应用RT-nest-PCR法检测慢性髓细胞白血病非亲缘异基因骨髓移植后微小残留病变

目的：应用筑巢式RT—PCR（RT—nest-PCR）法检测慢性髓细胞性白血病（CML）患者非亲缘异基因骨髓移植（URD）后微小残留病变（MRD），并探讨它与复发的相关性。方法：分别采用RT-nest—PCR法和常规细胞遗传学方法检测bcr／abl融合基因和Ph染色体。结果：20例CML行URD患者术前Ph染色体和bcr／abl融合基因检测均为阳性，移植术后30dPh染色体皆转为阴性，bcr／abl融合基因完全转阴时间为1～3个月，中位时间2个月；在随防的18例CML患者中，有16例患者bcr／abl融合基因完全转阴后没再转为阳性。在1例复发的CML患者中可见到血型、Ph染色体和bcr／abl融合基因动态变化，另1例CML患者在移植成功后的21个月和36个月检测到bcr／abl融合基因，经随访未见临床和血液学复发征象。结论：检测bcr／abl融合基因是目前观察CML患者非亲缘异基因骨髓移植后微小残留病变最敏感的方法之一，非亲缘异基因骨髓移植能最大限度地消除CML患者体内残留病变，患者能长期无病生存。     

Detection of minimal residual disease by polymerase chain reaction in Philadelphia chromosome-positive chronic myelogenous leukemia following interferon therapy.

The significance of the polymerase chain reaction (PCR) in the detection of minimal residual disease in Philadelphia chromosome (Ph')-positive chronic myelogenous leukemia (CML) following interferon therapy was investigated. Forty remission blood samples obtained at various remission time points from 29 patients in complete cytogenetic remission were analyzed. All 40 samples showed minimal residual Ph'-positive cells by PCR: 22 in remission for less than 12 months, 12 in remission for 12 to 24 months, four in remission for 25 to 60 months, and two in remission for more than 60 months. Of these 29 patients, seven relapsed at 4, 6, 9, 14, 17, 19, and 50 months after their first PCR-positivity during remission. One developed extramedullary myelopoiesis at 49 months after PCR-positivity. The remaining 21 patients remained in complete hematologic and cytogenetic remission with median follow-up of 13 months (range, 4 to 36 months) after PCR analysis. These findings indicate that PCR-positivity is not associated with immediate disease recurrence. Long-term follow-up is essential to determine the relevance of PCR-positivity, since late recurrence is observed in our study.     

Detection of minimal residual leukemia after bone marrow transplantation in patients with chronic myeloid leukemia using the polymerase chain reaction

We have utilized the polymerase chain reaction (PCR) to sensitively detect the minimal residual leukemia in 24 patients who were hematologically normal and were negative for Ph1 chromosome after bone marrow transplantation (BMT). None of the patient showed breakpoint cluster region (BCR) gene rearrangement by Southern blot analysis, however, PCR technique revealed the bcr/abl mRNA in 13 of 24 patients. In patients who received CY + TBI as a conditioning regimen, 6 out of 8 patients were detected bcr/abl mRNA by PCR. On the other hand, patients who received high dose AraC+ CY + TBI or busulfan + CY as a conditioning, bcr/abl mRNA was detected in 4 out of 9 patients and 1 out of 5 patients, respectively. There was no clear association between the presence or absence of graft versus host disease and the detection of bcr/abl mRNA by PCR.     

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.     

巢式聚合酶链反应检测慢性粒细胞白血病bcr／abl融合基因

目的 探讨Ph染色体和bcr/abl融合基因在慢性粒细胞白血病（CGL）的发病机制、诊断、治疗、预后判断的价值。方法 对46例CGL患者作巢式逆转录聚合酶链反应（RT-PCR）检测bcr/abl融合基因,同时对其中28例作细胞遗传学检查。结果 46例CGL患者中,44例bcr/abl融合基因阳性,阳性率为95.7%;28例CGL患者作细胞遗传学检查。26例Ph染色体阳性,阳性率为92.9%;2例Ph染色体阴性的CGL患者,用RT-PCR检测出ber/abl融合基因。结论 巢式RT-PCR是一种快速、敏感而准确的检测方法,可以为部分Ph染色体阴性的CGL患者提供分子生物学的诊断依据。     

Detection of residual leukemia by the polymerase chain reaction and sequence-specific oligonucleotide probe hybridization after allogeneic bone marrow transplantation for AKR leukemia: a murine model for minimal residual disease

Disease relapse after allogeneic bone marrow transplantation (BMT) is a major cause of treatment failure and is thought to evolve from clinically occult residual disease in the recipient. However, the demonstration of minimal residual disease (MRD) in individual patients is of uncertain prognostic significance because the detection of residual disease has not consistently correlated with subsequent relapse. Moreover, the optimal therapeutic approach in patients with MRD after allogeneic BMT is unknown. The study of these issues has been hindered by the lack of clinically relevant animal models. In this report, we characterize a novel murine model for the study of MRD after allogeneic BMT. This model was designed to simulate high-risk BMT in humans in which patients receive transplants in relapse and disease recurrence in the major cause of treatment failure. The H-2-compatible, mixed lymphocyte culture nonreactive murine strains, AKR (H-2k) and CBA (H-2k), were chosen to parallel marrow transplants from HLA-matched siblings, which represent the majority of allo-transplants in humans. Male AKR leukemia cells were used in female donor/host chimeras permitting the Y chromosome to serve as a leukemia-specific marker for MRD. Detection of residual male leukemia cells in the peripheral blood of the primary host was facilitated by use of the polymerase chain reaction (PCR) and sequence-specific oligonucleotide probe hybridization (SSOPH). Use of PCR/SSOPH was highly predictive of clinical outcome (relapse or cure) in animals receiving transplants (P < .00002) and detected disease recurrence earlier than comparative flow cytometric analysis studies. This murine model will be useful in evaluating the efficacy of therapeutic strategies aimed at reducing disease relapse posttransplant and can be adapted to other transplant murine tumor systems for the study of MRD.     

Use of the polymerase chain reaction to monitor engraftment following allogeneic bone marrow transplantation

Engraftment following allogeneic bone marrow transplantation (BMT) was assessed in three cases, two of which were sex-mismatched and one sex-matched. The polymerase chain reaction (PCR) was used to amplify the hypervariable region lying 3' to the apolipoprotein B gene on chromosome 2. Amplification of this region provided informative marker bands capable of distinguishing host/donor populations in each case. The method allowed rapid analysis of minimal numbers of cells from peripheral blood and bone marrow in the early stages following BMT and was predictive of either successful engraftment or graft failure.     

Cytogenetic conversion following allogeneic bone marrow transplantation for advanced chronic myelogenous leukemia

We performed a pilot study to test the effectiveness of allogeneic bone marrow transplantation in the treatment of chronic myelogenous leukemia. Five patients in the advanced stages of chronic myelogenous leukemia (four in blast crisis, one in accelerated phase) with abnormal chromosomes underwent matched-sibling allogeneic bone marrow transplantation after preparation with busulfan, vincristine, cyclophosphamide, and fractionated total body irradiation. Engraftment and conversion to normal chromosome patterns after transplantation occurred in all five patients. None of the patients reverted to an abnormal chromosome pattern of demonstrated clinical or hematologic evidence of recurrent disease during the course of this study; however, longest survival from transplant was 248 days. Allogeneic bone marrow transplantation can eradicate the abnormal clone even in far advanced chronic myelogenous leukemia and can provide normal hematopoiesis. We suggest that clinical complications of chemotherapeutic toxicity and infection were responsible for the short survival in this group of patients, and that these complications could be decreased by performing transplantation in the chronic phase or early accelerated phase of the disease.     

Minimal residual disease after allogeneic bone marrow transplantation for chronic myeloid leukaemia: a metaphase-FISH study

Metaphase-FISH was adopted for the detection of proliferating Philadelphia-positive (Ph⁺) residual leukaemic cells in 25 patients with chronic myeloid leukaemia treated with allogeneic bone marrow transplantation (BMT). Patients were followed up during their clinical remission for 4–50 months (median 17 months) after BMT. 80 bone marrow samples were studied. For most of the cases no fewer than 1000 metaphases were analysed. Six patients (24%) showed residual Ph⁺ cells during the first 6 months and two others by the end of the first year after BMT. Three patients relapsed during the study and in two of them residual Ph⁺ cells were detected during the first 6 months after BMT. In 17 patients no Ph⁺ cells were detected at any stage of follow-up and 16 (94.1%) of them continue in complete clinical and haematological remission. Our results indicate that metaphase-FISH is a reliable tool in the quantitation of proliferating residual leukaemic cells. We suggest that consecutive findings of equal amounts of residual leukaemic cells do not necessarily predict a relapse. However, their presence calls for follow-up at shorter intervals where an increasing number of these cells predicts an ensuing relapse.     

Detection and quantitation of genetically marked acute myeloid leukemia by competitive polymerase chain reaction after autologous bone marrow transplantation: a preclinical model for minimal residual disease

Preclinical models and methods aimed at detecting and quantitating minimal residual disease (MRD) after autologous bone marrow transplantation (BMT) for acute myeloid leukemia (AML) could facilitate assessment of innovative therapeutic strategies for their antileukemic potential. Among the various techniques exploited to identify MRD, polymerase chain reaction (PCR) proved to be a valuable tool in instances in which clonogeneic markers are involved during the evolution of disease. In human AML, however, detection of MRD by PCR is limited to a minority of subgroups, as clonospecific markers are absent or presently unknown. Although gene labeling has proved to be efficient in detecting marker-devoid leukemia cells in preclinical models, detection and quantitation by PCR have not yet been considered. We therefore developed an experimental model in which detection and quantitation of genetically marked murine AML cells are based on a highly sensitive two-step nested PCR and competitive PCR protocol, respectively. We further demonstrated its applicability to a murine syngeneic BMT model that was designed to monitor minimal numbers of gene-tagged AML cells at various time intervals after transplantation. Our results showed that detection and quantitation could reproducibly be achieved at levels as low as one in 10(6) and 10(5) cells, respectively.     

Reversal of severe bone marrow fibrosis and osteosclerosis following allogeneic bone marrow transplantation for chronic granulocytic leukaemia

S ummary . Severe marrow fibrosis and osteosclerosis gradually disappeared after a 33-year-old woman received an allogeneic bone marrow transplantation (BMT) as experimental treatment for chronic granulocytic leukaemia. Serial biopsies demonstrate gradual resolution of dense reticulin fibrosis, collagen fibrosis and osteosclerosis, and restoration of normal marrow architecture after transplantation. These changes correspond with histological and cytogenetic evidence of normal marrow engraftment and sustained complete remission from chronic granulocytic leukaemia. In this case severe marrow infiltration with reticulin and collagen fibrosis as well as severe derangement of marrow architecture and obliteration of the medullary cavity by osteosclerosis was an entirely reversible process after allogeneic bone marrow transplantation, and did not prevent successful engraftment, haemopoietic and cytogenetic reconstitution and complete remission from chronic granulocytic leukaemia.     

Detection of BCR/ABL RNA transcripts using the polymerase chain reaction is highly predictive for relapse in patients transplanted with unrelated marrow grafts for chronic myelogenous leukaemia

Relapse after allogeneic bone marrow transplantation (BMT) for chronic myelogenous leukaemia (CML) is thought to result from residual leukaemia cells which survive the intensive conditioning regimen and are not eradicated by donor-derived immune effector cells capable of mediating a graft-versus-leukaemia (GVL) effect. Early relapse can be detected using highly sensitive assays such as the polymerase chain reaction (PCR) which have been shown to have predictive value for subsequent relapse in selected patient populations. The validity of PCR for predicting CML relapse in unrelated marrow transplant recipients where the GVL effect appears to be augmented due to increased HLA disparity between donor and recipient, however, has not been well defined. In this study we assessed the prognostic value of PCR in a cohort of 52 patients transplanted with T-cell-depleted unrelated marrow grafts for CML. The actual probability of relapse at 3 years was 71% in patients with at least one positive assay versus 6% in patients with no positive assays post-transplant. Patients with one or more positive assays at any time post-transplant had a 56-fold increased risk of relapse which was significantly higher ( P  = 0.0002) than that observed in patients who remained persistently PCR negative. Moreover, PCR detected relapse a median of 5 months earlier than cytogenetic analysis in a subgroup of patients in whom concurrent sampling had been performed. These data validate the use of PCR as a prognostic test in this patient population and may help to identify a cohort of patients to be considered as candidates for pre-emptive adoptive immunotherapy.