Faculty Bibliography

Autoimmune phenomena are well-recognised complications of Waldenström's macroglobulinemia (WM) and IgM monoclonal gammopathy. Peripheral neuropathy and cold agglutinin hemolytic anemia are the most common reported and occur in 5-10% of patients. Autoimmune thrombocytopenia has been rarely reported in WM and its incidence is not known. In this study we report the case of a 67-year-old man who presented with autoimmune thrombocytopenia who was subsequently found to have WM. Laboratory investigation demonstrated that platelet-associated IgM (PAIgM) but not PAIgG was clearly elevated compared to normal controls. In addition the patient's serum reacted strongly with a panel of donor platelets analysed with an indirect platelet immunofluorescence assay utilising an anti-IgM secondary antibody. Glycoprotein specificity could not however be demonstrated by ELISA techniques for platelet glycoproteins IIbIIIa, IaIIa, IbIXa, and IV. We also reviewed the case records of 104 additional cases of WM diagnosed at our institution between 5/93 and 5/99. Three further cases with clinically significant autoimmune thrombocytopenia were identified. The overall incidence of autoimmune thrombocytopenia (4/105, 3.8%) in this cohort of patients was similar to the incidence of peripheral neuropathy (7/105, 6.7%) and cold agglutinins (3/105, 2.9%).

NAD(P)H:quinone oxidoreductase 1 (NQO1) is an enzyme that detoxifies quinones and reduces oxidative stress. A cysteine-to-threonine (C --> T) substitution polymorphism at nucleotide 609 of the NQO1 complementary DNA (NQO1 C609T) results in a lowering of NQO1 activity. Individuals homozygous for this mutation have no NQO1 activity, and heterozygotes have low to intermediate activity compared with people with wild type. DNA samples from 493 adult de novo acute leukemia patients and 838 matched controls were genotyped for NQO1 C609T. The majority of cases were diagnosed as acute myeloid leukemia (AML) (n = 420); 67 as acute lymphoblastic leukemia (ALL); and 6 as other forms of acute leukemia. The frequency of cases with low or null NQO1 activity (heterozygote + homozygous mutant) was significantly higher among total acute leukemia case subjects compared with their matched controls (odds ratio [OR] = 1.49; 95% confidence interval [CI], 1.17-1.89). Both ALL (OR = 1.93; 95% CI, 0.96-3.87) and AML case subjects (OR = 1.47; 95% CI, 1.13-1.90) exhibited a higher frequency of low or null NQO1 genotypes than controls. For de novo AML, the most significant effect of low or null NQO1 activity was observed among the 88 cases harboring translocations and inversions (OR = 2.39; 95% CI, 1.34-4.27) and was especially high for those harboring inv(16) (OR = 8.13; 95% CI, 1.43-46.42). These findings were confirmed in a second group of 217 de novo AML cases with known cytogenetics. Thus, inheritance of NQO1 C609T confers an increased risk of de novo acute leukemia in adults, implicating quinones and related compounds that generate oxidative stress in producing acute leukemia.

The majority of patients with multiple myeloma have translocations involving the immunoglobulin heavy chain switch regions on chromosome 14q32 and a promiscuous range of partner chromosomes. We describe a patient with an insertion of 132 bp of chromosome 22q12 sequence into the 5' region flanking S(mu) on chromosome 14q32. The 132 bp region from chromosome 22q12 contains the whole of exon 3 from a novel gene of unknown function in man. The significance of such insertional events remains unclear. The description of insertional events occurring as a result of abnormal switch recombination suggests that, in myeloma, dysregulation of oncogenes may occur by a mechanism other than chromosomal translocation.

The nature of the proliferating fraction in myeloma is still not known and understanding the characteristics of this fraction is central to the development of effective novel therapies. However, myeloma plasma cells typically show a very low rate of proliferation and this complicates accurate analysis. Although the level of CD45 and/or VLA-5 has been reported to identify proliferating 'precursor' plasma cells, there are discrepancies between these studies. We have therefore used a rigorous sequential gating strategy to simultaneously analyse cycle status and immunophenotype with respect to CD45, VLA-5 and a range of other integrin molecules. In 11 presentation myeloma patients, the proliferative fraction was distributed evenly between CD45+ and CD45- cells, however, cycling plasma cells were consistently VLA-5-. There was close correlation between the expression of VLA-5 and a range of other integrin molecules (CD11a, CD11c, CD103), as well as the immunoglobulin-associated molecules CD79a/b (Spearman, n = 10, P < 0.0001). In short-term culture, cells that were initially VLA-5-showed increasing VLA-5 expression with time. However, simultaneous analysis of the DNA-binding dye 7-amino-actinomycin D demonstrated that this was not as a result of differentiation, as VLA-5+ plasma cells were all non-viable. This was confirmed in freshly explanted plasma cells from nine patients. Discrete stages of plasma cell differentiation could not be distinguished by the level of CD45 or VLA-5 expression. The results indicate that there is a single stage of plasma cell differentiation, with the phenotype CD38+CD138+VLA-5-. These findings support the hypothesis that neoplastic bone marrow plasma cells represent an independent, self-replenishing population.

Initial studies with high-dose therapy (HDT) in myeloma suggest some beneficial effects of attaining a complete response (CR); however, the effect on survival is difficult to assess owing to inconsistencies in the definition of response between studies. We have analysed 96 newly diagnosed patients aged less than 65 years who received HDT and assessed the effect of response on survival using electrophoresis, immunofixation and fluorescent IgH polymerase chain reaction (PCR) to define CR. Patients received induction chemotherapy with C-VAMP (adriamycin, vincristine, methylprednisolone, cyclophosphamide) followed by melphalan 200 mg/m2 and reinfusion of peripheral blood stem cells. There was a high response to C-VAMP [CR = 24%, partial response (PR) = 64%], with all but one patient improving the depth of response after HDT (CR = 69%, PR = 31%). The progression-free survival (PFS) and overall survival (OS) were excellent at a median of 46.4 months and 72+ months. There was a trend towards an improved PFS in patients with an immunofixation-negative CR compared with patients with a PR (49.4 months, 41.14 months; P = 0.26). This was not evident when electrophoresis was used to define CR. The method used to define CR did not impact on the overall survival and fluorescent IgH PCR failed to add any additional prognostic information. This study supports the widespread use of the European Bone Marrow Transplantation group (EBMT) response criteria and suggests that immunofixation should be performed on all patients who become electrophoresis negative.

The antiangiogenic activity of thalidomide (Thal), coupled with an increase in bone marrow angiogenesis in multiple myeloma (MM), provided the rationale for the use of Thal in MM. Previously, the direct anti-MM activity of Thal and its analogues (immunomodulatory drugs, IMiDs) on MM cells was demonstrated, suggesting multiple mechanisms of action. In this study, the potential immunomodulatory effects of Thal/IMiDs in MM were examined. It was demonstrated that Thal/IMiDs do not induce T-cell proliferation alone but act as costimulators to trigger proliferation of anti-CD3-stimulated T cells from patients with MM, accompanied by an increase in interferon-gamma and IL-2 secretion. However, an increase in autologous T-cell killing of patient MM cells could not be demonstrated. A role for natural killer (NK)- and LAK-cell-mediated killing is suggested because IL-2-primed peripheral blood mononuclear cells (PBMCs) treated with Thal/IMiDs demonstrated significantly increased lysis of MM cell lines. Cold target inhibition assays suggested NK- rather than LAK-cell-mediated killing. Furthermore, this killing was not major histocompatibility complex-class restricted, and the depletion of CD56(+) cells blocked the drug-induced MM cell lysis. It was significant that increased killing of patient MM cells by autologous PBMCs treated with Thal/IMiDs was also observed. Although the in vivo relevance of NK-cell-mediated MM cell killing is unknown, phenotypic analysis performed in MM patients receiving Thal therapy demonstrated an increase in CD3(-)CD56(+) cells in patients responding to therapy. Thus in vitro and in vivo data support the hypothesis that Thal may mediate its anti-MM effect, at least in part, by modulating NK cell number and function.

In peripheral blood stem cell transplantation (PBSCT), the number of CD34+ cells transplanted has been shown to correlate well with both rapidity and durability of engraftment. However, it is clear that engraftment does not necessarily correlate with total CD34+ cell numbers in some patients. Consequently, there is increasing interest in evaluating the role of CD34+ subsets in haemopoietic recovery as a more accurate marker of harvest quality. We analysed the numbers of CD34+ cell subsets, namely Thy-1+, L-Selectin+ and CD38-, and correlated this with engraftment in 86 patients undergoing PBSCT. Adequate engraftment was defined as being a platelet count greater than 50 x 10(9)/l and a neutrophil count greater than 1.0 x 10(9)/l. CD34+L-Selectin+ provided the best prediction of engraftment rapidity, although the improvement over total CD34+ cell dose was minor. Only the dose of CD34+Thy-1+ cells transplanted correlated with durable engraftment. The probability of adequate 3-month engraftment increased with the dose of CD34+ cells transplanted, but 10% of patients receiving > 5 x 10(6)/kg still showed poor engraftment at 3 months. However, all patients receiving > 2.5 x 10(5)/kg CD34+Thy-1+ showed adequate engraftment at this time point. We also demonstrated that CD34+Thy-1+ progenitors were restricted to the bone marrow under normal conditions and, during stem cell mobilization, their kinetics generally paralleled total CD34+ numbers.

Polymorphisms within the phase II metabolizer enzymes GST T1, GST M1 and GST P1 affect the body's ability to detoxify a range of potential leukaemogens encountered in the environment. Using PCR, GST T1, GST M1 and GST P1 genotypes were determined in 557 adults with acute leukaemia and 952 age, sex and geographically matched controls. The strongest association with acute leukaemia was observed for the GST T1 null genotype, which occurred among 19% of cases and 14% of controls [odds ratio (OR) 1.45, 95% confidence interval (CI) 1.09-1.93]. A slightly higher proportion of cases (53%) than controls (49%) displayed the GST M1 null genotype, although the difference was not statistically significant (OR 1.22, 95% CI 0.98-1.52). No effect was observed for the GST P1 genotype and no interaction between the GST T1 and GST M1 genotypes was evident. Acute myeloid leukaemia (AML) was weakly associated with both GST T1 null (OR 1.32, 95% CI 0.97-1.79) and GST M1 null (OR 1. 24, 95% CI 0.98-1.56), whereas acute lymphoblastic leukaemia (ALL) was associated with GST T1 null (OR 3.28, 95% CI 1.31-8.26). No associations between smoking and disease risk in relation to GST T1 and GST M1 polymorphic status were found.