Leukemia Residual Disease Research Articles

Evaluation of MicroRNA as Minimal Residual Disease in Leukemia: Diagnostic and Prognostic Approach: A Review.

Various factors are effective in the development of minimal residual disease (MRD), one of which is MicroRNAs (miRNAs). miRNAs and their dysfunction in gene expression have influential role in the pathogenesis of leukemia. Nowadays, treatments that lead to the suppression or replacement of miRNAs have been developed. Focusing on the role of miRNAs in managing the treatment of leukemia, in this review article we have investigated the miRNAs and signaling pathways involved in the process of apoptosis and cell proliferation, as well as miRNAs with oncogenic function in malignant leukemia cells. Among the studied miRNAs, miR-99a, and miR-181a play an essential role in apoptosis, proliferation and oncogenesis via AKT, MAPK, RAS, and mTOR signaling pathways. miR-223 and miR-125a affect apoptosis and oncogenesis via Wnt/B-catenin, PTEN/PI3K, and STAT5/AKT/ERK/Src signaling pathways. miR-100 also affects both apoptosis and oncogenesis; it acts via IGF1 and mTOR signaling pathways.

Close interaction with bone marrow mesenchymal stromal cells induces the development of cancer stem cell-like immunophenotype in B cell precursor acute lymphoblastic leukemia cells.

Minimal residual disease of leukemia may reside in the bone marrow (BM) microenvironment and escape the effects of chemotherapeutic agents. This study investigated interactions between B cell precursor (BCP)-acute lymphoblastic leukemia (ALL) cells and BM mesenchymal stromal cells (BM-MSCs) in vitro. Five BCP-ALL cell lines established from pediatric patients and primary samples from a BCP-ALL patient were examined by flow cytometry and immunocytochemistry for expression of specific cell surface markers and cell adhesion proteins. The cell lines developed chemoresistance to commonly used anti-leukemic agents through adhesion to MSC-TERT cells in long-term culture. The change in chemosensitivity after adhering to BM-MSCs was associated with the expression of CD34, CD133, P-glycoprotein and BCRP/ABCG2, and downregulation of CD38. Similar phenotypic changes were observed in primary samples obtained by marrow aspiration or biopsy from a BCP-ALL patient. BM-MSC-adhering leukemia cells also showed deceleration of cell proliferation and expressed proteins in the Cadherin and Integrin pathways. These results suggest that BCP-ALL cells residing in the BM microenvironment may acquire chemoresistance by altering their phenotype to resemble that of cancer stem cells. Our results indicate that cell adhesion could be potentially targeted to improve the chemosensitivity of residual BCP-ALL cells in the BM microenvironment.

Detection of Fusion Genes to Determine Minimal Residual Disease in Leukemia Using Next-Generation Sequencing.

Measuring minimal residual disease (MRD), the persistence of leukemic cells after treatment, is important for monitoring leukemia recurrence. The current methods for monitoring MRD are flow cytometry, to assess aberrant immune phenotypes, and digital droplet PCR (ddPCR), to target genetic aberrations such as single-nucleotide variants and gene fusions. We present the performance of an RNA-based next-generation sequencing (NGS) method for MRD gene fusion detection compared with ddPCR. This method may have advantages, including the capacity to analyze different genetic aberrations and patients in 1 experiment. In particular, detection at the RNA level may be highly sensitive if the genetic aberration is highly expressed. We designed a probe-based NGS panel targeting the breakpoints of 11 fusion genes previously identified in clinical patients and 2 fusion genes present in cell lines. Blocking probes were added to prevent nonspecific enrichment. Each patient RNA sample was diluted in background RNA, depleted for rRNA and globin mRNA, converted to cDNA, and prepared for sequencing. Unique sequence reads, identified by unique molecular identifiers, were aligned directly to reference transcripts. The same patient and cell-line samples were also analyzed with ddPCR for direct comparison. Our NGS method reached a maximum sensitivity of 1 aberrant cell in 10000 cells and was mostly within a factor of 10 compared with ddPCR. Our detection limit was below the threshold of 1:1000 recommended by European Leukemia Net. Further optimizations are easy to implement and are expected to boost the sensitivity of our method to diagnostically obtained ddPCR thresholds.

Variations in the Genome: The Mutation Detection 2015 Meeting on Detection, Genome Sequencing, and Interpretation.

The content of the 13th Mutation Detection meeting (Leiden, April 2015) is summarized in this report. Topics discussed at the meeting included current challenges of clinical NGS, advances in bioinformatics, data quality control, single cell analysis and RNA sequencing, among others. Social, ethical and regulatory challenges of genomic data handling and data sharing were the focus of an expert panel debate. The 14th International Symposium on Variants in the Genome will take place in Santiago de Compostela, June 5-8, 2017. http://isv.variome.org.

Minimal residual disease of leukemia and the quality of cryopreserved human ovarian tissue in vitro

Auto-transplant of cryopreserved ovarian tissue in leukemia patients carries a risk to reintroduce malignant cells. Maturation of ovarian follicles in vitro is a promising strategy to overcome the leukemic cell contamination. The follicle development and survival in 14 cryopreserved ovarian tissues with leukemia-specific PCR marker was evaluated after 7 or 14 days culture. Minimal residual disease (MRD) quantification was assessed by real-time quantitative PCR in order to identify the MRD positive (n = 6) and negative (n = 8) samples and to monitor levels of MRD before and after culture. The morphology of ovarian follicles were studied by light microscopy. After culture, no statistical significant differences were detected in follicle densities between MRD positive- and negative samples. Ovarian MRD either decreased below undetectable or fluctuated near the baseline level after 7 and 14 days in culture. This study provides quantitative in vitro evidence that leukemia contamination does not affect the follicle survival in cryopreserved ovarian tissue.

Bendamustine for non-Hodgkin lymphoma

Interview by Louise Rishton, Commissioning EditorProfessor John Gribben is Chair of the International Workshop on non-Hodgkin Lymphoma and the Gordon Hamilton Fairley Chair of Medical Oncology at St. Bartholomew’s Hospital, Bart’s Cancer Institute, London, UK, a Cancer Research UK Centre of Excellence. His doctoral studies were performed at University College London, UK as the recipient of a Wellcome Trust Fellowship Award and he continued post-doctoral training with Professor Lee Nadler at the Dana-Farber Cancer Institute (Harvard Medical School, MA, USA). In 1992, Gribben was appointed to the Faculty at Harvard Medical School, where he remained as Associate Professor of Medicine and an Attending Physician at the Dana-Farber Cancer Institute and Brigham and Women’s Hospital (MA, USA), until returning to London in 2005. Gribben is a founding member of the CLL Research Consortium, Associate Editor of Blood and was elected a Fellow of the Academy of Medical Science. His primary research interests include the immunotherapy of cancer (including stem cell transplantation), the identification of B-cell-tumor antigens and the detection and treatment of minimal residual disease in leukemia and lymphoma.

Expression of WT1 gene in multiple myeloma patients at diagnosis: is WT1 gene expression a useful marker in multiple myeloma?

Monitoring patients with multiple myeloma during and after treatment for the presence of residual myeloma cells (minimal residual disease – MRD) has been shown to give a major insight into the effectiveness of treatment. It has been reported that Wilms' tumor gene (WT1) expression levels measured by real-time quantitative polymerase chain reaction was useful as an indicator of minimal residual disease in leukemia and myelodysplastic syndrome. The aim of this study was to measure levels of WT1 expression, in order to find a possible association between the expression of this gene and multiple myeloma at diagnosis. If an association was found, the WT1 gene could be evaluated as an MRD marker by comparison with other prognostic factors. We investigated peripheral blood WT1 expression level measured by real-time light cycler quantitative polymerase chain reaction in 50 newly diagnosed multiple myeloma patients. The normal WT1 gene copy number was found to be <23/μl cDNA and all patients with myeloma were found to have normal WT1-mRNA levels. On this basis WT1 expression analyses is unlikely to be a useful genetic marker for routine clinical use in multiple myeloma patients at diagnosis.

High WT1 gene expression before haematopoietic stem cell transplant in children with acute myeloid leukaemia predicts poor event‐free survival

WT1 gene expression has been proposed as a useful marker of minimal residual disease in leukaemia. Its utility in paediatric haematopoietic stem cell transplantation (HSCT) has not been studied. We studied the prognostic value of WT1 expression in peripheral blood prior to HSCT in 36 children with acute myeloid leukaemia (AML). Samples were obtained 2 weeks pre-transplant to determine the level of WT1 expression. WT1 expression was normalized using K562 cells as a control and a relative value of 0.5 was chosen as the cut-off point between high and low WT1 expression. The median level of pre-transplant WT1 expression in the 36 patients was 0.09 (range 0.0001-11.0), with 11 patients having WT1 >or= 0.5 and 25, WT1 < 0.5. After HSCT, 76% of patients with high pre-transplant WT1 expression relapsed, in contrast to 0% of the patients with low WT1 expression. Those with high WT1 expression had significantly lower 5-year event-free survival (EFS) (18%, 95% CI 0-40%) as compared to those with low WT1 expression (68%, 95% CI 50-86%, P = 0.007). Multivariate analysis showed that pre-transplant WT1 level is the only significant prognostic factor for the difference in EFS. Our finding suggests that elevated WT1 gene expression before HSCT in paediatric AML predicts relapse and poor long-term EFS. A larger prospective study is warranted to compare the value of high WT1 expression and other markers of minimal residue disease in predicting clinical outcomes after HSCT.

Application of PCR derived technology for diagnosis of hematological disease

Polymerase chain reaction (PCR) technique for the diagnosis of hematological disease brought about revolutionary progress. It has become indispensable and basic technology for genotyping of acute and chronic leukemia and diagnosis of myeloproliferative disorders, thalassemia, hemophilia and other genetic diseases. Based on the PCR technology, multiple nested-PCR, real-time fluorescence quantitative PCR (Q-PCR), denaturing high pressure liquid chromatography (DHPLC), capillary electrophoresis, the molecular hybridization and directly sequencing technique have been developed. The multip]e nested-PCR can be used to examine a variety of leukemia genes and confirm the type of leukemia. The application of Q-PCR has effectively solved the problem of pollution of the PCR and it can be used to monitor minimal residual disease of leukemia. Capillary electrophoresis is more suitable for gene fragment length change. DHPLC can be applied for high-throughput screening of gene mutation. Sequencing is a reliable method and it has been used for routine diagnosis. Key words: Hematologic diseases; Polymerase chain reaction

Detection of minimal residual disease in leukemia using flow cytometry

Detection of minimal residual disease in leukemia using flow cytometry

No Significant Expression of Wt1 Gene in Multiple Myeloma Patients at Diagnosis: Is Wt1 Gene Expression Useful Marker for Minimal Residual Disease in Multiple Myeloma?.

Monitoring of multiple myeloma patients during and after treatment for the presence of remaining myeloma cells (minimal residual disease, MRD) has been shown to give major insight into the effectiveness of treatment. However, so far applicabile genetic markers amenable to sensitive detection by PCR for MRD in myeloma patients. Previouse studies showed that Wilms tumor gene (WT1) expression level, measured by quantitative reverse transcriptase polymerase chain reaction (RT-PCR), was useful as an indicator of minimal residual disease in leukemia and myelodysplastic syndrome. In this study was to measure, by real-time polymerase chain reaction, levels of WT1 expression, in order to find a possible association between this gene and the minimall residual disease. We investigated peripheral blood WT1 expression level in 41 newly diagnosed multiple myeloma cases using quantitative reverse transcriptase polymerase chain reaction. All patients carried normal WT1-mRNA levels. WT1 expression analyse may be not useful genetic marker for monitoring MRD in routine clinical use of multiple myeloma patients.

Real-time PCR array for simultaneous detection of 37 kinds of fusion genes in leukemia

To develop a real-time PCR array for simultaneous quantitative detection of translocations/chromosomal aberrations in patients with leukemia, and to investigate the feasibility and utility thereof. By construction and optimization a set of specific primes (totally 82 primers), an array containing 66 parallel PCR reactions was developed. That array was used on the specimens of bone marrow or peripheral blood from 31 patients with leukemia to detect simultaneously 37 fusion genes and 4 proto-oncogene activations often occurring in patients with leukemia. Eva Green fluorescent dye method was chosen in the protocol. Relative quantification was performed by Ct analysis and the result was expressed as the ratio of the target gene versus the internal control gene (ABL). Six patients with chronic myelocytic leukemia (CML) among the 31 cases underwent prior to and after treatment so as to study the expression changes of fusion genes and/or proto-oncogene. The established PCR array showed high efficiency of amplification and good sensibility (232 copies/microl) in the fusion gene detected. The standard curve had a satisfying linear range (10(2) approximately 10(8) copies/microl), showing a good reproducibility. Fourteen fusion genes, including PML/RARalpha, PLZF/RARalpha, BCR/ABL, MLL/AF1, MLL/AF6, MLL/AF10, AML/Eto, CBFbeta/MYH11, TLS/ERG, TEL/AML1, MOZ/CBP, MLL/hCDCrel, LAF4/MLLT2, and FIP1L1/PDGFRalpha, and activation of all 4 proto-oncogenes were found in the 31 samples. In one patient, 5 fusion genes and activation of 2 proto-oncogenes were observed. Such results were compared with those of RT-nested PCR in 28 samples. The comparison showed that this array was a bit less sensitive than RT-nested PCR, however, without significant difference between them (P = 0.009). The expression of BCR/ABL fusion gene, WT1 gene, and EVI1 gene decreased after treatment in the 6 CML patients, which was in accordance with the clinical features. The PCR array newly-established successfully detects various leukemia related fusion genes and proto-oncogene activation. It is useful in molecule diagnosis and monitoring minimal residual disease in leukemia, and therapeutic effect monitoring.

Examination of Stability of Bone Marrow Blood RNA in the PAXgene Tube

The PAXgene RNA blood collection tube is used for RNA of peripheral blood (PB) and the stability of PB RNA in this tube has already been reported. However, the stability of bone marrow blood (BM) RNA in the PAXgene tube is unknown. Thus, we examined the stability of BM RNA in the PAXgene tubes. BM from leukemia patients was collected into PAXgene and EDTA tubes and stored at 4 degrees C for 5 days. RNA isolated from both tubes was analyzed by a quantitative reverse-transcription polymerase chain reaction (RT-PCR) analysis. Porphobilinogen deaminase (PBGD) mRNA of BM (as high-expression mRNA) and Wilms tumor suppressor (WT1) mRNA of BM (as low-expression mRNA) and very low copies of major BCR-ABL mRNA (as minimal residual disease of leukemia) of leukemia of BM were quantified by a LightCycler system. RNA yield from the PAXgene tubes and the intensity of 28S rRNA bands on RNA electrophoresis showed a degradation trend. However, the intensity of 18S rRNA bands from the PAXgene tubes remained. The expression of PBGD and WT1 of BM in the PAXgene tubes did not decrease for 5 days. The very low copies of major BCR-ABL mRNA in the PAXgene tubes were detectable on day 5 but those in the EDTA tubes were not detectable. Therefore, the PAXgene tube can be used for BM samples in a quantitative RT-PCR of the fusion gene transcripts of leukemia.

EQUAL-quant: An International External Quality Assessment Scheme for Real-Time PCR

Quantitative gene expression analysis by real-time PCR is important in several diagnostic areas, such as the detection of minimum residual disease in leukemia and the prognostic assessment of cancer patients. To address quality assurance in this technically challenging area, the European Union (EU) has funded the EQUAL project to develop methodologic external quality assessment (EQA) relevant to diagnostic and research laboratories among the EU member states. We report here the results of the EQUAL-quant program, which assesses standards in the use of TaqMan probes, one of the most widely used assays in the implementation of real-time PCR. The EQUAL-quant reagent set was developed to assess the technical execution of a standard TaqMan assay, including RNA extraction, reverse transcription, and real-time PCR quantification of target DNA copy number. The multidisciplinary EQA scheme included 137 participating laboratories from 29 countries. We demonstrated significant differences in performance among laboratories, with 20% of laboratories reporting at least one result lacking in precision and/or accuracy according to the statistical procedures described. No differences in performance were observed for the >10 different testing platforms used by the study participants. This EQA scheme demonstrated both the requirement and demand for external assessment of technical standards in real-time PCR. The reagent design and the statistical tools developed within this project will provide a benchmark for defining acceptable working standards in this emerging technology.

Wilms' tumour gene 1 (WT1) in human neoplasia

The transcription factor Wilms' tumour gene 1 (WT1) is important as a prognostic marker as well as in the detection and monitoring of minimal residual disease in leukaemia and myelodysplastic syndromes. Evidence has accumulated over the past decade to show that WT1 is a key molecule for tumour proliferation in a large number of human neoplasms most prominent in acute leukaemias, making it a suitable target for therapeutic strategies. Based on animal results, showing safety and efficacy of immunization with WT1 peptides and protein, early clinical trials in leukaemia have recently been initiated. The First International Conference on WT1 in Human Neoplasia was held in Berlin, March 11--12, 2004. This report reviews the current knowledge on the role of WT1 in tumour promotion and as a diagnostic and therapeutic target, and summarizes the data presented and discussed in this meeting.

Mitochondrial DNA (mtDNA) Sequence Heterogeneity among and within Single Human CD34 Cells, T Cells, B Cells and Granulocytes.

Abnormalities of mitochondrial DNA (mtDNA) are responsible for a variety of inherited syndromes and have been broadly implicated in aging, cancer, and autoimmunity diseases. Mutations in mtDNA have been reported in myelodysplasia and leukemia, although their pathogenic mechanism remains uncertain. We have described age-dependent accumulation of mtDNA mutations, leading to a high degree of mtDNA sequence heterogeneity among normal marrow and blood CD34 cells as well as in granulocytes (Shin M et al, Blood 101:3118[ 2003], 103:553 [2004], 103:4466 [2004]). In order to examine mtDNA heterogeneity in detail, we developed a method for analysis of the mtDNA control region from single cells that were sorted by flow cytometry. Highly purified populations of CD34 cells, T cells, B cells, and granulocytes were obtained from five healthy adult donors. The sequence of the individual cells' mtDNA was compared to the aggregate mtDNA for the respective cell type and differences were expressed as a measure of mtDNA heterogeneity among cells. Overall, heterogeneity was high: for circulating CD34 cells, 38±3.4%; for T cells, 37±14%; B cells, 36±10.8%; and for granulocytes, 48±7.2% (the value for granulocytes statistically differed from CD34 cells; p = 0.03). Most intercellular heterogeneity was due to polyC tract length variability; however, mtDNA base substitution mutations were also prevalent: 15±5.5% in CD34 cells; 15±9.0% in T cells, 15±6.7% in B cells; and 33±2.4% for granulocytes (granulocytes were significantly higher than other cells; p < 0.01). The higher rate of base substitution in granulocytes may reflect their greater exposure to reactive oxygen species. Surprisingly, for both polyC tract length differences and point mutations, the specific mtDNA abnormalities and the proportion of circulating cells characterized by these changes were similar among different cell lineages and relatively constant over time (~2 years) in the same donors. One inference from these results is that mtDNA heterogeneity during development is fixed in the primitive lymphohematopoietic stem cell compartment. In contrast to normal adults, circulating CD34 cells from patients obtained even years after successful allogeneic stem cell transplantation showed a remarkable level of mtDNA homogeneity, similar to the uniformity we have previously observed in cord blood CD34 cells and consistent with limited numbers of stem cells active in these individuals. Leukemic blast cells (from patients with AML-M2, AML evolving from CMML, and T-PLL) also showed a high degree of homogeneity. We propose that mtDNA sequence of single cells may be utilized as a natural genetic marker of hematopoietic progenitors and stem cells; to detect minimal residual disease in leukemia; and as a measure of the accumulation of mutagenic events in mammalian cells in vivo and in vitro.

Quantitative analysis of PRAME for detection of minimal residual disease in leukemia.

AbstractPreferentially expressed antigen of melanoma (PRAME) was first isolated using cDNA expression cloning techniques as a gene encoding a human melanoma antigen recognized by melanoma reactive cytotoxic T-cells (CTL) (1). This gene codes for a 509-amino-acid protein whose function has not yet been identified. PRAME is expressed in various types of cancer, including melanoma (97%), sarcoma (80%), small-cell lung cancer (70%), renal cell carcinoma (40%), and head and neck cancer (29%) (1,2). PRAME is also found in limited normal tissues, including endometrium and adrenal glands, and found highly expressed in testis.KeywordsAcute Lymphocytic LeukemiaChronic Myelogenous LeukemiaMinimal Residual DiseaseAcute Myelogenous LeukemiaPolymerase Chain Reaction CycleThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Monitoring minimal residual disease in leukemia using real-time quantitative polymerase chain reaction for Wilms tumor gene (WT1).

We previously showed that Wilms tumor gene (WT1) expression level, measured by quantitative reverse transcriptase polymerase chain reaction (RT-PCR), was useful as an indicator of minimal residual disease (MRD) in leukemia and myelodysplastic syndrome. However, in conventional quantitative RT-PCR (CQ-PCR), RT-PCR must be performed for various numbers of cycles depending on WT1 expression level. In the present study, we developed a new real-time quantitative RT-PCR (RQ-PCR) method for quantitating WT1 transcripts. Results of intraassay and interassay variability tests demonstrated that the real-time WT1 assay had high reproducibility. WT1 expression levels measured by the RQ- and the CQ-PCR methods were strongly correlated (r = 0.998). Furthermore, a strong correlation was observed among WT1 transcript values normalized with 3 different control genes (beta-actin, ABL, and glyceraldehyde-3-phosphate dehydrogenase) and between relative WT1 transcript values with WT1 expression in K562 cells as the reference and absolute WT1 transcript copy numbers per microgram RNA. When WT1 expression and minor bcr-abl expression were concurrently monitored in 2 patients with bcr-abl-positive acute lymphoblastic leukemia, both MRDs changed mostly in parallel, indicating the reliability and validity of our RQ-PCR method. In conclusion, this RQ-PCR method is convenient and reliable for monitoring MRD and enables routine clinical use of a WT1 assay.

A Novel Method to Compensate for Different Amplification Efficiencies between Patient DNA Samples in Quantitative Real-Time PCR

Quantification of residual disease by real-time polymerase chain reaction (PCR) will become a pivotal tool in the development of patient-directed therapy. In recent years, various protocols to quantify minimal residual disease in leukemia or lymphoma patients have been developed. These assays assume that PCR efficiencies are equal for all samples. Determining t(14;18) and albumin reaction efficiencies for sixteen follicular lymphoma patient samples revealed higher efficiencies for blood samples than for lymph node samples in general. However, within one sample both reactions had equivalent efficiencies. Differences in amplification efficiencies between patient samples (low efficiencies) and the calibrator in quantitative analyses result in the underestimation of residual disease in patient samples whereby the weakest positive patient samples are at highest error. Based on these findings for patient samples, the efficiency compensation control was developed. This control includes two reference reactions in a multiplex setting, specific for the beta-actin and albumin housekeeping genes that are present in a constant ratio within DNA templates. The difference in threshold cycle values for both reference reactions, ie, the Ct(2) value, is dependent on the amplification efficiency, and is used to compensate for efficiency differences between patient samples and the calibrator. The beta-actin reference reaction is also used to normalize for DNA input. Furthermore, the efficiency compensation control facilitates identification of patient samples that are so contaminated with PCR inhibitory compounds that different amplification reactions are affected to a different extent. Accurate quantitation of residual disease in these samples is therefore impossible with the current quantitative real-time PCR protocols. Identification and exclusion of these inadequate samples will be of utmost importance in quantitative retrospective studies, but even more so, in future molecular diagnostic analyses.

Quantitative monitoring of the PRAME gene for the detection of minimal residual disease in leukaemia.

PRAME (Preferentially expressed antigen of melanoma) has been previously identified as a melanoma antigen recognized by cytotoxic T cells (CTLs) and found to be expressed in a variety of cancer cells including leukaemic cells. We have screened 98 Japanese patients with leukaemia and lymphoma for expression of the PRAME gene using semiquantitative reverse transcription polymerase chain reaction (RT-PCR). Forty-one patients (42%) showed high levels of PRAME expression. Eight of these patients were then monitored using real-time PCR for a period of 10-37 months. Significant reductions in the PRAME expression were observed in all patients after chemotherapy. An increased expression was detected in the two patients who relapsed, one of which was before cytological diagnosis. These changes were correlated with those of other known genetic markers, such as the bcr-abl gene. Therefore, quantitative monitoring of the PRAME gene using real-time PCR method may be useful for detecting minimal residual disease and to predict subsequent relapse, especially in patients without known genetic markers. In addition, a PRAME-positive leukaemia cell line and fresh leukaemic cells were found to be susceptible to lysis by PRAME-specific CTLs established from a patient with melanoma, suggesting that the PRAME peptide can also be a target leukaemia antigen for T cells.