Early detection of leukaemic relapse after bone marrow transplantation using the polymerase chain reaction

We report a case of acute lymphoblastic leukaemia relapsing after allogeneic bone marrow transplantation in which the polymerase chain reaction (PCR) was used to assess chimeric status. This technique demonstrated the progressive reappearance of host cells prior to clinical relapse. The relapse was of host cell origin as shown by the presence of female (recipient) metaphases containing an abnormal chromosomal marker (iso 9q) which had also been present at initial diagnosis. The emergence of host cells in this case, detected only by PCR techniques but not by cytogenetic methods, appeared to herald overt relapse. PCR analysis provides a sensitive tool for detecting a progressive rise in host cell numbers which may predict clinical relapse.

Chimaerism following allogeneic bone marrow transplantation:detection of residual host cells using the polymerase chain reaction

Chimaerism was assessed in five recipients following sex mismatched allogeneic bone marrow transplantation. Techniques included karyotyping of bone marrow cells, dot blot DNA analysis of blood and bone marrow suspensions, and in vitro amplification of DNA by the polymerase chain reaction (PCR) using blood and bone marrow suspensions and stored bone marrow slides. Results of karyotypic analysis suggested complete chimaerism in four patients, while in one patient mixed chimaerism was detected. Mixed chimaerism was also detected, however, in a second patient using PCR and confirmed by dot blot analysis on all tissues examined. PCR is a sensitive tool for investigation of chimaerism following bone marrow transplantation. Since this technique does not require radioactivity, it is an attractive method for use in a clinical laboratory. This technique represents a further development in the use of DNA methodologies in the assessment of haematological disease.

A rapid dot-blot assay to assess chimerism following sex-mismatched bone marrow transplantation

Mixed chimerism may occur more frequently than previously thought following allogeneic bone marrow transplantation and may have implications in terms of relapse, graft-versus-host disease and immune reconstitution. DNA analysis using single or multilocus polymorphic probes cannot reliably discriminate between donor and recipient cells below a level of 10%. We used probe pHY2.1, a cloned segment of tandemly repeated DNA (2000 copies) on the long arm of chromosome Y. A dot blot procedure allowed us to immobilize DNA directly from 50 microliter of peripheral blood or bone marrow. Cross-reactivity was eliminated by hybridization at conditions of extreme stringency (65 degrees C, 50% formamide). Mixing experiments detected male DNA at a level of 0.1% after 10 h exposure. Five patients were studied serially post-bone marrow transplantation. One patient showed mixed chimerism for 12 months, one had complete autologous recovery and the remaining three showed complete engraftment. All results were verified by standard karyotyping on bone marrow cells. This technique is a simple, rapid and sensitive assay for chimerism following sex mismatched bone marrow transplantation.

High-dose therapy and autologous stem cell transplantation in first relapse for diffuse large B cell lymphoma in the rituximab era:an analysis based on data from the European Blood and Marrow Transplantation Registry

Autologous stem cell transplantation (ASCT) consolidation remains the treatment of choice for patients with relapsed diffuse large B cell lymphoma. The impact of rituximab combined with chemotherapy in either first- or second-line therapy on the ultimate results of ASCT remains to be determined, however. This study was designed to evaluate the benefit of ASCT in patients achieving a second complete remission after salvage chemotherapy by retrospectively comparing the disease-free survival (DFS) after ASCT for each patient with the duration of the first complete remission (CR1). Between 1990 and 2005, a total of 470 patients who had undergone ASCT and reported to the European Blood and Bone Transplantation Registry with Medical Essential Data Form B information were evaluated. Of these 470 patients, 351 (74%) had not received rituximab before ASCT, and 119 (25%) had received rituximab before ASCT. The median duration of CR1 was 11 months. The median time from diagnosis to ASCT was 24 months. The BEAM protocol was the most frequently used conditioning regimen (67%). After ASCT, the 5-year overall survival was 63% (95% confidence interval, 58%-67%) and 5-year DFS was 48% (95% confidence interval, 43%-53%) for the entire patient population. Statistical analysis showed a significant increase in DFS after ASCT compared with duration of CR1 (median, 51 months versus 11 months; P < .001). This difference was also highly significant for patients with previous exposure to rituximab (median, 10 months versus not reached; P < .001) and for patients who had experienced relapse before 1 year (median, 6 months versus 47 months; P < .001). Our data indicate that ASCT can significantly increase DFS compared with the duration of CR1 in relapsed diffuse large B cell lymphoma and can alter the disease course even in patients with high-risk disease previously treated with rituximab.

A HuGE Review and Meta-Analyses of Genetic Associations in New Onset Diabetes after Kidney Transplantation

PURPOSE: New onset diabetes after transplantation (NODAT) is a serious complication following solid organ transplantation. There is a genetic contribution to NODAT and we have conducted comprehensive meta-analysis of available genetic data in kidney transplant populations.

METHODS: Relevant articles investigating the association between genetic markers and NODAT were identified by searching PubMed, Web of Science and Google Scholar. SNPs described in a minimum of three studies were included for analysis using a random effects model. The association between identified variants and NODAT was calculated at the per-study level to generate overall significance values and effect sizes.

RESULTS: Searching the literature returned 4,147 citations. Within the 36 eligible articles identified, 18 genetic variants from 12 genes were considered for analysis. Of these, three were significantly associated with NODAT by meta-analysis at the 5% level of significance; CDKAL1 rs10946398 p = 0.006 OR = 1.43, 95% CI = 1.11-1.85 (n = 696 individuals), KCNQ1 rs2237892 p = 0.007 OR = 1.43, 95% CI = 1.10-1.86 (n = 1,270 individuals), and TCF7L2 rs7903146 p = 0.01 OR = 1.41, 95% CI = 1.07-1.85 (n = 2,967 individuals).

CONCLUSION: Evaluating cumulative evidence for SNPs associated with NODAT in kidney transplant recipients has revealed three SNPs associated with NODAT. An adequately powered, dense genome-wide association study will provide more information using a carefully defined NODAT phenotype.

Gene scanning of VDJH-amplified segments is a clinically relevant technique to detect contaminating tumor cells in the apheresis products of multiple myeloma patients undergoing autologous peripheral blood stem cell transplantation.

Contaminating tumour cells in apheresis products have proved to influence the outcome of patients with multiple myeloma (MM) undergoing autologous stem cell transplantation (APBSCT). The gene scanning of clonally rearranged VDJ segments of the heavy chain immunoglobulin gene (VDJH) is a reproducible and easy to perform technique that can be optimised for clinical laboratories. We used it to analyse the aphereses of 27 MM patients undergoing APBSCT with clonally detectable VDJH segments, and 14 of them yielded monoclonal peaks in at least one apheresis product. The presence of positive results was not related to any pre-transplant characteristics, except the age at diagnosis (lower in patients with negative products, P = 0.04). Moreover, a better pre-transplant response trended to associate with a negative result (P = 0.069). Patients with clonally free products were more likely to obtain a better response to transplant (complete remission, 54% vs 28%; >90% reduction in the M-component, 93% vs 43% P = 0.028). In addition, patients transplanted with polyclonal products had longer progression-free survival, (39 vs 19 months, P = 0.037) and overall survival (81% vs 28% at 5 years, P = 0.045) than those transplanted with monoclonal apheresis. In summary, the gene scanning of apheresis products is a useful and clinically relevant technique in MM transplanted patients.

The detection of contaminating clonal cells in apheresis products is related to response and outcome in multiple myeloma undergoing autologous peripheral blood stem cell transplantation.

In the present paper, we report on the use of the heteroduplex PCR technique to detect the presence of clonally rearranged VDJ segments of the heavy chain immunoglobulin gene (VDJH) in the apheresis products of patients with multiple myeloma (MM) undergoing autologous peripheral blood stem cell (APBSC) transplantation. Twenty-three out of 31 MM patients undergoing APBSC transplantation with VDJH segments clonally rearranged detected at diagnosis were included in the study. Samples of the apheresis products were PCR amplified using JH and VH (FRIII and FRII) consensus primers and subsequently analyzed with the heteroduplex technique, and compared with those obtained at diagnosis. 52% of cases yielded positive results (presence of clonally rearranged VDJH segments in at least one apheresis). The presence of positive results in the apheresis products was not related to any pretransplant characteristics with the exception of response status at transplant. Thus, while no one patient with positive apheresis products was in complete remission (CR), negative immunofixation, before the transplant, five cases (46%) with negative apheresis were already in CR at transplant (P = 0.01). The remaining six cases with heteroduplex PCR negative apheresis were in partial remission before transplant. Patients with clonally free products were more likely to obtain CR following transplant (64% vs 17%, P= 0.02) and a longer progression-free survival, (40 months in patients transplanted with polyclonal products vs 20 with monoclonal ones, P = 0.03). These results were consistent when the overall survival was considered, since it was better in those patients with negative apheresis than it was in those with positive (83% vs 36% at 5 years from diagnosis, P= 0.01). These findings indicate that the presence of clonality rearranged VDJH segments is related to the response and outcome in MM transplanted patients.

The influence of transforming growth factor-beta1 gene polymorphisms on the severity of gingival overgrowth associated with concomitant use of cyclosporin A and a calcium channel blocker

The purpose of this study was to determine whether the prevalence and severity of gingival overgrowth in renal transplant recipients concomitantly treated with cyclosporin and a calcium channel blocker was associated with functional polymorphisms within the signal sequence of the transforming growth factor-(TGF)beta1 gene.

Bioreactor Expansion of Human Adult Bone Marrow-Derived Mesenchymal Stem Cells

Supplementation of mesenchymal stem cells (MSCs) during hematopoietic stem cell transplantation (HSCT) alleviates complications such as graft-versus-host disease, leading to a speedy recovery of hematopoiesis. To meet such clinical demand, a fast MSCs expansion method is required. In the present study, we examined the feasibility of expanding MSCs from the isolated bone marrow mononuclear cells using a rotary bioreactor system. The cells were cultured in a rotary bioreactor with Myelocult� medium containing a combination of supplementary factors, including stem cell factor (SCF), interleukin 3 and 6 (IL-3, IL-6). After 8 days of culture, total cell numbers, Stro-1+CD44+CD34- MSCs and CD34+CD44+Stro-1- HSCs were increased 9, 29, and 8 folds respectively. Colony forming efficiency-fibroblast per day (CFE-F/day) of the bioreactor-treated cells was 1.44-fold higher than that of the cells without bioreactor treatment. The bioreactor-expanded MSCs showed expression of primitive MSCs markers endoglin (SH2) and vimentin, whereas markers associated with lineage differentiation including osteocalcin (osteogenesis), Type II collagen (chondrogenesis) and C/EBPα (adipogenesis) were not detected. Upon induction, the bioreactor-expanded MSCs were able to differentiate into osteoblasts, chondrocytes and adipocytes. Taken together, we conclude that the rotary bioreactor with the modified Myelocult� medium reported in this study may be used to rapidly expand MSCs.