Measuring response to therapy in AML: Difference from normal flow cytometry vs RQ-PCR.

Multiple technologies have been used to monitor response to therapy in acute myeloid leukemia (AML) to improve detection of leukemia over the standard of practice, morphologic counting of blasts. The two techniques most frequently used in a routine clinical setting, flow cytometry and RQ-PCR, differ in their targets, sensitivity, and ability to detect residual disease. Both flow cytometry and RQ-PCR detect the expression of abnormal gene products, at the protein level or RNA level, respectively. Flow cytometry can be applied to a broad range of AML cases while RQ-PCR is limited to specific genetic abnormalities identified in subsets of AML. This article compares the results when both techniques were used in a reference laboratory to monitor AML over the course of treatment, comparing quantitative and qualitative results.

Committed change of real-time quantitative PCR to droplet digital PCR for monitoring BCR::ABL1 transcripts in tyrosine kinase inhibitor treated CML.

OBJECTIVES: We performed a feasibility study of an FDA-approved commercial ddPCR assay to measure BCR::ABL1 in CML patients treated using TKI therapy. METHODS: Assay performance of standard RQ-PCR and commercially available FDA-approved ddPCR were compared to measure BCR::ABL1 p210 transcripts in RNA samples from 100 CML patients who received TKI therapy. RESULTS: %BCR::ABL1/ABL1IS levels obtained from both methods were not statistically significant difference after normalization with batch-specific conversion factor (p = 0.0651). The correlation and agreement of %BCR::ABL1/ABL1IS between the two assays were high. Molecular response stratification data showed 56% concordance between RQ-PCR and ddPCR, and 37% higher residual disease detection using ddPCR. Furthermore, 21.21% (7/33) of RQ-PCR undetectable samples were detected by ddPCR, representing high sensitivity to quantify the low abundance of BCR::ABL1 transcripts. CONCLUSION: ddPCR was proven to be a highly sensitive method with the potential to overcome some limitations of traditional RQ-PCR, and has the potential of being a valuable tool for monitoring BCR::ABL1 transcripts in CML during TKI therapy. (163 words).

Identification of rare atypical BCR-ABL1 transcript: A case report.

CML is characterized by the presence of a BCR-ABL1 fusion transcript. Several guidelines have been published for its detection and molecular monitoring. Here, a case is described of chronic myeloid leukemia presenting in the blast phase with a rare variant transcript, with a discussion on possible red flags in its detection and genetic testing and description of the patient's clinical characteristics. This case highlights the pitfalls of using real-time quantitative reverse-transcription polymerase chain reaction (RQ-PCR) for diagnosis of CML, especially when the clinical picture and the test results are discordant.

Identification and validation of the optimal reference genes for standardizing the gene expression profiling diagnostic panel of Ph-like B-lineage acute lymphoblastic leukemia.

Gene expression profiling is the criterion standard for recognizing Ph-like ALL signatures among B-ALLs. The prerequisite of GEP is the accurate normalization of target genes with stable expression of housekeeping genes in a quantitative PCR. HKGs exhibit differential expression in the different experimental conditions and affect the target genes' expression, leading to imprecise qPCR results. The selection of stable HKGs is crucial in GEP experiments, especially in identifying high-risk Ph-like ALL cases. We have evaluated the expression stability of nine HKGs (GAPDH, ACTB, GUSB, RNA18S, EEF2, PGK1, B2M, TBP and ABL1) in identified Ph-like ALLs and Ph-negative (n = 23 each) using six algorithms, 4 traditional softwares; geNorm, BestKeeper, NormFinder, Delta Cq value method, and two algorithms, RefFinderTM and ComprFinder. Further, we have validated the expression of 8 overexpressed normalized genes in Ph-like ALL cases (JCHAIN, CA6, MUC4, SPATS2L, BMPR1B, CRLF2, ADGRF1 and NRXN3). GeNorm, BestKeeper, NormFinder, Delta Cq value method, RefFinderTM and ComprFinder algorithm analysis revealed that EEF2, GAPDH, and PGK1 form the best representative HKGs in Ph-like ALL cases, while RNA18s, ß-actin, and ABL1 in Ph-negative ALLs. Lastly, we performed a correlation analysis and found that the combination of EEF2, GAPDH, and PGK1 represents the best combination with a very high correlation in Ph-like ALL cases. This is the first report that shows EEF2, GAPDH, and PGK1 are the best HKG genes and can be used in the diagnostic panel of Ph-like ALL cases using qPCR at baseline diagnosis.

Detection and identification of Mucorales and Aspergillus in paraffin-embedded samples by real-time quantitative PCR.

Background: In this study, we used real-time quantitative PCR (RQ-PCR) to rapidly detect Mucorales and Aspergillus in formalin-fixed, paraffin-embedded (FFPE) samples, targeting 18SrRNA gene and 28SrRNA gene. Identification of Mucorales and Aspergillus was analysed by combining Mucorales RQ-PCR (Mucorales18SrRNA and Mucorales28SrRNA) with Aspergillus RQ-PCR (Aspergillus18SrRNA and Aspergillus28SrRNA). Objectives: The aims of this study were to compare the diagnostic performances of four RQ-PCR assays as single and combined diagnostic and identification tools. Methods: We collected 12 control group samples and 81 experimental group samples diagnosed by histopathology, including mucormycosis (19 patients, 21 FFPE samples), aspergillosis (54 patients, 57 FFPE samples) and mucormycosis with aspergillosis (3 patients, 3 FFPE samples). All samples were detected by four RQ-PCR tests to compare and analyze diagnostic performance. Results: The sensitivities of Mucorales18SrRNA and Mucorales28SrRNA were both 75%, with the tests having specificities of 97.10% and 94.20%. The sensitivities of Aspergillus18SrRNA and Aspergillus28SrRNA were 73.33% and 65%, with the tests having specificities of 87.88% and 81.82%. The values of the evaluation indexes of the combined detection of Mucorales28SrRNA and Aspergillus18SrRNA (M28A18) were the highest with a kappa coefficient value of 0.353, followed by M18A18. M28A18 had a sensitivity of 67.90% and a specificity of 100%. Conclusions: We recommend using the combination of Mucorales RQ-PCR and Aspergillus RQ-PCR as a screening tool to detect samples suspected of mucormycosis and/or aspergillosis.

Optimizing Molecular Minimal Residual Disease Analysis in Adult Acute Lymphoblastic Leukemia.

Minimal/measurable residual disease (MRD) evaluation has resulted in a fundamental instrument to guide patient management in acute lymphoblastic leukemia (ALL). From a methodological standpoint, MRD is defined as any approach aimed at detecting and possibly quantifying residual neoplastic cells beyond the sensitivity level of cytomorphology. The molecular methods to study MRD in ALL are polymerase chain reaction (PCR) amplification-based approaches and are the most standardized techniques. However, there are some limitations, and emerging technologies, such as digital droplet PCR (ddPCR) and next-generation sequencing (NGS), seem to have advantages that could improve MRD analysis in ALL patients. Furthermore, other blood components, namely cell-free DNA (cfDNA), appear promising and are also being investigated for their potential role in monitoring tumor burden and response to treatment in hematologic malignancies. Based on the review of the literature and on our own data, we hereby discuss how emerging molecular technologies are helping to refine the molecular monitoring of MRD in ALL and may help to overcome some of the limitations of standard approaches, providing a benefit for the care of patients.

Minimal Residual Disease Analysis by Monitoring Immunoglobulin and T-Cell Receptor Gene Rearrangements by Quantitative PCR and Droplet Digital PCR.

Analysis of immunoglobulin and T-cell receptor gene rearrangements by real-time quantitative polymerase chain reaction (RQ-PCR) is the gold standard for sensitive and accurate minimal residual disease (MRD) monitoring; it has been extensively standardized and guidelines have been developed within the EuroMRD consortium ( www.euromrd.org ). However, new generations of PCR-based methods are standing out as potential alternatives to RQ-PCR, such as digital PCR technology (dPCR), the third-generation implementation of conventional PCR, which has the potential to overcome some of the limitations of RQ-PCR such as allowing the absolute quantification of nucleic acid targets without the need for a calibration curve. During the last years, droplet digital PCR (ddPCR) technology has been compared to RQ-PCR in several hematologic malignancies showing its proficiency for MRD analysis. So far, no established guidelines for ddPCR MRD analysis and data interpretation have been defined and its potential is still under investigation. However, a major standardization effort is underway within the EuroMRD consortium ( www.euromrd.org ) for future application of ddPCR in standard clinical practice.

Identification and validation of suitable housekeeping genes for gene expression studies in BCR-ABL1 positive B-lineage acute lymphoblastic leukemia.

BACKGROUND: The stability of the housekeeping gene (HKG) expression is an absolute prerequisite for accurate normalization of target gene expression in a quantitative real-time polymerase chain reaction (RQ-PCR). In RQ-PCR, the widely used normalization approach involves the standardization of target genes to the most stable HKG control genes. According to the recent literature, in different experimental conditions the HKGs exhibit either up or down-regulation and thus affecting the gene expression profiles of target genes which leads to erroneous results. This implies that it is very important to select the appropriate HKG and verify the expression stability of the HKG before quantification of the target gene. METHODS AND RESULTS: The present study aims to analyze six different HKGs for their expression profiles and stability in BCR-ABL1 negative cases and validate them in BCR-ABL1 positive cases, detected by multiplex reverse transcribed polymerase chain reaction (RT-PCR). Six commonly used reference genes (GAPDH, ABL1, RNA18S, ACTB, GUSB, and EEF2) were selected in this study. RQ-PCR was performed on 24 BCR-ABL1 negative cases and the outcomes were validated on 24 BCR-ABL1 positive cases. RefFinder™, a web-based composite software was used to check the stability of HKG genes by different algorithms and comprehensive ranking of each HKG gene in BCR-ABL1 negative cases and finally validated in BCR-ABL1 positive cases. CONCLUSIONS: It was found that RNA18S, ABL1 and GUSB are good stable HKG genes, which showed minimum variability in gene expression compared to GAPDH, EEF2, and ACTB, the most commonly used HKG.

PHi-RACE: PGIMER in-house rapid & cost effective classifier for the detection of BCR-ABL1-like acute lymphoblastic leukaemia in Indian patients.

For the detection of BCR-ABL1-like ALL cases, two methodologies, specifically Gene expression profiling (GEP) or Next-generation targeted sequencing (NGS) and TaqMan based low-density (TLDA) card, are being used. NGS is very costly and TLDA is not widely commercially available. In this study, we quantified the expression of 8 selected overexpressed genes in 536 B-ALL cases. We identified 26.67% (143/536) BCR-ABL1-like ALLs using hierarchical clustering and principal component analysis. BCR-ABL1-like ALL cases were significantly older at presentation (p = 0.036) and had male preponderance (p = 0.047) compared to BCR-ABL1-negative ALL cases. MRD-positivity and induction failure were more commonest in BCR-ABL1-like ALL cases (30.55 vs.19.35% in BCR-ABL1-negative ALL cases). Lastly, we built a PHi-RACE classifier (sensitivity = 95.2%, specificity= 83.7%, AUC= 0.927) using logistic regression to detect BCR-ABL1-like ALL cases promptly at diagnosis. This classifier is beneficial for hematologists in quick decision making at baseline to start tailored treatment regimes.

Patient specific real-time PCR in precision medicine - Validation of IG/TR based MRD assessment in lymphoid leukemia.

Detection of patient- and tumor-specific clonally rearranged immune receptor genes using real-time quantitative (RQ)-PCR is an accepted method in the field of precision medicine for hematologic malignancies. As individual primers are needed for each patient and leukemic clone, establishing performance specifications for the method faces unique challenges. Results for series of diagnostic assays for CLL and ALL patients demonstrate that the analytic performance of the method is not dependent on patients' disease characteristics. The calibration range is linear between 10-1 and 10-5 for 90% of all assays. The detection limit of the current standardized approach is between 1.8 and 4.8 cells among 100,000 leukocytes. RQ-PCR has about 90% overall agreement to flow cytometry and next generation sequencing as orthogonal methods. Accuracy and precision across different labs, and above and below the clinically applied cutoffs for minimal/measurable residual disease (MRD) demonstrate the robustness of the technique. The here reported comprehensive, IVD-guided analytical validation provides evidence that the personalized diagnostic methodology generates robust, reproducible and specific MRD data when standardized protocols for data generation and evaluation are used. Our approach may also serve as a guiding example of how to accomplish analytical validation of personalized in-house diagnostics under the European IVD Regulation.

[Comparison of prognostic significance between multiparameter flow cytometry and real-time quantitative polymerase chain reaction in the detection of minimal residual disease of Philadelphia chromosome-positive acute B lymphocytic leukemia before allogeneic hematopoietic stem cell transplantation].

Objective: To explore the different values of minimal residual disease (MRD) detection by multiparameter flow cytometry (MFC) and real-time quantitative polymerase chain reaction (RQ-PCR) before hematopoietic stem cell transplantation (HSCT) for predicting relapse, leukemia-free survival (LFS) , and overall survival (OS) in Philadelphia chromosome-positive ALL (Ph(+) ALL) . Methods: A retrospective study (n=280) was performed. MRD was determined using multiparameter flow cytometry and RQ-PCR. Results: MRD analysis with MFC and RQ-PCR of the BCR-ABL fusion transcript showed a strong correlation before transplantation. The positive rates of MRD detected by MFC and RQ-PCR before transplantation were 25.7% (72/280) and 60.7% (170/280) , respectively. MFC MRD-positive (MRDpos) Ph(+) ALL patients had a higher 3 year cumulative incidences of relapse (CIR) than did MFC MRD-negative (MRDneg) Ph(+) ALL patients (23.6%vs 8.6%; P<0.001) . However, the RQ-PCR MRDpos group had similar rates of 3 year OS, LFS, and NRM compared with those in the RQ-PCR MRDneg group. Moreover, patients with RQ-PCR MRD ≥1% experienced higher 3 year CIR (23.1%vs 11.4%; P=0.032) , lower LFS (53.8%vs 74.4%; P=0.015) , and OS (57.7% vs 79.1%; P=0.009) compared with the RQ-PCR MRD<1% group. Multivariate analyses confirmed the association of MFC MRD status and RQ-PCR MRD ≥1% with outcomes (P<0.05) . The sensitivity, specificity, positive predictive value (PPV) , and negative predictive value (NPV) of MFC detection MRD to predict recurrence were 48.50%, 77.56%, 23.62%, and 87.16%, respectively. Moreover, the sensitivity, specificity, PPV, and NPV were 23.00%, 88.59%, 17.15%, and 91.84%, respectively, when RQ-PCR MRD ≥1% was used to predict recurrence. Additionally, the sensitivity, specificity, PPV, and NPV were 54.29%, 73.88%, 45.7% and 91.87%, respectively, when MRD-positive status before transplantation (MFC MRDpos or RQ-PCR MRD ≥1%) was used to predict recurrence after transplantation. Conclusions: Both MFC and RQ-PCR detection of pretransplant MRD levels can predict the prognosis of Ph(+) B-ALL patients receiving allogeneic HSCT. MFC MRD-positive status before transplantation is the risk factor of leukemia recurrence after transplantation. The combined use of the two methods (MFC MRDpos or RQ-PCR MRD ≥1%) can improve the sensitivity, PPV, and NPV of predicting recurrence and help to better screen high-risk patients for intervention, thereby improving clinical efficacy.

No prognostic significance of normalized copy number of PML-RARA transcript at diagnosis in patients with acute promyelocytic leukemia.

BACKGROUND: Acute promyelocytic leukemia is a peculiar disease with few studies that have investigated the prognostic significance of PML/RARA transcript level at diagnosis. PATIENTS AND METHODS: This retrospective study included all cases diagnosed with acute promyelocytic leukemia over the period from June 2015 to March 2019. The normalized copy number (NCN) was tested by real-time polymerase chain reaction at diagnosis, and at the end of induction regimen. RESULTS: Our study included 83 de novo APL patients, 53 (63.9%) were adults and 30 (36.1%) were children. The median (range) age of our patients was 28.0 (1.0-70.0) years. The pediatric group had a significantly higher prevalence in males (p = 0.02), higher incidence of disseminated intravascular coagulopathy (p = 0.014), and high-risk groups (p = 0.017). At diagnosis, the median NCN (%) of the entire group at 22.5 was set as the cut off value. There was no significant association between NCN at diagnosis and other prognostic variables except for bone marrow promyelocytes (p = 0.006). High-risk group APL patients as well as those presenting with hemorrhage had an inferior overall survival (OS) (p = 0.007; p < 0.001) respectively. PML-RARA NCN at diagnosis did not have an impact on the OS or increased risk of relapse of our patients (p = 0.434; p = 0.721). CONCLUSION: the initial PML/RARA tumor burden is not a prognostic factor for APL. The initial TLC at 10x109/L cut off is the most important predictive for OS. Early detection and close monitoring are required to decrease the high rate of early deaths in developing countries.

PCR Technology to Identify Minimal Residual Disease.

Although new techniques (i.e., droplet digital-PCR, next-generation sequencing, advanced flow cytometry) are being developed, DNA-based allele-specific real-time quantitative (RQ)-PCR is still the gold standard for sensitive and accurate immunoglobulin/T cell receptor (IG/TR)-based minimal residual disease (MRD) monitoring, allowing the detection of up to 1 leukemic cell in 100,000 normal lymphoid cells. We herewith describe the standard PCR procedure which has been developed and standardized (with minor modification in single labs) through the last 20 years of activity of the EuroMRD Consortium, a volunteer activity of expert laboratories that is continuously providing education, standardization, quality control rounds, and guidelines for interpretation of RQ-PCR data.

Prognostic value of MRD monitoring based on BCR-ABL1 copy numbers in Philadelphia chromosome positive acute lymphoblastic leukemia.

Assessment of measurable residual disease (MRD) has emerged as a powerful prognostic tool in pediatric and adult acute lymphoblastic leukemia (ALL). In this single-centre retrospective study, we evaluated the prognostic relevance of MRD based on BCR-ABL1 copy numbers in Ph + ALL patients between 2006 and 2018. Molecular responses were evaluated at 3, 6, 9 and 12 months after the initiation of treatment. Patients who had their MRD assessed at three or more time points were categorized into MRD good risk or poor risk based on BCR-ABL1/ABL1 copy number ratio. MRD positive patients consistently showed a trend toward poor survival and on multivariate analysis, MRD poor risk patients had adverse outcomes when compared to MRD good risk patients in terms of overall (OS; p = .031) and event-free (EFS; p < .001) survival. In conclusion, molecular MRD based on BCR-ABL1 copy number ratio is an ideal prognostic indicator in Ph + ALL patients undergoing treatment.

Minimal Residual Disease in Acute Lymphoblastic Leukemia: Technical and Clinical Advances.

Introduction: Acute lymphoblastic leukemia (ALL) is the first neoplasm where the assessment of early response to therapy by minimal residual disease (MRD) monitoring has proven to be a fundamental tool to guide therapeutic choices. The most standardized methods to study MRD in ALL are multi-parametric flow cytometry (MFC) and polymerase chain reaction (PCR) amplification-based methods. Emerging technologies hold the promise to improve MRD detection in ALL patients. Moreover, novel therapies, such as monoclonal antibodies, bispecific T-cell engagers, and chimeric antigen receptor T cells (CART) represent exciting advancements in the management of B-cell precursor (BCP)-ALL. Aims: Through a review of the literature and in house data, we analyze the current status of MRD assessment in ALL to better understand how some of its limitations could be overcome by emerging molecular technologies. Furthermore, we highlight the future role of MRD monitoring in the context of personalized protocols, taking into account the genetic complexity in ALL. Results and Conclusions: Molecular rearrangements (gene fusions and immunoglobulin and T-cell receptor-IG/TR gene rearrangements) are widely used as targets to detect residual leukemic cells in ALL patients. The advent of novel techniques, namely next generation flow cytometry (NGF), digital-droplet-PCR (ddPCR), and next generation sequencing (NGS) appear important tools to evaluate MRD in ALL, since they have the potential to overcome the limitations of standard approaches. It is likely that in the forthcoming future these techniques will be incorporated in clinical trials, at least at decisional time points. Finally, the advent of new powerful compounds is further increasing MRD negativity rates, with benefits in long-term survival and a potential reduction of therapy-related toxicities. However, the prognostic relevance in the setting of novel immunotherapies still needs to be evaluated.

Ficolled bone marrow is superior to bone marrow buffy coat for detection of minimal residual disease in multiple myeloma.

OBJECTIVE: Buffy coat and ficoll of bone marrow (BM) are viable options for the study of minimal residual disease (MRD) in multiple myeloma (MM). As yet, there is no data about the superiority of either sample types. Herein, we aimed to address this issue. METHODS: Forty pairs of ficolled BMs and BM buffy coats of 19 MM patients were studied for MRD by allele-specific oligonucleotide real-time quantitative PCR, with patient-specific primers/probes whenever appropriate. RESULTS: There were 41 pairs of MRD data for comparison analysis due to one patient with biclonal disease. MRD levels in ficolls and buffy coats were highly concordant (rs = 0.936, P < 0.0001), with 31 (76%) and seven (17%) pairs being concomitantly MRD-positive or -negative. On the other hand, apart from the 16 pairs being both MRD-negative, or -positive but not quantifiable in ficolls and buffy coats, majority (n = 22, 88%) had higher MRD levels in ficolled BMs than BM buffy coats. Furthermore, in 17 pairs, in which MRD was quantifiable in both, MRD levels in ficolled BMs were 3.1 times those of BM buffy coats (median, 567/105 vs. 184/105, P = 0.001). CONCLUSION: Taken together, ficolled BM is more sensitive than BM buffy coat for MRD detection in MM, hence should be recommended.

Outcome and prognostic factors of children with Philadelphia chromosome-positive acute lymphoblastic leukemia treated with imatinib followed by allogeneic hematopoietic cell transplantation in first remission.

BACKGROUND: Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph+ ALL) is a subset of ALL with poor prognosis. Here, we analyzed the outcomes and prognostic factors of children with Ph+ ALL who received imatinib and chemotherapy followed by allogeneic hematopoietic cell transplantation (HCT) in first complete remission (CR). METHODS: Thirty-one Ph+ ALL patients (female 10) diagnosed from January 2005 to December 2016 were included in the study. All patients were treated with imatinib and chemotherapy before HCT. Bone marrow (BM) evaluations included real-time quantitative polymerase chain reaction (RQ-PCR) study of the BCR-ABL1 fusion transcript. All patients received HCT with total body irradiation (TBI)-based conditioning at a median of 6.4 (range, 4.2-47.1) months from diagnosis. RESULTS: Compared to values at diagnosis, the median decrement of RQ-PCR value post-consolidation, and prior to HCT was -3.7 Log and -4.8 Log, respectively. The 5-year event-free survival (EFS) and overall survival of the patients were 64.5±9.4% (20/31) and 75.0±8.3% (23/31) respectively. Events included relapse (N=5) and death in CR post-HCT (N=6). The 5-year incidence of molecular relapse was 30.9±9.1% (9/31). An RQ-PCR decrement of at least -4 Log post-consolidation significantly predicted lower incidence of molecular relapse: 7.7±7.7% for ≥-4 Log decrement, 50.0±13.8% for <-4 Log decrement (P=0.027). CONCLUSION: Decrement in RQ-PCR for the BCR-ABL1 transcript that was determined after consolidation was the only significant prognostic factor for incidence of molecular relapse. In the post-induction TKI initiation setting, steadfast imatinib treatment during consolidation may allow for optimum post-HCT outcomes.

MRD Detection in B-Cell Non-Hodgkin Lymphomas Using Ig Gene Rearrangements and Chromosomal Translocations as Targets for Real-Time Quantitative PCR.

Minimal residual disease (MRD) diagnostics is of high clinical relevance in patients with indolent B-cell non-Hodgkin lymphomas (B-NHL) and serves as a surrogate parameter to evaluate treatment effectiveness and long-term prognosis. MRD diagnostics performed by real-time quantitative PCR (RQ-PCR) is still the gold standard and currently the most sensitive and the most broadly applied method in follicular lymphoma (FL) and mantle cell lymphoma (MCL). Alternatively, droplet digital PCR (ddPCR) can be used for MRD monitoring in multiple myeloma, mantle cell lymphoma, and follicular lymphoma with comparable sensitivity, accuracy, and reproducibility.The most broadly applicable MRD target in B-NHL is the junctional regions of the rearranged immunoglobulin heavy chain gene (IGHV). Chromosomal translocations like the t(14;18) translocation in FL and t(11;14) translocation in MCL can be used as MRD target in selected lymphoma subtypes. In patients with B-cell chronic lymphocytic leukemia, both flow-cytometry and RQ-PCR are equally suited for MRD assessment as long as a sensitivity of 10-4 shall be achieved.MRD diagnostics targeting the IGHV gene is complex and requires extensive knowledge and experience because the junctional regions of each lymphoma have to be identified before the patient-specific RQ-PCR assays can be designed for MRD monitoring. In addition, the presence and load of somatic hypermutation (SHM) within the rearranged IG heavy variable (IGHV) gene occurring as during B-cell development of germinal center and post-germinal center lymphomas may hamper appropriate primer binding leading to false-negative results. The translocations mentioned above have the advantage that consensus forward primers and probes, both placed in the breakpoint regions of chromosome 18 in FL and chromosome 11 in MCL, can be used in combination with a reverse primer placed in the IGH joining region of chromosome 14. RQ-PCR-based methods can reach a good sensitivity (≤10-4). This chapter provides all relevant background information and technical aspects for the complete laboratory process from detection of the clonal IGHV gene rearrangement and the chromosomal translocations at diagnosis to the actual MRD measurements in clinical follow-up samples of B-NHL. However, it should be noted that MRD diagnostics for clinical treatment protocols has to be accompanied by regular international quality control rounds to ensure the reproducibility and reliability of the MRD results.

Minimal residual disease by flow cytometry and allelic-specific oligonucleotide real-time quantitative polymerase chain reaction in patients with myeloma receiving lenalidomide maintenance: A pooled analysis.

BACKGROUND: Minimal residual disease (MRD) is one of the most relevant prognostic factors in patients with multiple myeloma (MM); however, the impact of maintenance therapy on MRD levels remains unclear. Among patients with newly diagnosed MM (NDMM) who received lenalidomide maintenance until they developed disease progression, the role of MRD status as a predictor of progression-free survival (PFS) was evaluated by multiparameter flow cytometry (MFC) and allelic-specific oligonucleotide real-time quantitative polymerase chain reaction (ASO-RQ-PCR) analysis. METHODS: Seventy-three patients with NDMM enrolled in the RV-MM-EMN-441 (clinical trials.gov identifier, NCT01091831) and RV-MM-COOP-0556 (clinicaltrials.gov identifier, NCT01208766; European Myeloma Network EMN02/HO95 MM Trial) phase 3 trials who achieved at least a very good partial response after intensification/consolidation were included. The median patient age was 57 years (interquartile range, 53-61 years), and all patients received lenalidomide maintenance until they developed progression. MRD was evaluated on bone marrow after intensification/consolidation, after 6 courses of maintenance, and every 6 months thereafter until clinical relapse using both ASO-RQ-PCR (sensitivity, 10-5 ) and MFC (sensitivity, from 10-4 to 10-5 ). RESULTS: After intensification/consolidation, 33 of 72 patients (46%) achieved a molecular complete response (m-CR), and 44 of 70 (63%) achieved a flow complete response (flow-CR). Almost 27% of patients who were MRD-positive after consolidation became MRD-negative during maintenance. After a median follow-up of 38 months, PFS was prolonged in patients who achieved negative MRD status during maintenance according to results from both ASO-RQ-PCR analysis (hazard ratio, 0.29; 95% confidence interval, 0.14-0.62; P = .0013) and MFC (hazard ratio, 0.19; 95% confidence interval, 0.09-0.41; P < .001). The impact of negative MRD status on PFS was similar in all subgroups (ASCT and no-ASCT; International Staging System stages I, II, and III; high-risk and standard-risk cytogenetics), and the two techniques were highly correlated. CONCLUSIONS: MRD status is a stronger predictor of PFS than standard risk factors, and lenalidomide maintenance further increases the rate of negative MRD results.

Outcome and prognostic factors of children with Philadelphia chromosome-positive acute lymphoblastic leukemia treated with imatinib followed by allogeneic hematopoietic cell transplantation in first remission

BACKGROUND: Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph+ ALL) is a subset of ALL with poor prognosis. Here, we analyzed the outcomes and prognostic factors of children with Ph+ ALL who received imatinib and chemotherapy followed by allogeneic hematopoietic cell transplantation (HCT) in first complete remission (CR). METHODS: Thirty-one Ph+ ALL patients (female 10) diagnosed from January 2005 to December 2016 were included in the study. All patients were treated with imatinib and chemotherapy before HCT. Bone marrow (BM) evaluations included real-time quantitative polymerase chain reaction (RQ-PCR) study of the BCR-ABL1 fusion transcript. All patients received HCT with total body irradiation (TBI)-based conditioning at a median of 6.4 (range, 4.2–47.1) months from diagnosis. RESULTS: Compared to values at diagnosis, the median decrement of RQ-PCR value post-consolidation, and prior to HCT was −3.7 Log and −4.8 Log, respectively. The 5-year event-free survival (EFS) and overall survival of the patients were 64.5±9.4% (20/31) and 75.0±8.3% (23/31) respectively. Events included relapse (N=5) and death in CR post-HCT (N=6). The 5-year incidence of molecular relapse was 30.9±9.1% (9/31). An RQ-PCR decrement of at least −4 Log post-consolidation significantly predicted lower incidence of molecular relapse: 7.7±7.7% for ≥−4 Log decrement, 50.0±13.8% for <−4 Log decrement (P=0.027). CONCLUSION: Decrement in RQ-PCR for the BCR-ABL1 transcript that was determined after consolidation was the only significant prognostic factor for incidence of molecular relapse. In the post-induction TKI initiation setting, steadfast imatinib treatment during consolidation may allow for optimum post-HCT outcomes.