B. C. Millar

Allogeneic blood and bone-marrow stem-cell transplantation in haematological malignant diseases: a randomised trial

BACKGROUND Autologous transplantation with peripheral blood stem cells (PBSC) results in faster haematopoietic-cell repopulation than with bone marrow. We prospectively compared bone marrow and PBSC for allogeneic transplantation. METHODS Adult HLA-identical sibling donors provided bone marrow and lenograstim-mobilised PBSC. 39 patients with malignant haematological disorders were infused with either bone marrow (n=19) or PBSC (n=20) after standard conditioning regimens in a double-blind, randomised fashion. The identity of the infused products for all patients remained masked until 1 year after the last patient had received transplantation. FINDINGS The PBSC group had significantly faster neutrophil recovery to 0.5x10(9)/L (median 17.5 vs 23 days, p=0.002), and platelet recovery to 20x10(9)/L (median 11 vs 18 days, p<0.0001) and to 50x10(9)/L (median 20.5 vs 27 days, p=0.02) than the bone-marrow group. PBSC patients were discharged from hospital earlier than were bone-marrow patients (median 26 vs 31 days, p=0.01). At 4 weeks after transplantation, absolute lymphocytes (0.48 vs 0.63, p=0.08) and CD25 cells (0.04 vs 0.08, p=0.007) were higher in the PBSC group, and the proportion of patients with absolute lymphopenia (74% vs 33%, p=0.03) and CD4 lymphopenia (59% vs 24%, p=0.05) was significantly higher in the bone-marrow group. There was no significant difference in the occurrence of acute or chronic graft-versus-host disease and overall survival. The probability of relapse was significantly higher in the bone-marrow group than in the PBSC group (p=0.01); all five relapses occurred among bone-marrow recipients. INTERPRETATION Our small study indicates that PBSCs are better than bone marrow for allogeneic transplantation from HLA-identical siblings in terms of faster haematopoietic and immune recovery, and have the potential to reduce disease recurrence.

Influence of collection and separation of blood samples on plasma IL-1, IL-6 and TNF-α concentrations

The anticoagulant used for the collection of blood was found to influence in vitro cytokine production in whole blood. Lithium heparin in certain collection tubes was found to contain endotoxin and induced cytokine synthesis in a time-dependent manner whereas endotoxin-free lithium heparin did not. No induction of cytokine occurred in the presence of EDTA which was also able to inhibit endotoxin-induced cytokine synthesis. Synthesis or absence of cytokine correlated with the induction of messenger RNA. Investigation of the kinetics of cytokine induction in whole blood revealed that tumour necrosis factor alpha (TNF) was detectable after 2 h of incubation at 37 degrees C and interleukin-1 beta (IL-1 beta) and interleukin-6 (IL-6) after 3 h. In certain samples IL-1 and IL-6 were detectable in plasma separated immediately from blood collected into endotoxin-free lithium heparin, presumably reflecting in vivo synthesis, and similar concentrations were detected after 3 h of incubation of whole blood at 37 degrees C. These data indicate that as long as blood is collected into endotoxin-free anticoagulant then cytokine measurements will reflect the in vivo status.

A low CD34+ cell dose results in higher mortality and poorer survival after blood or marrow stem cell transplantation from HLA-identical siblings : should 2 x 106 CD34+ cells/kg be considered the minimum threshold?

We studied the effect of the CD34+ cell dose on transplant-related mortality (TRM) and survival in 39 patients randomized to receive lenograstim-mobilized PBSCT (n= 20) or BMT (n = 19) from HLA-identical siblings. Both marrow and blood were harvested, and one infused in a double-blind fashion. The median nucleated (7.0 vs 3.2 × 108/kg; P < 0.0001), cd34+ (3.7 vs 1.5 × 106/kg; P = 0.002), CFU-GM (42 vs 19 × 104/kg; P= 0.002), and CD3+ (1.9 vs 0.3 × 108/kg; P < 0.0001) cell doses with pbsct were higher. thirteen patients (6 bmt and 7 pbsct) experienced trm at 15–733 days (median 57); 10 of 20 receiving <2 × 106 CD34+ cells/kg compared with three of 19 receiving ⩾2. Eight of 20 patients receiving <2 × 106 CD34+ cells/kg are alive compared with 14 of 19 receiving ⩾2. In Cox analysis, CD34+ cell dose ⩾2 × 106/kg was associated with lower TRM (RR 0.2, P = 0.01), and higher overall (RR 3.7, P = 0.01) and event-free (RR 3.2, P = 0.02) survival. Other cell populations and the source of stem cells did not affect TRM or survival. We conclude that 2 × 106 CD34+ cells/kg may be the ideal minimum cell dose for allogeneic transplantation although lower doses do not preclude successful therapy. Since the likelihood of obtaining this threshold CD34+ cell number is significantly greater from blood than marrow, PBSCT may be preferable to marrow for allografts from HLA-identical siblings. Bone Marrow Transplantation (2000) 26, 489–496.

Outcome assessment of a population-based group of 195 unselected myeloma patients under 70 years of age offered intensive treatment

A single centre series of 195 consecutive newly diagnosed untreated myeloma patients under 70 years, seen between September 1986 and March 1994, were analysed to assess the impact of current intensive treatment methods upon remission rate, response rate and subsequent outcome. They were predominantly an unselected population based group of patients (other than by age) that could be used by purchasers of health care as a model for outcome assessment. All patients were scheduled to receive a care plan which included a sequential package of treatment consisting initially of courses of infusional chemotherapy using vincristine, adriamycin and methyl prednisolone (VAMP) and 90 of these also received cyclophosphamide (C-VAMP). Thirty-eight patients received verapamil in addition to C-VAMP(V-C-VAMP). After VAMP all patients were planned to receive high-dose treatment with melphalan and an autograft (marrow or blood) and 112 received this treatment; a further 29 patients received modified high-dose treatment with melphalan alone (23) or busulfan (6) and 54 (28%) did not proceed to high-dose treatment because of refusal, resistant disease, poor performance or treatment-related death. The patients who received melphalan or busulfan alone instead of high-dose melphalan/autografts did so because of increasing age (P = 0.001) and a raised creatinine (P = 0.05). The complete remission rate was 53% for the whole group and 74% for those receiving high-dose melphalan and an autograft. From July 1988, the sequential therapy package included continuous three times weekly interferon (IFN) after high-dose treatment as maintenance therapy, initially as part of a controlled randomised trial and then for all suitable patients. Fifty-seven patients received IFN. The median overall survival (OS) and progression-free survival (PFS) from first treatment for the whole group of 195 patients is 4.5 years and 25 months, respectively. The 112 patients receiving the melphalan autografts fared significantly better than the rest of the patients with OS and PFS (from high-dose treatment) of 6.6 years and 27 months, respectively (P < 0.005), and the 57 patients also receiving ifn have a os yet to reach a median at 8 years and a pfs of 44 months, significantly better than non ifn high-dose patients (P < 0.0036). however, although we showed benefit for selected patients in studies and trials (particularly with ifn) during the 8-year period of the series, this did not translate into overall pfs benefit in our study for unselected cohorts of patients for 1986–1988 (64 patients) 1989–1992 (100 patients) and 1992–1994 (31 patients) in spite of progressive increases in the proportion of patients receiving ifn (respectively 6, 35 and 58%). this is likely to be due to the dilution of benefit to specific patients by the overall number of patients involved. outcome data from unselected patients are now expected by purchasers and presented in this way, help qualify the activity impact of advances made from research trials for the treatment of population-based cancer problems.

A comparison of vincristine and doxorubicin infusional chemotherapy with methylprednisolone (VAMP) with the addition of weekly cyclophosphamide (C‐VAMP) as induction treatment followed by autografting in previously untreated myeloma

In a sequential nonrandomized study, 204 consecutive unselected patients aged < 70 years received induction chemotherapy with infusional vincristine and adriamycin with oral methyl prednisolone (VAMP; n=75) or with additional cyclophosphamide, C‐VAMP (n=129). 38/129 C‐VAMP patients also received verapamil during induction as part of a controlled trial with the aim to overcome drug resistance. A median of five courses (range 1–11) of chemotherapy were required before maximal response was attained and this was similar in both groups. An over‐all response rate of 71% was noted at the end of induction. The complete remission (CR) rate with C‐VAMP was 24%, which was significantly higher (P=0.04) than the CR rate with VAMP alone (8%). The addition of verapamil did not alter the response rate of C‐VAMP. Compliance to VAMP was overall 83% and not affected by the addition of cyclophosphamide. The proportion of patients going on to receive high‐dose chemotherapy and an autograft was the same for VAMP and C‐VAMP treated patients (71%). The median overall survival (OS) and progression‐free survival (PFS) for the whole group were 4.4 years and 2.0 years and no difference in outcome was observed between the different treatment groups. Therefore the addition of weekly cyclophosphamide to VAMP induction therapy has significantly improved the response rates of previously untreated myeloma patients. C‐VAMP was not more toxic and did not compromise the chances of receiving an autograft. Verapamil was without influence on any parameters in this study.

Inhibitory effect of simvastatin on the proliferation of human myeloid leukaemia cells in severe combined immunodeficient (SCID) mice

SCID mice were inoculated intravenously with cells from the human HL60 myeloblastic leukaemia cell line and then treated with the 3‐hydroxy‐3‐methylglutaryl coenzyme A (HMG CoA) reductase inhibitor, simvastatin, by subcutaneous continuous infusion. The effect of the drug was measured by subsequent colony formation of surviving HL60 cells in vitro and flow cytometry. The number of clonogenic HL60 cells was reduced in the bone marrow of mice that received simvastatin compared with control mice by 65% and 68% in two separate experiments. The number of clonogenic, normal, murine, bone marrow progenitor cells concomitantly exposed to simvastatin in vivo, was not affected in either experiment. Flow cytometric analysis of bone marrow and spleen cells confirmed these results by showing that simvastatin had reduced the percentage of human leukaemia cells in these tissues by 70% and 88% respectively.  The data show that the reported selective effect of simvastatin against acute myeloid leukaemia cells in vitro, can be extended to this in vivo model. HL60 bears an N‐ras mutation. In further in vitro studies, ketoconazole, an inhibitor of cholesterol biosynthesis post farnesyl pyrophosphate synthesis, had a similar effect to simvastatin on HL60 colony development. Furthermore, the clonogenicity of a population of N‐ras mutated, primary acute myeloid leukaemia (AML) cells was no more sensitive to simvastatin than a population without the mutation. The data suggest that the inhibition of AML cell proliferation by simvastatin may be independent of the RAS signalling pathway.

Comparison of marrow and blood cell yields from the same donors in a double-blind, randomized study of allogeneic marrow vs blood stem cell transplantation.

Forty healthy adult donors underwent marrow (BM) as well as peripheral blood (PBSC) stem cell collections for their HLA-identical adult siblings with hematologic malignancies. BM was harvested on day 1 (target 3 × 108 nucleated cells/kg, 10 μg/kg lenograstim (glycosylated G-CSF) administered on days 2–6, and a single leukapheresis performed on day 6. The blood volume processed was the higher of 200% donor blood volume or 10 liters. The total nucleated cell (TNC) yields from PBSC were 1.1- to 4.3-fold higher than BM (median 7.0 vs 3.1 × 108/kg, P 7lt; 0.0001). Although bm contained a higher proportion of cd34+cells (1.3% vs 0.7%, P < 0.0001) and a comparable proportion of cd3+ cells (median 29% vs 26%, P = 0.4), the absolute numbers of CD34+ and CD3+ cells and their subsets were several times higher in PBSC. There was a poor correlation between BM and PBSC CD34 and TNC numbers, but a significant correlation between BM and PBSC CD3 numbers. Only five of 40 BM harvests contained ⩾2 × 106 CD34+ cells/kg compared with 35 of 40 PBSC harvests (P < 0.0001). we conclude that the numbers of progenitor and immunocompetent cells in pbsc are several times higher than in bm. it is possible to collect adequate numbers of progenitor cells from blood after lenograstim stimulation more frequently than from marrow, and donors yielding low quantities of progenitor cells from bm usually deliver better quantities from pbsc. Bone Marrow Transplantation (2000) 25, 501–505.

The importance of CD34 + /CD33 − cells in platelet engraftment after intensive therapy for cancer patients given peripheral blood stem cell rescue

The study was designed to determine whether the number of CD34+/CD33− cells given at autologous peripheral blood stem cell (PBSC) rescue after intensive therapy for cancer was a better predictor of platelet engraftment than the total number of CD34+ cells infused. Comparison between the total number of CD34+ cells/kg infused with the number of CD34+/CD33− cells/kg infused showed that, generally, 2 × 106 total CD34+ cells contained 1.38 × 106 CD34+/CD33− cells. There was poor correlation between the number of CD34+/CD33− and CD34+/ CD33+ cells in the graft (r = 0.332). Engraftment times for platelets and neutrophils were evaluated in 68 patients. There was no significant difference between the times for platelets to reach >25 × 109/l or neutrophils to reach >0.5 × 109/l among patients who received > or <2 × 106 total CD34+ cells or > or <1.38 × 106 CD34+/CD33− cells although the latter was consistently the better predictor. Platelet recovery to >50 × 109/l and >100 × 109/l was delayed significantly in patients who received <1.38 × 106 CD34+/CD33−/kg infused (P < 0.02 and P < 0.05, respectively). The number of cd34+/ CD33− cells/kg infused was a stronger predictor of platelet recovery than the total number of CD34+ cells infused (P < 0.05 for platelets >50 or >100 × 109/l). Although platelet recovery was delayed significantly in patients who had <4 × 104 granulocyte–macrophage colony-forming units (CFU-GM)/kg infused, the time delay between receipt of PBSCs and availability of the colony counts limits the use of this assay to patients who do not require stem cells to be given immediately. Our data suggest that the number of CD34+/CD33− cells given at PBSC rescue provide information about the quality of the graft necessary for long-term platelet engraftment. However, since the percentage of CD34+/CD33− cells shows considerable inter-patient variation, measurement of this cell population may be important in patients who experience poor stem cell mobilization or when a target dose of 2 × 106 total CD34+ cells/kg is not achieved.

Comparison of marrow vs blood-derived stem cells for autografting in previously untreated multiple myeloma

Sixty-three new untreated patients with multiple myeloma under the age of 70 years received C-VAMP induction treatment followed by high-dose intravenous melphalan (200 mg m(-2)) and autologous stem cell transplant, either with marrow [autologous bone marrow transplants (ABMT), n = 26] or with granulocyte colony-stimulating factor (G-CSF)-mobilized stem cells from the blood [peripheral blood stem cell transplants (PBSCT), n = 37]. This was a sequential study and the two groups were not significantly different for all known prognostic variables. The complete remission (CR) rate after high-dose treatment was the same for both groups [ABMT 84% and PBSCT 70%; P = not significant (NS)]. Neutrophil recovery to 0.5 x 10(9) l(-1) occurred at a median of 22 days in the ABMT patients compared with 19 days for the PBSCT patients (P = NS). Platelet recovery to 50 x 10(9) l(-1) was significantly faster in PBSCT patients (19 days vs 33 days; P = 0.0015), and the PBSCT patients spent fewer days in hospital (median 20 vs 27 days; P = 0.00001). There was no difference in the two groups with respect to starting interferon (58 days for ABMT vs 55 days for PBSCT), and tolerance to interferon was identical. The median overall survival (OS) and progression-free survival (PFS) for the PBSCT patients has not yet been reached. The OS in the ABMT patients at 3 years was 76.9% (95% CI 60-93%) compared with 85.3% (95% CI 72-99%) in the PBSCT patients (P = NS), and the PFS at 3 years in the ABMT patients was 53.8% (95% CI 34-73%) and in the PBSCT patients was 57.6% (95% CI 34-81%) (P = NS). The probability of relapse at 3 years was 42.3% in the ABMT arm compared with 40% in the PBSCT patients (P = NS). Thus, PBSCT patients had a faster engraftment and a shorter stay in hospital than ABMT; the survival outcome and probability of relapse was the same for both groups.

Interleukin-6 is a cofactor for the growth of myeloid cells from human bone marrow aspirates but does not affect the clonogenicity of myeloma cells in vitro.

Summary Several groups have claimed that IL‐6 is a growth factor for human myeloma cells in vitro. Bone marrow aspirates from 30 patients at different stages of treatment with VAMP/high dose melphalan, were examined for myeloma colony formation (MY‐CFU,) using a clonogenic assay in vitro. Myeloma cells from 16/30 patients produced MY‐CFUc in our assay system, which uses heavily irradiated HL60 cells as an underlay in soft agar. These heavily irradiated cells were shown to be essential for the inhibition of granulocyte‐macrophage colonies (GM‐CFUc). The addition of recombinant human IL‐6 (10 ng/plate) reduced the number of bone marrow samples which produced MY‐CFUc from 16 to six. Furthermore, the addition of antibody to IL‐6 (1 μg/plate) failed to inhibit MY‐CFUc from 6/7 samples.