异基因造血干细胞移植后早期嵌合状态研究

目的:建立多重PCR扩增短串联重复序列(STR)结合毛细管电泳,定量检测异基因造血干细胞移植(Allo-HSCT)后受者体内供、受体来源血细胞嵌合率的方法及灵敏度。并探讨该方法的早期定量检测对Allo-HSCT后白血病早期复发进行干预的指导意义。方法:将2名健康成人静脉血分离单个核细胞,并按供、受体单个核细胞不同百分比制成模拟嵌合体标本并提取基因组DNA,用多重PCR扩增后,进行毛细管电泳,确定基因位点及相应峰面积。15例Allo-HSCT患者移植前采集供、受者外周血,移植后采集受者外周血,分别提取DNA,进行PCR扩增和毛细管电泳,确定STR基因位点及峰面积,计算混合样本中2单个样本DNA含量百分比及Al-lo-HSCT患者嵌合率。结果:①当混合样本中某一细胞比例为4%以上时,即可从电泳图中明确区分混合样本中该单个样本的基因型,且扩增前细胞百分率与扩增后两者峰面积比值呈直线相关;②所有患者在移植后2周内均出现供者来源的细胞,13例患者在术后30d内均达稳定嵌合。2例患者移植后出现不稳定混合嵌合状态或复发,经干预治疗后转为完全嵌合状态。结论:STR毛细管电泳PCR方法非常敏感,能应用于Allo-HSCT后早期嵌合体的定量检测。嵌合状态的早期定量检测对了解Allo-HSCT后的移植物的植入状态和指导早期干预治疗均有重要参考价值。     

Strategies and clinical implications of chimerism diagnostics after allogeneic hematopoietic stem cell transplantation

Analysis of donor chimerism has become a routine method for the documentation of engraftment after allogeneic hematopoietic stem cell transplantation (HSCT). In recent years several groups have also focused on the application of this technique for the detection of relapsing disease after allogeneic HSCT. This review addresses technical issues (sensitivity, specificity) and discusses the advantages and limitations of methods currently used for chimerism analysis and their usefulness for the detection of MRD. In addition, the potential impact of novel procedures, e.g. subset chimerism or real-time PCR-based procedures, is discussed.     

Monthly prospective analysis of hematopoietic chimerism after allogeneic hematopoietic cell transplantation

Summary:Hematopoietic chimerism (HpC) was assayed monthly using a sensitive, polymerase chain reaction (PCR) -based method in consecutive patients. Between January 1998 and April 2002, 181 patients underwent non-T cell depleted allogeneic hematopoietic cell transplantation (HCT). A total of 163 patients were evaluable for HpC at 1 month (11 early deaths; no informative band for HpC analysis/no genomic DNA in seven). In all, 53 of 163 patients (33%, median recipient DNA of 15% (range 5-95)), 39 of 151 patients (26%), and 27 of 142 patients (19%) showed mixed chimerism (MC) at 1, 2, and 3 months after HCT, respectively. Conditioning regimen (busulfan-fludarabine-ATG vs BuCy, relative risk 3.99 (95% CI 1.16-10.92)), neutrophil engraftment (>/=day 17 vs /=5% recipient DNA at 1 month. Five patients experienced secondary graft failure. All five patients showed MC at 1 month with median recipient DNA of 40%. None of the 109 patients with complete chimerism experienced graft failure (P=0.002). Our study showed that MC shown on monthly analysis of HpC after allogeneic HCT is a significant predictor of secondary graft failure.Bone Marrow Transplantation (2003) 32, 423-431. doi:10.1038/sj.bmt.1704147     

Human pulmonary chimerism after hematopoietic stem cell transplantation

Many of the body's tissues once thought to be only locally regenerative may, in fact, be actively replaced by circulating stem cells after hematopoietic stem cell transplantation. Localization of donor-derived cells ("chimerism") has recently been shown to occur in the lungs of mice after either hematopoietic stem cell transplantation or infusion of cultured marrow. To determine whether tissues of the human lung might be similarly derived from extrapulmonary sources, we examined lung specimens from a retrospective cohort of female allogeneic hematopoietic stem cell transplant recipients who received stem cells from male donors. Tissue samples from three such patients who had undergone diagnostic lung biopsy or autopsy were examined. Slides were stained by immunohistochemistry for cytokeratin (epithelium) and platelet endothelial cell adhesion molecule, CD31 (PECAM) (endothelium) and were imaged and then examined by fluorescent in situ hybridization analysis to identify male cells. The resulting overlapping in situ hybridization and immunohistochemistry images were examined for the presence and, if present, cell type of donor cells in the lung. We found significant rates of epithelial (2.5-8.0%) and endothelial (37.5-42.3%) chimerism. These results suggest that significant chimerism of the human lung may follow hematopoietic stem cell transplantation and that adult human stem cells could potentially play a therapeutic role in treatment of the damaged lung.     

Analysis of chimerism in patients with hematologic malignancies after allogeneic hematopoietic stem cell transplantation

Serial monitoring of chimerism after allogeneic hematopoietic stem cell transplantation (HSCT) can be performed easily and rapidly using PCR-based assays analyzing informative tandem repeat genetic markers. Sequential analysis of individual chimerism status was performed in 34 patients who underwent myeloablative allo-HSCT using a commercial multiplex short tandem repeat (STR) kit. Mixed chimerism (MC) was found in 14 of the patients for more than one month. The incidence of MC seemed to be dependent on the type of disease or pretransplantation regimen. There was no significant difference in relapse rates between MC and complete donor chimerism (CC) in all patients. However, the relapse rate was significantly higher in MC than in CC among patients with acute leukemia. The severity of acute graft-versus-host disease (aGVHD) was significantly reduced in the patients with MC. Most of the MC patients with hematologic malignancies had transient mixed T-lymphoid chimerism, and CC was achieved within 6 months after HSCT in such cases. Patients with MC beyond 6 months after HSCT and patients with reappearance of autologous signals (MC after CC) may have an enhanced risk of relapse.     

A single nucleotide polymorphism at chromosome 2q21.3 (LCT -13910C>T) associates with clinical outcome after allogeneic hematopoietic stem cell transplantation

A single nucleotide polymorphism (SNP) responsible for lactase persistence (LCT -13910C>T) changes intestinal microflora. Considering the influence of bacterial microflora on various immune effects, we tested DNA from 111 recipients/donors and analyzed whether this SNP interferes with survival and the incidence of acute graft-versus-host disease (aGVHD) after allogeneic hematopoetic stem cell transplantations (HSCT). Median overall survival (OS) was significantly longer when donors had a CC genotype (not reached after 133 vs 11.1 months, P = .004). Multivariate analysis identified a donor T allele (hazard ratio 2.63, 95% confidence interval 1.29-5.33, P = .008) as independent risk factor for death. Surprisingly, recipient genotypes did not influence outcome and there were no differences regarding aGVHD. Transplantation-related mortality (TRM), relapse and pneumonia were significantly less frequent in patients with CC donors. These findings add to the growing list of non-HLA polymorphisms with impact on outcome after allogeneic HSCT.     

四核苷酸重复序列估价异基因外周血干细胞移植后嵌合状态

目的 确定适合于检测中国人群异基因外周血干细胞移植后嵌合状态的5个四核苷酸重复序列（或称位点）,探讨嵌合状态与预后的关系。方法 PCR扩增140份脐带血和17对移植供受者基因组DNA,电泳,银染,据所得片段大小考察5个位点在人群中的多态性及移植后嵌合状态,结合临床分析。结果 位点多态性：各位点发现7个以上等位基因（7－12）,16种以上基因型（16－28）,杂合度仅CSF1PO低于70％。移植患者中完全嵌合状态（CC)12例,1例死于移植物抗宿主病;混合嵌合状态（MC）1例死亡;受者型1例死亡;MC经二次移植转为CC1例无复发。2例嵌合情况不同。结论 上述5位点多态性较强,可成功检测大多数嵌合状态。移植患者随着体内受者细胞成分的增加,预后渐差。     

Heart failure after allogeneic hematopoietic stem cell transplantation

Background

Heart failure (HF) occurring after allogeneic hematopoietic stem cell transplantation (allo-HSCT) is rare but severe. We examine the role of pre-HSCT therapeutic exposures, conditioning regimens, pre-HSCT comorbidities, severe transplant-related complications, and post-HSCT cardiovascular risk factors in the development of heart failure after allo-HSCT.

Methods

A nested case-control study was designed. Cases with HF and controls matched for age, year of allo-HSCT, and length of follow-up were identified from a cohort of 2455 patients who underwent allo-HSCT between 2000 and 2011 for hematologic malignancies.

Results

Forty-two patients suffered from HF; mean age at presentation was 35 years (± 14 years) and mean time to presentation was 5 months (± 9 months) post-HSCT. The number of pre-HSCT cycles of chemotherapy was significantly greater (7 vs. 5 courses, P = 0.023). Cases were significantly more likely to have severe acute GVHD (≥ grade III), hemorrhagic cystitis (≥ grade 2), and multiple severe transplant-related complications compared with controls (42.9% vs. 20.4%, P = 0.008). Multivariate analysis revealed that pre-HSCT cycles of chemotherapy of ≥ 5 courses (OR = 3.5, P = 0.003) and two or more severe transplant-related complications (OR = 3.6, P = 0.003) were independently associated with HF.

Conclusions

These results identify the individuals who are at higher risk of developing HF after allo-HSCT. We should pay more attention to these patients and more active management would be reasonable.     

Pulmonary function after allogeneic hematopoietic stem cell transplantation

This study was undertaken to identify the factors influencing pulmonary function in patients who underwent hematopoietic stem cell transplantation (HCT). Pulmonary function tests were evaluated before and after HCT in 51 adult patients who underwent HCT between 1993 and 1998. The patients with hematologic malignancies were given total body irradiation. Graft-versus-host disease (GVHD) prophylaxis consisted of cyclosporine A and short-term methotrexate. Six patients suffered from acute GVHD above grade II and 27 patients suffered from chronic GVHD. The post-transplant % diffusing capacity (%DLco) 100 days after HCT was significantly lower than pretransplant values (82 +/- 21% versus 71 +/- 15%, p < 0.01). The %DLco at 100 days was significantly lower in patients with chronic GVHD than in patients without chronic GVHD (66 +/- 16% versus 77 +/- 9%, p < 0.05). These findings suggested chronic GVHD is related to the decreased %DLco values observed 100 days after HCT.     

Pulmonary complications after allogeneic hematopoietic stem cell transplantation

We investigated the occurrence of pulmonary complications in patients who underwent allogeneic hematopoietic stem cell transplantation at our institution. Pulmonary complications were observed in 12 out of 60 patients. Interstitial pneumonia developed in 12 cases: 7 idiopathic, 2 cytomegalovirus-associated, 1 P. carinii, 1 HSV, and 1 HHV-6-associated. HSV- and HHV-6-associated pneumonias were exhibited 100 days after transplantation. PCR analysis was diagnostically useful for detection of viral DNA in bronchial alveolar lavage fluid. Respiratory disease with airway obstruction was observed in 4 patients with chronic graft-versus-host disease, and all 4 had a history of interstitial pneumonia. Three patients died of respiratory failure. Mycobacicrium avium complex was detected in 2. Exacerbation of respiratory failure may be associated with mycobacterial infection.     

Molecular assessment of erythroid lineage chimerism following nonmyeloablative allogeneic stem cell transplantation

OBJECTIVE: Nonmyeloablative conditioning regimens for allogeneic stem cell transplantation are now commonly used in the treatment of patients with hematologic malignancies. Since this treatment often results in the establishment of mixed hematopoietic chimerism, this approach may also prove to be useful in the treatment of nonmalignant disorders, such as sickle cell disease and thalassemia major. To apply this approach to these diseases, it will be necessary to determine the levels of donor erythropoiesis required to correct hemolysis and ameliorate disease symptoms. Current methods for measuring hematopoietic chimerism are based on DNA polymorphisms that distinguish recipient from donor. These methods accurately measure donor leukocyte engraftment but do not quantify the relative contributions of recipient and donor erythropoiesis following transplant. METHODS: To specifically measure erythroid-lineage chimerism, we used pyrosequencing of the sickle cell mutation to quantify the relative levels of normal and sickle beta-globin mRNA in patient samples. Results of beta-globin RNA chimerism were compared to assessment of beta-globin DNA chimerism as well as analysis of short tandem repeat (STR) polymorphisms, cytogenetics, and hemoglobin electrophoresis. RESULTS: Donor engraftment was measured in two adult patients following nonmyeloablative stem cell transplant for sickle cell disease. In Patient 1, 25 to 30% of peripheral leukocytes were donor derived after day 41. In contrast, more than 55% of peripheral blood beta-globin mRNA was of donor origin, and these results correlated with posttransplant clinical improvement. Patient 2 achieved 40 to 50% donor leukocyte engraftment from day 33 onward. This was associated with 70 to 100% peripheral blood donor beta-globin mRNA. CONCLUSIONS: These studies demonstrate that relatively low levels of donor leukocyte engraftment can be associated with higher levels of donor erythropoiesis and with significant clinical improvement. Pyrosequencing of lineage-specific mRNA directly measures functional reconstitution of donor cells and provides valuable information that can affect clinical decisions in patients with nonmalignant diseases following allogeneic transplant.     

Acute eosinophilic pneumonia after allogeneic hematopoietic stem cell transplantation

A 55-year old woman with multiple myeloma was treated with hematopoietic stem cell transplantation (HSCT). She developed cutaneous and hepatic graft-vs-host disease (GVHD). Sixty-five days after HSCT, acute respiratory failure occurred. A thoracic computed tomography scan showed bilateral patchy infiltrates. Bronchoalveolar lavage revealed 40% eosinophils on differential cell count with no infectious pathogens. These findings were in favor of acute eosinophilic pneumonia. High-dose steroid treatment was started, which had a rapid and lasting favorable course. After HSCT, clinicians should be aware that acute eosinophilic pneumonia mimics infectious pneumonitis and can be associated with GVHD.     

Human metapneumovirus infection after allogeneic hematopoietic stem cell transplantation

Background

The clinical characteristics of human metapneumovirus (hMPV)-associated lower respiratory tract infection (LRTI) after allogeneic hematopoietic stem cell transplantation (HSCT) is not well described. We describe the clinical course in eight HSCT recipients suffering from hMPV infection.

Methods

We prospectively included all patients with hMPV-associated LRTI after allogeneic HSCT during a period of 1?year. hMPV was diagnosed by multiplex polymerase chain reaction (PCR) from bronchoalveolar lavage (BAL).

Results

Eight patients with hMPV-associated LRTI were identified from 93 BAL samples. Three of the eight patients had co-infections with other pathogens. The median age of the patients was 45?years [interquartile range (IQR) 36.8?C53.5], the median time posttransplant was 473?days (IQR 251?C1,165), 5/8 patients had chronic graft-versus-host disease (cGvHD), and 6/8 patients received immunosuppression. Chest computed tomography (CT) scanning showed a ground-glass pattern in 7/8 patients. Seven of eight patients required hospitalization due to severe symptoms and hypoxemia. All were treated with intravenous immunoglobulin (IVIG), which was combined with oral ribavirin in six patients. The mortality rate was 12.5?% (1/8).

Conclusions

hMPV-associated LRTI in allogeneic HSCT recipients are not uncommon and present with unspecific respiratory symptoms, ground-glass pattern in CT scanning, and co-infection.     

Airflow obstruction after myeloablative allogeneic hematopoietic stem cell transplantation

Despite advances in the management of myeloablative allogeneic hematopoietic stem cell transplants, airflow obstruction (AFO) remains a significant complication. We conducted a 12-year study to examine the recent epidemiology of AFO and its associated mortality. Using the rate of percent predicted FEV1 decline after transplant, we defined AFO as a more than 5% per year decline in percent predicted FEV1 with the lowest post-transplant FEV1/FVC ratio less than 0.8. New obstruction was more frequent than previous estimates (26% overall, 32% among patients with chronic graft-versus-host disease [GVHD]) and was significantly associated with older age at transplant, lower pretransplant FEV1/FVC ratio, history of both acute and chronic GVHD, and respiratory viral infection within the first 100 days after transplant. AFO was associated with significant attributable mortality rates of 9% at 3 years, 12% at 5 years, and 18% at 10 years after transplant, which were much higher for the subpopulation of patients with chronic GVHD (22% at 3 years, 27% at 5 years, and 40% at 10 years). These results suggest that the incidence of AFO may have been underestimated previously, and its presence significantly increases the mortality of long-term survivors of myeloablative allogeneic hematopoietic stem cell transplant patients.