ALBERT

Description: Studies were done of cell production by marrow in diffusion chambers implanted in the peritoneal cavity of rabbits subjected to various stimuli to hematopoiesis. In chambers in neutropenic hosts and in hosts injected with endotoxin, animals presumed to have an increased stimulus to granulopoiesis, there was increased production of granulocytes but there was also increased production of red cells. Although red cell production was decreased in chambers in polycythemic hosts, granulocyte production was not different from that in controls. Stimulation of erythropoiesis by erythropoietin injections or by exposure to hypoxia increased red cell production by marrow in the implanted diffusion chambers without diminishing granulopoiesis. Only in chambers in hosts made anemic by bleeding was there an increase in red cell production accompanied by a decrease in granulocyte production. In these anemic hosts induction of neutropenia led to an increase in granulopoiesis without any depression of erythropoiesis.

Description: By CD40 crosslinking in the presence of cytokines leukemic cells can be modified into good antigen presenting cells (APC) expressing costimulatory molecules CD40, CD80, CD86, and CD83. Previously, primary alloreactive T cell responses from HLA-matched donors have been generated using these leukemic-APC as stimulator cells against acute and chronic myeloid leukemia (AML&CML), and acute and chronic lymphocytic leukemias (ALL&CLL). However, the likelihood of generating a good immune response is highly unpredictable and long-term culture in the presence of high dose IL-2 is needed to enrich for leukemia-reactive T cells. Since the length of the in-vitro culture period has been shown to be inversely correlated with the potential of cells to survive and expand in-vivo, we developed a method facilitating early activation, detection and rapid isolation of leukemia-reactive T cells based on their interferon-gamma (IFNg) secretion using the cytokine capture assay (Miltenyi). In order to enrich for leukemia-reactive T cells and to synchronize the production of IFNg, T cells were first stimulated with the leukemic APC with addition of low dose IL-2 (10 IU/mL) at day 7, resulting in re-entry of the majority of the T cells into a quiescent state after 14 days of culture. Then, the cells were specifically restimulated resulting in synchronized production of IFNg and allowing efficient isolation. Using this method we were able to isolate T cell populations containing a high frequency of leukemia-reactive T cells against CLL, ALL, AML, and CML in 11 donor/patient pairs. Using a CFSE-based cytotoxicity assay (Jedema, Blood2004; 103: 2677) as read-out we were able to demonstrate 20–80% lysis of the primary leukemic blasts by the IFNg+ T cells at very low E/T ratios (3/1-0.3/1) in the majority of the responses, whereas the IFNg- fractions induced only 5–30% lysis. Single cell sorting of the IFNg producing T cells revealed that 15–30% of the T cell clones was capable of exerting minor antigen specific cytotoxic activity against the patient cells in an HLA-restricted fashion. However, in individual cases despite minor antigen disparities between donor and patient no specific anti-leukemia immune response could be detected. Prior to exposure to the leukemic-APC in-vivo activated T cells were observed in the responder T cell population of these donors that contained a high frequency of regulatory T cells defined as CD4+/CD25+, CD4+/CD152+, and CD8+/CD28−. We hypothesized that these regulatory T cells might actively inhibit the induction of an anti-leukemic T cell response. Therefore, in a donor/CLL patient pair, in which we were not able to induce a cytotoxic immune response against the CLL-APC we removed the in-vivo activated T cells from the responder material prior to the initial activation with the CLL-APC. Whereas no cytotoxic activity could be isolated from unmodified responder material (only 1/288 clones was cytotoxic), the IFNg+ T cells isolated from the response induced after depletion of the in-vivo activated T cells was capable of exerting massive cytotoxicity against both the primary CLL (55%) and the CLL-APC (70%). Single cell cloning of this response revealed that 35/129 T cell clones (〉25%, 26 CD8+, 9 CD4+) exerted HLA-restricted CLL-specific cytotoxicity. From these results we conclude that the likelihood of generating a primary anti-leukemic immune response is not only determined by the frequency of precursor CTLs, but also by the frequency of inhibitory regulatory T cells at the onset of the immune response.

Description: The use of intensive antileukemic treatment is less widely accepted in high-risk MDS pts compared to de novo AML, due to the reported inferior results. It is questionable whether the poorer outcome reflects an intrinsic property of the involved stem cell or a higher frequency of poor prognostic factors. The purpose of this analysis is to identify disease-specific prognostic factors for outcome of young (aged

Description: During the period from January 1970 until December 1973, therapy was started in 41 previously untreated adolescents and adults with acute lymphoblastic leukemia. Induction therapy was started with vincristine and prednisone in all patients, resulting in complete remission in 19 and death due to infection during the first month in one case. After 3 wk on these two drugs, the addition of daunorubicin was required in the remaining 21 patients. Fifteen of these obtained remission, one died during induction therapy, and five patients were unresponsive to this therapy, as well as to all subsequent induction schemes. The overall remission rate was 83%. Significantly higher initial leukocyte counts were found in the group treated with vincristine, prednisone, and daunorubicin. Meningeal leukemia prophylaxis, by either periodic methotrexate injections given intrathecally or a combination of cranial irradiation and intrathecally administrated methotrexate, was administered in 29 therapy responders. The median duration of complete remission obtained with various maintenance therapy schemes was 13 mo. No differences were seen in the results obtained in patients between 14 and 20 yr of age and older patients. Twenty-two patients relapsed within 2–37 mo. Relapses were confined to the central nervous system in two cases, to the bone marrow in 18, and to the bone marrow and CNS simultaneously in two. A second remission was obtained in 17 cases (77%). The median survival time of the whole group was 27 mo, as compared with 32 mo for therapy responders and 7 mo for the nonresponders. The percentage and duration of remission and the survival time in our group of adolescents and adults were comparable to those currently being achieved in other centers, but not as good as those reported for children treated with the same protocol.

Description: Studies were done of cell production by marrow in diffusion chambers implanted in the peritoneal cavity of rabbits subjected to various stimuli to hematopoiesis. In chambers in neutropenic hosts and in hosts injected with endotoxin, animals presumed to have an increased stimulus to granulopoiesis, there was increased production of granulocytes but there was also increased production of red cells. Although red cell production was decreased in chambers in polycythemic hosts, granulocyte production was not different from that in controls. Stimulation of erythropoiesis by erythropoietin injections or by exposure to hypoxia increased red cell production by marrow in the implanted diffusion chambers without diminishing granulopoiesis. Only in chambers in hosts made anemic by bleeding was there an increase in red cell production accompanied by a decrease in granulocyte production. In these anemic hosts induction of neutropenia led to an increase in granulopoiesis without any depression of erythropoiesis.

Description: Gemtuzumab ozogamicin, a conjugate of a humanized anti-CD33 monoclonal antibody linked to the cytotoxic antibiotic calicheamicin, has shown significant antileukemic activity in relapsed AML with a favorable toxicity profile. The EORTC-LG performed a phase II study to investigate the activity and toxicity of GO used alone and in combination with conventional chemotherapy for remission induction in medically fit elderly pts (age between 61 and 75 years; WHO PS 0-1) with untreated AML. GO was administered iv over 2 hrs at the FDA-approved dose of 9 mg/m2. A second dose was given on day 15 in the absence of progressive disease or excessive toxicity and response was assessed four weeks later. This was to be followed by a course of conventional chemotherapy with mitoxantrone, cytarabine and etoposide (MICE regimen) and no further treatment in complete responders. Between 09/00 and 10/01 64 pts were enrolled , 57 of whom were evaluable for response and toxicity. The median age was 68 yrs (range 61–73); 43 pts had primary AML; CD33 positivity (≥20% marrow blasts) was documented in 44 pts (85%). GO therapy was completed by 47 of the 57 pts (82%): primary reasons 10 pts did not receive the scheduled second dose were disease progression (4), early death (2), toxicity (2) and protocol violation (2). The rate of initial response to GO was 35.1% (95% CI, 22.9%–48.9%) with 13 pts achieving complete remission (CR) and 7 CRp (complete remission with incomplete platelet recovery); 6 additional pts entered partial remission (PR). There were 3 (5.3%) toxic deaths (1 ARDS, 1 VOD, 1 infection) and 28 pts had resistant disease. Severe myelosuppression was the primary toxicity to GO. Grade 3 or 4 non-hematologic toxicities in more than 5% of pts during GO therapy included febrile neutropenia (39%), infection (28%), elevations of bilirubin, SGPT and creatinine of 8%, 6% and 6%, respectively. A clinical picture compatible with hepatic VOD developed in 3 pts (5%) resulting in 2 deaths (1 early, 1 in CRp). No baseline characteristics predicted for response to GO, but pts expressing CD33 in 〉80% marrow blasts tended to respond more favourably to the immunoconjugate (CR+CRp 7/14, 50%). Altogether, 51 pts survived GO therapy and 38 of them (75%) went on to receive a course of MICE after a median interval of 49 days (range 12–77) from the first dose of GO. Excessive toxicity (11 pts), protocol violation (1 pt) and treatment refusal (1 pt) were the main reasons for not starting chemotherapy. The ultimate best response rate was 54.4% (31/57; CR 35.1% and CRp 19.3%) increasing up to 65.8% among the 38 pts who were able to complete the whole induction sequence. Five pts died of toxicity during the MICE segment (2 VOD, 1 infection, 1 heart failure, 1 multi-organ failure) for an overall treatment related mortality of 14.1% (8/57). With a median follow-up of 3 yrs, 2-year overall survival (OS) and disease-free survival (DFS) estimates were 11.0% (SE=4.1%) and 12.1% (SE=3.5%), respectively. We conclude that: 1) single agent, standard dose GO has significant activity in untreated AML of the elderly with an acceptable safety profile; 2) sequential combination with standard chemotherapy is feasible and may improve treatment outcome in this poor risk disease, but myelotoxicity and liver dysfunction are of concern. An ongoing randomized trial will determine the contribution of GO given at reduced dose (6 mg/m2) to the observed antileukemic effect and toxicity.