The use of the real-time reverse-transcription polymerase-chain reaction (RT-PCR) method to quantify BCR-ABL transcripts before and after allogeneic transplant was prospectively studied in 65 patients with chronic myeloid leukemia (CML). The expression of the BCR-ABL transcript was determined and normalized using the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) housekeeping gene product as an endogenous reference. In the single step real-time PCR assay, tenfold serial dilutions of cDNA of the K5652 cell line remained positive down to 100 pg cDNA only. However, molecular relapses of CML after transplant were only safely detectable when a nested real-time PCR assay was performed, which was able to detect 1-10 pg cDNA from a tenfold serial dilution. The median normalized BCR-ABL transcript level was measured as 0.004% in 17 patients with a molecular relapse, 0.4% in 7 patients with a cytogenetic relapse, 2.6% in 36 patients with a stable phase of CML, and 36% in 5 patients with a relapse in a blast crisis. The analyzed median normalized amount of BCR-ABL transcript differed significantly (P<0.001) between the various disease stages. In ten CML patients with relapse, the real-time PCR method was used to monitor the response of various immunotherapies as donor leukocyte infusions, withdrawal of immunosuppression, or interferon-alpha application. The results of the quantitative evaluation of BCR-ABL transcripts reflected very well the clinical effect of the different applied immunotherapies. The new real-time PCR method seems to be a suitable technique for the early detection of relapse after allogeneic transplant in patients with the BCR-ABL transcript. Its ability to distinguish between molecular and cytogenetic relapse (P<0.001) allows early therapeutic decisions.

We compared the biological effects of the CXC chemokine SDF-1␣ on immunomagnetically purified CD34 ؉ cells isolated from human normal bone marrow (NBM), leukapheresis products (LP) and patients with chronic myeloid leukaemia (CML). LP CD34 ؉ cells showed a significantly stronger migration response to SDF-1␣ (100 ng/ml) than CD34 ؉ cells isolated from the peripheral blood (PB) of CML patients (P Ͻ 0.05). The chemotactic response to SDF-1␣ was also reduced in CML BM CD34 ؉ cells in comparison to NBM CD34 ؉ cells but the observed differences were not statistically significant. In analogy to normal CD34 ؉ cells circulating CML PB CD34 ؉ cells were less responsive to SDF-1␣ than their BM counterparts (P Ͻ 0.05). Furthermore, SDF-1␣ elicited similar concentrationdependent growth suppressive effects on normal and CML CD34 ؉ cells (P Ͼ 0.05) in colony-forming cell assays. We then demonstrated that SDF-1␣ triggers intracellular calcium increases in CD34 ؉ cells and there were no differences in the time course and dose response characteristics of normal and CML CD34 ؉ cells. The reduced migration response to SDF-1␣ in CML CD34 ؉ cells was not due to a down-regulation of the SDF-1␣ receptor CXCR-4 as flow cytometric analysis revealed similar CXCR-4 expression levels on NBM, LP, CML PB and CML BM CD34 ؉ cells (P Ͼ 0.05). Finally, no differences in the modulation of CXCR-4 levels in response to SDF-1␣ and serum were observed in CML and normal CD34 ؉ cells. Our data suggest that the impaired chemotactic response of CML CD34 ؉ cells to SDF-1␣ is not caused by a lack or complete uncoupling of CXCR-4, but may be due to an intracellular signalling defect downstream of the receptor. Leukemia (2000) 14, 1652-1660.

Summary:Keywords: AML; inv(16); CBF␤/MYH11; MRD; BMTWe evaluated the occurrence of the CBF␤/MYH11 fusion transcripts by PCR analysis in 10 patients with Acute myelomonocytic leukemia with bone marrow eosinoinv(16)(p13;q22) acute myeloid leukemia (AML) who philia (AML-M4Eo), according to the French-Americanunderwent allogeneic bone marrow transplantation British (FAB) classification, is a distinct subtype of AML. (BMT) (n = 5), peripheral blood progenitor cell trans-AML-M4Eo is often associated with rearrangements of plantation (PBPCT) (n = 3), or autologous transplanchromosome 16, mostly involving 16p13 and 16q22, leadtation (n = 2). In addition to the analysis of minimal ing either to a pericentric inversion, inv(16)(p13q22) or, residual disease (MRD), the chimerism status of patients less commonly, to a translocation between the homologous after allogeneic transplant was studied by PCR. The chromosomes, t(16;16)(p13;q22). The pericentric inversion CBF␤/MYH11 fusion trancript was not detectable in six inv(16)(p13q22) is one of the most frequently occurring of seven patients who remained in remission after allochromosomal rearrangements detected in this neoplasm, geneic BMT or PBPCT. Two of these patients in which has been reported to account for approximately 16% remission were monitored for 50 months and 64 months of all AML. undergo allogeneic BMT have a significantly lower risk of transcript was detectable. In one patient in relapse, the relapse than their counterparts treated with chemotherapy fusion transcript was not only detectable in blood and alone. On the other hand allogeneic BMT is associated with bone marrow, but also in a cerebrospinal fluid sample more treatment-related mortality than autologous BMT or prior to transplant. Two patients who received autologchemotherapy alone. ous BMT were monitored for CBF␤/MYH11 transcripts Several reports suggest that the CBF␤/MYH11 fusion 3 months after BMT. The CBF␤/MYH11 was detected mRNA can often be detected in patients in long-term in these patients. Both patients subsequently relapsed 3 remission after chemotherapy. [4][5][6][7] To date, other than single months and 23 months post-autologous BMT. The case reports, studies of the detection of the CBF␤/MYH11 results study show that analysis of the CBF␤/MYH11 fusion transcripts after allogeneic BMT do not exist. institution. In addition to the analysis of minimal residual Germany

Summary:We studied the immunomodulating effect of withdrawal of immunosuppression with cyclosporin A (CsA) in 42 patients with leukemic relapse of chronic myelogenous leukemia (CML) (n ‫؍‬ 24), acute myeloid leukemia (AML) (n ‫؍‬ 13) and acute lymphoblastic leukemia (ALL) (n ‫؍‬ 5) after allogeneic unmanipulated bone marrow (BMT) or peripheral blood stem cell transplantation (PBSCT). Response to CsA withdrawal was monitored molecularly by the polymerase chain reaction for elimination of CML cells containing the bcr-abl messenger RNA (mRNA) transcript (n ‫؍‬ 24), or mll-af4 mRNA transcript characteristic of leukemic cells with a 11q23 chromosomal abnormality (n ‫؍‬ 1). Rapid tapering of CsA resulted in subsequent achievement of cytogenetic remission in 11 of 14 CML patients (79%) who relapsed in early disease phase (n ‫؍‬ 9 cytogenetic relapse, n ‫؍‬ 2 hematological relapse) after a median of 57 days. Three of 13 AML patients and one of five ALL patients achieved complete remission. CsA withdrawal was accompanied by the development of acute graft-versushost disease (GVHD) grade II in most of the 24 patients with CML. Two patients who achieved remission of AML or ALL died from severe GVHD grade III-IV. We calculated a probability of 84% for achieving and remaining in remission with early relapse of CML 4 years after relapse post BMT, whereas patients with AML have only a probability of about 10% of achieving and remaining in remission after 3 years. Patients with advanced CML and ALL had no chance of achieving and remaining in remission in the same time period.

Summary:In order to evaluate the risk of cytomegalovirus (CMV) associated disease after allogeneic stem cell transplantation (SCT), 158 consecutive patients at risk for infection were analyzed. BMT was performed in 101 patients and peripheral blood stem cell transplantation (PBSCT) in 57 patients. CMV antigenemia was found in 57 cases (56%) after BMT and 27 cases (47%) after PBSCT, respectively. CMV antigenemia resistant to a 14-day course of GCV was found in 26 patients (26%) after BMT but in only four patients (7%) after PBSCT (P Ͻ 0.01). Eighteen patients (11%) developed CMV disease, 14 post BMT and four post PBSCT. Lethal CMVrelated interstitial pneumonia (CMV-IP) occurred in 13 cases of whom 12 patients were bone marrow recipients (P = 0.04). The subgroup of seronegative patients with a CMV seropositive donor had a significantly lower risk of developing CMV antigenemia, GCV-resistant CMV antigenemia (P Ͻ 0.01) and CMV-related disease (P = 0.01). In conclusion, the incidence of persistent CMV antigenemia and CMV-IP was significantly reduced when allogeneic transplantation was performed with peripheral blood stem cells instead of bone marrow. These findings suggest that our previous in vitro data on improved immune reconstitution after allogeneic PBSCT as compared to allogeneic BMT have clinical relevance. Bone Marrow Transplantation (2000) 25, 665-672. Keywords: CMV infection; CMV interstitial pneumonia; pp65 antigenemia; BMT; PBSCT; immune reconstitution Cytomegalovirus (CMV)-related disease is one of the most frequent infectious complications after allogeneic stem cell transplantation (SCT). 1,2 Despite new therapeutic options, symptomatic CMV infection still has a high mortality rate, especially when interstitial pneumonia (CMV-IP) develops. 3,4 Well-known risk factors of CMV infection and related diseases are acute graft-versus-host disease (aGVHD) and positive serology for CMV of recipient and/or donor before transplantation. 1,3 There is still controversy about an increased incidence of CMV infection or CMV-related disease after allogeneic bone marrow transplantation (BMT) with a matched unrelated donor (MUD) instead of an identical sibling donor (ISD). 5,6 The transmission of CMV infection from seropositive blood products can be neglected since white blood cell filtration is routinely used. 3,7,8 Prophylaxis with ganciclovir (GCV) given to all seropositive patients leads to a decreased incidence and severity of CMV infection but this therapeutic option is associated with neutropenia and in consequence bacterial and fungal infections are more frequent in these patients. 9,10 Pre-emptive therapy based on screening for culture-proven CMV infection, CMV antigenemia by leukocyte expression of pp65 matrix protein, or CMV-DNA by PCR testing reduces the incidence of CMV diseases. 11-19 Viral load appears to be proportional to CMV antigenemia in the systemic circulation. Thus, CMV antigenemia-triggered preemptive treatment lowers the duration and side-effects of antiviral therapy after SCT which is considered ...

Summary:Two hundred and ten bronchoalveolar lavage (BAL) samples were obtained from 50 patients 10 days before and on defined days after allogeneic bone marrow transplantation (BMT). The samples were examined for human cytomegalovirus (HCMV) and human herpesvirus-6 (HHV-6) by polymerase chain reaction (PCR). Fifteen patients (30%) had a positive result for HCMV in at least one sample and 25 (50%) were positive for HHV-6 in at least one sample. Five patients developed HCMV-associated interstitial pneumonia (HCMV-IP) within 100 days after allogeneic BMT. Four of these patients were positive for both HCMV and HHV-6. Conspicuous HHV-6 positivity was detected in BAL samples obtained because of respiratory symptoms. No association was found between detection of HHV-6 and acute graft-versus-host disease. Engraftment failure or a delay in engraftment was observed in none of the 50 patients. The data from this study indicate that HHV-6 is a pathogen in HCMV-associated, as well as in non-HCMV-associated infectious lung disease after BMT. Bone Marrow Transplantation (2000) 26, 639-644.

Summary:The detection of chimerism, residual molecular and cytogenetic disease following transplantation of peripheral blood stem cells (PBSCT) with a nonmyeloablative conditioning (n = 9) and the transplantation of highly purified CD34 ؉ stem cells (CD34 ؉ PBSCT) (n = 16) were compared to unmanipulated bone marrow transplantation (BMT) (n = 69) and unmanipulated PBSCT (n = 50) after myeloablative conditioning in patients with first chronic phase of chronic myelogenous leukemia (CML) (n = 137), second chronic phase of CML (n = 4), acute lymphoblastic leukemia (n = 2) and acute myeloid leukemia (n = 1). A molecular relapse (MR) as defined by two consecutive positive polymerase chain reaction assays for the detection of M-bcr-abl transcripts (n = 141) and cbf␤-myh11 transcripts (n = 1) in a 4-week interval was found in 10 of 16 patients (63%) after CD34 ؉ PBSCT, and in 27 of 69 patients (39%) after BMT, whereas only three of 50 patients (6%) after PBSCT (P Ͻ 0.001) and one of eight patients (13%) after PBSCT with reduced conditioning suffered from a MR. A cytogenetic relapse occurred in five of 16 patients (31%) after CD34 ؉ PBSCT and 21 of 69 patients (30%) after BMT (NS) compared to two of 50 patients (4%) after PBSCT and none of the eight patients after PBSCT with reduced conditioning (P Ͻ 0.05). The lowest treatment-related mortality was seen in the 16 patients after CD34 ؉ PBSCT, who are all currently alive with a median follow-up of 15 months, whereas the survival rate for BMT, PBSCT and PBSCT with reduced conditioning were 65%, 63% and 58%, respectively. Multivariate analysis including all potential influential factors of post-transplant residual disease recurrence showed that patients after CD34 ؉ PBSCT had a significantly higher risk (two times) to develop a MR than patients after BMT (P Ͻ 0.03), whereas patients after unmanipulated PBSCT had a significant lower risk (eight times) for the occurrence of a MR post

Myelodysplastic syndromes (MDSs) often occur in older adults with significant comorbidities. Therefore, a reduced-toxicity conditioning regimen may be more suitable than standard conditioning regimens before allogeneic blood stem cell transplantation. Here, we retrospectively compare the outcome of a treosulfan-based conditioning regimen with standard myeloablative TBIbased conditioning regimens in patients (pts) with MDS. A total of 48 pts with MDS were included in the study, of which 29 (60%) pts received TBI-based and 19 (40%) pts received a treosulfan-based conditioning regimen. A significantly lower relapse incidence (5% vs 34% at 3 years, P ¼ 0.019) resulting in a better, but not statistically significant relapse-free survival (RFS) (57% vs 31%, P ¼ 0.086) was observed after treosulfan-based conditioning. In pts with increased risk for significant side effects due to comorbidities (haematopoietic stem cell transplantation specific comorbidity index), the estimated 3-year RFS was significantly better in the treosulfan group: 54% (95% confidence interval (CI), 17-90%) compared with pts in the TBI group: 11% (95% CI, 0-44%; log-rank test P ¼ 0.0455). Treosulfan-based conditioning therapy is a feasible and effective regimen for pts with MDS, especially in pts with preexisting comorbidities.