CD34+-selected peripheral blood progenitor cell transplantation in patients with multiple myeloma: tumour cell contamination and outcome.

Thirty-six patients with multiple myeloma (23 PR1, nine PR2, four stable disease) were entered into a pilot study evaluating the use of CD34+-selected peripheral blood progenitor cell transplantation (PBPCT) following high-dose melphalan alone or high-dose melphalan and total body irradiation. Peripheral blood progenitor cells (PBPCs) were mobilized with cyclophosphamide and granulocyte colony stimulating factor (G-CSF). CD34+ selection using the Cellpro Ceprate-SC system was performed in 22 cases with an adequate yield in 20. 10 patients failed to mobilize sufficient cells to permit selection and in four cases selection was not performed for other reasons. 16 patients therefore received unselected PBPC. Tumour cell contamination was evaluated by IgH gene fingerprinting (fpPCR). Harvested PBPC were fpPCR positive in 13/20 CD34+-selected cases and remained positive after selection in seven. Harvested PBPC were studied in 9/16 patients receiving unselected cells; fpPCR was positive in five and negative in four. There was no difference in event-free survival (EFS) between the CD34+-selected group and the unselected group (median 21 and 26 months, respectively, P=ns). The CD34+-selection process therefore reduced contamination but did not eliminate it completely, and in this small non-randomized study there was no apparent clinical benefit of CD34+ selection.

High-dose therapy including carboplatin adjusted for renal function in patients with relapsed or refractory germ cell tumour: outcome and prognostic factors.

Thirty-one consecutive patients with relapsed or refractory GCT received an HDT schedule including carboplatin, the dose of which was adjusted to measured glomerular filtration rate. There was one HDT-associated death (3%), due to acute renal failure. The 3-year probability of overall and disease-free survival for 21 patients with primary refractory disease or responsive relapse was 60% and 42%, respectively, while none of ten patients with refractory relapse have survived disease free.

When to harvest peripheral-blood stem cells after mobilization therapy: prediction of CD34-positive cell yield by preceding day CD34-positive concentration in peripheral blood.

PURPOSE: To evaluate whether the CD34+ yield from a single peripheral-blood stem-cell (PBSC) harvest could be predicted by measurement of the patient's circulating WBC and CD34+ cell concentrations on the day before harvest. PATIENTS AND METHODS: Thirty-nine patients with hematologic or nonhematologic malignancy underwent 41 stem-cell mobilization episodes with cytotoxic chemotherapy and/or granulocyte colony-stimulating factor (G-CSF), and a total of 63 leukapheresis procedures were performed. Peripheral-blood samples were analyzed for WBC and CD34+ cell concentration both on the day before and the day of leukapheresis. RESULTS: The median WBC and CD34+ concentrations on the day preceding leukapheresis were 10.0 x 10(9)/L (range, 0.4 to 44.4) and 24.9 x 10(6)/L (range, 0.1 to 349.4), respectively. On the day of harvest, the corresponding figures were 15.1 x 10(9)/L (range, 1.5 to 52.6) and 29.3 x 10(6)/L (range, 0.1 to 543.1), respectively. The median CD34+ cell number collected in a single leukapheresis was 2.6 x 10(6)/kg body weight (range, 0.1 to 26.1). Both the preceding day (r = .84, P < .001) and harvest day (r = .95, P < .001) CD34+ circulating concentrations correlated significantly with the number of CD34+ cells per kilogram collected at leukapheresis. The correlation between CD34+ cells per kilogram collected and harvest day WBC count was also significant (r = .43, P <.001), but with the preceding day WBC count was nonsignificant. CONCLUSION: The number of CD34+ cells harvested in a single leukapheresis can be predicted by measurement of the preceding day peripheral-blood circulating CD34+ concentration, and on the basis of these data a table of probable CD34+ cell yield has been constructed. This correlation may facilitate the efficient organization of leukapheresis procedures.

Quantitation of multidrug resistant MDR1 transcript in acute myeloid leukaemia by non-isotopic quantitative cDNA-polymerase chain reaction.

Drug resistance in acute myeloid leukaemia (AML) may be caused by overexpression of the P glycoprotein (PGP), an efflux pump encoded by the multidrug resistance mdr 1 gene. Previous studies have suggested that increased PGP expression in the leukaemic blasts is of prognostic significance, and that use of PGP antagonists may be beneficial in treatment. We describe preliminary results with a non-isotopic quantitative MDR 1 cDNA-PCR assay, using an artificial RNA construct sharing primer recognition sites with the target MDR 1 mRNA (MDR 1 nucleic acids 483-504 and 624-644) as an internal control. KB 3.1 parent and KB 8.5 MDR positive cell lines expressed 0.004 and 1.96 molecules MDR 1 mRNA/pg total RNA. Semiquantitative screening of 60 RNA samples from 53 AML cases detected MDR 1 transcript ranging from 0 to 1.81 molecules per pg RNA. The median value at presentation (33 patients) was 0.055 and was higher in 14 patients at relapse (0.13) and in seven patients with refractory disease (0.14). Quantitation of MDR 1 transcript in serial samples in seven treated patients between presentation and relapse showed the decrease in three patients (0.18-0.02 x) to be as marked as the increase in three other patients (3-16 x). The method described is well suited for the study of clinical samples because it is sensitive, specific, rapid and requires small amounts of clinical material.

Sustained trilineage response in a patient with ALG-resistant severe aplastic anaemia after treatment with G-CSF, erythropoietin and cyclosporin A: association of recovery with marked elevation of serum alkaline phosphatase.

Aplastic anaemia is characterized by multilineage bone marrow failure resulting in pancytopenia. We have successfully treated a young woman with severe aplastic anaemia (SAA) who was resistant to antilymphocyte globulin (ALG) and corticosteroids, with a combination therapy consisting of erythropoietin, cyclosporin A and granulocyte-colony stimulating factor (G-CSF). The patient received erythropoietin and CSA for a period of 10 months without success before G-CSF treatment was started. After 6 weeks of G-CSF therapy she responded with a sustained trilineage recovery. This suggests that immunosuppression together with haemopoietic growth factors may be an effective treatment in patients with SAA who are ALG resistant and cannot be treated by BMT.

Flow cytometric assessment of multidrug resistance (MDR) phenotype in acute myeloid leukaemia.

Forty one patients with acute myeloid leukemia (AML), including 27 at presentation and 14 relapsed or resistant cases, were assessed for laboratory evidence of the MDR phenotype. Leukaemic cells from all 41 cases were studied by immunocytochemistry using the JSB-1 monoclonal antibody and simultaneously by reverse transcription polymerase chain reaction (RT-PCR) to evaluate expression of the mdr 1 gene. Cells from 32/41 cases were also assessed for daunorubicin (DNR) accumulation and retention by flow cytometry (FC). Nineteen of the 41 (46%) patients were positive for MDR by JSB-1 immunocytochemistry (11/27 [41%] at presentation and 8/14 [57%] relapsed or resistant cases). Nine of the 19 (47%) P-gp positive, de novo patients achieved complete remission. 22 patients were negative by JSB-1 immunocytochemistry (16/27 [59%] at presentation and 6/14 [43%] of the relapsed or resistant cases) and 11/22 (50%) P-gp negative patients achieved a complete remission. Of the 32 patients assessed by FC, 7 (22%) were positive for the MDR phenotype with increased DNR accumulation and retention in the presence of the MDR reversing agent verapamil (VPM). 6/7 of the FC positive cases were also JSB-1 positive, and 6 had additional poor risk features. Of the twenty five FC negative patients, 6 had received previous chemotherapy and 15 (60%) achieved complete remission. Mdr 1 mRNA levels were increased in all seven FC positive cases whereas only 7/19 JSB-1 positive cases had raised mdr 1 mRNA levels. These results suggest that the assessment of MDR status by immunocytochemistry using JSB-1 is not predictive of response to chemotherapy. Flow cytometric analysis of blast cells appears to correlate well with mdr 1 mRNA levels and may be a better predictor of treatment outcome.

Multidrug Resistance in Acute Leukaemia: A Comparative Study of Immunocytochemical and RNA Slot Blot Detection of P-glycoprotein Expression.

We have analysed and compared the applicability of mRNA quantitation and immunocytochemistry to the detection of the multidrug resistant gene mdr1 and its protein product P-glycoprotein (PGP) in patients with acute leukaemia. Elevated mdr1 mRNA was detected in 12 of 55 patients and PGP expression by immunocytochemistry in 10 of 27. Raised mdr1 transcript was associated with immunocytochemical positivity in 6 of 8 cases studied. 4 of 10 cases of immunocytochemical positivity were not associated with elevated mdr1 mRNA. Immunocytochemistry showed PGP expression to be heterogeneous within blast populations. In 4 cases PGP positivity was most marked in differentiated leukaemic subpopulations. PGP expression was increased in secondary acute myeloid leukaemia (AML) compared to other disease groups. No association with disease stage was detected except within the secondary leukaemias. In 40 patients studied mdr3 mRNA was raised in one patient with AML and in two remission marrows. These results indicate that further prospective studies in acute leukaemias are required using combined immunocytochemical and RNA quantitation of PGP expression.

Prolonged remission of Epstein-Barr virus associated lymphoma secondary to T cell-depleted bone marrow transplantation.

High grade non-Hodgkin's lymphoma developed 42 days after allogeneic T cell-depleted bone marrow transplantation (BMT) for idiopathic aplastic anaemia. DNA hybridization studies confirmed clonality and incorporation of Epstein-Barr virus (EBV) genome. Prolonged remission followed low dose chemotherapy, local radiotherapy and early withdrawal of cyclosporin.