Stem cell transplantation in poor-risk chronic lymphocytic leukemia: assessment of post-transplant minimal residual disease using four- and six-color flow cytometry and allele-specific RQ-PCR

A total of 178 bone marrow samples were taken for minimal residual disease (MRD) analysis after 34 stem cell transplantations for poor-risk chronic lymphocytic leukemia, and 86 of them were analyzed in parallel by flow cytometry and allele-specific oligonucleotide-PCR (ASO-PCR). ASO primer was successfully designed for all patients whose frozen diagnosis samples were available. Flow cytometry and ASO-PCR were concordant, i.e. both either positive or both negative, in 78% of the analyses. Flow cytometry did not detect MRD in any of the samples that were PCR-negative cases. In contrast, ASO-PCR detected MRD in samples that were negative for MRD by flow cytometry in 22% of the analyses. In one patient, the immunophenotype but not the IgV_H gene sequence had changed during a course of the disease, and MRD could not be followed by flow cytometry. In the remaining cases, the discrepancy was due to a higher sensitivity of ASO-PCR. Autologous stem cell transplantation resulted in clinical complete response in 87% (20/23) of the patients. By flow cytometry, 35% (8/23) of autotransplanted patients became MRD-negative, but only 12.5% (2/16) PCR-negative (sensitivity of ASO-PCR <0.001 and <0.01, respectively). All allotransplanted patients achieved or maintained hematological CR, and five out of nine patients (56%) became PCR-negative (sensitivity of PCR between <0.001 and <0.003), two of them having non-myeloablative conditioning. None of the patients who became PCR-negative after allogeneic transplantation have relapsed.     

Novel flow-cytometric analysis based on BCD5+ subpopulations for the evaluation of minimal residual disease in chronic lymphocytic leukaemia

We describe a new flow-cytometric analysis using quadruple labelling with anti-CD19, CD20, CD5, CD79b monoclonal antibodies and sequential gating. We determined a novel criteria defined by BCD5+CD79b-/low/total BCD5+ cells ratio (BCD5+R), and compared it with the previous definition of phenotypic remission, based on CD19+CD5+ coexpression, and with complementarity-determining region 3 polymerase chain reaction (CDR3 PCR) and clonotypic PCR (cPCR). A series of 54 peripheral blood samples from 21 chronic lymphocytic leukaemia (CLL) patients in complete haematological remission and a series of 16 from normal volunteers were analysed. In normal controls, the BCD5+R was always < 0.2. The sensitivity of the BCD5+R was 1 x 10-4vs 5 x 10-2 for CDR3 PCR and 1 x 10-5 for cPCR. Among the 54 CLL samples, 35 had a BCD5+R < 0.2 and showed polyclonal CDR3 PCR, whereas the cPCR was positive in 12 out of 20 tested. In the remaining 19 samples, BCD5+R was > 0.2, CDR3 PCR was monoclonal in 16 out of 19 and cPCR positive in 14 out 14 tested, including one out of three samples with polyclonal CDR3 amplification. Even though cPCR remains the most sensitive method to evaluate MRD, this new, sensitive and specific flow cytometric parameter, the BCD5+R, is more suitable than CDR3 PCR for routine clinical MRD assessment in CLL.     

Multiplex technologies for the assessment of minimal residual disease and low-level mutation detection in leukaemia: mass spectrometry versus next-generation sequencing

With the focus of leukaemia management shifting to the implications of low-level disease burden, increasing attention is being paid on the development of highly sensitive methodologies required for detection. There are various techniques capable of identification of measurable residual disease (MRD) either evidencing as relevant mutation detection [e.g. nucleophosmin 1 (NPM1) mutation] or trace levels of leukaemic clonal populations. The vast majority of these methods only permit detection of a single clone or mutation. However, mass spectrometry and next-generation sequencing enable the interrogation of multiple genes simultaneously, facilitating a more complete genomic profile. In the present review, we explore the methodologies of both techniques in conjunction with the important advantages and limitations associated with each assay. We also highlight the evidence and the various instances where either technique has been used and propose future strategies for MRD detection.     

Minimal residual disease detection of myeloma using sequencing of immunoglobulin heavy chain gene VDJ regions

After therapy or stem cell transplantation, multiple myeloma patients achieving complete response or stringent complete response can still have a significant risk of disease relapse. This highlights the importance of using highly sensitive laboratory methods for minimal residual disease detection and prognostication. Older methods such as allele-specific oligonucleotide real time quantitative polymerase chain reaction and fluorescent polymerase chain reaction have their drawbacks. Meanwhile, the recent generation of multiparametric flow cytometry and next-generation sequencing (NGS)-based detection methods currently offer the highest technical sensitivities, and are likely to gain more widespread use and be recognized as the standard of care for disease monitoring in myeloma patients.     

Relationship between minimal residual disease measured by multiparametric flow cytometry prior to allogeneic hematopoietic stem cell transplantation and outcome in children with acute lymphoblastic leukemia

Background

The presence of minimal residual disease detected by polymerase chain reaction techniques prior to allogeneic hematopoietic stem cell transplantation has proven to be an independent prognostic factor for poor outcome in children with acute lymphoblastic leukemia.

Design and Methods

The aim of this study was to ascertain whether the presence of minimal residual disease detected by multiparametric flow cytometry prior to allogeneic hematopoietic stem cell transplantation is related to outcome in children acute lymphoblastic leukemia. Minimal residual disease was quantified by multiparametric flow cytometry at a median of 10 days prior to hematopoietic stem cell transplantation in 31 children (age range, 10 months to 16 years) with acute lymphoblastic leukemia. Thirteen patients were transplanted in first remission. Stem cell donors were HLA-identical siblings in 8 cases and matched unrelated donors in 23. Twenty-six children received a total body irradiation-containing conditioning regimen. According to the level of minimal residual disease, patients were divided into two groups: minimal residual disease-positive (≥0.01%) (n=10) and minimal residual disease-negative (<0.01%) (n=21).

Results

Estimated event-free survival rates at 2 years for the minimal residual disease-negative and -positive subgroups were 74% and 20%, respectively (P=0.004) and overall survival rates were 80% and 20%, respectively (P=0.005). Bivariate analysis identified pre-transplant minimal residual disease as the only significant factor for relapse and also for death (P<0.01).

Conclusions

The presence of minimal residual disease measured by multiparametric flow cytometry identified a group of patients with a 9.5-fold higher risk of relapse and a 3.2-fold higher risk of death than those without minimal residual disease. This study supports the strong relationship between pre-transplantation minimal residual disease measured by multiparametric flow cytometry and outcome following allogeneic hematopoietic stem cell transplantation and concur with the results of previous studies using polymerase chain reaction techniques.     

HIV/AIDS患者免疫重建的影响因素与促进免疫重建的治疗策略

HIV/AIDS患者免疫重建的研究是近年来AIDS研究领域的热点、难点之一.本文就目前HIV/AIDS患者抗病毒治疗后免疫重建的概念和特点、异常的免疫重建,免疫重建的影响因素和预测指标,促进免疫重建的治疗策略3个方面进行综述.     

The prognostic impact of minimal residual disease in patients with chronic lymphocytic leukemia requiring first-line therapy

A proportion of patients with chronic lymphocytic leukemia achieve a minimal residual disease negative status after therapy. We retrospectively evaluated the impact of minimal residual disease on the outcome of 255 consecutive patients receiving any front-line therapy in the context of a detailed prognostic evaluation, including assessment of IGHV, TP53, NOTCH1 and SF3B1 mutations. The median follow-up was 73 months (range, 2–202) from disease evaluation. The median treatment-free survival durations for patients achieving a complete response without or with minimal residual disease, a partial response and no response were 76, 40, 11 and 11 months, respectively (P<0.001). Multivariate analysis revealed that three variables had a significant impact on treatment-free survival: minimal residual disease (P<0.001), IGHV status (P<0.001) and β₂-microglobulin levels (P=0.012). With regards to overall survival, factors predictive of an unfavorable outcome were minimal residual disease positivity (P=0.014), together with advanced age (P<0.001), unmutated IGHV status (P=0.001), TP53 mutations (P<0.001) and elevated levels of β₂-microglobulin (P=0.003). In conclusion, for patients requiring front-line therapy, achievement of minimal residual disease negativity is associated with significantly prolonged treatment-free and overall survival irrespective of other prognostic markers or treatment administered.     

以降钙素基因高度甲基化为分子基因标志检测白血病微量残留病

目的 探讨以降钙素（CT）基因高度甲基化为白血病克隆的分子基因标志,检测白血病微量残留病（MRD）,并预测预后的可能性。方法 用限制性内切酶消化DNA〈应用聚合酶链反应（PCR）技术检测载9例急性白血病（AL）,其中急性淋巴细胞白血病（ALL）14例,急性非淋巴细胞白血病（ANLL）15例和8例慢性粒细胞白血病（CML）急变患者的CT基因的甲基化程度,对CT基因高度甲基化阳性者,以CT基因高度甲基化为白血病克隆的分子基因标志,应用PCR技术定期追踪检测骨髓MRD。结果 ALL中CT基因高度甲基化阳性率为857%（12/14）,ANLL为60%（9/15）,CML急变为100%（8/8）,而对照组无一例阳性,20例AL和CML急变患者完全缓解（CR）后均存在MRD,CR后MRD持续阳性或转阴后再次出现阳性,提示将发生骨髓复发,能提早2-11个月预示疾病的复发,MRD较早转阴并持续长期阴性者可能获得长期自下而上度。结论 以CT基因高度甲基化为白血病克隆的分子基因标志,应用PCR技术能起到监测白血病MRD的作用。为预测白血病的预后开辟了一条新途径。     

Stem cells and immune reconstitution in AIDS

The hematopoietic system generally has reserve sufficient to tolerate significant insult and regenerative capacity to overcome most damage due to infectious agents. However, HIV infection results in a progressive decline in hematopoietic function and even in the context of potent, anti-retroviral therapy is able to only incompletely reconstitute immune function. The ability of the immune system to respond to HIV itself remains compromised, a defect that leaves infected individuals with a lifelong dependence on medications. The capability of stem cells and the thymus to restore function and their limitations in the context of HIV infection are discussed in this review.     

Monitoring minimal residual disease in the bone marrow using next generation sequencing

Achieving minimal residual disease (MRD) negativity in the bone marrow is one of the strongest prognostic factors in multiple myeloma. Consequently, MRD testing is routinely performed in clinical trials and moving towards standard of care. This review focuses on the role of next generation sequencing (NGS) of tumor-specific immunoglobulin V(D)J sequences for MRD tracking. The immunoglobulin variable regions are ideal targets for tracking, because every tumor cell shares an identical gene sequence, which is stable over time and generally distinct from the immunoglobulin sequences of normal B-cells. Several excellent assays for NGS-based MRD testing are available, both commercial and community-based, including one that is FDA-approved. These assays can achieve the gold standard analytical sensitivity of one tumor cell per million (10⁻⁶), requiring a minimum input of 3 million bone marrow cells. On-going clinical trials will outline how MRD testing should be used to inform dynamic risk-adopted therapy.     

Patient‐tailored analysis of minimal residual disease in acute myeloid leukemia using next‐generation sequencing

Next‐generation sequencing techniques have revealed that leukemic cells in acute myeloid leukemia often are characterized by a limited number of somatic mutations. These mutations can be the basis for the detection of leukemic cells in follow‐up samples. The aim of this study was to identify leukemia‐specific mutations in cells from patients with acute myeloid leukemia and to use these mutations as markers for minimal residual disease. Leukemic cells and normal lymphocytes were simultaneously isolated at diagnosis from 17 patients with acute myeloid leukemia using fluorescence‐activated cell sorting. Exome sequencing of these cells identified 240 leukemia‐specific single nucleotide variations and 22 small insertions and deletions. Based on estimated allele frequencies and their accuracies, 191 of these mutations qualified as candidates for minimal residual disease analysis. Targeted deep sequencing with a significance threshold of 0.027% for single nucleotide variations and 0.006% for NPM1 type A mutation was developed for quantification of minimal residual disease. When tested on follow‐up samples from a patient with acute myeloid leukemia, targeted deep sequencing of single nucleotide variations as well as NPM1 was more sensitive than minimal residual disease quantification with multiparameter flow cytometry. In conclusion, we here describe how exome sequencing can be used for identification of leukemia‐specific mutations in samples already at diagnosis of acute myeloid leukemia. We also show that targeted deep sequencing of such mutations, including single nucleotide variations, can be used for high‐sensitivity quantification of minimal residual disease in a patient‐tailored manner.