Clinical relevance of minimal residual disease monitoring in non-Hodgkin's lymphomas: a critical reappraisal of molecular strategies.

Although current treatments can induce clinical complete remissions in the vast majority of patients with indolent lymphoma, most of them actually relapse, because of the persistence of residual tumor cells which are undetectable using conventional diagnostic procedures. Polymerase chain reaction (PCR)-based methods are increasingly used for minimal residual disease detection (MRD), and provide useful prognostic information. In this review, current approaches for MRD detection in indolent lymphomas are summarized. In addition, the prognostic aspects of molecular monitoring after transplantation procedures are discussed. The experience accumulated over the past decade shows that PCR analysis has a prognostic impact in several therapeutic programs including conventional and high-dose regimens. Major advantages coming from the introduction of molecular monitoring in clinical programs have been: (1) a rapid evaluation of the anti-tumor activity of innovative treatments; and (2) an early identification of patients with a high-risk of disease recurrence.     

Circulating tumor DNA-based molecular residual disease detection for treatment monitoring in advanced melanoma patients

Background

Immune checkpoint inhibitors (ICIs) have substantially improved overall survival in patients with advanced melanoma; however, the lack of biomarkers to monitor treatment response and relapse remains an important clinical challenge. Thus, a reliable biomarker is needed that can risk-stratify patients for disease recurrence and predict response to treatment.

Methods

A retrospective analysis using a personalized, tumor-informed circulating tumor DNA (ctDNA) assay on prospectively collected plasma samples (n = 555) from 69 patients with advanced melanoma was performed. Patients were divided into three cohorts: cohort A (N = 30), stage III patients receiving adjuvant ICI/observation; cohort B (N = 29), unresectable stage III/IV patients receiving ICI therapy; and cohort C (N = 10), stage III/IV patients on surveillance after planned completion of ICI therapy for metastatic disease.

Results

In cohort A, compared to molecular residual disease (MRD)-negative patients, MRD-positivity was associated with significantly shorter distant metastasis-free survival (DMFS; hazard ratio [HR], 10.77; p = .01). Increasing ctDNA levels from the post-surgical or pre-treatment time point to after 6 weeks of ICI were predictive of shorter DMFS in cohort A (HR, 34.54; p < .0001) and shorter progression-free survival (PFS) in cohort B (HR, 22; p = .006). In cohort C, all ctDNA-negative patients remained progression-free for a median follow-up of 14.67 months, whereas ctDNA-positive patients experienced disease progression.

Conclusion

Personalized and tumor-informed longitudinal ctDNA monitoring is a valuable prognostic and predictive tool that may be used throughout the clinical course of patients with advanced melanoma.     

Aberrant stabilization of c-Myc protein in some lymphoblastic leukemias.

Overexpression of the c-Myc oncoprotein is observed in a large number of hematopoietic malignancies, and transgenic animal models have revealed a potent role for c-Myc in the generation of leukemias and lymphomas. However, the reason for high c-Myc protein levels in most cases is unknown. We examined whether aberrant protein stabilization could be a mechanism of c-Myc overexpression in leukemia cell lines and in primary bone marrow samples from pediatric acute lymphoblastic leukemia (ALL) patients. We found that c-Myc protein half-life was prolonged in the majority of leukemia cell lines and bone marrow samples tested. There were no mutations in the c-myc gene in any of the leukemia cell lines that could account for increased c-Myc stability. However, abnormal phosphorylation at two conserved sites, Threonine 58 and Serine 62, was observed in leukemia cell lines with stabilized c-Myc. Moreover, stabilized c-Myc from the ALL cell lines showed decreased affinity for glycogen synthase kinase3beta, the kinase that phosphorylates c-Myc at Threonine 58 and facilitates its degradation. These findings reveal that deregulation of the c-Myc degradation pathway controlled by Serine 62 and Threonine 58 phosphorylation is a novel mechanism for increased expression of a potent oncoprotein known to be involved in hematopoietic malignancies.     

Molecular monitoring of minimal residual disease in acute leukemia

Treatment of patients with acute leukemia is based on antineoplastic drug therapy (mainly chemotherapy) and/or immunotherapy, such as allogeneic stem cell transplantation, both associated with the risk of severe toxicity, including treatment-related mortality. Therefore, the extent of therapy should ideally be adapted to the patient’s individual relapse risk. The latter can be estimated taking into account leukemia subtype as well as conventional and molecular cytogenetics, as determined at the time of diagnosis. Furthermore, particularly in acute lymphoblastic leukemia (ALL), early and subsequent assessment of treatment response is routinely incorporated into the global risk stratification. Multiparameter flow cytometry and molecular methods allow for the detection of minimal residual disease that remains obscure to conventional cytology. While molecular monitoring of the treatment efficacy has entered clinical routine in chronic myelogenous leukemia, acute promyelocytic leukemia and ALL, this concept is still evolving in acute myeloid leukemia. This short review is aimed to give an overview of current methods as well as established and candidate indications of molecular disease monitoring in patients with acute leukemia.     

Clonality profile in relapsed precursor-B-ALL children by GeneScan and sequencing analyses. Consequences on minimal residual disease monitoring.

Detection of minimal residual disease (MRD), using immunoglobulin (Ig) and T-cell receptor (TCR) gene rearrangements as clone-specific targets, represents the most recent development in diagnosis and treatment of acute lymphoblastic leukaemia (ALL). Nevertheless, risk of false-negative results, due to secondary or ongoing rearrangements of Ig/TCR genes during the disease course, might hamper MRD detection. Therefore, to gain extensive information on clonal stability, we performed PCR-GeneScan analysis of Ig/TCR gene rearrangements at diagnosis and subsequent relapse in bone marrow samples from 53 childhood precursor-B-ALL patients. In addition, sequencing analysis of junctional regions at diagnosis and relapse provided a detailed insight in the stability and changes of Ig/TCR gene rearrangements during the disease course. At least one stable clonal Ig/TCR target was found in 94% of patients. In three patients complete differences in Ig/TCR rearrangements between diagnosis and relapse were observed, suggesting relapse with a new clone. At relapse, 71% of diagnostic clonal PCR targets was conserved. Since the comparison of Ig/TCR gene rearrangements at diagnosis and relapse in our precursor-B-ALL patients did not show significant difference in the stability of different clonal PCR targets (IGH, 70%; IGK, 71%; TCRD, 67%; TCRG, 75%), we conclude that there is no 'preferential' clone-specific target for MRD monitoring.     

International standardized approach for flow cytometric residual disease monitoring in chronic lymphocytic leukaemia.

The eradication of minimal residual disease (MRD) in chronic lymphocytic leukaemia (CLL) predicts for improved outcome. However, the wide variety of MRD techniques makes it difficult to interpret and compare different clinical trials. Our aim was to develop a standardized flow cytometric CLL-MRD assay and compare it to real-time quantitative allele-specific oligonucleotide (RQ-ASO) Immunoglobulin heavy chain gene (IgH) polymerase chain reaction (PCR). Analysis of 728 paired blood and marrow samples demonstrated high concordance (87%) for patients off-therapy. Blood analysis was equally or more sensitive than marrow in 92% of samples but marrow analysis was necessary to detect MRD within 3 months of alemtuzumab therapy. Assessment of 50 CLL-specific antibody combinations identified three (CD5/CD19 with CD20/CD38, CD81/CD22 and CD79b/CD43) with low inter-laboratory variation and false-detection rates. Experienced operators demonstrated an accuracy of 95.7% (specificity 98.8%, sensitivity 91.1%) in 141 samples with 0.01-0.1% CLL. There was close correlation and 95% concordance with RQ-ASO IgH-PCR for detection of CLL above 0.01%. The proposed flow cytometry approach is applicable to all sample types and therapeutic regimes, and sufficiently rapid and sensitive to guide therapy to an MRD-negativity in real time. These techniques may be used as a tool for assessing response and comparing the efficacy of different therapeutic approaches.     

Targeting oncogenic fusion genes in leukemias and lymphomas by RNA interference

Leukemias and lymphomas are often characterized by non-random chromosomal translocations that, at the molecular level, induce the activation of specific oncogenes or create novel chimeric genes. They have frequently been regarded as optimal targets for gene-silencing approaches because of the large body of evidence that these single abnormalities directly initiate and maintain the malignant process. Herein, we discuss RNA interference (RNAi)-based approaches for targeting the fusion sites of chromosomal translocations as a future treatment option in leukemias and lymphomas.     

Quantitative monitoring of cyclin D1 expression: a molecular marker for minimal residual disease monitoring and a predictor of the disease outcome in patients with mantle cell lymphoma

In mantle cell lymphoma (MCL), minimal residual disease (MRD) is an indicator of the disease outcome. Quantitative methods used so far do not provide a suitable molecular marker in 30-70% patients with MCL (depending on the technique used). We tested cyclin D1 as a marker for quantitative MRD monitoring. The real-time PCR of cyclin D1 mRNA was performed in 144 bone marrow (BM) specimens including 95 BMs from MCL patients, 39 BMs from patients with other B-cell non-Hodgkin's lymphomas and 10 BMs from healthy volunteer donors. In 73 BMs obtained from 20 MCL patients we examined the cyclin D1 level during the treatment and follow-up period. We detected a cyclin D1 overexpression exclusively in BMs infiltrated with MCL, including minimal residual infiltration. Dynamics of cyclin D1 correlated with the patient's clinical status in 69/73 BMs. Individual monitoring of patients during the disease course showed cyclin D1 quantitative changes accompanying either the disease relapse or a successful treatment response or the disease-free survival (remission) and it showed a predictive significance. Cyclin D1 detection is a promising approach for the quantitative MRD monitoring in MCL patients, and the individual monitoring of the cyclin D1 dynamics represents a suitable indicator of the disease course.     

Extensive junctional diversity of gamma delta T-cell receptors expressed by T-cell acute lymphoblastic leukemias: implications for the detection of minimal residual disease.

Rearrangements, junctional regions and expression of T-cell receptor (TcR)-gamma and TcR-delta genes were analyzed in thirteen TcR-gamma delta + T-cell acute lymphoblastic leukemias (T-ALL). All TcR-gamma genes were rearranged except for one deleted allele and one allele in germline configuration. The expressed TcR-gamma protein chains showed a preference for V gamma l (11/13), J gamma 2.3 (7/13) and C gamma 2 (8/13). Furthermore, the TcR-gamma combinatorial repertoire appears to be even more limited by the fact that particular V gamma genes are preferentially used in TcR-gamma 1 or TcR-gamma 2 derived receptors, i.e. in disulfide-linked or non-disulfide-linked TcR-gamma delta receptors. The combinatorial repertoire of the expressed TcR-delta genes was homogeneous, as all thirteen T-ALL expressed V delta 1-D delta-J delta 1-C delta protein chains. In contrast to the limited combinatorial repertoire of the TcR-gamma and TcR-delta genes, the junctional diversity of both TcR genes was extensive due to insertion of N-region, P-region, and D delta gene nucleotides in addition to deletion of nucleotides by trimming. Using polymerase chain reaction (PCR)-mediated amplification and subsequent direct sequencing, we determined the junctional region sequences of all TcR-gamma and TcR-delta rearrangements. Sequence analysis showed that in the TcR-gamma junctional regions insertion varied from 0 to 25 nucleotides (average 8.0) and deletion from 1 to 27 nucleotides (average 8.7). In TcR-delta junctional regions, nucleotide insertion varied from 5 to 47 nucleotides (average 25.5) and deletion from 0 to 29 nucleotides (average 6.2) per junctional region. In general, the N-region nucleotides were the most prominent element in the junctional regions, i.e. 97% of the TcR-gamma and 55% of the TcR-delta junctional regions. D delta genes contributed significantly (40%) to the TcR-delta junctional diversity, whereas P-regions were hardly found in both TcR genes. Altogether, the junctional regions of TcR-delta genes were far more diverse than the junctional regions of TcR-gamma genes. With respect to the new methods of detecting minimal residual disease (MRD) in lymphoid malignancies utilizing PCR-mediated amplification of the junctional regions of TcR genes, our data indicate that this MRD-PCR analysis will generally be more sensitive if TcR-delta instead of TcR-gamma junctional-region-specific probes are used.     

Acute promyelocytic leukemia: a model for the role of molecular diagnosis and residual disease monitoring in directing treatment approach in acute myeloid leukemia.

Acute promyelocytic leukemia (APL) is characterized by a number of features that underpin the need for rapid and accurate diagnosis and demand a highly specific treatment approach. These include the potentially devastating coagulopathy, sensitivity to anthracycline-based chemotherapy regimens, as well as unique responses to all-trans retinoic acid and arsenic trioxide that have revolutionized therapy over the last decade. The chromosomal translocation t(15;17) which generates the PML-RARalpha fusion gene has long been considered the diagnostic hallmark of APL; however, this abnormality is not detected in approximately 10% cases with successful karyotype analysis. In the majority of these cases, the PML-RARalpha fusion gene is still formed, resulting from insertion events or more complex rearrangements. These cases share the beneficial response to retinoids and favorable prognosis of those with documented t(15;17), underscoring the clinical relevance of molecular analyses in diagnostic refinement. In other cases of t(15;17) negative APL, various chromosomal rearrangements involving 17q21 have been documented leading to fusion of RARalpha to alternative partners, namely PLZF, NPM, NuMA and STAT5b. The nature of the fusion partner has a significant bearing upon disease characteristics, including sensitivity to retinoids and arsenic trioxide. APL has provided an exciting treatment model for other forms of AML whereby therapeutic approach is directed towards cytogenetically and molecularly defined subgroups and further modified according to response as determined by minimal residual disease (MRD) monitoring. Recent studies suggest that rigorous MRD monitoring, coupled with pre-emptive therapy at the point of molecular relapse improves survival in the relatively small subgroup of PML-RARalpha positive patients with 'poor risk' disease. Advent of 'real-time' quantitative RT-PCR technology seems set to yield further improvements in the predictive value of MRD assessment, achieve more rapid sample throughput and facilitate inter- and intra-laboratory standardization, thereby enabling more reliable comparison of data between international trial groups.     

实时荧光定量聚合酶链反应动态监测慢性粒细胞白血病治疗后微小残留病的研究

 【摘要】 目的 建立慢性粒细胞白血病（CML）实时荧光定量聚合酶链反应（RQ-PCR）检测方法,并研究应用此方法检测CML治疗后微小残留病（MRD）对评价MRD的意义。方法 应用RQ-PCR对80例CML患者初诊时和（或）治疗后的骨髓标本进行分析,检测bcr-abl融合基因的转录水平,回顾性分析临床资料,根据治疗方案不同分为异基因造血干细胞移植组（34例）、甲磺酸伊马替尼组（33例）、羟基脲组（13例）为,监测CML患者治疗前后bcr-abl融合基因变化。结果　RQ-PCR检测初诊CML患者bcr-abl阳性平均值为6847.67拷贝／万个细胞,加速期检测平均值为306 176.08拷贝／万个细胞;异基因造血干细胞移植组移植后1个月检测平均值为944.33拷贝／万个细胞,移植后6个月复测平均值2.37拷贝／万个细胞,移植后12个月复测平均值0.29拷贝／万个细胞,移植后24个月检测不到bcr-abl融合基因;甲磺酸伊马替尼组服用3个月后检测平均值3720.23拷贝／万个细胞,服用12个月＜0.10拷贝／万个细胞;羟基脲组用药0个月平均值7290.11拷贝／万个细胞,服用9个月后检测平均值3143.24拷贝／万个细胞。基因造血干细胞移植组、甲磺酸伊马替尼组相同时间bcr-abl水平差异无统计学意义（t＝1.74,P＝0.17）,羟基脲组与前两组间比较差异有统计学意义（t＝3.74.P＝0.01;t＝2.97,P＝0.02）。疾病进展时可检测到bcr-abl转录水平的上升;加速期患者的转录本水平明显高于慢性期患者。结论 RQ-PCR作为CML治疗后MRD检测的方法,可以评价治疗效果,预测疾病复发,早期给予干预治疗,有重要临床应用价值。