123 may be a promising strategy for the preferential ablation of AML cells.

Use of the anti-idiotype antibody vaccine TriAb after autologous stem cell transplantation in patients with metastatic breast cancer.

Between April 1997 and March 1998 we evaluated the immune response and outcome in 11 chemosensitive patients who were treated with the anti-idiotype antibody vaccine TriAb after recovery from intensive therapy and autologous stem cell transplant (ASCT). Triab was commenced after recovery from the acute effects of ASCT; a minimum interval of 1 month was required from completion of consolidation radiotherapy, if given. Nine patients (82%) manifest anti-anti-idiotype antibody (Ab3) responses post ASCT. The maximal Ab3 response was seen after a median of 10 doses (range 5-20), which corresponded to a median of 14 months (range 5-19) post ASCT. Evidence of a T cell proliferative response was seen in eight patients; the response was modest in most of these. At a median follow-up of 24 months (range 22-33) after ASCT, four patients are alive without evidence of disease progression. All four of these patients were in the subgroup with more vigorous immune responses. Subsequent efforts have been directed toward the achievement of higher levels of immune responses more rapidly post ASCT. Bone Marrow Transplantation (2000) 26, 729-735.

Activity of single-agent melphalan 220-300 mg/m2 with amifostine cytoprotection and autologous hematopoietic stem cell support in non-Hodgkin and Hodgkin lymphoma.

High-dose chemotherapy using melphalan (HDMEL) is an important component of many conditioning regimens that are given before autologous hematopoietic stem cell transplantation (AHSCT). In contrast to the situation in myeloma, and to a lesser degree acute leukemia, only a very limited published experience exists with the use of HDMEL conditioning as a single agent in doses requiring AHSCT for lymphoma, both Hodgkin lymphoma (HL) and especially non-Hodgkin lymphoma (NHL). Thus, we report results of treating 26 lymphoma patients (22 with NHL and four with HL) with HDMEL 220-300 mg/m(2) plus amifostine (AF) cytoprotection and AHSCT as part of a phase I-II trial. Median age was 51 years (range 24-62 years); NHL histology was varied, but was aggressive (including transformed from indolent) in 19 patients, indolent in two patients and mantle cell in one. All 26 patients had been extensively treated; 11 were refractory to the immediate prior therapy on protocol entry and two had undergone prior AHSCT. All were deemed ineligible for other, 'first-line' AHSCT regimens. Of these 26 patients, 22 survived to initial tumor evaluation on D +100. At this time, 13 were in complete remission, including four patients who were in second CR before HDMEL+AF+AHSCT. Responses occurred at all HDMEL doses. Currently, seven patients are alive, including five without progression, with a median follow-up in these latter patients of D +1163 (range D +824 to D +1630); one of these patients had a nonmyeloablative allograft as consolidation on D +106. Conversely, 14 patients relapsed or progressed, including five who had previously achieved CR with the AHSCT procedure. Two patients, both with HL, remain alive after progression; one is in CR following salvage radiotherapy. Six patients died due to nonrelapse causes, including two NHL patients who died while in CR. We conclude that HDMEL+AF+AHSCT has significant single-agent activity in relapsed or refractory NHL and HL. This experience may be used as a starting point for subsequent dose escalation of HDMEL (probably with AF) in established combination regimens.

Interim analysis of the use of the anti-idiotype breast cancer vaccine 11D10 (TriAb) in conjunction with autologous stem cell transplantation in patients with metastatic breast cancer.

The anti-idiotype monoclonal antibody breast cancer vaccine 11D10 (TriAb) was administered before and after autologous stem cell transplantation (ASCT) in 45 patients with metastatic breast cancer whose disease was responsive to conventional chemotherapy. Evidence of a positive anti-anti-idiotype antibody (Ab3) humoral response was noted at a median of 1.76 months post-ASCT (range, before ASCT-6 months) with this strategy. Maximal Ab3 levels and idiotype-specific T-cell proliferative responses were observed at a median of 3 and 4 months, respectively, after ASCT. The achievement of rapid immune responses after ASCT, during a known period of decreased immunoresponsiveness, opens the possibility of an additional antitumor effect at a time when the tumor burden is relatively small. Moreover, in this interim analysis, patients with the most vigorous humoral and cellular immune responses had a significant improvement in progression-free survival. Further follow-up and evaluation of this approach is warranted.

Expression of tumor-suppressor genes interferon regulatory factor 1 and death-associated protein kinase in primitive acute myelogenous leukemia cells.

Previous studies indicate that human acute myelogenous leukemia (AML) arises from a rare population of leukemic stem cells. Cells of this nature can initiate and maintain leukemic cell growth in both long-term cultures and nonobese diabetic/severe combined immune-deficient mice. To characterize the biology of primitive AML cells, gene expression screens were performed with 7 primary AML and 3 normal specimens. For each sample, stem cell populations (CD34(+)/CD38(-)) were isolated and used to synthesize radiolabeled complementary DNA (cDNA). AML vs normal probes were then hybridized to cDNA arrays containing genes related to cancer and apoptosis. Of approximately 1400 genes analyzed, 2 tumor-suppressor genes were identified that were overexpressed in all 7 of the AML CD34(+)/CD38(-) cell populations: death-associated protein kinase and interferon regulatory factor 1. Expression of each gene was confirmed by reverse-transcription polymerase chain reaction and immunoblot analysis. It is proposed that tumor-suppressor proteins play a role in the biology of primitive AML cells. (Blood. 2001;97:2177-2179)

G-CSF Primed Autologous Marrow Harvest and Transplantation in Cytapheresis "Mobilization Failure" Patients: A Descriptive Analysis; Bone Marrow Transplantation.

Fifteen cancer patients, deemed blood HSC "mobilization failures" due to CD34 + cell yields of < 0.5 x 10(6) /kg from two consecutive daily cytaphereses, underwent G-CSF primed autologous bone marrow harvest in an attempt to obtain adequate hematopoietic support for subsequent autotransplantation. CD34 + cell yields from the primed marrow harvest were variable; however, some patients had > 5-fold increases in CD34 + cell yields in the marrow compared to cytapheresis, and 4 patients had CD34 + cell yields of > 1.0 (i.e., 1.2, 1.44, 1.61 and 2.45) x 10(6) /kg from the primed marrow harvest. None of the five patients previously exposed to stem cell toxins or fludarabine achieved > 0.85 x 10(6) /kg CD34 + cells with the primed marrow harvest. A significant difference was noted between G-CSF primed blood and marrow for CD34 + cells but not for GM-CFU ( p = 0.011 and p = 0.135, respectively, paired t-test). All evaluable patients engrafted; a median ANC > 0.5 x 10(9) /L recovery was achieved on D + 12 (range + 9 to + 17) in 12 of 13 evaluable patients - one died on D + 9 without recovery. The last day of platelet transfusion occurred at a median D + 13 (range + 8 to > + 66); only one patient remained platelet transfusion-dependent beyond D + 34. As anticipated, patients with higher numbers of CD34 + cells transplanted had somewhat more rapid recoveries. Although stem cell damage is obviously a key factor in mobilization failure patients, these findings raise the possibility that poor mobilization, at least in some patients, results from a mechanism other than, or in addition to, simple stem cell damage. Moreover, they raise the issue of the minimum number of marrow CD34 + - or more arguably other - cells needed for adequate short- and long-term reconstitution. The role of G-CSF in this situation, especially regarding dose and/or schedule, is intriguing but remains to be clarified. G-CSF primed marrow harvest is a potential option in certain poor mobilizers but, as fully expected, is frequently inadequate. Whether such is preferable to "steady-state" marrow harvest, continued or repeated G-CSF primed cytapheresis (with or without chemotherapy), or primed marrow with G-CSF in other schedules - or with other cytokines - is unclear and will be the subject of further study.

Nuclear factor-kappaB is constitutively activated in primitive human acute myelogenous leukemia cells.

Human acute myelogenous leukemia (AML) is thought to arise from a rare population of malignant stem cells. Cells of this nature, herein referred to as leukemic stem cells (LSCs), have been documented for nearly all AML subtypes and appear to fulfill the criteria for stem cells in that they are self-renewing and give rise to the cells found in many leukemic populations. Because these cells are likely to be critical for the genesis and perpetuation of leukemic disease, the present studies sought to characterize unique molecular properties of the LSC population, with particular emphasis on the transcription factor, nuclear factor-kappaB (NF-kappaB). Previous experiments have shown that unstimulated human CD34(+) progenitor cells do not express NF-kappaB. In contrast, primary AML CD34(+) cells display readily detectable NF-kappaB activity as assessed by electrophoretic mobility shift assay and gene expression studies. Furthermore, detailed analyses of enriched AML stem cells (CD34(+)/CD38(-)/CD123(+)) indicate that NF-kappaB is also active in the LSC population. Given the expression of NF-kappaB in leukemic, but not normal primitive cells, the hypothesis that inhibition of NF-kappaB might induce leukemia-specific apoptosis was tested by treating primary cells with the proteasome inhibitor MG-132, a well-known inhibitor of NF-kappaB. Leukemic CD34(+)/CD38(-) cells displayed a rapid induction of cell death in response to MG-132, whereas normal CD34(+)/CD38(-) cells showed little if any effect. Taken together, these data indicate that primitive AML cells aberrantly express NF-kappaB and that the presence of this factor may provide unique opportunities to preferentially ablate LSCs.

Adenovirus infections in hematopoietic stem cell transplant recipients.

We report a 12% incidence of adenovirus infections among 532 recipients of hematopoietic stem cell transplant (HSCT) from January 1986 through March 1997. The median time from day of stem cell infusion to first positive culture was 41 days. Recipients of allogeneic stem cells, as opposed to autologous stem cell recipients, were more likely to have a culture positive for adenovirus (16% vs. 3%; P<.0001). Pediatric patients were also more likely than adults to have a positive culture (23% vs. 9%; P<.0001). Among stem cell recipients with partially matched related donors, pediatric recipients appear to be at significantly greater risk for infection than adult recipients (P<.001). Positive cultures were associated with evidence of invasion in 64% of cases (41 of 64). A multiple logistic regression analysis showed that isolating adenovirus from more than 1 site correlated with greater risk for invasive infections (P=.002). Invasive infections were associated with poorer chance of survival.

Amifostine and autologous hematopoietic stem cell support of escalating-dose melphalan: a phase I study.

This study was conducted to define a new maximum tolerated dose and the dose-limiting toxicity (DLT) of melphalan and autologous hematopoietic stem cell transplantation (AHSCT) when used with the cytoprotective agent amifostine. Fifty-eight patients with various types of malignancy who were ineligible for higher-priority AHSCT protocols were entered on a phase I study of escalating doses of melphalan beginning at 220 mg/m(2) and advancing by 20 mg/m(2) increments in planned cohorts of 4 to 8 patients until severe regimen-related toxicity (RRT) was encountered. In all patients, amifostine 740 mg/m(2) was given on 2 occasions before the first melphalan dose (ie, 24 hours before and again 15 minutes before). AHSCT was given 24 hours after the first melphalan dose. Melphalan was given in doses up to and including 300 mg/m(2). Hematologic depression was profound, although it was rapidly and equally reversible at all melphalan doses. Although mucosal RRT was substantial, it was not the DLT, and some patients given the highest melphalan doses (ie, 300 mg/m(2)) did not develop mucosal RRT. The DLT was not clearly defined. Cardiac toxicity in the form of atrial fibrillation occurred in 3 of 36 patients treated with melphalan doses >/=280 mg/m(2) and was deemed fatal in 1 patient given melphalan 300 mg/m(2). (Another patient with a known cardiomyopathy was given melphalan 220 mg/m(2) and died as a result of heart failure but did not have atrial fibrillation.) Another patient given melphalan 300 mg/m(2) died of hepatic necrosis. The maximum tolerated dose of melphalan in this setting was thus considered to be 280 mg/m(2), and 27 patients were given this dose without severe RRT. Moreover, 38 patients were evaluable for delayed toxicity related to RRT; none was noted. Tumor responses have been noted at all melphalan doses and in all diagnostic groups, and 21 patients are alive at median day +1121 (range, day +136 to day +1923), including 16 without evidence of disease progression at median day +1075 (range, day +509 to day +1638). Amifostine and AHSCT permit the safe use of melphalan 280 mg/m(2), an apparent increase over the dose of melphalan that can be safely administered with AHSCT but without amifostine. Further studies are needed not only to confirm these findings, but also to define the antitumor efficacy of this regimen. Finally, it may be possible to evaluate additional methods of further dose escalation of melphalan in this setting.