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.

Self-reported smoking histories were collected during face-to-face interviews with 807 patients with acute leukaemia and 1593 age- and sex-matched controls. Individuals who had smoked regularly at some time during their lives were more likely to develop acute leukaemia than those who had never smoked (odds ratio (OR) = 1.2, 95% confidence interval (CI) 1.0-1.4). The association was strongest for current smokers, defined here as smoking 2 years before diagnosis (OR = 1.4, 95% CI 1.1-1.7). With respect to the numbers of years smoked, risk estimates were raised in all groups except those who had smoked for fewer than 10 years. Similarly, the odds ratio decreased as the number of years 'stopped smoking' increased, falling to one amongst those who had given up smoking for more than 10 years. No significant linear trends were found, however, with either the numbers of years smoked or the numbers of years stopped smoking, and no significant differences were found between AML and ALL.

BACKGROUND:The capacity of the polymerase chain reaction (PCR) to detect very low numbers of cells bearing a t(14;18) translocation has led to its application in assessment of the results of treatment for follicular lymphoma, and suggestions that therapy might be guided by molecular studies. To test the reliability of PCR a collaborative study was undertaken to compare results from different laboratories in Europe and North America. METHODS:Twenty laboratories with records of publication in molecular diagnostics were sent blood from normal donors with varying numbers of t(14;18)-bearing cells added from a cell line with a translocation in the major breakpoint region (MBR) of the bcl-2 gene. Samples contained 1000, 100, 10, 1 or 0 cells per ml of whole blood and were sent blinded in duplicate. PCR methodology varied widely, with the total number of amplification cycles between 30 and 70, and 13 different primers used for the MBR region. Twelve laboratories used nested PCR and eight single round amplification. RESULTS:The sensitivity of nested and single round PCR was similar at 100 cells/ml but below this the nested method proved significantly more sensitive. The false positive rate was 28%, with 11 samples from 9 laboratories reported as positive when no t(14;18) cells were added. PCR product size and sequence analysis showed that false positives were due to contamination from cell-line DNA rather than background translocations in the donors. There was no significant difference in false positive rates between nested and single round techniques. CONCLUSION/CONCLUSIONS:The polymerase chain reaction to detect bcl-2-IgH rearrangements is presently carried out with widely disparate results. Further effort is required to bring forward a standard PCR protocol which can be re-tested in different laboratories to improve accuracy and reproducibility. The application of quantitative techniques such as real-time PCR may resolve many of the problems presently encountered.

Reduction of 5,10-methylenetetrahydrofolate (methyleneTHF), a donor for methylating dUMP to dTMP in DNA synthesis, to 5-methyltetrahydrofolate (methylTHF), the primary methyl donor for methionine synthesis, is catalyzed by 5,10-methylenetetrahydrofolate reductase (MTHFR). A common 677 C --> T polymorphism in the MTHFR gene results in thermolability and reduced MTHFR activity that decreases the pool of methylTHF and increases the pool of methyleneTHF. Recently, another polymorphism in MTHFR (1298 A --> C) has been identified that also results in diminished enzyme activity. We tested whether carriers of these variant alleles are protected from adult acute leukemia. We analyzed DNA from a case-control study in the United Kingdom of 308 adult acute leukemia patients and 491 age- and sex-matched controls. MTHFR variant alleles were determined by a PCR-restriction fragment length polymorphism assay. The MTHFR 677TT genotype was lower among 71 acute lymphocytic leukemia (ALL) cases compared with 114 controls, conferring a 4.3-fold decrease in risk of ALL [odds ratio (OR = 0.23; 95% CI = 0.06-0.81]. We observed a 3-fold reduction in risk of ALL in individuals with the MTHFR 1298AC polymorphism (OR = 0.33; 95% CI = 0.15-0.73) and a 14-fold decreased risk of ALL in those with the MTHFR 1298CC variant allele (OR = 0.07; 95% CI = 0.00-1.77). In acute myeloid leukemia, no significant difference in MTHFR 677 and 1298 genotype frequencies was observed between 237 cases and 377 controls. Individuals with the MTHFR 677TT, 1298AC, and 1298CC genotypes have a decreased risk of adult ALL, but not acute myeloid leukemia, which suggests that folate inadequacy may play a key role in the development of ALL.

Peripheral blood T cells in patients with paroxysmal nocturnal hemoglobinuria (PNH) comprise a mixture of residual normal and glycosylphosphatidylinositol (GPI)-deficient PNH cells. Using multicolor flow cytometry, we demonstrated significant differences between the proportions of naive and memory cells within these populations. PNH T cells comprise mainly naive cells (CD45RA(+)CD45R0(-)), whereas normal T cells in the same patients were predominantly memory (CD45RA(-)CD45R0(+)) cells. Functional analyses showed that GPI-deficient CD45RA(+) T cells can convert to a CD45R0(+) phenotype. We present data from a PNH patient in remission for 20 years who still had significant numbers of GPI-deficient T cells; these showed a normal distribution of naive and memory components. The predominantly naive phenotype of GPI-deficient T cells seen in PNH patients with active disease likely reflects the phenotype of recent normal thymic emigrants. In patients where hematopoiesis was predominantly derived from the PNH stem cell, absolute numbers of both naive PNH CD4(+) cells and CD8(+) cells show an inverse correlation with patient age, implying this age-related decline in T-cell production is secondary to a decrease in thymic activity rather than a stem cell defect.

Paroxysmal nocturnal haemoglobinuria (PNH) cells are deficient in glycosylphosphatidylinositol (GPI) linked antigens due to a somatic mutation of the PIG-A gene in a haemopoietic stem cell. It appears that a PNH clone reaches detectable proportions only when there is selection in its favour. GPI-deficient T lymphocytes have been identified in patients treated with CAMPATH-1H, a monoclonal antibody against the GPI-linked CD52 molecule. CAMPATH-1H selects for cells that are deficient in CD52 (such as PNH-like cells) promoting the development of a PNH-like clone (analogous to PNH). We report that 10/15 patients with chronic lymphocytic leukaemia developed PNH-like lymphocytes after therapy with CAMPATH-1H. The remaining five patients developed no PNH-like cells at any stage, including one patient who received 12 weeks of therapy. The inactivating PIG-A mutation has been identified in one patient. This mutation was detectable by an extremely sensitive mutation-specific PCR-based analysis in the patient's mononuclear cells prior to CAMPATH-1H therapy. The frequency and phenotype of GPI-deficient lymphocytes after CAMPATH-1H and the detection of a PIG-A mutation in the lymphocytes prior to CAMPATH-1H therapy indicated that such mutations were present in a very small proportion of cells prior to selection in their favour by CAMPATH-1H. This suggests that a large proportion of individuals have cells with PIG-A mutations that are not detectable by flow cytometry and thus may have the potential to develop PNH.

Immunoglobulin class switching occurs as a result of recombination between pairs of switch region sequences located 5' to each constant heavy chain gene except Cdelta. In the B cell neoplasm multiple myeloma, tumour cells have generally undergone class switching and often contain oncogenic sequences translocated into switch regions, presumably as a result of aberrant switch recombination. We have developed a method (LDV-PCR) which combines long distance PCR with one-sided vectorette PCR that is capable of detecting and isolating both normal and aberrant switch recombination breakpoints from multiple myeloma cell lines and primary multiple myeloma tumour material. Using LDV-PCR we have directly cloned the translocation breakpoints present in two multiple myeloma cell lines and isolated a normal productive switch recombination event from a primary tumour. Furthermore, we have isolated a novel translocation t(14;22)(q32;q12) from a primary tumour sample and have demonstrated that internal deletions within switch regions can occur in multiple myeloma cells. Compared to a Southern blotting approach, LDV-PCR is simpler and more rapid to perform, allows the simultaneous detection and isolation of recombination events, and can also be applied to amounts of DNA which are too low to permit the conventional cloning of recombination breakpoints.

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.

A collaborative study was carried out of the descriptive epidemiology of the lymphomas from seven countries across Europe in the period 1985-1992. Careful attention was paid to sources of information and the data quality in close collaboration with expert histopathologists. The data were classified as non-Hodgkin's lymphoma (NHL) and Hodgkin's disease (HD). An attempt was made to put the data into a modified version of the Revised European American Lymphoma (REAL) classification. We observed an overall rise in total NHL throughout the time period in all European countries but no such trend in HD. The increase in NHL overall being 4.2% per annum, representing an increase of 4.8% in males and 3.4% in females per annum, was only marked in middle and old age. Such increases were observed in all participating areas except in Burgundy. Different countries, however, have different base rates, the rates being highest in Scandinavia and the Netherlands. The analysis by subcategory classification suggested that the increase in NHL was confined to the follicle centre cell type, extranodal B-cell, nodal T-cell and nodal lymphomas not otherwise specified, categories. These new observations present a picture of real increase in case incidence with no obvious explanation. The increases in NHL do not appear to be due solely to better diagnoses. Pending other explanations or refutation, these present a compelling picture of an inexorable rise in incidence of this disease.