Faculty Bibliography

OBJECTIVE:To examine the safety and efficacy of granulocyte colony-stimulating factor (G-CSF) alone for the mobilization of peripheral blood progenitor cells in patients with resistant active rheumatoid arthritis (RA). METHODS:Five patients with resistant active RA were studied. A dose of 5 microg/kg of G-CSF (Filgrastim) was given subcutaneously each day for 5 days, and the number of stem cells mobilized into the peripheral blood was assessed by daily CD34 counts. RA disease activity was assessed by standard clinical methods. RESULTS:The absolute numbers of peripheral blood CD34+ cells peaked on day 4, with a mean value of 0.025 x 10(9)/liter (range 0.013-0.048 x 10(9)/liter). There was no significant change in disease activity during the study or in the month following therapy. CONCLUSION/CONCLUSIONS:Using G-CSF alone, CD34+ progenitor peripheral blood cells were mobilized in numbers suitable for leukopheresis. G-CSF therapy was well-tolerated in patients with active RA, and was not associated with a flare during treatment or in the month following treatment.

PURPOSE/OBJECTIVE:A pilot study to validate the use of CD34+ selection (Ceprate SC) of blood stem-cell collection in patients with advanced follicular lymphoma receiving myeloablative chemoradiotherapy. PATIENTS AND METHODS/METHODS:Seventeen patients were entered onto the protocol. Thirteen of 17 patients have undergone transplantation; the median age is 42.5 years (range, 33 to 51), seven of 13 are stage IVB, two stage IVA, three stage IIIB, and one stage IIB. All except two patients were treated after first or subsequent relapses after receiving cyclophosphamide, doxorubicin, vincristine, and prednisone (CHOP) chemotherapy to achieve a good partial (six of 13) or complete (seven of 13) response before stem-cell mobilization with cyclophosphamide 3 g/m2 and filgrastim 300 microg once daily. RESULTS:Eleven of 13 patients had a detectable t(14;18) by nested polymerase chain reaction (PCR). Median CD34+ count before selection was 2.9 x 10(6)/kg (range, 1.17 to 11.3) and after CD34+ selection was 1.54 x 10(6)/kg (range, 0.88 to 7.6) with a median CD34+ yield of 62.4% (range, 17% to 95%) and purity of 60% (range, 39.3% to 73%). Of the 11 patients known to have t(14;18), 10 had PCR-detectable contamination of stem-cell harvests that became PCR negative in six of the 10 after CD34+ selection. Engraftment was rapid with a median day to absolute neutrophil count (ANC) greater than 0.5 x 10(9)/L of 13 days (range, 11 to 21) and platelet count greater than 20 x 10(9)/L of 14 days (range, 10 to 44). With a median follow-up duration of 15 months, three patients have remained persistently PCR-positive, two of whom received PCR-positive stem cells. Two have relapsed. Of the seven patients who received PCR-negative stem cells, five have had no PCR-detectable disease in posttransplant bone marrow samples. CONCLUSION/CONCLUSIONS:Longer follow-up duration is required to determine the significance of these findings, but we have confirmed the feasibility of CD34+ selected cells to deplete peripheral-blood stem cells of tumor cells from patients undergoing high-dose therapy for follicular lymphoma.

The sensitivity of detection of residual disease by two IgH PCR strategies, fluorescent framework 3 (Ffr3) and allele-specific oligonucleotide probing (ASOP), was compared in 57 'remission' BM samples obtained from 19 children with B-lineage acute lymphoblastic leukaemia (ALL). Oligonucleotide probing was more sensitive than FFr3 PCR in 10/16 cases, achieving a sensitivity of 0.01% or greater in 15/16 cases. Comparable sensitivities were obtained in the six remaining cases; the FFr3 PCR achieving a sensitivity of 0.1% or greater in 14/16 cases. 39/57 'remission' BM samples analysed showed no evidence of MRD by either technique although 18 were positive by ASOP and 14 positive by FFr3 PCR. The level of disease was estimated to be 0.01% or less in the four false negative samples.

The aim of this study was to develop a flow cytometric test to quantitate low levels of circulating myeloma plasma cells, and to determine the relationship of these cells with disease stage. Cells were characterized using five-parameter flow cytometric analysis with a panel of antibodies, and results were evaluated by comparison with fluorescent consensus-primer IgH-PCR. Bone marrow myeloma plasma cells, defined by high CD38 and Syndecan-1 expression, did not express CD10, 23, 30, 34 or 45RO, and demonstrated weak expression of CD37 and CD45. 65% of patients had CD19- 56+ plasma cells, 30% CD19- 56(low), and 5% CD19+ 56+, and these two antigens discriminated myeloma from normal plasma cells, which were all CD19+ 56(low). Peripheral blood myeloma plasma cells had the same composite phenotype, but expressed significantly lower levels of CD56 and Syndecan-1, and were detected in 75% (38/51) of patients at presentation, 92% (11/12) of patients in relapse, and 40% (4/10) of stem cell harvests. Circulating plasma cells were not detectable in patients in CR (n = 9) or normals (n = 10), at a sensitivity of up to 1 in 10,000 cells. There was good correlation between the flow cytometric test and IgH-PCR results: myeloma plasma cells were detectable by flow cytometry in all PCR positive samples, and samples with no detectable myeloma plasma cells were PCR negative. Absolute numbers decreased in patients responding to treatment, remained elevated in patients with refractory disease, and increased in patients undergoing relapse. We conclude that flow cytometry can provide an effective aternative to IgH-PCR that will allow quantitative assessment of low levels of residual disease.

We have developed a competitor-based RT-PCR technique which will detect and quantitate the CBFbeta/MYH11 transcripts associated with inv(16)(q22;p13) and have used it to study presentation and follow-up samples of acute myeloid leukaemia (AML). The levels of the leukaemia-specific transcripts are expressed as a ratio to a ubiquitously expressed mRNA species (Abl) which controls for RNA degradation. This technique has been applied to 75 consecutive patients presenting with either de novo AML or tMDS; 6/75 patients analysed were positive for the inv(16), all were confirmed by conventional cytogenetics. The inv(16) has a strong association with M4Eo, but we found only 2/6-positive patients to have this diagnosis (two patients with M2, one patient M1 and one patient had MDS). At presentation the levels of CBFbeta/MYH11 transcripts were 0.1-10/Abl transcript (mean 3.3/Abl transcript). Seventeen follow-up samples were available on 5/6 of these patients, and on two further patients in whom stored material was available. Following the first cycle of chemotherapy the level of transcripts was at least 10(-2) lower (0.1-10 x 10(-2)/abl transcript) than their presentation sample. Subsequent samples on these patients when in remission gave transcript levels in the range (1.0 x 10(-4) - 2 x 10(-3)/abl transcript), and three long-term follow-up samples were negative. We have developed a quantitative test which opens the possibility of predicting relapse by detecting changes in the numbers of leukaemia-specific transcripts.

AIM/OBJECTIVE:To assess whether p53 gene mutation is important in the pathogenesis and progression of multiple myeloma. METHODS:Thirty eight DNA samples (derived predominantly from bone marrow) obtained from 31 patients with multiple myeloma were examined for mutations in p53 exons 5-9 by polymerase chain reaction single strand conformation polymorphism. Twenty three samples were analysed at the time of diagnosis (one patient had plasma cell leukaemia), three in plateau phase, and 12 at relapse (one plasma cell leukaemia and one extramedullary relapse). RESULTS:One p53 mutation was detected in this group of patients (3.2%). This was seen in the diagnostic bone marrow sample of a 35 year old man with stage IIA disease and occurred in exon 6 as a result of a silent A to G transition at codon 213 (CGA-->CGG), a polymorphism that has been reported in about 3% of breast and lung tumours. CONCLUSIONS:p53 gene mutations are rare events in multiple myeloma and would seem to be of limited value as a prognostic factor.

We have carried out an analysis of 44 patients undergoing allogeneic PBSC transplants from fully HLA-matched related donors with particular emphasis on engraftment kinetics and the incidence and severity of GVHD. The recipients had a median age of 37 years (range 5-56 years), 16 patients had standard-risk disease and 28 had poor-risk disease. GVHD prophylaxis was with cyclosporin A and methotrexate (n = 41), cyclosporin A alone (n = 2) or cyclosporin A and methyl-prednisolone (n = 1). Stem cells were mobilised using G-CSF, collecting a median of 5.75 x 10(6) CD34+ cells/kg recipient weight (range 0.94-35 x 10(6) CD34+ cells/kg). Engraftment times to a neutrophil count > 0.5 x 10(9)/1 and platelets > 20 x 10(9)/1 were achieved at a median of day +14 (range 10-25) and day +14 (range 9-130) respectively. Patients receiving > or = 4 x 10(6) CD34+ cells/kg had significantly accelerated neutrophil and platelet engraftment and this number of CD34+ cells would appear to be a prerequisite for maximum engraftment using PBSC. Acute GVHD occurred in 25 of 43 evaluable patients although in only 12 was this clinically significant (grades II-IV). Chronic GVHD has occurred in 17 out of 36 evaluable patients, there was no significant difference between the standard- and poor-risk groups in incidence of either acute or chronic GVHD. In conclusion, these results confirm the feasibility of using PBSC for allogeneic transplantation without evidence for increased risk of either acute or chronic GVHD and provide further evidence supporting the potential of PBSC to replace bone marrow as the major source of haemopoietic cells for allogeneic transplantation.

Non-Hodgkin's lymphoma (NHL) is not a uniform disease entity, and in order to investigate the reported changes in incidence we have set up a study in seven population-based cancer registries in Europe. The study is designed to look at changes in the incidence of total NHL and disease subgroups using standard definitions and methodology. The registries are based in Leeds, Dijon, Kuopio, Odense, Florence, Eindhoven, and Ragussa. The classification system we have used is based on the REAL classification and has utility for epidemiological studies. We have used it to convert data sets which have utilized both local cases and the ICD-O classification. In order to improve data reproducibility, CLL/LL, myeloma/MGUS, lymphoblastic disease, and Hodgkin's disease have been excluded because of the difficulty in defining incident cases accurately. The preliminary results of this study show that there is still an upward trend in incidence rate and that in Yorkshire this is 3% per annum in total NHL. The subgroups which are increasing are extranodal and nodal peripheral T-cell lymphoma. Similar increases in incidence have been reported for the other registries. We conclude that there is a continued upward trend in incidence of NHL, the causes of which are uncertain.