Flow Cytometric MRD Detection in Selected Mature B-Cell Malignancies.

The quantification of submicroscopic minimal residual disease (MRD) after therapy proved to have independent prognostic significance in many mature B-cell malignancies. With the advent of routine benchtop cytometers capable of simultaneously analyzing ≥8 colors and with improved standardization, flow cytometry has become the method of choice for MRD assessments in some lymphoma entities. Herein we describe general aspects of flow cytometric standardization. Chronic lymphocytic leukemia (CLL) and multiple myeloma (MM) are used as examples to explain the technical standardization of flow cytometry for MRD detection according to EuroFlow strategies. MRD data acquisition and detailed analysis in MM and CLL is a particular focus of this chapter.

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

Minimal residual disease (MRD) diagnostics is of high clinical relevance in patients with indolent B-cell non-Hodgkin lymphomas (B-NHL), B-cell chronic lymphocytic leukemia (CLL), and multiple myeloma and serves as a surrogate parameter to evaluate treatment effectiveness and long-term prognosis. Real-time quantitative PCR (RQ-PCR) targeting circulating lymphoma cells is still the gold standard for MRD detection in indolent B-NHL 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, CLL, and FL with comparable sensitivity, accuracy, and reproducibility.The most broadly applicable MRD target in B-NHL is the junctional regions of the rearranged immunoglobulin heavy (IGH) and light chain genes. Complete and incomplete IGH and additionally IG kappa light chain rearrangements can be used as targets for MRD. Next-generation sequencing (NGS) of IG-rearrangements (IG-NGS) as new sequencing-based technology can overcome the limitation of PCR-based approaches and has a potential for higher sensitivity. Chromosomal translocations like the t(14;18)(q32;q21) translocation associated with IGH::BCL2 fusion in FL and t(11;14)(q13;q32) translocation in MCL leading to the IGH::CCND1 fusion can be used as MRD target in selected lymphoma subtypes. In patients with CLL, both flow-cytometry and RQ-PCR are equally suited for MRD assessment as long as a sensitivity of 10-4 is achieved.MRD diagnostics targeting the IG loci is complex and requires extensive knowledge and experience because the junctional regions of each clonal rearranged gene have to be identified before the patient-specific PCR assays can be designed for MRD monitoring. In addition, the presence and load of somatic hypermutation within the rearranged IGH gene occurring during B-cell development of germinal center and post-germinal center B-cell 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. PCR-based methods using allele-specific primers can reach a high sensitivity of up to 10-5. This chapter provides all relevant background information and technical aspects for the complete laboratory process from detection of the clonal IG gene rearrangements 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. This is available by the EuroMRD network ( https://euromrd.org ), a subgroup of ESHLO ( https://eslho.org ).

Immunoparesis recovery in newly diagnosed transplant ineligible multiple myeloma patients, an independent prognostic factor that complements minimal residual disease.

Information on the prognostic value of immunoparesis (IP) recovery in multiple myeloma (MM) patients has been only generated in some observational and retrospective studies. We have evaluated the prognostic impact of IP recovery and its association with minimal residual disease (MRD) in a series of 113 newly diagnosed transplant-ineligible (NDTI) patients, that received fix duration treatment (18 cycles of VMP/lenalidomide-dexamethasone) within the PETHEMA/GEM2010MAS65 trial and who achieved CR or VGPR. Immunoglobulin levels were measured at diagnosis, at the end of treatment (after cycle 18th) and during subsequent follow up whereas MRD was analyzed only at the end of the treatment (after cycle 18th). We found that patients who had IP at diagnosis and recovered it during or after treatment had longer progression free survival (PFS) [p < 0.001; HR 0.32 (0.19-0.52)] and longer overall survival (OS) [p = 0.007; HR 0.40 (0.20-0.80)] compared to those who failed to recover it. When we analyzed IP recovery in MRD negative patients, we found that those cases with IP recovery had longer PFS [p = 0.007; HR 0.31 (0.13-0.76)] and longer OS [p = 0.012; HR 0.21 (0.06-0.80)] as compared to MRD negative patients but without IP recovery. In conclusion, IP recovery confers better prognosis in NDTI-MM patients with fixed duration treatment who achieve CR or VGPR and the prognostic value of MRD can be complemented when combined with IP recovery.

Maintenance therapy promotes profound organ and haematologic response in light-chain amyloidosis patients not undergoing autologous stem cell transplantation.

BACKGROUND: Evidence on maintenance therapy following frontline induction is sparse in primary light-chain amyloidosis (AL), especially for those who do not undergo autologous haematopoietic stem cell transplantation (ASCT). METHODS: We enrolled primary AL patients who achieved at least haematologic very good partial response (VGPR) by the 4th month at the frontline from December 2008 to June 2023 at Zhongshan Hospital, Fudan University. Those who followed maintenance therapy were identified as the maintenance group (n = 44), whereas those entering the observational phase were classified as the observational group (n = 24). RESULTS: After 7.2(interquartile range, 4.7-18.6) months of maintenance therapy, 9(20.5%), 14(31.8%) and 5 (11.4%) patients achieved improvement in haematologic, cardiac and renal response respectively. Five (11.4%) patients had minimal residual disease (MRD) response conversion from positive to negative based on maintenance therapy. In the observation group, none of the patients had haematologic response improvement, with 3(12.5%) and 1(4.2%) patient showing cardiac and renal response improvement during follow-up. CONCLUSION: This study identified the clinical benefits of maintenance therapy in patients with AL who did not undergo ASCT in real-world practice.

The treatment response evaluation through the combination of contrast-enhanced ultrasound and squamous cell carcinoma antigen in cervical cancer.

Background: The evaluation of the treatment response after concurrent chemotherapy and radiotherapy (CCRT) for locally advanced cervical cancer is closely related to the formulation of treatment strategies. Magnetic resonance imaging (MRI) is a recommended method for efficacy evaluation; however, a unified consensus has not yet been reached on its use, and it has its limitations. This study aimed to evaluate the diagnostic value of a combination of contrast-enhanced ultrasound (CEUS) parameters and the squamous cell carcinoma antigen (SCC-Ag) to establish another efficient and feasible examination method. Methods: The data of 94 patients with cervical cancer who underwent transvaginal contrast-enhanced ultrasound (TV-CEUS) from October 2020 to March 2023 were retrospectively collected. Based on the inclusion and exclusion criteria, 70 patients diagnosed with cervical squamous cell carcinoma (SCC) who underwent CCRT were selected for inclusion in the study. The patients were divided into the residual disease (RD) group (comprising 26 patients) and the complete response (CR) group (comprising 44 patients) according to the diagnostic standard. Data on the grayscale echogenicity, color Doppler flow imaging (CDFI), CEUS parameters, and the SCC-Ag of all the patients were collected by two experienced radiologists. Inter-observer reliability was assessed using the intraclass correlation coefficient (ICC). Receiver operating characteristic (ROC) curves were created based on the non-parametric U-test or t-test results for the two groups. Delong's test was used to compare the area under the curve (AUC) between different ROC curves. A subgroup analysis was conducted based on the patient's age, tumor diameter, and disease stage. Results: The ICCs between the two observers ranged from 0.915 and 0.947. Hypervascular hyper-enhancement in the arterial phase, hypo-enhancement in the venous phase, and the SCC-Ag differed significantly between the RD and CR groups (P<0.05). The AUC of the ROC curve combining these indicators was 0.890 [95% confidence interval (CI): 0.792-0.989], which was higher than the AUC of any indicator alone (P<0.05). The subgroup analysis showed that the AUCs of the patients aged ≥53 and <53 years were 0.922 (95% CI: 0.816-1.00) and 0.896 (95% CI: 0.782-1.00), respectively, those of the patients with stage II, III, and IV were 0.881 (95% CI: 0.732-1.00), 0.955 (95% CI: 0.894-1.00), and 1.000 (95% CI: 1.00-1.00), respectively, and those of the patients with a tumor diameter ≤10 mm, 10 mm < tumor diameter (post) <20 mm, and tumor diameter (post) ≥20 mm were 0.976 (95% CI: 0.910-1.00), 0.883 (95% CI: 0.763-1.00), and 1.00 (95% CI: 1.00-1.00) respectively. Conclusions: Transvaginal ultrasound (TVUS), TV-CEUS, and the SCC-Ag can be used in combination to evaluate the patient response to CCRT in locally advanced cervical SCC. This integrated approach enhanced the accuracy of the diagnosis of residual lesions and may be helpful in treatment plan optimization.

[Basics of FCM analysis for leukemia diagnosis].

Flow cytometry (FCM) remains an essential test in the diagnosis of leukemia despite advances in genomic testing. However, the role of FCM results as a risk factor is already extremely limited. International diagnostic criteria for leukemia already prioritize diagnosis based on genetic abnormalities, with FCM diagnosis only serving as an aid to morphological diagnosis for subtypes without genetic abnormalities. However, rapid lineage diagnosis of leukemia by FCM remains important for selecting initial treatment. FCM is also an important tool for evaluating response to molecular targeted therapy, which requires repeated measurements and rapid results. Furthermore, FCM enables prediction of specific genetic abnormalities by immunophenotypic patterns, which could make it useful for verifying the clinical impact of genetic abnormalities detected by multi-gene panel testing.

Measurable residual disease assessment prior to allogeneic hematopoietic stem cell transplantation in acute myeloid leukemia and myelodysplastic syndromes: a 20-year monocentric study.

Patients with acute myeloid leukemia (AML) or myelodysplastic syndromes (MDS) who undergo allogeneic hematopoietic stem-cell transplantation (alloHSCT) can have divergent survival outcomes while all in morphological complete remission (CR). Techniques of measurable residual disease (MRD) have allowed us to refine their prognosis in two categories: MRD-positive and MRD-negative patients. We conducted a monocentric retrospective study (01/2000-12/2020) to assess the prognosis of pretransplant MRD status measured by multiparametric flow cytometry (MFC) and molecular biology assessed by PCR. 192 patients were included. The median follow-up period was 77 months. Among patients undergoing alloHSCT in CR, overall survival (median-OS: 130.6 vs. 16.0 months, P < 0.001), disease-free survival (median-DFS: 109.6 vs. 7.1 months, P < 0.001) and cumulative incidence of relapse (12-month CIR: 7.3% vs. 33.7%, P < 0.0001) were significantly different between MRD-negative and MRD-positive patients. Patients with discordant intermethod results had intermediate DFS. MRD-negative patients according to molecular PCR-based techniques, WT1 overexpression and MFC had longer median-DFS, compared to MRD-positive patients (P = 0.001, P < 0.001, P < 0.001, respectively). Looking into subgroups, MRD-positive patients among the ELN2017 adverse-category (P < 0.0001), myeloablative and reduced-intensity conditioning regimens (P < 0.0001, P = 0.005), < 60-year patients (P < 0.001) and AML patients (P < 0.001) were associated with lower DFS. This difference was not found in ≥ 60-year patients (P = 0.27) and MDS patients (P = 0.70). MRD-positive patients within the favorable/intermediate ELN2017 category trended toward lower DFS (P = 0.05). We confirmed that MRD status prior to alloHSCT is a strong prognostic factor for OS, DFS and CIR. Combining MFC and molecular-PCR techniques to assess MRD seems primordial as inter-method discordance can be consequential.

Impact of pre-infusion disease burden on outcomes in pediatric relapsed/refractory B-cell lymphoblastic leukemia following anti-CD19 CAR T-cell therapy.

Anti-CD19 chimeric antigen receptor (CAR) T-cell therapies have demonstrated high efficacy in pediatric patients with relapsed/refractory (R/R) B-cell acute lymphoblastic leukemia (B-ALL). Despite this success, the challenge of post-infusion relapse persists. In our study, we evaluate 116 children with R/R B-ALL who received anti-CD19 CAR T-cell therapy at our center. All patients were included in the response analysis and assessed for survival and toxicity. The CR rate was 98.3%, with 90.5% achieving minimal residual disease negative (MRD)- CR by day 28 (d28). The overall survival (OS) and event-free survival (EFS) were 69.3%±4.5% and 59.0%±4.6%, respectively, with a median follow-up duration of 47.9 months. The patients with pre-infusion MRD ≥ 1% was associated with lower 4-year OS (p = 0.006) and EFS (p = 0.027) comparing to those with MRD < 1%. The incidences of grade ≥ 3 cytokine release syndrome (CRS) and neurotoxicity were21.6 and 5.0%, respectively. Therefore, pre-infusion disease burden is a predictor of long-term outcome following anti-CD19 CAR T-cell therapy for pediatric R/R B-ALL.

Are Positive Biopsy Margins in Melanoma Significant?: A Cohort Study of Micro- Versus Macroscopic Margin Status and Their Impact on Residual Disease and Survival.

BACKGROUND: Presence of positive biopsy margins in melanoma can provoke anxiety over potential disease progression from delays to surgical excision, but their impact on outcomes is unknown. We aimed to compare the presence of residual melanoma in the surgical excision specimen and survival between patients with negative, microscopically positive, and macroscopically positive biopsy margins. METHODS: Patients with cutaneous melanoma who underwent surgical excision over a 13-year period were included. Biopsy characteristics, residual disease in the surgical specimen, and overall and recurrence-free survival were compared between patients with negative, microscopically positive (only scar visible), and macroscopically positive (visible remaining melanoma) biopsy margins. RESULTS: Of 901 patients, 42.4%, 33.3%, and 24.3% had negative, microscopically positive, and macroscopically positive margins, respectively. The incidence of residual invasive melanoma in the surgical specimen varied (P < 0.001), occurring in 5.5%, 17.0%, and 74.9% of patients, respectively. Both microscopically and macroscopically positive margins were associated with residual disease (P < 0.001) but only the latter predicted worse overall (P = 0.013) and recurrence-free survival (P = 0.009). Kaplan-Meier estimated survival was comparable between those with negative and microscopically positive margins, but overall (P = 0.006) and recurrence-free survival (P = 0.004) were significantly worse in the macroscopically positive margin group. These patients had worse prognosis melanoma, with 33.8% being stage III disease, and 23.2% having positive sentinel lymph nodes. CONCLUSIONS: Patients and physicians may be reassured in the presence of microscopically positive biopsy margins which are not associated with worse survival, However, patients with macroscopically positive margins have poorer prognosis and should be treated within an acceptable time frame.

Clinical validation of a tissue-agnostic genome-wide methylome enrichment molecular residual disease assay for head and neck malignancies.

BACKGROUND: Outcomes for patients with locally advanced head and neck cancer (HNC) treated with curative intent remain disappointing, with 5-year survival rates at 50%. Most recurrences occur within the first 2 years after treatment, providing a window of opportunity to identify patients with molecular residual disease (MRD). A tissue-agnostic test for MRD detection in patients with human papillomavirus (HPV) positive and negative HNC, where tissue is often scarce, is needed. PATIENTS AND METHODS: Patients with stage I-IVB HNC, including patients positive and negative for HPV, were enrolled and peripheral blood plasma was collected longitudinally at diagnosis and ∼3, 12, and 24 months after curative intent treatment. The full cohort includes 325 patients with 1155 samples. Samples were split into distinct sets to train and validate a classifier capable of identifying MRD using a tissue-agnostic genome-wide methylome enrichment platform. The primary endpoint was recurrence-free survival (RFS). RESULTS: With a median follow-up of 60 months, patients in the blinded validation set with MRD positivity experienced significantly worse RFS with a hazard ratio (HR) of 35.7 [95% confidence interval (CI) 10.8-117.8; P < 0.0001]. For patients with HPV negativity, HR was 42.3 (95% CI 9.8-182.3; P < 0.0001); for patients with HPV-positive oropharyngeal cancer, HR was 24.1 (95% CI 3.0-196.8; P < 0.0001). Moreover, the lead time between MRD positivity and clinical recurrence was up to 14.9 months, with a mean lead time of 4.1 months. Surveillance sensitivity was 91% (95% CI 77% to 97%) and specificity was 88% (95% CI 80% to 93%). CONCLUSIONS: Here we validate the clinical performance characteristics of a tissue-agnostic genome-wide methylome enrichment assay for MRD detection in patients with HNC. The MRD detection test showed high sensitivity for identifying recurrence at high specificity across different anatomical sites, HPV status, and treatment regimens, highlighting the broad applicability for MRD detection in patients with HNC.

Real-World Evidence on Prognostic Value of MRD in Multiple Myeloma Using Flow Cytometry.

Minimal residual disease (MRD) is one of the most important prognostic factors in multiple myeloma (MM) and a valid surrogate for progression-free survival (PFS) and overall survival (OS). Recently, MRD negativity was approved as an early clinical endpoint for accelerated drug approval in MM. Nevertheless, there is limited evidence of MRD utility in real-world setting. In this retrospective multicenter study, we report outcomes of 331 newly diagnosed MM patients with MRD evaluation at Day+100 after autologous stem cell transplantation using flow cytometry with a median limit of detection of 0.001%. MRD negativity was reached in 47% of patients and was associated with significantly prolonged median PFS (49.2 months vs. 18.4 months; hazard ratios (HR) = 0.37; p < 0.001) and OS (not reached vs. 74.9 months; HR = 0.50; p = 0.007). Achieving MRD negativity was associated with PFS improvements regardless of age, International Staging System (ISS) stage, lactate dedydrogenase (LDH) level, or cytogenetic risk. Importantly, MRD positive patients benefited from lenalidomide maintenance versus no maintenance (18-months PFS: 81% vs. 46%; HR = 0.24; p = 0.002) while in MRD negative patients such benefit was not observed (p = 0.747). The outcomes of our real-world study recapitulate results from clinical trials including meta-analyses and support the idea that MRD positive patients profit more from lenalidomide maintenance than MRD negative ones.

Prognostic impact of microscopic residual disease after neoadjuvant chemotherapy in patients undergoing interval debulking surgery for advanced ovarian cancer.

PURPOSE: To determine the prognostic impact of microscopic residual disease after neoadjuvant chemotherapy (NACT) in patients undergoing interval debulking surgery (IDS) for advanced epithelial ovarian cancer (AEOC). METHODS: Patients affected by FIGO stage IIIC-IV ovarian cancer undergoing IDS between October 2010 and April 2016 were selected. Progression-free survival (PFS) and overall survival (OS) were estimated using the Kaplan-Meier analysis. RESULTS: In total, 98 patients were identified. Four patients (4.1%) were considered inoperable. Overall, 67 patients (out of 94; 71.3%) had macroscopic disease, equating Chemotherapy Response Score (CRS) 1 and 2, 7 (7.4%) had microscopic residuals, equating CRS3, rare CRS2, while 20 (21.3%) had both microscopic and macroscopic disease. Median OS and PFS were, respectively, 44 and 14 months in patients with no macroscopic residual disease (RD = 0) compared to 25 and 6 months, in patients with RD > 0 (OS: p = 0.001; PFS: p = 0.002). The median PFS was 9 months compared to 14 months for patients with more or less than 3 areas of microscopic disease at final pathologic evaluation (p = 0.04). The serum Ca125 dosage after NACT was higher in patients with RD > 0 compared to those without residue (986.31 ± 2240.7 µg/mL vs 215.72 ± 349.5 µg/mL; p = 0.01). CONCLUSION: Even in the absence of macroscopic disease after NACT, the persistence of microscopic residuals predicts a poorer prognosis among AEOC patients undergoing IDS, with a trend towards worse PFS for patients with more than three affected areas. Removing all fibrotic residuals eventually hiding microscopic disease during IDS represents the key to improving the prognosis of these patients.

Safety and efficacy of consolidative stereotactic radiotherapy for oligo-residual EGFR-mutant non-small cell lung cancer after first-line third-generation EGFR-tyrosine kinase inhibitors: a single-arm, phase 2 trial.

Background: Prospective data is limited on the efficacy and safety of consolidative stereotactic radiotherapy (SRT) in metastatic epidermal growth factor receptor (EGFR)-mutant non-small cell lung cancer (NSCLC) patients harboring oligo-residual disease (ORD) after first-line third-generation EGFR-tyrosine kinase inhibitors (TKIs). Methods: In this single-arm, phase II trial, 61 patients from two academic centers were enrolled from March 2021 to March 2023. All these patients had metastatic EGFR-mutant NSCLC and harbored ORD after first-line third-generation EGFR-TKIs. Consolidative SRT was performed and EGFR-TKIs were not held during SRT. The primary endpoint was progression-free survival (PFS) and the secondary endpoints included overall survival and treatment-related adverse events (TRAEs). A prespecified propensity score matched (PSM) comparison was conducted with a contemporary cohort of patients who developed ORD but received EGFR-TKIs alone. This trial was registered with ClinicalTrails.gov, NCT04764214. Findings: All patients received consolidative SRT. With a median follow-up of 21.1 months, the median PFS was 29.9 (80% CI 22.4-32.4) months and the lower boundary exceeded the predefined threshold, meeting the primary endpoint. TRAEs occurred in 43 (70%) patients, with pneumonitis (27.9%) and esophagitis (26.2%) being the most common toxicities. Four patients (6.6%) reported grade ≥3 TRAEs, each for pneumonitis, esophagitis, leukopenia, and cranial radiation necrosis. PSM analysis showed significantly prolonged PFS in EGFR-TKI + SRT group compared to EGFR-TKI group (HR 0.46, 80% CI 0.20-0.61; p = 0.002). Interpretation: Consolidative SRT is associated with an encouraging PFS in first-line third-generation EGFR-TKI-treated metastatic NSCLC patients harboring ORD, with generally acceptable toxicities. Further confirmatory studies are warranted. Funding: Hui Lan Public Welfare and the Chinese Society of Clinical Oncology Foundation.

Minimal Residual Disease Detection with Urine-derived DNA Is Prognostic for Recurrence-free Survival in Bacillus Calmette-Guérin-unresponsive Non-muscle-invasive Bladder Cancer Treated with Nadofaragene Firadenovec.

BACKGROUND AND OBJECTIVE: Urinary tumor DNA (utDNA) profiling identifies mutations associated with urothelial carcinoma and can be used to detect minimal residual disease (MRD). We evaluate the utility of utDNA profiling to predict treatment failure in bacillus Calmette-Guérin-unresponsive high-grade (HG) non-muscle-invasive bladder cancer (NMIBC) treated with nadofaragene firadenovec. METHODS: Urine was collected from participants prior to induction (n = 32) and at their 3-mo evaluation (n = 18) in the parallel-arm, phase 2 study (NCT01687244) of nadofaragene firadenovec. The UroAmp MRD assay (Convergent Genomics, South San Francisco, CA, USA) was used to perform utDNA testing. Risk of HG NMIBC recurrence was determined using two algorithm versions, and recurrence-free survival (RFS) was assessed using a Kaplan-Meier analysis. KEY FINDINGS AND LIMITATIONS: TP53, TERT, PIK3CA, ARID1A, PLEKHS1, ELF3, and ERBB2 were the most prevalently mutated genes. With pretreatment urine, the validated MRD algorithm resulted in 12-mo RFS of 56% for negative and 22% for positive patients (p = 0.097). The experimental, enhanced algorithm classified two additional patients as positive, giving RFS of 71% for negative and 20% for positive patients (p = 0.012). With 3-mo urine, both algorithms gave RFS of 100% for negative and 38% for positive patients (p = 0.038). Longitudinal utDNA testing classified patients as negative (7%), complete responders (13%), partial responders (27%), unresponsive (20%), and expanding (33%). CONCLUSIONS AND CLINICAL IMPLICATIONS: Urinary MRD testing after nadofaragene firadenovec induction provided statistically significant prognostication of recurrence among phase 2 trial participants. PATIENT SUMMARY: By analyzing urine-borne tumor DNA, we can help determine which patients with high-grade non-muscle-invasive bladder cancer are at the greatest risk of recurrence when receiving second-line therapy.

Longitudinal monitoring of circulating tumor DNA to detect relapse early and predict outcome in early breast cancer.

PURPOSE: Detection of molecular residual disease (MRD) allows for the identification of breast cancer patients at high-risk of recurrence, with the potential that early initiation of treatment at early stages of relapse could improve patient outcomes. The Invitae Personalized Cancer Monitoring™ assay (PCM) is a newly developed next-generation sequencing approach that utilizes up to 50 patient-specific, tumor-informed DNA variants, to detect circulating tumor DNA (ctDNA). The ability of the PCM assay to detect MRD before clinical relapse was evaluated. METHODS: The cohort included 61 female patients with high-risk breast cancer who underwent neoadjuvant chemotherapy. Plasma samples were collected before and during neoadjuvant therapy, after surgery and during monitoring. PCM was used to detect ctDNA at each time point. RESULTS: The sensitivity to detect ctDNA in plasma from patients who relapsed during the monitoring phase was 76.9% (10/13). Specificity and positive predictive values were both 100% with all (10/61, 16%) of the patients who had ctDNA detected during the monitoring phase subsequently relapsing. Detection of ctDNA during monitoring was associated with a high-risk of future relapse (HR 37.2, 95% CI 10.5-131.9, p < 0.0001), with a median lead-time from ctDNA detection to clinical relapse of 11.7 months. CONCLUSION: PCM detected ctDNA in patients who relapsed with a long lead-time over clinical relapse, shows strong association with relapse-free survival and may be used to identify patients at high-risk for relapse, allowing for earlier intervention.

A new face in the treatment of Philadelphia chromosome-positive acute lymphoblastic leukaemia.

The prognosis for patients with relapsed/refractory or measurable (minimal) residual disease-positive Philadelphia chromosome (Ph)-positive acute lymphoblastic leukaemia (ALL) is poor. Currently, ponatinib is the only approved tyrosine kinase inhibitor (TKI) that is effective for Ph-positive ALL with the T315I mutation. Although the report by Liu et al. is a retrospective observational study, it offers prospects for the efficacy of chemotherapy combined with the novel third-generation TKI, olverembatinib, in these conditions, which may be validated in future prospective clinical trials. Commentary on: Liu et al. Efficacy and safety of olverembatinib in adult BCR::ABL1-positive ALL with T315I mutation or relapsed/refractory disease. Br J Haematol 2024 (Online ahead of print). doi: 10.1111/bjh.19804.

Microfluidic Affinity Selection of B-Lineage Cells from Peripheral Blood for Minimal Residual Disease Monitoring in Pediatric B-Type Acute Lymphoblastic Leukemia Patients.

Assessment of minimal residual disease (MRD) is the most powerful predictor of outcome in B-type acute lymphoblastic leukemia (B-ALL). MRD, defined as the presence of leukemic cells in the blood or bone marrow, is used for the evaluation of therapy efficacy. We report on a microfluidic-based MRD (MF-MRD) assay that allows for frequent evaluation of blood for the presence of circulating leukemia cells (CLCs). The microfluidic chip affinity selects B-lineage cells, including CLCs using anti-CD19 antibodies poised on the wall of the microfluidic chip. Affinity-selected cells are released from the capture surface and can be subjected to immunophenotyping to enumerate the CLCs, perform fluorescence in situ hybridization (FISH), and/or molecular analysis of the CLCs' mRNA/gDNA. During longitudinal testing of 20 patients throughout induction and consolidation therapy, the MF-MRD performed 116 tests, while only 41 were completed with multiparameter flow cytometry (MFC-MRD) using a bone marrow aspirate, as standard-of-care. Overall, 57% MF-MRD tests were MRD(+) as defined by CLC numbers exceeding a threshold of 5 × 10-4%, which was determined to be the limit of quantitation. Above a threshold of 0.01%, MFC-MRD was positive in 34% of patients. The MF offered the advantage of the opportunity for efficiently processing small volumes of blood (2 mL), which is important in the care of pediatric patients, especially infants. The minimally invasive means of blood collection are of high value when treating patients whose MRD is typically tested using an invasive bone marrow biopsy. MF-MRD detection can be useful for stratification of patients into risk groups and monitoring of patient well-being after completion of treatment for early recognition of potential impending disease recurrence.

Measurable Residual Disease Testing in Multiple Myeloma Following T-Cell Redirecting Therapies.

Several novel T-cell-based therapies have recently become available for multiple myeloma (MM). These T-cell redirecting therapies (TRTs) include chimeric antigen receptor T-cells (CAR-T) and bispecific antibodies (BiAbs). In both clinical trial and real-world data, these therapies have demonstrated high rates of deep clinical response, and some are now approved for second-line treatment for relapsed MM. The deep and sustained clinical responses these therapies are capable of inducing will require sophisticated response monitoring to provide meaningful information for patient care. Obtaining measurable residual disease (MRD) negativity has been validated as an independent positive prognostic marker for progression-free survival (PFS) and overall survival (OS) in both newly diagnosed and relapsed refractory patients with multiple myeloma. Assessment for MRD negativity was performed in all of the trials for FDA-approved TRT. Here, we summarize pertinent data for MRD assessment following TRT in MM and provide a rationale and structured framework for conducting MRD testing post TRT.

The significance of MRD-based strategy by dynamic assessment to guide treatment decisions in B-ALL - the enlightenment provided by demonstrating survival differences in the retrospective study.

PURPOSE: The study aimed to access the impact of related factors on the long-term survival of patients with B-cell acute lymphoblastic leukemia (B-ALL) by analyzing clinical characteristics of B-ALL patients, and observed the significance of dynamic measurable residual disease (MRD) assessment for the prognosis and treatment selection of B-ALL patients, aiming to deepen the understanding of the disease and improve the survival prognosis of B-ALL patients. METHODS: The clinical characteristics of 65 patients with B-ALL were collected to calculate the median overall survival (OS) and median disease-free survival (PFS), and to evaluate the significance of survival analysis guided by dynamic MRD assessment. RESULTS: The survival analysis based on dynamic MRD assessment suggested that B-ALL patients who maintained MRD negative status during dynamic assessment after induction chemotherapy had better survival outcomes in the comparison of progression-free survival between subgroups, with statistically significant differences (P = 0.0002 (HR: 0.26, 95% CI: 0.13-0.51)). High-risk B-ALL patients who maintained negative MRD status during dynamic assessment after induction chemotherapy had longer median progression - free survival, and the survival difference between subgroups was statistically significant (P = 0.0016 (HR: 0.28, 95% CI: 0.09-0.48)). CONCLUSION: Dynamic MRD assessment had significant clinical value: maintaining negative MRD status during dynamic assessment can improve the prognosis and survival of B-ALL patients; dynamic MRD assessment after induction chemotherapy can help guide subsequent treatment, which could provide reference for the advancement of future treatment strategies.

Feasibility of ctDNA in detecting minimal residual disease and predicting recurrence for colorectal cancer liver metastases.

Introduction: Approximately 50% of patients diagnosed with colorectal cancer develop colorectal cancer liver metastases (CRLM). Although curative intent liver resection provides 5-year survival of 40-50%, up to 70% of patients develop recurrence of CRLM. Detection of minimal residual disease (MRD) is essential for timely, optimized treatment. This study evaluated the feasibility and utility of using circulating tumor DNA (ctDNA) to identify MRD and predict disease recurrence. Methods: Patients with CRLM that underwent liver resection and had known KRAS or PIK3CA mutations were retrospectively identified. Serial blood samples were collected every 3 months following surgery for disease surveillance. ctDNA was isolated from the samples and analyzed with digital PCR (dPCR). Results: KRAS and PIK3CA mutations were identified by dPCR in 29 patients over 115 timepoints. In patients with detectable ctDNA at time of liver resection, 81% (13/16) developed disease recurrence, while 46% (6/13) of the patients with undetectable ctDNA recurred (p=0.064). Presence of ctDNA was detected in 27.6% (8/29) of the initial postoperative samples. Radiologic recurrence was later diagnosed in 100% (8/8) of these patients, while 52% (11/21) who had undetectable ctDNA postoperatively recurred (p=0.026). Detectable ctDNA postoperatively was associated with a shorter disease-free survival (DFS) of 9 months vs 13 months in patients who had undetectable ctDNA (HR 2.95, 95% CI 1.16-7.49; p=0.02). Conclusion: Liquid biopsy using dPCR can identify low levels of ctDNA, enabling early detection of disease recurrence. Additionally, the presence of ctDNA postoperatively was predictive of recurrence. This study corroborates current literature and provides rational for moving toward a clinical trial using ctDNA and dPCR to detect MRD after CRLM resection.