B. Glass

Early stem cell transplantation for chronic lymphocytic leukaemia: a chance for cure?

B-cell chronic lymphocytic leukaemia (CLL) cannot be cured by conventional therapy. To improve the prognosis of patients with CLL, we have designed a sequential treatment strategy that comprises intensive chemotherapy for mobilization of peripheral blood progenitor cells (PBPCs) and induction of minimal disease, followed by high-dose radiochemotherapy with stem cell reinfusion and post-transplant molecular monitoring by polymerase chain reaction (PCR) amplification of the complementary determining region III (CDRIII) gene. In a prospective study, we have evaluated this protocol in 18 patients with CLL, also including early stages of the disease. The median age was 49 (29-61) years; Binet stages were A, six; B, nine; and C, three. Adverse prognostic factors [high lymphocyte count and/or diffuse bone marrow (BM) infiltration] were present in 16 out of 18 patients. All patients showed a clone-specific molecular marker as demonstrated by PCR amplification of CDRIII rearrangements. For stem cell mobilization and reduction of tumour load, one to two cycles of Dexa-BEAM chemotherapy were administered, resulting in minimal disease (circulating lymphoma cells <1 x 10(9) l(-1); BM infiltration <20%; lymphomas <2 cm) in 16 out of 18 patients, including four patients who already had minimal disease before Dexa-BEAM. Stem cell harvesting was successful in 14 patients. All grafts [three BM, 11 peripheral blood (PB)] were purged from leukaemic cells using immunomagnetic methods. Thirteen patients having achieved minimal disease were reinfused with purged autologous stem cells (ASC) after preparation with total body irradiation and cyclophosphamide. Engraftment was delayed in patients receiving BM (n = 3) but prompt [neutrophils >0.5 x 10(9) l(-1) after 10 (9-13) days, platelets >20 x 10(9) l(-1) after 11 (9-214) days] in patients restored with PBPCs (n = 10). Procedure-related deaths did not occur. Although the results of CDRIII PCR suggest persistence or recurrence of the leukaemic clone in at least three cases, to date only one patient has relapsed, whereas all others survive without clinical evidence of disease with a maximum follow-up of 48 months. We conclude that sequential high-dose therapy using Dexa-BEAM and autologous stem cell transplantation is a safe and highly effective treatment for patients with CLL. However, a longer follow-up is needed to assess whether definite cures can be achieved using this strategy.

Graft-versus-leukaemia activity can be predicted by natural cytotoxicity against leukaemia cells

We have investigated graft‐versus‐leukaemia (GVL) effects after allogeneic bone marrow transplantation (BMT), using three murine leukaemia models, A20 (B lymphocytic), WEHI‐3 (myelomonocytic) and PU5‐1R (myeloid). Injection of leukaemia cells in a high number (106 cells) into syngeneic Balb/c mice (H‐2d) invariably led to death with a median survival time of 22u2003d (A20), 18u2003d (WEHI‐3) and 45u2003d (PU5‐1R). A lower tumour load of A20 (5u2003×u2003105 cells) was used in some experiments resulting in a leukaemic death rate of 94%. Lethal total‐body irradiation followed by syngeneic BMT prolonged survival (Pu2003<u20030.05) for animals bearing the leukaemia A20 and WEHI‐3 but was unsuccessful for animals injected with cells from the monocytic leukaemia PU5‐1R. Graft‐versus‐host (GVH)‐nonreactive marrow of (C57u2003×u2003Balb/c)F1 mice (H2bxd) exerted a significant GVL‐effect with reduced relapse rate and improved survival in mice receiving the leukaemia cell line A20. In animals with low tumour load a significant reduction of the relapse rate from 82% following syngeneic BMT to 47% following allogeneic, GVH‐nonreactive BMT could be achieved. Depletion of natural killer (NK) cells from the GVL‐reactive semi‐allogenic bone marrow graft enhances the relapse rate of the leukaemia A20 to 65%. In mice bearing the leukaemias WEHI‐3 or PU5‐1R allogeneic GVH‐nonreactive BMT did not improve survival compared to syngeneic BMT. Transplantation of GVH‐reactive bone marrow from DBA mice (MHC identical to Balb/c, minor difference) caused only a limited and insignificant reduction of relapse rate for animals with the leukaemia A20. These in vivo data are in close correlation with in vitro natural killer cell (NK) activity of the donor strains against the respective leukaemia targets. Depletion of NK cells from the GVL‐reactive (C57u2003×u2003Balb/c)F1 bone marrow resulted in a significant loss of GVL activity. We conclude that NK cells are involved in graft‐versus‐leukaemia effects independent of graft‐versus‐host disease (GVHD).

Allogeneic MHC-mismatched activated natural killer cells administered after bone marrow transplantation provide a strong graft-versus-leukaemia effect in mice.

Allogeneic lymphocytes administered with an unmanipulated bone marrow transplant provide a strong antileukaemic effect, the so‐called graft‐versus‐leukaemia (GVL) effect. On the other hand, T‐cell‐mediated graft‐versus‐host‐disease (GVHD) observed after transplantation of unmanipulated BM graft causes substantial morbidity and mortality. The aim of the present study was to determine the antileukaemic potential of enriched IL‐2 activated NK cells administered 2u2003h after BMT. Balb/c (H‐2d) mice were given a dose of A20 (H‐2d, B‐cell leukaemia) cells 2u2003d prior to lethal total body irradiation (TBI) and transplantation of either syngeneic or allogeneic anti‐Thy1.2 (CD90) depleted bone marrow cells. Either syngeneic (Balb/c, H‐2d) or allogeneic (C57BL/6, H‐2b) enriched and IL‐2 (200u2003U/ml for 24 h) activated NK cells were given 2u2003h after BMT. Injection of A20 leukaemia into normal Balb/c recipients led to death after a median of 14u2003d. A lethal dose of TBI followed by either syngeneic or allogeneic Thy1.2‐depleted BMT resulted in a modest antileukaemic effect. The adoptive transfer of syngeneic enriched and IL‐2 preincubated NK cells given at time of BMT exerted a significantly better GVL effect. However, the infusion of allogeneic enriched NK cells resulted in a stronger GVL effect. These results clearly demonstrate that allogeneic NK cells are superior to syngeneic NK cells in their potential to eradicate residual leukaemia cells after BMT without mediating clinical overt GVHD. This experimental setting may offer a strategy for treatment of haematological malignancies in a phase of minimal residual disease.

Myeloablative radiochemotherapy followed by reinfusion of purged autologous stem cells for Waldenström's macroglobulinaemia

Waldenströms macroglobulinaemia (WM) is an incurable lymphoproliferative disorder. The purpose of this study was to investigate the role of autologous peripheral blood stem cell transplantation (ASCT) for the treatment of WM. Seven patients (untreated or after first‐line therapy) with symptomatic WM underwent two or three cycles of Dexa‐BEAM chemotherapyu2003+u2003G‐CSF with stem cell harvesting and proceeded to total body irradiation and high‐dose cyclophosphamide followed by reinfusion of ex‐vivo B‐cell‐depleted stem cells. Engraftment was prompt, and procedure‐related deaths did not occur. A strong reduction or normalization of BM infiltration and serum IgM levels occurred in all evaluable patients, but immunofixation electrophoresis revealed persistent paraproteinaemia in five of them. With 3–30 months of follow‐up, all patients are alive without clinical or serological signs of disease progression. This pilot trial shows for the first time that high‐dose radiochemotherapy with purged stem cells is effective and may improve the course of patients with WM. In the majority of cases, however, complete eradication of the disease does not appear to be possible with ASCT alone.

Autografting of highly purified peripheral blood progenitor cells following myeloablative therapy in patients with lymphoma: a prospective study of the long-term effects on tumor eradication, reconstitution of hematopoiesis and immune recovery.

In a prospective study, we have investigated CD34+ selection of peripheral blood progenitor cells (PBPC) for autotransplantation in patients with lymphoma. Twenty-six consecutive patients (10 follicular lymphomas, seven mantle cell lymphomas, seven B-CLL, two immunocytomas) were mobilized using chemotherapy plus G-CSF. Sufficient numbers of PBPC could be collected from 24 patients and were immunoselected with the semiautomated Isolex 300 (n = 17) or the fully integrated Isolex 300i (n = 7) devices. The selection products were assayed by PCR amplification of clonal CDRIII or t(14;18) rearrangements for residual tumor cell content. Residual disease and long-term hematopoietic and immune recovery were studied by assessing the following parameters at 3, 6, and 12 months post-transplant: CDRIII or t(14;18) PCR, platelet count, lymphocyte subsets, serum IgG, serum IgA, and measles titer. With the Isolex 300 device 26% (10–65) of input CD34+ cells were recovered with a median purity of 89.2% (49.4–98.9) after CD34+ selection. The Isolex 300i device allowed significantly better recoveries (46% (22–86)) and purities of CD34+ cells (98.8% (92.2–99.2)). The overall purging efficacy was 3.2 (0.6–5.1) log. Twenty patients have been reinfused with CD34+ selected grafts after myeloablative preparation. Rapid engraftment occurred in all patients. With a median follow-up of 28 (19–42) months, 14 patients are alive without clinical or molecular evidence of disease recurrence, whereas five have relapsed and one additional patient shows persistent presence of the disease-specific molecular marker without clinical progression. Cellular and serological parameters of hematopoietic and immune functions were largely normal at 12 months post-transplant including the measles titer which was present in all patients. Kinetics of immunohematopoietic recovery were similar to those of 12 control patients who had received unmanipulated PBPC during the same time period except for the recovery of CD4+ CD45RA+ T cells which was significantly delayed in the CD34+ group. During the first year post-transplant, transient monoclonal or oligoclonal gammopathies were observed in seven of 16 study patients. We conclude that CD34+ selection with the Isolex system allows preparation of highly purified CD34+ fractions and effective tumor cell depletion. The CD34+ products can be reinfused safely after myeloablative treatment and result in sustained hematopoietic and immune recovery. The fact that all patients retained their specific measles immunity suggests that myeloablative treatment with reinfusion of highly purified CD34+ PBPC is not immunoablative.

Gaft-versus-leukemia activity after bone marrow transplantation does not require graft-versus-host disease

SummaryClinical data have suggested that graft-versus-host disease (GVHD) plays a crucial role in the antileukemic effects of bone marrow grafts. We investigated (a) whether bone marrow cells unable to induce GVHD can effect graft-versus-leukemia (GVL) activity and (b) whether such antileukemic capacity depends on the presence of T lymphocytes in the graft. Balb/c mice were inoculated with A 20 cells, a B-cell lymphoma/leukemia of Balb/c origin. Four weeks after tumor inoculation the animals were lethally irradiated and received a bone marrow graft. Cells from (Balb/c × C57) F1 or (C3H × Balb/c) F1 hybrids were transplanted into parental-strain Balb/c mice. Since lymphocytes from F1 hybrids are unable to cause graft-versus-host reactivity against a parental-strain animal, we used this experimental setting to explore GVL effects in a GVHD-free system. In vitro incubation with monoclonal anti-Thy-1.2 antibody plus complement was used to eliminate Thy-1+ cells. After syngeneic transplantation, the death rate due to leukemia remained unchanged (91%) compared with that among untreated animals (86%). Following transplantation of F1 marrow cells of either (C57 × Balb/c) F1 or (C3 H × Balb/c) F1 origin, death rates of 40% and 50% were observed; these were significantly lower. Depletion of Thy 1+ cells from bone marrow graft caused only a slight increase in the leukemic death rate after transplantation of bone marrow of (C57 × Balb/c) F1 hybrid origin (50%), but a high leukemic death rate was seen after transplantation of (C3H × Balb/c) F1 bone marrow (100%). Additional experiments with fully allogeneic, T-cell-depleted C57 bone marrow transplantation suggest an antileukemic effect that is comparable to that seen after transplantation of unmanipulated F1 bone marrow. Taken together, our results indicate that GVL activity can be dissociated from graft-versus-host reaction.

A prospective study of positive/negative ex vivo B-cell depletion in patients with chronic lymphocytic leukemia

OBJECTIVEnAutologous peripheral blood stem cell (PBSC) transplantation is increasingly being used in patients with chronic lymphocytic leukemia (CLL). As the autografts are frequently contaminated with large numbers of tumor cells, we have prospectively investigated the feasibility and efficacy of ex vivo double purging of PBSC grafts in an open, nonrandomized, single-center phase I/II clinical study.nnnMATERIALS AND METHODSnTwenty consecutive patients with poor-risk CLL underwent uniform stem cell mobilization with chemotherapy and granulocyte colony-stimulating factor (G-CSF). Double B-cell depletion of the harvested PBSC products was performed using immunomagnetic CD34(+) cell selection (Isolex300i Nexell, Irvine, CA) followed by a negative step with anti-CD19/20/23/37-labeled immunomagnetic beads. The purified PBSC were reinfused after myeloablative treatment with TBI/CY.nnnRESULTSnA total of 25 separation runs was accomplished using collection products containing 3.4% (1.1-8.1) CD34(+) cells and 1.2% (0.1-42) CD19(+)CD5(+) CLL cells. After double selection, 33% (15-67) CD34(+) cells were recovered with a purity of 98.8% (89.1-99. 8). CLL cells were undetectable by high-resolution flow cytometry in 15 of 25 final products; median purging efficacy was 5 (4.1-6) log. The CD34(+) content of the 20 final grafts was 4.6 (2.2-6.5) x 10(6)/kg. Rapid and durable engraftment developed in all cases. With a median follow-up of 20 (6-29) months, 17 patients live in complete clinical remission, two have recurrent disease, and one patient died due to pulmonary embolism five months after transplant. Persistence of the leukemic clone on the molecular level was demonstrated by dot blotting with clone-specific CDR3 probes in an additional five patients. Serious or unexpected infectious complications did not occur.nnnCONCLUSIONSnPositive/negative purging with the Isolex system allows preparation of highly purified CD34(+) fractions and up to six log of tumor cell depletion in patients with B-CLL and can be safety reinfused after myeloablative therapy without affecting hematopoietic engraftment.

Autologous stem cell transplantation for follicular lymphoma: no benefit for early transplant?

Abstract. Autologous stem cell transplantation (SCT) is widely used as salvage treatment for patients with relapsed follicular lymphoma (FL). Although SCT can induce prolonged remissions, it does not appear to be curative in the vast majority of patients. The purpose of this study was to investigate if incorporation of SCT into first-line therapy can improve its efficacy. Fifty-five patients underwent sequential high-dose therapy as up-front (n=33) or salvage treatment (n=22) for advanced stage FL at our institution. Treatment consisted of intensive chemotherapy with dexamethasone, carmustine (BCNU), etoposide, cytarabine, and melphalan (Dexa-BEAM) for mobilization of peripheral stem cells and reduction of tumor load, followed by one of three different myeloablative regimens and SCT. With a median follow-up of 4xa0years, projected event-free survival (EFS) and overall survival (OS) at 4xa0years post transplant was 59% and 84%, respectively, with no evidence of plateau in the survival curves. By univariate and multivariate analysis weighing age, sex, stage, BM and extranodal involvement, timing of transplant, ex vivo purging, and conditioning regimen [total body irradiation (TBI) vs non-TBI], the only significant factor predicting for superior EFS and OS was up-front vs salvage transplant (4-year EFS 76% vs 38%, p=0.02; 4-year OS 92% vs 73%, p=0.033). However, when calculated from diagnosis, EFS and OS of the up-front and salvage groups were virtually identical, implying that the longer survival post SCT in the up-front group was completely compensated by the longer interval between diagnosis and transplant in the salvage group. Median OS from diagnosis was 13.5xa0years. Except for one case of anaplastic large cell lymphoma, secondary neoplasms have not occurred to date. In conclusion, our data indicate that SCT might improve the prognosis of patients with disseminated FL, although it is probably not curative even if applied early during the course of the disease. The optimum timing of SCT remains to be determined by the ongoing randomized multicenter trial of the German Low-grade Lymphoma Study Group. The impact of radiotherapy on the success of SCT does not seem to be as essential as originally believed.

Sequential high-dose therapy and autologous stem cell transplantation for treatment of mantle cell lymphoma

BACKGROUNDnMantle cell lymphoma (MC) is not curable with conventional chemotherapy. To improve the prognosis of patients with this disease, we prospectively studied an intensive sequential therapy consisting of the Dexa-BEAM regimen (dexamethasone, BCNU, etoposide, ara-C, melphalan) followed by myeloablative therapy with autologous stem cell reinfusion.nnnPATIENTS AND METHODSnNine consecutive patients with stage III/IV MC were included. Two had untreated disease, four were in first remission, whereas three had more advanced disease. All patients underwent one to two cycles of Dexa-BEAM chemotherapy to reduce the tumor load and to mobilize peripheral blood progenitor cells (PBPC). Subsequently, patients were treated with high-dose radiochemotherapy followed by PBPC reinfusion and were prospectively analyzed for residual disease by clinical methods as well as by PCR amplification clonal CDRIII rearrangements.nnnRESULTSnWith an overall response rate of 100%, the initial Dexa-BEAM cycles effectively reduced the tumor load. All patients proceeded to high-dose therapy and subsequent stem cell rescue. Engraftment was prompt, and procedure-related deaths did not occur. With a median follow-up of 12 (3-33) months post transplant, all patients are alive in continuing clinical and molecular remission.nnnCONCLUSIONSnSequential intensive therapy consisting of Dexa-BEAM and high-dose radiochemotherapy appears to be a highly effective treatment for patients with MC. However, the data are still preliminary, and larger patient numbers and a longer follow-up are required.

Reduced-intensity allogeneic stem cell transplantation as salvage treatment for patients with indolent lymphoma or CLL after failure of autologous SCT

Reduced-intensity allogeneic stem cell transplantation as salvage treatment for patients with indolent lymphoma or CLL after failure of autologous SCT