Wild-type Blocking PCR Combined with Sanger Sequencing for Detection of Low-frequency Somatic Mutation.

When monitoring minimal residual disease (MRD) after tumor treatment, there are higher requirements of the lower limit of detection than when detecting for drug resistance mutations and circulating tumor cell mutations during therapy. Traditional Sanger sequencing has 5%-20% wild-type mutation detection, so its limit of detection cannot meet the corresponding requirements. The wild-type blocking technologies that have been reported to overcome this include blocker displacement amplification (BDA), non-extendable locked nucleic acid (LNA), hot-spot-specific probes (HSSP), etc. These technologies use specific oligonucleotide sequences to block wild-type or recognize wild-type and then combine this with other methods to prevent wild-type amplification and amplify mutant amplification, leading to characteristics like high sensitivity, flexibility, and convenience. This protocol uses BDA, a wild-type blocking PCR combined with Sanger sequencing, to optimize the detection of RHOA G17V low-frequency somatic mutations, and the detection sensitivity can reach 0.5%, which can provide a basis for MRD monitoring of angioimmunoblastic T-cell lymphoma.

Prognostic significance and treatment strategies for IKZF1 deletion in pediatric B-cell precursor acute lymphoblastic leukemia.

BACKGROUND: The predictive importance of IKZF1del in pediatric B-cell precursor acute lymphoblastic leukemia (BCP-ALL) has shown variability across different studies. Thus, the optimal treatment approach for children with IKZF1del BCP-ALL remains contentious, with the ongoing debate surrounding the use of IKZF1del-based high-risk stratification versus a minimal residual disease (MRD)-guided protocol. METHODS: IKZF1 status was reliably determined in 804 patients using multiplex ligation-dependent probe amplification (MLPA) data obtained from four hospitals in Fujian, a province of China. In the Chinese Children Leukemia Group (CCLG)-ALL 2008 cohort, IKZF1 status was included in the risk assignment, with all IKZF1del patients receiving a high-risk regimen. Conversely, in the Chinese Children's Cancer Group (CCCG)-ALL 2015 cohort, IKZF1del was not incorporated into the risk assignment, and patients were treated based on an MRD-guided risk stratification protocol. RESULTS: IKZF1del was found in 86 patients (86/804, 10.7%) overall and in 30 (30/46, 65.2%) BCR::ABL1-positive patients. Overall, IKZF1del was a poor prognostic predictor for patients, though the significance diminished upon age adjustment, white blood cell (WBC) count at diagnosis, treatment group, and MRD status. In the CCLG-ALL 2008 cohort, IKZF1del conferred a notably lower 5-year overall survival (OS) and event-free survival (EFS) and a significantly higher 5-year cumulative incidence of relapse (CIR) than IKZF1wt. In the CCLG-ALL 2015 cohort, IKZF1del conferred a lower 5-year OS and EFS and a higher 5-year CIR than IKZF1wt, but the differences were insignificant. The IKZF1del patients treated with higher intensity chemotherapy (CCLG-ALL 2008 high-risk regimen) had a markedly lower 5-year OS and EFS compared with those treated with the MRD-guided protocol (CCCG-ALL 2015 protocol). Furthermore, patients treated with the CCLG-ALL 2008 high-risk regimen experienced a higher frequency of serious adverse events (SAEs), especially infection-related SAEs, compared with those treated with the CCCG-ALL 2015 MRD-guided protocol. CONCLUSIONS: The prognostic effect of IKZF1del may vary in different protocols. Compared with higher intensity chemotherapy, the MRD-guided protocol may be a more effective approach to treating BCP-ALL with IKZF1del in children.

Ultra-sensitive molecular residual disease detection through whole genome sequencing with single-read error correction.

While whole genome sequencing (WGS) of cell-free DNA (cfDNA) holds enormous promise for detection of molecular residual disease (MRD), its performance is limited by WGS error rate. Here we introduce AccuScan, an efficient cfDNA WGS technology that enables genome-wide error correction at single read-level, achieving an error rate of 4.2 × 10-7, which is about two orders of magnitude lower than a read-centric de-noising method. The application of AccuScan to MRD demonstrated analytical sensitivity down to 10-6 circulating variant allele frequency at 99% sample-level specificity. AccuScan showed 90% landmark sensitivity (within 6 weeks after surgery) and 100% specificity for predicting relapse in colorectal cancer. It also showed 67% sensitivity and 100% specificity in esophageal cancer using samples collected within one week after surgery. When AccuScan was applied to monitor immunotherapy in melanoma patients, the circulating tumor DNA (ctDNA) levels and dynamic profiles were consistent with clinical outcomes. Overall, AccuScan provides a highly accurate WGS solution for MRD detection, empowering ctDNA detection at parts per million range without requiring high sample input or personalized reagents.

Evaluation of a New Closed-System Automated RT-qPCR Assay for the Rapid Detection and Monitoring of Common Nucleophosmin Mutations in Patients with Acute Myeloid Leukemia.

Quantitative assessment of nucleophosmin 1 (NPM1) mutation status is integral to evaluating measurable residual disease (MRD) in NPM1-mutated acute myeloid leukemia (AML) patients. In a retrospective study, leftover peripheral blood (PB) specimens (n = 40) which were collected for routine clinical diagnostic evaluations of AML disease burden were tested by both a novel automated RT-qPCR quantitative NPM1 assay (Xpert NPM1 mutation assay) and the NPM1 mutA, mutB&D MutaQuant kit. Based on a Deming regression analysis, there was a high correlation (slope = 0.92; intercept = 0.12; Pearson's r = 0.982) between the quantitative results of the Xpert NPM1 mutation assay and the NPM1 mutA, mutB&D MutaQuant kit. The Xpert test quantitative results are thus highly correlated with the comparator method and the former has potential as a useful alternative for the monitoring of AML patients with a known NPM1 mutation.

Tissue-Free Liquid Biopsies Combining Genomic and Methylation Signals for Minimal Residual Disease Detection in Patients with Early Colorectal Cancer from the UK TRACC Part B Study.

PURPOSE: The absence of postoperative circulating tumor DNA (ctDNA) identifies patients with resected colorectal cancer (CRC) with low recurrence risk for adjuvant chemotherapy (ACT) de-escalation. Our study presents the largest resected CRC cohort to date with tissue-free minimal residual disease (MRD) detection. EXPERIMENTAL DESIGN: TRACC (tracking mutations in cell-free tumor DNA to predict relapse in early colorectal cancer) included patients with stage I to III resectable CRC. Prospective longitudinal plasma collection for ctDNA occurred pre- and postsurgery, post-ACT, every 3 months for year 1 and every 6 months in years 2 and 3 with imaging annually. The Guardant Reveal assay evaluated genomic and methylation signals. The primary endpoint was 2-year recurrence-free survival (RFS) by postoperative ctDNA detection (NCT04050345). RESULTS: Between December 2016 and August 2022, 1,203 were patients enrolled. Plasma samples (n = 997) from 214 patients were analyzed. One hundred forty-three patients were evaluable for the primary endpoint; 92 (64.3%) colon, 51 (35.7%) rectal; two (1.4%) stage I, 64 (44.8%) stage II, and 77 (53.8%) stage III. Median follow-up was 30.3 months (95% CI, 29.5-31.3). Two-year RFS was 91.1% in patients with ctDNA not detected postoperatively and 50.4% in those with ctDNA detected [HR, 6.5 (2.96-14.5); P < 0.0001]. Landmark negative predictive value (NPV) was 91.2% (95% CI, 83.9-95.9). Longitudinal sensitivity and specificity were 62.1% (95% CI, 42.2-79.3) and 85.9% (95% CI, 78.9-91.3), respectively. The median lead time from ctDNA detection to radiological recurrence was 7.3 months (IQR, 3.3-12.5; n = 9). CONCLUSIONS: Tissue-free MRD detection with longitudinal sampling predicts recurrence in patients with stage I to III CRC without the need for tissue sequencing. The UK TRACC Part C study is currently investigating the potential for ACT de-escalation in patients with undetectable postoperative ctDNA, given the high NPV indicating a low likelihood of residual disease.

Identification of Therapy-Induced Clonal Evolution and Resistance Pathways in Minimal Residual Clones in Multiple Myeloma through Single-Cell Sequencing.

PURPOSE: In multiple myeloma (MM), therapy-induced clonal evolution is associated with treatment resistance and is one of the most important hindrances toward a cure for MM. To further understand the molecular mechanisms controlling the clonal evolution of MM, we applied single-cell RNA sequencing (scRNA-seq) to paired diagnostic and posttreatment bone marrow (BM) samples. EXPERIMENTAL DESIGN: scRNA-seq was performed on 38 BM samples from patients with monoclonal gammopathy of undetermined significance (n = 1), MM patients at diagnosis (n = 19), MM posttreatment (n = 17), and one healthy donor (HD). The single-cell transcriptome data of malignant plasma cells (PC) and the surrounding immune microenvironment were analyzed. RESULTS: Profiling by scRNA-seq data revealed three primary trajectories of transcriptional evolution after treatment: clonal elimination in patients with undetectable minimal residual disease (MRD-) and clonal stabilization and clonal selection in detectable MRD (MRD+) patients. We noted a metabolic shift toward fatty acid oxidation in cycling-resistant PCs, whereas selective PCs favored the NF-κB pathway. Intriguingly, when comparing the genetic and transcriptional dynamics, we found a significant correlation between genetic and nongenetic factors in driving the clonal evolution. Furthermore, we identified variations in cellular interactions between malignant PCs and the tumor microenvironment. Selective PCs showed the most robust cellular interactions with the tumor microenvironment. CONCLUSIONS: These data suggest that MM cells could rapidly adapt to induction treatment through transcriptional adaptation, metabolic adaptation, and specialized immune evasion. Targeting therapy-induced resistance mechanisms may help to avert refractory disease in MM.

CoHIT: a one-pot ultrasensitive ERA-CRISPR system for detecting multiple same-site indels.

Genetic testing is crucial for precision cancer medicine. However, detecting multiple same-site insertions or deletions (indels) is challenging. Here, we introduce CoHIT (Cas12a-based One-for-all High-speed Isothermal Test), a one-pot CRISPR-based assay for indel detection. Leveraging an engineered AsCas12a protein variant with high mismatch tolerance and broad PAM scope, CoHIT can use a single crRNA to detect multiple NPM1 gene c.863_864 4-bp insertions in acute myeloid leukemia (AML). After optimizing multiple parameters, CoHIT achieves a detection limit of 0.01% and rapid results within 30 minutes, without wild-type cross-reactivity. It successfully identifies NPM1 mutations in 30 out of 108 AML patients and demonstrates potential in monitoring minimal residual disease (MRD) through continuous sample analysis from three patients. The CoHIT method is also competent for detecting indels of KIT, BRAF, and EGFR genes. Integration with lateral flow test strips and microfluidic chips highlights CoHIT's adaptability and multiplexing capability, promising significant advancements in clinical cancer diagnostics.

Breast Cancer Plasticity after Chemotherapy Highlights the Need for Re-Evaluation of Subtyping in Residual Cancer and Metastatic Tissues.

This research paper presents a novel approach to identifying biomarkers that can be used to prognosticate patients with triple-negative breast cancer (TNBC) eligible for neoadjuvant therapy. The study utilized survival and RNA sequencing data from a cohort of TNBC patients and identified 276 genes whose expression was related to survival in such patients. The gene expression data were then used to classify patients into two major groups based on the presence or absence of Wingless/Integrated-pathway (Wnt-pathway) and mesenchymal (Mes) markers (Wnt/Mes). Patients with a low expression of Wnt/Mes-related genes had a favorable outcome, with no deaths observed during follow-up, while patients with a high expression of Wnt/Mes genes had a higher mortality rate of 50% within 19 months. The identified gene list could be validated and potentially used to shape treatment options for TNBC patients eligible for neoadjuvant therapy providing valuable insights into the development of more effective treatments for TNBC. Our data also showed significant variation in gene expression profiles before and after chemotherapy, with most tumors switching to a more mesenchymal/stem cell-like profile. To verify this observation, we performed an in silico analysis to classify breast cancer tumors in Prediction Analysis of Microarray 50 (PAM50) molecular classes before treatment and after treatment using gene expression data. Our findings demonstrate that following drug intervention and metastasis, certain tumors undergo a transition to alternative subtypes, resulting in diminished therapeutic efficacy. This underscores the necessity for reevaluation of patients who have experienced relapse or metastasis post-chemotherapy, with a focus on molecular subtyping. Tailoring treatment strategies based on these refined subtypes is imperative to optimize therapeutic outcomes for affected individuals.

FLT3-TKD Measurable Residual Disease Detection Using Droplet Digital PCR and Clinical Applications in Acute Myeloid Leukemia.

The tyrosine kinase domain of the FMS-Like tyrosine kinase 3 (FLT3-TKD) is recurrently mutated in acute myeloid leukemia (AML). Common molecular techniques used in its detection include PCR and capillary electrophoresis, Sanger sequencing and next-generation sequencing with recognized sensitivity limitations. This study aims to validate the use of droplet digital PCR (ddPCR) in the detection of measurable residual disease (MRD) involving the common FLT3-TKD mutations (D835Y, D835H, D835V, D835E). Twenty-two diagnostic samples, six donor controls, and a commercial D835Y positive control were tested using a commercial Bio-rad® ddPCR assay. All known variants were identified, and no false positives were detected in the wild-type control (100% specificity and sensitivity). The assays achieved a limit of detection suitable for MRD testing at 0.01% variant allelic fraction. Serial samples from seven intensively-treated patients with FLT3-TKD variants at diagnosis were tested. Five patients demonstrated clearance of FLT3-TKD clones, but two patients had FLT3-TKD persistence in the context of primary refractory disease. In conclusion, ddPCR is suitable for the detection and quantification of FLT3-TKD mutations in the MRD setting; however, the clinical significance and optimal management of MRD positivity require further exploration.

Ultrasensitive plasma-based monitoring of tumor burden using machine-learning-guided signal enrichment.

In solid tumor oncology, circulating tumor DNA (ctDNA) is poised to transform care through accurate assessment of minimal residual disease (MRD) and therapeutic response monitoring. To overcome the sparsity of ctDNA fragments in low tumor fraction (TF) settings and increase MRD sensitivity, we previously leveraged genome-wide mutational integration through plasma whole-genome sequencing (WGS). Here we now introduce MRD-EDGE, a machine-learning-guided WGS ctDNA single-nucleotide variant (SNV) and copy-number variant (CNV) detection platform designed to increase signal enrichment. MRD-EDGESNV uses deep learning and a ctDNA-specific feature space to increase SNV signal-to-noise enrichment in WGS by ~300× compared to previous WGS error suppression. MRD-EDGECNV also reduces the degree of aneuploidy needed for ultrasensitive CNV detection through WGS from 1 Gb to 200 Mb, vastly expanding its applicability within solid tumors. We harness the improved performance to identify MRD following surgery in multiple cancer types, track changes in TF in response to neoadjuvant immunotherapy in lung cancer and demonstrate ctDNA shedding in precancerous colorectal adenomas. Finally, the radical signal-to-noise enrichment in MRD-EDGESNV enables plasma-only (non-tumor-informed) disease monitoring in advanced melanoma and lung cancer, yielding clinically informative TF monitoring for patients on immune-checkpoint inhibition.

Evaluation of molecular residual disease in operable non-small cell lung cancer with gene fusions, MET exon skipping or de novo MET amplification.

Gene fusions and MET alterations are rare and difficult to detect in plasma samples. The clinical detection efficacy of molecular residual disease (MRD) based on circulating tumor DNA (ctDNA) in patients with non-small cell lung cancer (NSCLC) with these mutations remains unknown. This prospective, non-intervention study recruited 49 patients with operable NSCLC with actionable gene fusions (ALK, ROS1, RET, and FGFR1), MET exon 14 skipping or de novo MET amplification. We analyzed 43 tumor tissues and 111 serial perioperative plasma samples using 1021- and 338-gene panels, respectively. Detectable MRD correlated with a significantly higher recurrence rate (P < 0.001), yielding positive predictive values of 100% and 90.9%, and negative predictive values of 82.4% and 86.4% at landmark and longitudinal time points, respectively. Patients with detectable MRD showed reduced disease-free survival (DFS) compared to those with undetectable MRD (P < 0.001). Patients who harbored tissue-derived fusion/MET alterations in their MRD had reduced DFS compared to those who did not (P = 0.05). To our knowledge, this is the first comprehensive study on ctDNA-MRD clinical detection efficacy in operable NSCLC patients with gene fusions and MET alterations. Patients with detectable tissue-derived fusion/MET alterations in postoperative MRD had worse clinical outcomes.

Molecular, clinical, and therapeutic determinants of outcome in NPM1-mutated AML.

ABSTRACT: Although NPM1-mutated acute myeloid leukemia (AML) carries a generally favorable prognosis, many patients still relapse and die. Previous studies identified several molecular and clinical features associated with poor outcomes; however, only FLT3-internal tandem duplication (ITD) mutation and adverse karyotype are currently used for risk stratification because of inconsistent results and uncertainty about how other factors should influence treatment, particularly given the strong prognostic effect of postinduction measurable residual disease (MRD). Here, we analyzed a large group of patients with NPM1 mutations (NPM1mut) AML enrolled in prospective trials (National Cancer Research Institute [NCRI] AML17 and AML19, n = 1357) to delineate the impact of baseline molecular and clinical features, postinduction MRD status, and treatment intensity on the outcome. FLT3-ITD (hazard ratio [HR], 1.28; 95% confidence interval [CI], 1.01-1.63), DNMT3A (HR, 1.65; 95% CI, 1.32-2.05), WT1 (HR, 1.74; 95% CI, 1.27-2.38), and non-ABD NPM1mut (HR, 1.64; 95% CI, 1.22-2.21) were independently associated with poorer overall survival (OS). These factors were also strongly associated with MRD positivity. For patients who achieved MRD negativity, these mutations (except FLT3-ITD) were associated with an increased cumulative incidence of relapse (CIR) and poorer OS. However, apart from the few patients with adverse cytogenetics, we could not identify any group of MRD-negative patients with a CIR >40% or with benefit from allograft in first remission. Intensified chemotherapy with the FLAG-Ida (fludarabine, cytarabine, granulocyte colony-stimulating factor, and idarubicin) regimen was associated with improved outcomes in all subgroups, with greater benefits observed in the high-risk molecular subgroups.

Minimal Residual Disease using a Plasma-Only Circulating Tumor DNA Assay to Predict Recurrence of Metastatic Colorectal Cancer Following Curative Intent Treatment.

PURPOSE: Minimal residual disease (MRD) detection can identify the recurrence in patients with colorectal cancer (CRC) following definitive treatment. We evaluated a plasma-only MRD assay to predict recurrence and survival in patients with metastatic CRC who underwent curative intent procedures (surgery and/or radiotherapy), with or without (neo)adjuvant chemotherapy. The primary objective of this study was to assess the correlation of postprocedure tumor cell-free DNA detection status with radiographic disease recurrence. EXPERIMENTAL DESIGN: Preprocedure and postprocedure longitudinal samples were collected from 53 patients and analyzed with a multiomic MRD assay detecting circulating tumor DNA (ctDNA) from genomic and epigenomic signals. Preprocedure and postprocedure ctDNA detection correlated with recurrence-free and overall survival (OS). RESULTS: From 52 patients, 230/233 samples were successfully analyzed. At the time of data cutoff, 36 (69.2%) patients recurred with median follow-up of 31 months. Detectable ctDNA was observed in 19/42 patients (45.2%) with ctDNA analyzed 3 weeks postprocedure. ctDNA detection 3 weeks postprocedure was associated with shorter median recurrence-free survival (RFS; HR, 5.27; 95% CI, 2.31-12.0; P < 0.0001) and OS (HR, 12.83; 95% CI, 3.6-45.9; P < 0.0001). Preprocedure ctDNA detection status was not associated with RFS but was associated with improved OS (HR, 4.65; 95% CI, 1.4-15.2; P = 0.0111). Undetectable ctDNA preprocedure had notable long-term OS, >90% 3 years postprocedure. CONCLUSIONS: In this cohort of oligometastatic CRC, detection of ctDNA preprocedure or postprocedure was associated with inferior outcomes even after accounting for known prognostic clinicopathologic variables. This suggests ctDNA may enhance current risk stratification methods helping the evaluation of novel treatments and surveillance strategies toward improving patient outcomes.

Analysis of measurable residual disease by IG/TR gene rearrangements: quality assurance and updated EuroMRD guidelines.

Minimal/measurable residual disease (MRD) diagnostics using real-time quantitative PCR analysis of rearranged immunoglobulin and T-cell receptor gene rearrangements are nowadays implemented in most treatment protocols for patients with acute lymphoblastic leukemia (ALL). Within the EuroMRD Consortium, we aim to provide comparable, high-quality MRD diagnostics, allowing appropriate risk-group classification for patients and inter-protocol comparisons. To this end, we set up a quality assessment scheme, that was gradually optimized and updated over the last 20 years, and that now includes participants from around 70 laboratories worldwide. We here describe the design and analysis of our quality assessment scheme. In addition, we here report revised data interpretation guidelines, based on our newly generated data and extensive discussions between experts. The main novelty is the partial re-definition of the "positive below quantitative range" category by two new categories, "MRD low positive, below quantitative range" and "MRD of uncertain significance". The quality assessment program and revised guidelines will ensure reproducible and accurate MRD data for ALL patients. Within the Consortium, similar programs and guidelines have been introduced for other lymphoid diseases (e.g., B-cell lymphoma), for new technological platforms (e.g., digital droplet PCR or Next-Generation Sequencing), and for other patient-specific MRD PCR-based targets (e.g., fusion genes).

Evaluation of next-generation sequencing for measurable residual disease monitoring in three major fusion transcript subtypes of B-precursor acute lymphoblastic leukaemia.

The use of next-generation sequencing (NGS) for monitoring measurable residual disease (MRD) in acute lymphoblastic leukaemia (ALL) has been gaining traction. This study aimed to investigate the utility of NGS in MRD monitoring for the three major fusion transcript (FT) subtypes of B-precursor ALL (B-ALL). The MRD results for 104 bone marrow samples from 56 patients were analysed through NGS and real time quantitative reverse transcription PCR (RT-qPCR) for the three major FTs: BCR::ABL1, TCF3::PBX1, and ETV6::RUNX1. To validate the NGS approach, NGS-MRD was initially compared with allele-specific oligonucleotide-qPCR-MRD, and the coefficient of determination was good (R2=0.8158). A subsequent comparison of NGS-MRD with FT-MRD yielded a good coefficient of determination (R2=0.7690), but the coefficient varied by subtype. Specifically, the R2 was excellent for TCF3::PBX1 ALL (R2=0.9157), good for ETV6::RUNX1 ALL (R2=0.8606), and subpar for BCR::ABL1 ALL (R2=0.5763). The overall concordance between the two methods was 83.7%, and an excellent concordance rate of 95.8% was achieved for TCF3::PBX1 ALL. Major discordance, which was defined as a >1 log difference between discordant NGS-MRD and FT-MRD, occurred in 6.7% of the samples, with all but one sample being BCR::ABL1 ALL. Among the four non-transplanted patients with BCR::ABL1-MRD (+)/NGS-MRD (-), three did not relapse after long-term follow-up. Our finding indicates that NGS-MRD has a better prognostic impact than RT-qPCR-MRD in ETV6::RUNX1 and BCR::ABL1 ALL, whereas in TCF3::PBX1 ALL, both methods exhibit comparable efficacy.

[The value of minimal residual disease and IKZF1 deletion for predicting prognosis in adult patients with B-cell acute lymphoblastic leukemia].

Objective: To reassess the prognostic value of minimal residual disease (MRD) and IKZF1 gene deletions in adults with B-cell acute lymphoblastic leukemia (B-ALL) who received pediatric-specific chemotherapy regimens during the Nanfang Hospital PDT-ALL-2016 trial. Methods: We retrospectively analyzed the prognosis of 149 adult patients with B-ALL who were admitted to Nanfang Hospital from January 2016 to September 2020. Prognostic factors were identified using Cox regression models. Results: The complete remission rate was 93.2% in 149 patients, with a 5-year overall survival (OS) rate of (54.3±5.0) % and a cumulative incidence of relapse (CIR) of (47.5±5.2) %. The Cox regression analysis revealed that MRD positivity at day 45 (MRD(3)) after induction therapy was independently associated with relapse risk (HR=2.535, 95%CI 1.122-5.728, P=0.025). Deletion of IKZF1 gene was independently associated with mortality risk (HR=1.869, 95%CI 1.034-3.379, P=0.039). Based on MRD(3) and IKZF1 gene status, we categorized adult patients with B-ALL into the low-risk (MRD(3)-negative and IKZF1 gene deletion-negative) and high-risk (MRD(3)-positive and/or IKZF1 gene wild type) groups. The 5-year OS and CIR rates were (45.5±6.0) % vs (69.4±8.6) % (P<0.001) and (61.6±8.3) % vs (25.5±6.5) % (P<0.001), respectively, in the high-risk and low-risk groups, respectively. The multivariate analysis showed that the high-risk group was an independent risk factor for OS (HR=3.937, 95%CI 1.975-7.850, P<0.001) and CIR (HR=4.037, 95%CI 2.095-7.778, P<0.001) . Conclusion: The combined use of MRD and IKZF1 gene in prognostic stratification can improve clinical outcome prediction in adult patients with B-ALL, helping to guide their treatment.

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.

Flow cytometric minimal residual disease measurement accounting for cytogenetics in children with non-high-risk acute lymphoblastic leukemia treated according to the ALL-MB 2008 protocol.

BACKGROUND: Quantitative measurement of minimal residual disease (MRD) is the "gold standard" for estimating the response to therapy in childhood B-cell precursor acute lymphoblastic leukemia (BCP-ALL). Nevertheless, the speed of the MRD response differs for different cytogenetic subgroups. Here we present results of MRD measurement in children with BCP-ALL, in terms of genetic subgroups with relation to clinically defined risk groups. METHODS: A total of 485 children with non-high-risk BCP-ALL with available cytogenetic data and MRD studied at the end-of-induction (EOI) by multicolor flow cytometry (MFC) were included. All patients were treated with standard-risk (SR) of intermediate-risk (ImR) regimens of "ALL-MB 2008" reduced-intensity protocol. RESULTS AND DISCUSSION: Among all study group patients, 203 were found to have low-risk cytogenetics (ETV6::RUNX1 or high hyperdiploidy), while remaining 282 children were classified in intermediate cytogenetic risk group. For the patients with favorable and intermediate risk cytogenetics, the most significant thresholds for MFC-MRD values were different: 0.03% and 0.04% respectively. Nevertheless, the most meaningful thresholds were different for clinically defined SR and ImR groups. For the SR group, irrespective to presence/absence of favorable genetic lesions, MFC-MRD threshold of 0.1% was the most clinically valuable, although for ImR group the most informative thresholds were different in patients from low-(0.03%) and intermediate (0.01%) cytogenetic risk groups. CONCLUSION: Our data show that combining clinical risk factors with MFC-MRD measurement is the most useful tool for risk group stratification of children with BCP-ALL in the reduced-intensity protocols. However, this algorithm can be supplemented with cytogenetic data for part of the ImR group.