Faculty Bibliography

BACKGROUND:PBSC are increasingly being used as the source of stem cells in allogeneic transplantation. An increased incidence of chronic GvHD has been suggested following unmanipulated allogeneic PBSC transplantation (PBSCT), however, how this affects overall survival is not yet clear. Our aim was to study the impact of chronic GvHD on survival and relapse following allogeneic PBSCT. METHODS:We have analyzed data from 73 patients undergoing HLA-matched allogeneic PBSCT. GvHD prophylaxis was with CYA and MTX in 97% of patients. We have studied the incidence of chronic GvHD and its affect on relapse and survival in these patients. All patients were at least 100 days post-transplant at the time of analysis. RESULTS:Seventy-three patients were evaluable for analysis of chronic GvHD. The overall incidence of chronic GvHD was 55% (limited in 18% and extensive in 37%). Overall median survival was 991 days, with a 4 year survival rate of 48%. Twelve patients relapsed. Patients with chronic GvHD had a significantly lower incidence of disease relapse (p = 0.005) with a relapse probability of 8% at 3 years, compared with 40% in patients with no chronic GvHD. In addition, the extent of chronic GvHD had a marked effect on survival, patients with limited chronic GvHD had a 4 year survival rate of 83%, compared with 45% in patients with extensive chronic GvHD and 38% in patients with no chronic GvHD. This difference was primarily due to the low incidence of relapse and low mortality seen in patients with limited chronic GvHD. DISCUSSION/CONCLUSIONS:The presence and extent of chronic GvHD is an important predictor of outcome following allogeneic PBSCT, in that patients who developed either limited or extensive chronic GvHD had a low risk of disease relapse.

IgM paraproteinemia is considered to be the major defining feature of Waldenström's macroglobulinemia (WM), but it may also occur in other B-cell lymphoproliferative disorders. In this study we have reviewed the final pathological diagnosis of 106 patients with IgM paraproteinemia investigated in our laboratories between April 1993 and May 1999. In 22 of the 106 patients (20.8%), there was no clinical or laboratory evidence of an underlying lymphoproliferative disorder, and a diagnosis of monoclonal gammopathy of undetermined significance (MGUS) was therefore made. In 60 cases (56.6%), a diagnosis of WM was made, while in the remaining 24 patients, the final diagnosis was chronic lymphocytic leukemia (n = 10), diffuse large B-cell lymphoma (n = 5), extranodal marginal-zone lymphoma (n = 3), follicular lymphoma (n = 3), and mantle-cell lymphoma (n = 3). The median paraprotein concentration in patients with WM, MGUS, and "other" lymphoproliferative disorders was 13 g/L (range, 2-54), 6 g/L (range, 3-30), and 4.5 g/L (range, 3-61), respectively. It is clear that IgM paraproteins are demonstrable in all subtypes of peripheral B-cell disorders and, although paraprotein concentrations are generally higher in WM, there is considerable overlap. Immunophenotypic criteria are therefore essential for the accurate diagnosis of WM.

Peripheral blood B cells in patients with paroxysmal nocturnal hemoglobinuria (PNH) comprise variable mixtures of normal B cells produced before the onset of disease and glycosylphosphatidylinositol (GPI)-deficient B cells derived from the PNH hematopoietic stem cell. In a detailed phenotypic analysis of 29 patients with PNH, this study shows consistent phenotypic differences between PNH B cells and residual normal B cells. In the majority of patients with active disease, PNH B cells comprised mainly naive cells with a CD27(-)IgM(+)IgD(strong+)IgG(-) phenotype. The proportion of CD27(+) memory cells within this compartment was related to disease duration (Spearman [r(s)] 0.403; P =.030). In PNH patients with predominantly GPI-deficient hematopoiesis, that is, a large granulocyte PNH clone, the residual normal B cells had a predominantly memory (CD27(+)) phenotype. Furthermore, the majority of these memory B cells were not immunoglobulin (Ig) class switched and had an IgM(+)IgD(+)IgG(-) phenotype. Using PNH as a novel model with which to study B lymphopoiesis, this study provides direct evidence that production of new naive B cells occurs throughout life and that the major population of long-lived memory B cells are IgM(+)IgD(+). Moreover, studies of GPI(-) B cells in 2 patients in remission from PNH suggest that the life span of a B-cell clone can be more than 24 years.

We have genotyped over 550 cases of acute leukaemia and 950 matched controls from a population-based case-control study, to investigate the impact cytochrome P450s 2D6, 2C19 and 1A1 have on susceptibility to adult acute leukaemia. Analysis included potential associations between polymorphic status and acute myeloid leukaemia (AML), acute lymphoblastic leukaemia (ALL), plus the FAB and cytogenetic subtypes therein. A significant increased risk was found for CYP2D6 poor metabolizer phenotype and acute leukaemia [odds ratio (OR) = 1.69, 95% confidence interval (CI) 1.17-2.43], a risk also found in AML and ALL. No interaction was found with smoking. However, a significant age-related association between CYP2D6 polymorphism and acute myeloid leukaemia implied that the excess risk was confined to persons aged 40 years and over (OR 2.38, 95% CI 1.53-3.71). Amongst AML cases, increased odds ratios were observed in both de-novo (OR 1.54, 95% CI 1.02-2.32) and secondary leukaemia (OR 2.83, 95% CI 0.91-8.77), and among patients with a chromosomal abnormality (OR 2.00, 95% CI 1.11-3.61). An increased risk was found for the CYP2C19 poor metabolizer phenotype (OR 1.68, 95% CI 0.97-2.92) which was also present in AML and ALL. For this CYP450 locus, an increased risk was suggested in secondary leukaemia (OR 2.67, 95% CI 0.44-16.3) and amongst AML cases with a chromosomal abnormality (OR 6.72, 95% CI 2.22-20.4). No difference in CYP1A1 genotype distribution was found for acute leukaemia, AML, ALL or any other diagnostic classification group used. No significant interactions between CYP2D6, CYP2C19 or CYP1A1 were found.

B-cell chronic lymphocytic leukemia (CLL) can be classified as typical or atypical based on morphologic and immunophenotypic features. The relationship between these 2 groups is uncertain, and there is some evidence they may be different entities. We used comparative genomic hybridization (CGH) to explore the cytogenetic relationship between typical and atypical B-cell CLL. Results showed a similar pattern of chromosome gains and losses detected in typical and atypical B-cell CLL, suggesting they are related disorders. Gain on chromosome 12 material occurred in cases that were apparently normal by interphase fluorescence in situ hybridization (FISH). The common region mapped to chromosome 12q21. Gains on chromosome 4 were present in 74% (32) of cases analyzed and were confirmed by interphase FISH in 30% (13) of cases. We previously have shown the strong association between trisomy 12 as detected by FISH and CD11a expression in atypical B-cell CLL. In the present study, CGH demonstrated additional gains on 12p and 12q outside the common amplified region of 12q21 in these patients.

One of the most common translocations in acute myeloid leukemia (AML) is the t(8;21), which produces the fusion gene AML1-MTG8. We have developed a sensitive competitive reverse transcriptase-polymerase chain reaction (RT-PCR) assay for AML1-MTG8 transcripts, coupled with a competitive RT-PCR for the ABL transcript as a control to accurately estimate the level of amplifiable RNA. We have shown that AML1-MTG8 and ABL transcripts have equal degradation rates. Thus, this method is useful for multicenter studies. We studied 25 patients with t(8;21) AML by means of serial analysis done on bone marrow (BM) and peripheral blood (PB) samples from 21 patients. Our analysis showed that, in general, a successful induction chemotherapy produces a reduction of 2 to 3 log in the level of AML1-MTG8, followed by a further 2 to 3 log after consolidation/intensification chemotherapy. Levels up to 1 x 10(3) and 1 x 10(2) molecules/microg of RNA in BM and PB, respectively, were compatible with durable remission. On the other hand, 5 patients with levels of 0.71 x 10(5) to 2.27 x 10(5) molecules/microg of RNA in BM and 2.27 x 10(3) to 2.27 x 10(4) molecules/microg of RNA in PB had hematologic relapse within 3 to 6 months. Our data indicate that serial quantitation of AML1-MTG8 transcripts is useful in identifying patients at high risk of relapse and may offer an opportunity for clinical intervention to prevent hematologic relapse. This approach was applied successfully in a patient who had an allogeneic BM transplantation. We also suggest that PB may be used an alternative to BM for quantitating AML1-MTG8 transcripts.

The aim of this study was to investigate the combination of fludarabine phosphate, dexamethasone, cytosine arabinoside and cis-platinum (FLUDAP) in the treatment of patients with relapsed/refractory aggressive non-Hodgkin's lymphoma (NHL). This regimen comprises: dexamethasone 100 mg/d continuous infusion (cont. inf.) d1-3; cytosine arabinoside (ara-C) 1 g/m2/d cont. inf. d 2,3; fludarabine phosphate 30 mg/m2 short inf. 4hr prior to each 24hr ara-C inf.; cis-platinum 50 mg/m2 4hr inf. at the start of each 24hr ara-C inf. G-CSF (lenograstim, Granocyte) is given at 263 microg s.c. daily from day 7 until the neutrophil count reaches 1.0x10(9)/l. The regimen repeats at 21 day intervals. A total of 33 patients were registered (median age 47 years; 24 males, 9 females); the majority (73%) were refractory to their previous treatment and most had advanced disease by Ann Arbor stage. Thirteen (39%) of the 33 enrolled patients (52% of the 25 fully evaluable patients who received at least 2 courses of FLUDAP) responded to treatment. A maximum response of complete remission was achieved in 5 patients, good partial remission in 3, and partial remission in 5. Twelve patients went on to successful stem cell supported intensification therapy. Median survival times were higher in the responding patients, and in those patients transplanted post-FLUDAP. The toxicity associated with the FLUDAP regimen was generally predictable; frequently reported severe events included haematological toxicity and infection. In conclusion, the FLUDAP regimen shows promise as a salvage regimen and increases the available therapeutic options in the treatment of recurrent/refractory aggressive NHL.

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.

A novel nonmyeloablative conditioning regimen was investigated in 44 patients with hematologic malignancies. The median patient age was 41 years. Many of the patients had high-risk features, including 19 patients with a previous failed transplant. Recipient conditioning consisted of CAMPATH-1H, 20 mg/day on days -8 to -4; fludarabine, 30 mg/m(2) on days -7 to -3; and melphalan, 140 mg/m(2) on day -2. Thirty-six recipients received unmanipulated granculocyte colony-stimulating factor-mobilized peripheral blood stem cells from HLA-identical siblings, and 8 received unmanipulated marrow from matched unrelated donors. GVHD prophylaxis was with cyclosporine A alone for 38 patients and cyclosporine A plus methotrexate for 6 sibling recipients. Forty-two of the 43 evaluable patients had sustained engraftment. Results of chimerism analysis using microsatellite polymerase chain reaction indicate that 18 of 31 patients studied were full-donor chimeras while the other patients were mixed chimeras in one or more lineages. At a median follow-up of 9 months (range 3 to 29 months), 33 patients remain alive in complete remission or with no evidence of disease progression. Seven patients relapsed or progressed post-transplantation, and 4 of them subsequently died. Four patients died of regimen-related complications. There were no cases of grades III-IV acute GVHD. Only 2 patients developed grade II acute GVHD, and only 1 had chronic GVHD. The estimated probability of nonrelapse mortality was 11%. Although longer follow-up is needed to establish the long-term remission rates, this study demonstrates that this nonmyeloablative preparative regimen is associated with durable engraftment, minimal toxicity, and low incidence of GVHD.