Faculty Bibliography

Peripheral blood progenitor cells used during high dose treatments for malignancy may be contaminated with tumour cells that could later contribute to recurrence. CD34+ selected harvests still contain tumour cells and an additional negative selection may be capable of reducing this contamination. We have assessed a two-stage technique in which a CD34+ selection is followed by a tumour specific depletion stage using a B cell or breast cancer specific antibody panel. Initial small-scale selections on 11 patients with NHL and breast cancer showed that cell loss was greatest following the CD34+ selection with a median yield of 38.8 per cent (range 17. 2-56.4 per cent). The addition of the depletion stage resulted in a minimal loss of CD34+ cells with a yield for this step of 94.2 per cent (range 77.5-99.3 per cent). Clinical scale selections were performed on seven patients with CLL and a median of 2.8x10(6)/kg CD34+ cells (range 1.5-6.1x10(6)/kg) were collected. Cell recovery was 53.3 per cent following CD34+ selection and 76.9 per cent following the tumour specific depletion stage, resulting in a final product containing a median of 1.0x10(6)/kg CD34+ cells (range 0. 55-2.0x10(6)/kg). All unmanipulated harvests were heavily contaminated with tumour cells (median contamination 10.2 per cent, range 2.0-83.1 per cent) as measured by flow cytometry and a median 4.7 log (range 3-5 log) tumour cell purge was produced following two-stage selection. Six of the patients have received cells manipulated in this way with median engraftment times of neutrophils>0.5x10(9)/l=16 days (range 13-20 days) and platelets>20x10(9)/l=16.5 days (range 11-42 days). At a median follow-up of 25 months, these transplanted patients remain well and in molecular complete remission.

PURPOSE/OBJECTIVE:To determine the effect of polymorphic variations in the tumor necrosis factor alpha (TNFalpha) and lymphotoxin alpha (LTalpha) genes on the predisposition to myeloma and the effect of these polymorphisms on response to treatment and overall survival. PATIENTS AND METHODS/METHODS:Genotype distribution was determined in 63 patients with monoclonal gammopathy of uncertain significance (MGUS) and 198 patients with myeloma and compared with that in 250 age- and sex-matched population-based controls. The effect on treatment response and survival was determined in 171 myeloma patients treated with either conventional or high-dose chemotherapy. RESULTS:Comparison of the extended TNFalpha/LTalpha haplotype in the myeloma cases and controls showed a significant excess of high-producer alleles in the cases. The double heterozygotes TNF1/2 and LT10.5/5.5 were present in 35.8% of cases but in only 18% of the controls; this presence was associated with a significant increased risk of myeloma (odds ratio, 2.05; 95% confidence interval, 1.26 to 3.35). A similar odds ratio was seen in the MGUS cases, suggesting that this genotype is associated with the initiation of plasma-cell disorders rather than the progression of MGUS to myeloma. The median overall survival time of myeloma patients was 53.8 months and showed no difference with regard to TNFalpha/LTalpha polymorphic status. A trend toward an improved progression-free survival was apparent in cases with a high-producer haplotype, although this effect was seen only in patients receiving high-dose chemotherapy. CONCLUSION/CONCLUSIONS:Individuals with polymorphisms associated with a high production of TNFalpha/LTalpha are at a significantly increased risk of developing MGUS and myeloma. The impact of polymorphic status on overall survival is minimal, although there is a trend toward an increased progression-free survival in the high-producer group.

Primary amyloidosis (AL), like multiple myeloma (MM), results from a clonal proliferation of plasma cells. Recent detection of Kaposi's sarcoma-associated herpesvirus (KSHV) gene sequences in MM patients, although controversial, suggested that KSHV may also be present in AL. In the present study, we assayed for KSHV gene sequences in patients with primary AL independently in 2 laboratories. Nested polymerase chain reaction (PCR) was performed on DNA isolated from 21 bone marrow (BM) core biopsy samples to amplify orf26 and orf72, 2 regions of the KSHV genome. Eighteen of 21 (86%) BM core biopsy samples were KSHV PCR positive. BM aspirates from 16 of these 21 AL patients were cultured for 4-6 weeks to generate long term bone marrow stromal cells (LT-BMSCs), and 13 of 16 (81%) LT-BMSCs were also KSHV PCR positive. Results in all but 1 sample were consistent in the 2 laboratories. Sequencing of the PCR products in the 2 laboratories confirmed 94-98% and 95-98% homology to the published orf 26 and orf 72 KSHV gene sequences respectively, with interpatient base pair differences. Despite the presence of KSHV gene sequences, only 4/18 (22%) KSHV PCR positive patients demonstrated KSHV lytic antibodies by immunoblot assay. A sensitive assay performed on the BCBL-1 cell line confirmed the presence of KSHV at a very low copy number in AL. PCR using patient specific light chain gene primers also amplified DNA isolated from 2 AL BM core biopsies and 3 AL LT-BMSCs which were KSHV PCR positive, suggesting the presence of clonotypic cells. Our results therefore demonstrate KSHV gene sequences albeit at a very low copy number in the majority of BM core biopsies and LT-BMSCs from AL patients, and serological responses in only a minority of cases. Ongoing studies to identify viral transcripts and gene products will determine the biological relevance of KSHV in AL disease pathogenesis.

Multiple myeloma remains an incurable disease, and a new perspective on the approach to therapy is required. The aim of this review is to focus on a number of key areas where recent advances in the biology of the disease have not only yielded an understanding of the disease pathogenesis but have also suggested novel treatment approaches. Factors mediating myeloma cell growth, survival and the complex interaction of myeloma cells with the bone marrow microenvironment have provided a framework for the rational design of therapeutic agents. The development of such biologically based treatments which target both the tumour cell and the microenvironment, in order to achieve more complete and selective eradication of myeloma cells and the maintenance of minimal residual disease states, may ultimately lead to improved disease-free survival and potentially a cure.

We developed a simple and rapid method to enrich tumor cells within bone marrow (BM) aspirates from patients with multiple myeloma (MM). Thirty patients with a median of 50% (8-85%) MM cells by morphology and 55% (6--85%) MM cells identified by CD38+CD45-cell surface phenotype were studied. BM mononuclear cells (BMMCs) were isolated by Ficoll Hypaque sedimentation and incubated with a cocktail of mouse monoclonal antibodies (mAbs) directed against CD3 (T cells); CD11b and CD14 (monocytes); CD33 (myeloid cells), CD45 and CD45RA (leucocyte common antigen); CD32 as well as glycophorin A. After the addition of anti-mouse Fc Ig-coated immunomagnetic beads, mAb-bound cells were removed in a magnetic field. The residual cell populations were enriched for MM cells, evidenced by >95% plasma cell morphology and >95% CD38+CD45RA-cell surface phenotype. Since this method requires only two short incubations, cell losses were minimal and the yield of MM cells was therefore high (>95%). Viability of the MM-cell enriched fractions was 99%, and these cells were functional in assays of proliferation, cell cycle analysis and immunoglobulin secretion. This immunomagnetic bead depletion method therefore permits the ready isolation of homogeneous populations of patient MM cells for use in both cellular and molecular studies.

Studies utilizing flow cytometry and PCR have shown that the B-cell compartment in myeloma contains cells which are clonally related to the myelomatous plasma cells. Current data, however, remains inconclusive regarding the extent of this involvement. By combining fluorescent immunophenotyping, tyramine signal amplification and fluorescence in-situ hybridization (FICTION-TSA), we have used the presence of numerical chromosomal abnormalities within plasma cells as a clonal marker to examine the CD20+ B-cell compartment for the presence of aneuploidy. A series of 54 cases of myeloma were screened for the presence of numerical abnormalities of chromosomes 3 and 11. FICTION-TSA was performed on 13 cases with either trisomy 3 or 11 and on a control group of six cases known to be disomic for the two chromosomes. B-cell numbers were reduced in the myeloma cases compared to the normal controls (median 1.8% v 3.0%, P = 0.05). In the cases with a chromosomal marker, three signals were seen in a median of 1.88% of CD20+ B cells compared to 2.58% within the control group. Comparison of the two groups using a Wilcoxon-Mann-Whitney U test showed no statistical significant difference. Using this data set, it was possible to exclude a 3.03% expansion of clonally related B cells (95% confidence level). We conclude that the B-cell compartment in myeloma does not represent the major site of clonal expansion, and if clonally related cells are present then the numbers are few.

There is a wide variation in the degree of marrow and blood involvement between patients with multiple myeloma. Both of these parameters are known to be highly significant prognostic factors, and the differences between patients may be due to variable expression of adhesion molecules. To test this we used three-colour flow cytometry to study three adhesion molecules associated with myeloma, namely CD38, CD56 and CD138. The level of expression of these molecules was compared with the distribution of myeloma plasma cells in bone marrow (n=59) and peripheral blood (n=26) in patients at presentation or relapse. The extent of marrow infiltration on the trephine biopsy correlated inversely with CD56 expression (Mann-Whitney U Test, P=0.022); there was no difference in CD38 or in CD138 expression. CD56 expression also correlated inversely with the number of circulating plasma cells (linear regression, R2=0.4268, slope=-0.58, P=0.0003). Peripheral blood plasma cell numbers correlated weakly with bone marrow plasmacytosis, and inversely with CD38 expression. The level of CD56 expression by neoplastic plasma cells was assessed in 37 patients over a median of 11 months (range 6-25). There was no significant change in expression (Wilcoxon Signed Rank, P=0.6271). We conclude that plasma cell CD56 expression is constant over the course of the disease; unlike CD138 expression, it is significantly linked to the degree of both bone marrow and peripheral blood involvement.

Liposomal encapsulation of anthracyclines is a potential method of drug targeting, altering both the antitumour activity and side-effect profile of anthracyclines. Liposomal daunorubicin (daunoxome) shows both altered pharmacokinetics and a potential for reducing dose-limiting cardiotoxicity compared to conventional daunorubicin. Anthracyclines have a common role in the treatment of multiple myeloma, a prevalent and fatal haematological malignancy. Avoiding cumulative anthracycline toxicity in these patients is important. There is also a need for more effective relapse schedules given that many patients have chemosensitive disease at relapse. We have analysed daunoxome in vitro in myeloma cell lines using a thymidine-based cytotoxicity assay and show superior efficacy compared to a pegylated liposomal doxorubicin derivative. Subsequently we have treated seven relapsed myeloma patients with a regime consisting of oral CCNU 25-50 mg/m2 on day 1, 4 days of oral dexamethasone 10 mg/m2 and intravenous daunoxome (liposomal daunorubicin) given for 4 days (total 100 mg/m2). The main toxicity was myelosuppression but non-haematological toxicity was minimal and the regime was well tolerated. Four out of seven of these heavily pretreated patients responded. Together with the in vitro data on its cytotoxicity in myeloma and its favourable pharmacokinetic profile further studies of liposomal daunorubicin in myeloma would be warranted.