Faculty Bibliography

We have used dogs to study gene transfer into hematopoietic stem cells, because of the applicability of results in dogs to human transplantation and the availability of canine disease models that mimic human diseases. Previously we reported successful gene transfer into canine marrow repopulating cells, however, gene transfer efficiency was low, usually below 0.1% (Kiem et al, Hum Gene Ther 1996; 7: 89). In this study we have used CD34-enriched marrow cells to study different retroviral pseudotypes for their ability to transduce canine hematopoietic repopulating cells. Cells were divided into two equal fractions that were cocultivated for 72 h with irradiated packaging cells producing vector with different retroviral pseudotypes (GALV, amphotropic or 10A1). The vectors used contained small sequence differences to allow differentiation of cells genetically marked by the different vectors. Nonadherent and adherent cells from the cultures were infused into four dogs after a myeloablative dose of 920 cGy total body irradiation. Polymerase chain reaction (PCR) analysis of DNA from peripheral blood and marrow after transplant showed that the highest gene transfer rates (up to 10%) were obtained with the GALV-pseudotype vector. Gene transfer levels have remained stable now for more than 18 months. Southern blot analysis confirmed the high gene transfer rate. Interference studies on canine D17 cells revealed that 10A1 virus behaved like an amphotropic virus and was not able to use the GALV receptor. In summary, our results show improved gene transfer into canine hematopoietic repopulating cells when CD34-enriched cells are transduced by cocultivation on a GALV-pseudotype packaging cell line in combination with a GALV-pseudotype vector. Furthermore, these results demonstrate that the monoclonal antibody to canine CD34 used in this study is able to enrich for hematopoietic repopulating cells.

BACKGROUND AND OBJECTIVE/OBJECTIVE:All culture systems exploring the early (pre-CFU) hematopoietic compartment are generally complex, time-consuming and unsuitable for routine application. The aim of our study was to develop a stroma-free culture system to quantify early bone marrow (BM) myeloid progenitor cells. DESIGN AND METHODS/METHODS:Low density, progenitor cell enriched BM cells underwent a double cytotoxic treatment with CD38 and CD33 monoclonal antibodies + rabbit complement, which depleted 99% of CFU-GM and BFU-E. Then they were cultured, both in agar and in limiting-dilution liquid culture, in the presence of 5637 cell line supernatant (containing GM-CSF, G-CSF and interleukin 1 ), stem cell factor (SCF) and interleukin 3 (IL3). RESULTS:The largest number (median 14.9 on 1x10(5) cells) and size (>50,000 cells) of myelomonocytic cell clones from CD33Eth /CD38Eth progenitors was reached after 3-4 weeks of liquid culture. SCF, but not IL3, was essential for that growth. The frequency of CD33-/ CD38- progenitors grown in liquid culture was approximately three times greater than the LTC-IC frequency in the same cell suspension. An average 93% of CD33-/CD38- progenitors displayed HLA-DR antigens and 43% generated secondary CFU-GM. In the BM of 9/10 patients, previously exposed to chemotherapy, CD33-/CD38- progenitor frequency was quite low (median 0.9 on 1x10(5) cells), in spite of normal cellularity and morphology and sustained disease remission. INTERPRETATION AND CONCLUSIONS/CONCLUSIONS:CD33-/CD38- progenitors can be grown and quantified in a stroma-free culture system in a relatively short time. The test can reveal long-lasting, subclinical BM damage induced by chemotherapy and could also be valuable for estimating the amount of early myeloid progenitors for transplantation purposes.

This trial was designed to compare foscarnet with ganciclovir as pre-emptive therapy for CMV infection in patients undergoing allogeneic hemopoietic stem cell transplant (HSCT). Thirty-nine patients were randomized to receive foscarnet 90 mg/kg every 12 h (n = 20) or ganciclovir 5 mg/kg every 12 h (n = 19) for 15 days at the time of development of CMVAg-emia. Primary-end points of the study were (1) outcome of CMVAg-emia; (2) progression to CMV disease; and (3) side-effects of treatment. The secondary end-point was transplant-related mortality (TRM). The two groups were comparable for diagnosis, status of disease, donor type, acute graft-versus-host (aGVHD) prophylaxis, interval between HSCT and CMVAg-emia and number of CMVAg positive cells; the donor and recipient age were borderline older in the foscarnet group. Increments of serum creatinine in the foscarnet group, and cytopenia in the ganciclovir group were controlled by reducing the administered dose: in the first 15 days of therapy 9/20 foscarnet and 10/19 ganciclovir patients had a dose reduction greater than 20% (P = 0.43). Clearance of CMVAg-emia was faster in the foscarnet group although with borderline statistical significance. Failures of treatment occurred in 3/20 patients in foscarnet group vs 8/19 patients in ganciclovir group (P= 0.06): causes of failure were the need for combination therapy to control antigenemia (1/20 vs 5/19), and reactivation during treatment for 2 vs 3 patients, respectively. CMV disease was diagnosed in 1 vs 2 patients (P = 0.5) who subsequently died. The actuarial 1-year TRM was 25 vs 12%, respectively (P = 0.3). This study suggests that foscarnet and ganciclovir are both effective for pre-emptive therapy of CMVAg-emia, although the number of failures would seem to be slightly higher in the ganciclovir patients. Side-effects are seen in both groups and can be managed with appropriate dose reduction.

In vitro priming of T cell with horse antilymphocyte globulin (HALG) results in cytokine release, and this has been associated with its clinical efficacy in patients with severe aplastic anaemia (SAA). Rabbit antithymocyte globulin (RATG) has been studied less extensively. In this study we compare the in vitro priming effect of HALG and RATG on purified normal marrow T cells: end-points of the study were 1) levels of TNF-alpha (TNF-alpha), IFN-gamma (IFN-gamma) GM-CSF in T cell supernatants, and 2) effect of T cell supernatants on colony formation with or without exogenous GM-CSF TNF-alpha, IFN-gamma and GM-CSF levels were comparable for HALG, RATG and phytohaemagglutinin (PHA). T cell supernatants showed comparable enhancement of colony formation in the presence of recombinant human GM-CSF (rhGM-CSF) and supported colony forming unit granulomacrophage (CFU-GM) growth in the absence of growth factor. This study shows that horse and rabbit derived ALG/ATG and PHA have a comparable in vitro priming effect on T cells: both agents should probably be tested for their clinical efficacy in SAA patients.

Using a polyclonal antiserum against canine CD34, we previously found that CD34 is expressed on canine bone marrow progenitor cells in a manner analogous to that found in humans. To further characterize CD34+ cells and to facilitate preclinical canine stem cell transplant studies, monoclonal antibodies (MoAbs) were raised to CD34. A panel of 10 MoAbs was generated that reacted with recombinant CD34 and with CD34+ cell lines and failed to react with CD34- cell lines. Binding properties of five purified MoAbs were determined by BIAcore analysis and flow cytometric staining, and several MoAbs showed high affinity for CD34. Two antibodies, 1H6 and 2E9, were further characterized, and in flow cytometry studies typically 1% to 3% of stained bone marrow cells were CD34+. Purified CD34+ bone marrow cells were 1.8- to 55-fold enriched for colony-forming unit-granulocyte-macrophage and for long-term culture initiating cells as compared with bone marrow mononuclear cells, whereas CD34- cells were depleted of progenitors. Three autologous transplants were performed with CD34+ cell fractions enriched by immunomagnetic separation. After marrow ablative total body irradiation (920 cGy), prompt hematopoietic recovery was seen with transplanted cell doses of </=1.1 x 10(7) /kg that were 29% to 70% CD34+. Engraftment kinetics were similar to those of dogs previously transplanted with approximately 10- to 100-fold more unmodified autologous marrow cells. This suggests that CD34+ is a marker not only of canine bone marrow progenitors but also for cells with radioprotective or marrow repopulating function in vivo. MoAbs to CD34 will be valuable for future studies of canine hematopoiesis and preclinical studies concerning stem cell transplantation, gene therapy, and ex vivo progenitor cell expansion.

Attempts to increase dose intensity have been hampered by hematologic toxicity. To address this issue, we designed a study to determine whether the reinfusion of PBPC significantly reduces the toxicity of multicyclic dose-intensive chemotherapy. Thirty refractory patients, median age 63, received CY 3 g/m2 plus melphalan 60 mg/m2 followed by PBPC and G-CSF (CM regimen). CY (at day 0) and G-CSF were used to mobilize PBPC harvested by a single leukapheresis at day 10. Melphalan was infused at day 11. PBPC were kept unprocessed at 4 degrees C for 48 h and reinfused at day 12. This regimen was repeated three times every 6 months. Outcomes were compared with those of 30 similar patients treated with melphalan 30 mg/m2 followed by G-CSF only, and repeated every 2 months for a total of six cycles. In patients receiving CY plus melphalan followed by PBPC reinfusion, the median duration of neutropenia (ANC < 500/microliters) and thrombocytopenia (platelets < 2500 microliters) was only 5 and 2 days respectively, and did not increase after the subsequent courses. Hematologic toxicity was quite similar to that observed after melphalan 30 mg/m2 plus G-CSF. The CM regimen was followed by 30% complete remission and 86% response > 50%, melphalan 30 mg/m2 by no complete remissions and 38% response > 50%. Patients receiving CM regimen showed a longer progression-free survival (22 vs 10 months, P < 0.01). The dose intensity of melphalan can be doubled by reinfusing PBPC without increasing toxicity. The combination of CY and melphalan followed by PBPC improves response rate and outcome when compared to low-dose melphalan.

An in vitro synergism between different inducers of AML cell differentiation has been previously observed. Therefore, we treated 53 myelodysplastic (MDS) patients with a low dose combination of cis-retinoic acid (cRA, 20-40 mg/day) and 1,25 alpha (OH)2 cholecalciferol [(OH)2D3, 1-1.5 micrograms/day] +/- intermittent 6-thioguanine (30 mg/m2/day). The latter was reserved for patients with bone marrow (BM) blast excess (> or = 5%). The treatment was well tolerated, without major toxicity. Among 25 patients with BM blasts less than 5%, we observed one complete, eight partial and four minor responses (response rate 52%) with a median response duration of 8 months (2 +/- 24). Median survival, which did not correlate with response, is projected at 76 months. Thirty-one patients with BM blast excess (> or = 5%), including three of the previous group who progressed to refractory anemia with excess of blasts (RAEB), were treated with the three-drug protocol. One complete, 12 partial and six minor responses were obtained (response rate 61%) with a median response duration of 6 months (2-29+). A significant difference in survival (P < 0.005) was observed between the 19 responders (median 25 months) and the 12 non-responders (median 9 months). A reduction in the transfusion need was observed in 41% of the transfusion-dependent patients with blast excess and in 53% of those without blast excess. Therefore, combined differentiating therapy seems more effective than previously reported single agent treatments and should be considered for a larger randomized study to assess its actual impact on survival of MDS patients.

Biological heterogeneity is a characteristic of multiple myeloma. A dysregulated cytokine network underlies the various phases of the disease. Numerous cytokines, either promoting or inhibiting plasma cell growth, are involved in tumor control. Interferon-gamma (IFN-gamma) showed the most powerful inhibiting activity on myeloma cell proliferation. This effect was demonstrated on IL-6 dependent myeloma cell lines, but not on IL-6 independent ones. It was also evident on fresh explanted bone marrow myeloma cells. The antiproliferative effect of IFN-gamma seems mainly due to the inhibition of IL-6, the central myeloma growth factor. IL-6 inhibition may occur at various levels: a downregulation of IL-6 receptor has been reported, and also a block of the IL-6 signal transduction pathway via interaction with cytoplasmic proteins such as p91 has been suggested. Our findings showed that IFN-gamma strongly inhibited myeloma cell proliferation to the same extent as dexamethasone (DEX), whereas interferon-alpha (IFN-alpha) inhibited Ig secretion. The combined use of interferons (IFNs) showed inhibitory activities both on proliferation and Ig synthesis that paralleled the effects of DEX. In some cases, IFN-gamma was also shown to augment monoclonal immunoglobulin secretion suggesting a possible differentiating activity on plasma cells. The in vitro data encouraged pilot studies to evaluate the in vivo antitumor effects of IFN-gamma.(ABSTRACT TRUNCATED AT 250 WORDS)

Retinoic acid has been shown to induce growth inhibition in a variety of cell types including human myeloma cell lines. Bone marrow plasma cells from 31 multiple myeloma (MM) patients were cultured to investigate the activity of 13-cis-retinoic acid (cRA), all-trans-retinoic acid (tRA), interferon-alpha (IFN-alpha), interferon-gamma (IFN-gamma), and dexamethasone (DEX), alone or in combination, on in vitro proliferation and immunoglobulin (Ig) secretion. Both cRA and tRA inhibited proliferation: the labelling index (LI) of treated cultures/controls, was 0.47 +/- 0.05 (mean +/- standard error mean, M +/- SEM) P < 0.0001, and 0.67 +/- 0.04 (M +/- SEM), P < 0.0001, respectively. The inhibitory effect of cRA was significantly superior to tRA (P = 0.0129) and IFN-alpha, similar to IFN-gamma and DEX. The combinations of cRA + IFN alpha, tRA + IFN-gamma, tRA + DEX did not show any synergistic effect on myeloma proliferation. In contrast, the combination cRA + DEX (0.29 +/- 0.04, M +/- SEM) markedly increased the effect of both cRA and DEX used as single agents. Ig synthesis was not significantly affected by CRA, tRA, IFN-gamma and the combination tRA + IFN-gamma. As expected, only IFN-alpha (P = 0.002) and DEX (P < 0.001) inhibited Ig production. The combinations cRA + IFN-alpha, cRA + DEX and tRA + DEX decreased Ig secretion to the same extent as IFN-alpha and DEX alone respectively. In conclusion, our data indicate that tRA and especially cRA strongly inhibited plasma cell proliferation but had no effect on Ig synthesis. The combination of cRA + DEX showed the highest degree of inhibitory activity of all cytokines, alone or in combination.

BACKGROUND:In myeloma patients resistance to both melphalan- and doxorubicin-containing regimens has been related to very short survival (approximately 6 months). The development of effective regimens combined with a low toxicity rate is mandatory in this patient subgroup. METHODS:Fourteen resistant myeloma patients were treated with cyclophosphamide (a total of 3.6 g/m2 was delivered in 2 doses on days 1 and 3) and prednisone (2 mg/kg, days 1-4) every month for 4 cycles. G-CSF support was administered to reduce myelosuppression. RESULTS:This combination was well tolerated. Granulocyte levels fell below 0.1 x 10(9)/L in all patients after a median of 9 days (range 8-11), followed by recovery to 0.5 x 10(9)/L after a median of 12 days from the start of treatment (range 10-13 days). Platelets never fell below 50 x 10(9)/L. All patients were treated on an outpatient basis and only 2 required hospitalization for major complications (pneumonia and heart failure). Response to cyclophosphamide was observed in 6/14 patients: 2 achieved complete remission, 4 showed a 50% or greater reduction of the M-component. Five patients are still in remission after 2, 6, 7, 9 and 10 months; 1 relapsed after 10 months. All patients except one are alive 4-16 months from the start of treatment. CONCLUSIONS:This schedule may represent a new approach for resistant myeloma, and its very low toxicity allows it to be delivered on an outpatient basis.