The minimal residual disease (MRD) in hematological malignancies]

Molecular genetic and cytoimmunological markers have been applied for the detection of minimal residual disease (MRD) in hematological malignancies. These markers include surface markers or rearranged T-cell receptor and immunoglobulin genes in the lymphoid malignancies and fused genes associated with chromosomal translocations such as BCR-ABL in t(9;22) or PML-RAR alpha in t(15;17) in myeloid malignancies. The expression of the WT1 gene is recognized as the universal tumor marker for a wide variety of hematological malignancies. Using these sensitive markers, a tumor cell in 10,000 to 1,000,000 normal cells can be detected. By examining a large number of patients, it has been shown that the MRD in the early phase of chemotherapy has a correlation with the prognosis of childhood ALL. Based on these observations, a new strategy of chemotherapy in which the post-remission therapy is modified based on the MRD results has begun. The amount of tumor cells contaminated in the autologous stem cell grafts in ALL patients might be related to the prognosis. The diagnosis of MRD will be used as an important routine examination in chemotherapy for leukemia/lymphoma patients.     

Detection of minimal residual disease (MRD) after bone marrow transplantation (BMT) by multi-parameter flow cytometry (MPFC)

Multi-parameter flow cytometry (MPFC) was used to detect minimal residual disease (MRD) following bone marrow transplantation (BMT) in 21 patients. Bone marrow (BM)was analyzed pre-transplant and 3-4 months post-BMT while the patients were in clinical and morphological remission. MRD was detected by identifying cells with aberrant antigen expression and/or leukemia-associated phenotype (LAP) using MPFC. Prior to BMT, 8 out of 21 patients exhibited normal antigen expression based on normal BM samples while 13 BM aspirates had abnormal MPFC. Pre-BMT MPFC was abnormal in all 10 patients who were not in complete remission (CR) (>5% blasts in BM) as well as 3 patients acute lymphoblastic leukemia (ALL) who were in CR. In BM from ALL patients, an abnormal uniform B cell population was observed however antigen expression patterns varied greatly between patients. BM from acute myeloblastic leukemia (AML) patients showed an abnormal distribution of CD34+ cells. In addition, a correlation was observed between pre-BMT cytogenetics and MPFC. Only 2 out of 8 (25%) patients with normal MPFC pre-autologous bone marrow transplantation (ABMT) relapsed (AML), while 6 out of 13 (46%) patients with abnormal pre-BMT MPFC relapsed including 2 out of 3 patients who were transplanted in clinical CR. Pre-BMT MPFC may thus be an effective tool for detection of MRD by detection of a pre-transplant MPFC abnormality.     

Real-time t(11;14) and t(14;18) PCR assays provide sensitive and quantitative assessments of minimal residual disease (MRD).

Non-Hodgkin's lymphoma (NHL) arises as a clonal transformation of normal B and T cell differentiation and is often characterized by a higher incidence of specific chromosomal translocations. We have developed real-time TaqMan PCR assays directed toward two of these tumor-associated DNA markers, the t(14;18)(q32;q21.3) at the major breakpoint region of the bcl-2 gene and the t(11;14)(q13;q32) at the bcl-1 major translocation cluster. During analysis of serial dilutions of t(14;18)-positive DNA, the t(14;18) real-time PCR was at least as sensitive as nested PCR and demonstrated enhanced quantitative potential. Moreover, in a blinded comparison of the t(14;18) real-time PCR and a clinically validated nested PCR protocol using 134 cell line and patient DNA samples, the real-time PCR detected the translocation in 30.0% more cases than nested PCR. Both the t(14;18) and t(11;14) real-time PCR assays were used to quantitate minimal residual disease (MRD) in an NHL clinical trial assessing the safety and efficacy of a tumor-purging protocol in autologous stem cell transplantation. The assays were also used to evaluate disease depletion in an ex vivo tumor spiking model in which normal peripheral blood was spiked with tumor cell lines and processed according to the clinical purging method. PCR data from both the clinical trial and the ex vivo model demonstrated a 4 to 6 log reduction in tumor cells during CD34+ and CD34+ Thy-1+ enrichment. Because the t(14;18) and t(11;14) real-time PCR assays are very sensitive, quantitative, rapid, and require no post-PCR manipulation, they may serve as practical alternatives to nested PCR.     

Serial minimal residual disease (MRD) analysis as a predictor of response duration in Philadelphia-positive acute lymphoblastic leukemia (Ph+ALL) during imatinib treatment.

Patients with refractory or relapsed Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph+ALL) rarely have prolonged responses to salvage therapy, including imatinib, resulting in a short opportunity for potentially curative stem cell transplantation. To identify minimal residual disease (MRD) parameters predictive of imminent relapse, we quantitated Bcr-Abl expression by real-time PCR in peripheral blood (PB) and bone marrow (BM) of 24 Ph+ALL patients after achieving a complete response and MRD minimum. The ratio of Bcr-Abl and glyceraldehyde-3-phosphate dehydrogenase copies, magnitude of increase and velocity of increase were evaluated regarding subsequent time intervals to relapse, death or censoring. High Bcr-Abl levels >/=5 x 10(-4) in PB (n=23) and >/=10(-4) in BM (n=18) were significantly associated with short time periods to relapse. Bcr-Abl increases >2 logarithmic units (log) in PB, but not in BM preceded short-term relapse. The velocity of Bcr-Abl increases predicted response duration in PB (cutoff: 1.25 log/30 days) and BM (0.6). Bcr-Abl level and velocity of increase in BM as well as magnitude of increase in PB correlated with remaining periods of survival and predicted relapse within 2 months in nine of 10, 10 of 11 and four of four patients, respectively. Thus, these MRD parameters may guide timing and intensity of therapeutic modifications.     

Minimal residual disease (MRD) in follicular lymphoma in the era of immunotherapy with rituximab

The t(14;18)-translocation can be detected by PCR analysis in more than 90% of cytogenetically t(14;18)-positive follicular lymphomas (FLs), thus providing an easily accessible marker for molecular disease monitoring. Various technical aspects of the detection of residual lymphoma cells as well as the prognostic and clinical significance of the detection of minimal residual disease (MRD) after radiotherapy, chemotherapy and therapy with the monoclonal antibody rituximab are discussed. Up to now the comparability of the different studies investigating minimal residual disease in follicular lymphoma patients is hampered by the use of a variety of PCR techniques. A more standardized quantitative approach based on the real-time PCR technique will provide a powerful tool for the evaluation and optimization of therapy for each individual patient.     

Managing minimal residual malignant disease

So-called complete remission of acute leukemia and other tumors frequently leaves minimal residual disease cells, sometimes causing an inflammatory response and often relapse. Although the remaining cells often have long generation times or may even be in G0 condition, they may form a new tumor mass. This can be explained by cell kinetics alone, or by an escape from immunologic or other control mechanisms, or by a new promoting event. Maintenance chemotherapy beyond the first 6 months seemed only to have an effect where induction chemotherapy was weak and palliative, but not since it has become intensive and sometimes curative: it only increases the relapse incubation time, not the survival. Relapse cells may be refractory to cytostatics previously not given, in part because of alternative metabolic pathways or enzyme induction, in part because of tumor cell progression, and any chemotherapy seems to be able to play a significant role in this progression. The result is a relatively high residual tumor mass and a short duration of rare second remissions. Adjuvant immunotherapy, despite controversies about its results, seems to work in some tumors, but the statistical methods applied and the monitoring have been criticized. The strategy proposed for potentially 'curable' tumors (leukemias, lymphomas, embryomas) with short cell generation times is therefore intensive remission induction aiming at the smallest possible residual disease cell number. It is conceivable that this induction might mean accepting risks comparable to those accepted for transplant patients. Manipulation of the bioimmunological response can be effective even against cells in the G0 phase. New methods are proposed to manage residual disease of solid tumors with long generation times, and only maintenance chemotherapy active on cells with a long generation time is suggested.     

乳腺癌间质型循环肿瘤细胞对术后微小残留病灶的评估价值

目的 探讨间质型循环肿瘤细胞（MCTC）检测辅助乳腺癌患者术后微小残留病灶（MRD）和复发监测的评估价值。方法研究入组2019 年10 月至2021 年4 月临沂市肿瘤医院125例准备手术治疗的乳腺癌患者，使用CytoSorter微流控纳米芯片技术富集检测MCTC，分析术前和术后MCTC数量与乳腺癌的关系。结果125例乳腺癌患者中，术前和术后MCTC检出率分别为81.6%（102/125）和68.8%（86/125），术前和术后MCTC数量差异有统计学意义（P=0.000 2），手术治疗后MCTC下降（P=0.000 2）。乳腺癌术前术后MCTC水平与临床病理特征的关系结果显示，术前MCTC水平与肿瘤直径（P＜0.001）、病理分期（P＜0.001）、T分期（P＜0.001）、N分期（P＜0.001）、Ki67表达（P＜0.001）、HER2表达（P=0.033）、分子分型（P=0.004）相关。乳腺癌患者的复发率为32.8%（41/125），单因素分析结果显示肿瘤病灶直径越大、病理分期越晚、T分期越高、淋巴结转移、Ki67高表达、术前和术后MCTC数量越多的乳腺癌患者越容易发生术后复发。Cox多因素回归分析结果显示，影响乳腺癌患者术后复发的因素包括病理分期、N分期、淋巴结转移、ER表达、PR表达、HER2表达、分子分型、术前和术后MCTC数量。MCTC与乳腺癌患者手术治疗后复发关系分析，无论术前还是术后，MCTC≥4个/4 ml的乳腺癌患者手术治疗后复发率高于MCTC＜4个/4 ml的乳腺癌患者，差异有统计学意义（P＜0.001）。术前MCTC检测诊断乳腺癌患者手术治疗后复发的灵敏度、特异度分别为64.3%（18/28）、76.3%（74/97）；术后MCTC检测诊断乳腺癌患者手术治疗后复发的灵敏度、特异度分别为100.0%（11/11）、73.7%（84/114）。结论术前和术后MCTC检测可能辅助乳腺癌术后MRD评估，MCTC≥4个/4 ml的乳腺癌患者术后可能具有较高的复发风险。     

Minimal residual disease (MRD) in non‐Hodgkin lymphomas: Interlaboratory reproducibility on marrow samples with very low levels of disease within the FIL (Fondazione Italiana Linfomi) MRD Network

In 2009, the four laboratories of the Fondazione Italiana Linfomi (FIL) minimal residual disease (MRD) Network started a collaborative effort to harmonize and standardize their methodologies at the national level, performing quality control (QC) rounds for follicular lymphoma (FL) and mantle cell lymphoma (MCL) MRD assessment. In 16 QC rounds between 2010 and 2017, the four laboratories received 208 bone marrow (BM) samples (126 FL; 82 MCL); 187 were analyzed, according to the EuroMRD Consortium guidelines, by both nested (NEST) polymerase chain reaction (PCR) and real‐time quantitative (RQ) PCR for BCL2/IGH MBR or IGHV rearrangements. Here, we aimed at analyzing the samples that challenged the interlaboratory reproducibility and data interpretation. Overall, 156/187 BM samples (83%) were concordantly classified as NEST+/RQ+ or NEST?/RQ? by all the four laboratories. The remaining 31 samples (17%) resulted alternatively positive and negative in the interlaboratory evaluations, independently of the method and the type of rearrangement, and were defined “borderline” (brd) samples: 12 proved NEST brd/RQ brd, 7 NEST?/RQ brd, 10 NEST brd/RQ positive not quantifiable (PNQ), and 2 NEST brd/RQ?. Results did not change even increasing the number of replicates/sample. In 6/31 brd samples, droplet digital PCR (ddPCR) was tested and showed no interlaboratory discordance. Despite the high interlaboratory reproducibility in the MRD analysis obtained and maintained by the QC round strategy, samples with the lowest MRD levels can still represent a challenge: 17% (31/187) of our samples showed discordant results in interlaboratory assessments, with 6.4% (12/187) remained brd even applying the two methods. Thus, although representing a minority, brd samples are still problematic, especially when a clinically oriented interpretation of MRD results is required. Alternative, novel methods such as ddPCR and next‐generation sequencing have the potential to overcome the current limitations.     

Surgical strategies and minimal residual disease detection.

The ultimate goal in the treatment of cancer patients is the elimination of all tumor cells. A cure by surgery alone is possible only if the tumor is still confined locally. Detection of disseminated tumor cells may help to individually tailor the surgical procedure to each patient: extended or limited resections may be indicated depending on the individual state of tumor cell dissemination. In cases of systemic tumor cell dissemination, surgery alone cannot cure the patient. Thus, by detecting disseminated tumor cells, patients with a higher risk for relapse, who might benefit from multimodal therapeutic regimes, could be defined. A second aspect is the possibility of tumor cell shedding induced by manipulation during surgical procedures, which could be demonstrated for several tumor entities. Intraoperative tumor cell dissemination could be prevented by alternative operative strategies. In addition, perioperative antibody or cytotoxic therapy may prevent tumor cell implantation. Well-designed clinical studies are now of major importance to evaluate the clinical impact of individualized patient management and altered surgical procedures.     

多发性骨髓瘤的微小残留病检测

多发性骨髓瘤（MM）是浆细胞在骨髓中异常增生的一种血液系统恶性肿瘤,好发于老年人,且发病率明显上升。21世纪以来,MM患者的治疗取得了前所未有的进展。有效疗法的增加及更加深入缓解治疗组合的广泛应用,使得MM治疗的目标需要重新评估。微小残留病（MRD）阴性状态的实现变得越来越重要,有些观点甚至支持MRD是治疗的主要终点。     

Clinical significance of minimal residual disease in leukemia

Considerable progress has been made in the treatment of acute and chronic leukemias. Remission rates are generally high and cure rates of up to 80% can be achieved in children with acute lymphoblastic leukemia (ALL). However, in many patients the disease will ultimately recur. In most if not all of these patients, relapse is thought to result from subclinical levels of residual leukemia, termed minimal residual disease (MRD). Therefore, the study of MRD holds a significant potential to understand the biology of relapse and remission and to design new therapies to improve the cure rate of patients. A major goal of these studies is to be able and identify patients at a defined risk of relapse which can lead to risk-adapted therapy approaches. Laboratory assays such as polymerase chain reaction (PCR) and multicolor flow cytometry are sensitive enough to detect one leukemic cell in up to 104-105 normal cells and have become ideal tools to monitor MRD. Especially PCR has been used extensively. Although a wealth of data has been generated, some questions remain as to the impact of monitoring MRD on clinical outcome and are the object of this review.     

Assessing minimal residual disease in chronic lymphocytic leukemia

New treatment approaches have substantially improved response rates in chronic lymphocytic leukemia. Accurate assessment of effective combination chemoimmunotherapy requires more sensitive measures of response, and a variety of techniques to measure minimal residual disease (MRD) have been developed. Because many studies demonstrate that MRD eradication is associated with prolonged treatment-free survival, detection of MRD is becoming a standard component of clinical trials. Quantitative approaches using polymerase chain reaction or multiparameter flow cytometry are preferable because they allow comparison of efficacy between different studies. In most clinical settings, the levels of chronic lymphocytic leukemia always increase from the first detection of MRD; the exception is allogeneic transplantation, in which there may be stable MRD levels or delayed MRD eradication. MRD analysis in the peripheral blood also may be used during therapy to predict eventual response and potentially to guide therapy to achieve the optimal outcome.     

Chimerism and minimal residual disease monitoring after reduced intensity conditioning (RIC) allogeneic transplantation.

Since graft-versus-leukemia (GVL) is the main weapon for disease eradication after reduced intensity conditioning (RIC) allogeneic SCT, the availability of sensitive and specific techniques to monitor changes in tumor load after transplant are especially helpful. These minimal residual disease techniques would allow an early intervention in the event of low tumor burden, for which immunotherapy is highly effective. Some authors have found an association between persistence of MRD, mixed chimerism and risk of relapse. Nevertheless, data from the literature remain contradictory and further correlations should be established, especially in RIC transplants. In this study we have analyzed the impact of MRD and chimerism monitoring on the outcome of 34 patients undergoing RIC allogeneic SCT who were considered poor candidates for conventional transplantation due to advanced age or other concurrent medical conditions. At day +100 25 (75%) patients reached complete remission (CR), there were five (15%) partial responses and three patients progressed. Incidence of grade 2-4 aGVHD and extensive cGVHD were 35% and 58%, respectively. Sixteen percent of patients developing aGVHD relapsed as compared to 47% in those without aGVHD (P = 0.03) and also 10% of patients developing cGVHD relapsed as compared to 50% relapses in those without cGHVD (P = 0.03). Four patients (12%) died due to early (n = 1) and late (n = 3) transplant-related mortality. After a median follow-up of 15 months, 24 out of the 34 patients remain alive. Projected overall survival and disease-free survival at 3 years are 68% and 63%, respectively. Early chimerism analysis showed 67% of patients with complete chimerism (CC) in bone marrow (BM), 86% in peripheral blood (PB), 89% in granulocytes and 68% in T lymphocytes. On day +100, these figures were 68%, 79%, 90% and 73%, respectively, and on day +180 there were 83% patients with CC in BM, 100% in PB, 100% in granulocytes and 100% in T lymphocytes. We observed a trend to a higher incidence of relapse in patients with mixed chimerism (MC) as compared to patients with CC. MRD monitoring by flow cytometry and/or RT-PCR analysis was performed in 23 patients. MRD assessment on days +21 to +56 after transplant allowed identification of patients at risk of relapse. In this sense, seven out of 12 patients (58.3%) who had positive MRD on days +21 to +56 relapsed as compared to none out of 11 patients who had negative MRD (P = 0.002). Of the seven patients with criteria to monitor MRD who relapsed after transplant, all but one remained MRD positive until relapse. By contrast, 10 patients remained MRD negative and all of them are in continuous CR. In nine additional patients, persistence of MRD or mixed chimerism was observed after transplant and withdrawal of cyclosporin with or without DLI was performed. Only two out of these nine patients relapsed. MRD clearance was preceded by CC and GVHD. In conclusion, in our study we found that RIC allogeneic transplantation can be used in patients considered poor candidates for conventional transplantation due to advanced age or other concurrent medical conditions with both low toxicity and low transplant-related mortality. Simultaneous studies of both chimerism and MRD are a useful tool in order to predict risk of relapse in patients undergoing RIC transplants and so can be helpful for individualizing treatment strategies after transplant.     

Eradication of minimal residual disease in chronic lymphocytic leukemia

Although fludarabine-based regimens and monoclonal antibodies are able to induce high overall and complete remission rates in patients with chronic lymphocytic leukemia (CLL), a major impact on overall survival or even potential cure by these treatments is still lacking. Increased sensitivity and specificity of flow cytometry and polymerase chain reaction techniques enable us to detect few CLL cells in peripheral blood and bone morrow. This has brought significant advancement into the evaluation of response quality of CLL treatment. The eradication of minimal residual disease (MRD) below measurable levels seems to be critical to overcome recurring clonal expansion resulting in disease progression or relapse. Several studies suggest that achieving MRD negativity in patients with CLL provokes prolonged response duration and survival. Thus, elimination of MRD should become a surrogate end point of modern clinical trials and a goal in CLL. This review summarizes the current status of and future strategies for MRD eradication in CLL.