Circulating tumor plasma cells and peripheral blood measurable residual disease assessment in multiple myeloma patients not planned for upfront transplant.

Circulating tumor plasma cells (CTPCs) provide a noninvasive alternative for measuring tumor burden in newly diagnosed multiple myeloma (NDMM). Moreover, measurable residual disease (MRD) assessment in peripheral blood (PBMRD) can provide an ideal alternative to bone marrow MRD, which is limited by its painful nature and technical challenges. However, the clinical significance of PBMRD in NDMM still remains uncertain. Additionally, data on CTPC in NDMM patients not treated with transplant are scarce. We prospectively studied CTPC and PBMRD in 141 NDMM patients using highly sensitive multicolor flow cytometry (HS-MFC). PBMRD was monitored at the end of three cycles (PBMRD1) and six cycles (PBMRD2) of chemotherapy in patients with detectable baseline CTPC. Patients received bortezomib-based triplet therapy and were not planned for an upfront transplant. Among baseline risk factors, CTPC ≥ 0.01% was independently associated with poor progression-free survival (PFS) (hazard ratio [HR] = 2.77; p = 0.0047) and overall survival (OS) (HR = 2.9; p = 0.023) on multivariate analysis. In patients with detectable baseline CTPC, undetectable PBMRD at both subsequent time points was associated with longer PFS (HR = 0.46; p = 0.0037), whereas detectable PBMRD at any time point was associated with short OS (HR = 3.25; p = 0.004). Undetectable combined PBMRD (PBMRD1 and PBMRD2) outperformed the serum-immunofixation-based response. On multivariate analysis, detectable PBMRD at any time point was independently associated with poor PFS (HR = 2.0; p = 0.025) and OS (HR = 3.97; p = 0.013). Thus, our findings showed that CTPC and PBMRD assessment using HS-MFC provides a robust, noninvasive biomarker for NDMM patients not planned for an upfront transplant. Sequential PBMRD monitoring has great potential to improve the impact of the existing risk stratification and response assessment models.

15-color highly sensitive flow cytometry assay for post anti-CD19 targeted therapy (anti-CD19-CAR-T and blinatumomab) measurable residual disease assessment in B-lymphoblastic leukemia/lymphoma: Real-world applicability and challenges.

OBJECTIVES: Measurable residual disease (MRD) is the most relevant predictor of disease-free survival in B-cell acute lymphoblastic leukemia (B-ALL). We aimed to establish a highly sensitive flow cytometry (MFC)-based B-ALL-MRD (BMRD) assay for patients receiving anti-CD19 immunotherapy with an alternate gating approach and to document the prevalence and immunophenotype of recurrently occurring low-level mimics and confounding populations. METHODS: We standardized a 15-color highly-sensitive BMRD assay with an alternate CD19-free gating approach. The study included 137 MRD samples from 43 relapsed/refractory B-ALL patients considered for anti-CD19 immunotherapy. RESULTS: The 15-color BMRD assay with CD22/CD24/CD81/CD33-based gating approach was routinely applicable in 137 BM samples and could achieve a sensitivity of 0.0005%. MRD was detected in 29.9% (41/137) samples with 31.7% (13/41) of them showing <.01% MRD. Recurrently occurring low-level cells that showed immunophenotypic overlap with leukemic B-blasts included: (a) CD19+CD10+CD34+CD22+CD24+CD81+CD123+CD304+ plasmacytoid dendritic cells, (b) CD73bright/CD304bright/CD81bright mesenchymal stromal/stem cells (CD10+) and endothelial cells (CD34+CD24+), (c) CD22dim/CD34+/CD38dim/CD81dim/CD19-/CD10-/CD24- early lymphoid progenitor/precursor type-1 cells (ELP-1) and (d) CD22+/CD34+/CD10heterogeneous/CD38moderate/CD81moderate/CD19-/CD24- stage-0 B-cell precursors or ELP-2 cells. CONCLUSIONS: We standardized a highly sensitive 15-color BMRD assay with a non-CD19-based gating strategy for patients receiving anti-CD19 immunotherapy. We also described the immunophenotypes of recurrently occurring low-level populations that can be misinterpreted as MRD in real-world practice.

Treatment and follow-up of children with chronic myeloid leukaemia in chronic phase (CML-CP) in the tyrosine kinase inhibitor (TKI) era-Two decades of experience from the Tata Memorial Hospital paediatric CML (pCML) cohort.

Tyrosine kinase inhibitors (TKIs) have drastically improved the outcomes of pCML (paediatric CML) but data on long-term off-target toxicities of TKIs in children are scarce. In this single-centre, retrospective cum prospective study of pCML in chronic phase, we report our experience of treating 173 children with imatinib and following them for long-term toxicities. Mean (SD) time to attain CHR, CCyR and MMR were 3.05 (2.1), 10.6 (8.4) and 43.4 (31.8) months respectively. DMR was not attained in 59 (34%) patients at last follow-up. Ten patients were switched to second-generation TKIs (2G-TKIs; nilotinib = 1/dasatinib = 9) due to poor/loss in response, of which seven had kinase domain mutations. Three patients progressed to the blastic phase. At a median follow-up of 84 (3-261) months, the 5-year EFS and OS for the entire cohort were 96.9% (95% CI: 93.4-100) and 98.7% (95% CI: 96.9-100) respectively. Screening for long-term toxicities revealed low bone density and hypovitaminosis D in 70% and 80% respectively. Other late effects included short stature (27%), delayed puberty (15%), poor sperm quality (43%) and miscellaneous endocrinopathies (8%). Children younger than 5 years at diagnosis were more susceptible to growth and endocrine toxicities (p = 0.009). Regular monitoring for long-term toxicities, timely intervention and trial of discontinuation whenever feasible are likely to improve the long-term outlook of pCML.

Long-Term Cryopreservation of Peripheral Blood Stem Cell Harvest Using Low Concentration (4.35%) Dimethyl Sulfoxide with Methyl Cellulose and Uncontrolled Rate Freezing at -80 °C: An Effective Option in Resource-Limited Settings.

Long-term cryopreservation of peripheral blood stem cells (PBSCs) is highly useful in the setting of tandem/multiple transplantations or treatment of relapse in the autologous hematopoietic stem cell transplantation (HSCT) setting. Even in allogeneic HSCT, donor lymphocyte infusions may be stored for months to years if excess stem cells are collected from donors. Cryopreservation is a delicate, complex, and costly procedure, and higher concentrations of dimethyl sulfoxide (DMSO), a commonly used cryoprotectant, can be toxic to cells and cause adverse effects in the recipient during infusions. In this study, we examined the effect of long-term cryopreservation using 4.35% DMSO (as final concentration) with methyl cellulose and uncontrolled rate freezing in a mechanical freezer (-80 °C) on the viability and colony-forming ability of CD34+ human PBSCs. For patients undergoing autologous HSCT, PBSCs were cryopreserved using DMSO (final concentration of 4.35%) with methyl cellulose. The post-thaw viability of PBSCs was determined using Trypan blue exclusion and flow cytometry-based 7-amino-actinomycin-D (FC-7AAD) methods. Concentrations of CD34+ stem cells and immune cell subsets in post-thaw PBSC harvest samples were assessed using multicolor flow cytometry, and the clonogenic potential of post-thaw stem cells was studied using a colony-forming unit (CFU) assay. CD34+ stem cell levels were correlated with the prestorage CD34 levels using the Pearson correlation test. The viability results in the Trypan blue dye exclusion method and the flow cytometry-based method were compared using Bland-Altman plots. We studied 26 PBSC harvest samples with a median cryopreservation duration of 6.6 years (range, 3.8 to 11.5 years). The median viability of post-thaw PBSCs was >80% using both methods, with a weak agreement between them (r = .03; P = .5). The median CD34+ stem cell count in the post-thaw samples was 9.13 × 106/kg (range, .44 to 26.27 × 106/kg). The CFU assay yielded a good proliferation and differentiation potential in post-thaw PBSCs, with a weak correlation between granulocyte macrophage CFU and CD34+ stem cell levels (r = .4; P = .05). Two samples that had been cryopreserved for >8 years showed low viability. Cryopreservation of PBSCs using 4.35% DMSO with methyl cellulose and uncontrolled freezing in a mechanical freezer at -80 °C allows the maintenance of long-term viability of PBSC for up to 8 years.

Treating relapsed B cell-precursor ALL in children with a setting-adapted mitoxantrone-based intensive chemotherapy protocol (TMH rALL-18 PROTOCOL) - experience from Tata Memorial Hospital, India.

The outlook of relapsed ALL in low- and middle-income countries (LMICs) is dismal due to high treatment-related toxicities and inadequate resources. We report our experience of using a locally adapted mitoxantrone-based protocol for non-high risk (HR) relapsed B-ALL (rALL). A retrospective cum prospective study of standard and intermediate risk (SR and IR) rALL patients treated on TMH rALL-18 protocol (adapted from COG/UKALLR3/Int-Re-ALL protocols) between November 2018 and January 2021 was analyzed. The protocol comprising of 7 blocks of multi-agent chemotherapy including mitoxantrone in induction followed by local irradiation and maintenance, underwent serial modifications based on our experience with initial patients. Eighty-two patients (SR rALL, 3; IR rALL, 79) were treated on TMH rALL-18 protocol. Of 321 grade 3/4 reported toxicities, around 43% (138 toxicities) were noted during induction. Induction chemotherapy was outpatient-based; however, 68 patients (82.9%) required supportive care admissions. Twelve out of 19 patients with gram negative bacilli sepsis (included 7 MDRO) died during reinduction. Five remission deaths were seen during block 3 after which cytarabine was dose reduced (3 g to 2 g/m2). Post-reinduction minimal residual disease was negative in 54 (80.6%) out of 67 evaluable patients. At a median follow-up of 24 months (95% CI 22-27), the estimated 2-year event-free and overall survival of the entire cohort was 58% (95% CI 48.1-69.9) and 60.3% (95% CI 50.5-72). Until the time, targeted therapies are freely accessible in LMICs, strengthening supportive care as well as local adaptation of protocols that strike a fine balance between efficacy and tolerability are mandated.

Pediatric cancer-associated thrombosis: Analysis from a tertiary care cancer center in India.

BACKGROUND AND AIMS: Thrombotic events (TEs) have been extensively studied in adult cancer patients, but data in children are limited. We prospectively analyzed pediatric cancer-associated thrombosis (PCAT) in children with malignancies. METHODS: Children below 15 years of age with confirmed malignancies, treated at a large tertiary cancer center in India from July 2015 to March 2020 developing any TE were eligible. A standardized approach for detection and management was followed. Data were collected after informed consent. RESULTS: Of 6132 eligible children, 150 (2.44%) had 152 TEs, with median age 8.5 years and male:female of 1.83:1. Most TEs occurred on chemotherapy: 111 (74.0%). The most common site was central nervous system (CNS) 59 (39.3%), followed by upper-limb venous system 37 (24.7%). Hemato-lymphoid (HL) malignancies were more prone to PCAT than solid tumors (ST) (incidence 3.23% vs. 1.58%; odds ratio [OR] = 2.06, 95% confidence interval [CI] [1.36-2.88]; p < .001). Malignancies associated with PCAT were acute lymphoblastic leukemia (ALL) 2.94%, acute myeloid leukemia (AML) 6.66%, and non-Hodgkin lymphomas 5.35%. Response imaging done in 106 (70.7%) children showed complete to partial resolution in almost 90% children. Death was attributable to TE in seven (4.66%) children. Age above 10 years (OR 2.33, 95% CI [1.59-3.41]; p < .001), AML (OR 4.62, 95% CI [1.98-10.74]; p = .0062), and non-Hodgkin lymphoma (OR 4.01, 95% CI [1.15-14.04]; p = .029) were significantly associated with TEs. In ALL, age more than 10 years (OR 1.86, 95% CI [1.06-3.24]; p < .03), T-ALL (OR 3.32, 95% CI [1.69-6.54]; p = .001), and intermediate-risk group (OR 4.97, 95% CI [1.12-22.02]; p = .035) were significantly associated with thrombosis. The 2-year event-free survival (EFS) for HL malignancies with PCAT was 55.3% versus 72.1% in those without PCAT (p = .05), overall survival (OS) being 84.6% versus 80.0% (p = .32). CONCLUSION: Incidence of PCAT was 2.4%, and occurred predominantly in older children with hematolymphoid malignancies early in treatment. Most resolved completely with low molecular weight heparin (LMWH) and mortality was low. In hematolymphoid malignancies, PCAT reduce EFS, highlighting the need for prevention.

Expression levels and patterns of B-cell maturation antigen in newly diagnosed and relapsed multiple myeloma patients from Indian subcontinent.

BACKGROUND: Many novel therapies are being evaluated for the treatment of Multiple myeloma (MM). The cell-surface protein B-cell maturation antigen (BCMA, CD269) has recently emerged as a promising target for CAR-T cell and monoclonal-antibody therapies in MM. However, the knowledge of the BCMA expression-pattern in myeloma patients from the Indian subcontinent is still not available. We present an in-depth study of BCMA expression-pattern on abnormal plasma cells (aPC) in Indian MM patients. METHODS: We studied BM samples from 217 MM patients (211-new and 6-relapsed) with a median age of 56 years (range, 30-78 years & M:F-2.29) and 20 control samples. Expression levels/patterns of CD269 (clone-19f2) were evaluated in aPCs from MM patients and in normal PCs (nPC) from uninvolved staging bone marrow samples (controls) using multicolor flow cytometry (MFC). Expression-level of CD269 was determined as a ratio of mean fluorescent intensity (MFI-R) of CD269 in PCs to that of non-B-lymphocytes and expression-pattern (homogenous/heterogeneous) as coefficient-of-variation of immunofluorescence (CVIF). RESULTS: Median (range) percentage of CD269-positive abnormal-PCs in total PCs was 71.6% (0.49-99.29%). The MFI-R (median, range) of CD269 was significantly higher in aPCs (4.13, 1.12-26.88) than nPCs (3.33, 1.23-12.87), p < .0001. Median (range) MFI of CD269 at diagnosis and relapse were 2.39 (0.77-9.57) and 2.66 (2.15-3.23) respectively. CD269 levels were similar at diagnosis and relapse, p = .5529. CONCLUSIONS: We demonstrated that BCMA/CD269 is highly expressed in aPCs from a majority of MM patients, both at diagnosis and relapse. Thus, BCMA is a valuable target for therapy for Indian MM patients.

Critical Role of Flow Cytometric Immunophenotyping in the Diagnosis, Subtyping, and Staging of T-Cell/NK-Cell Non-Hodgkin's Lymphoma in Real-World Practice: A Study of 232 Cases From a Tertiary Cancer Center in India.

Background: T-cell/NK-cell non-Hodgkin's lymphoma (T/NK-NHL) is an uncommon heterogeneous group of diseases. The current classification of T/NK-NHL is mainly based on histopathology and immunohistochemistry. In practice, however, the lack of unique histopathological patterns, overlapping cytomorphology, immunophenotypic complexity, inadequate panels, and diverse clinical presentations pose a great challenge. Flow cytometric immunophenotyping (FCI) is a gold standard for the diagnosis, subtyping, and monitoring of many hematological neoplasms. However, studies emphasizing the role of FCI in the diagnosis and staging of T/NK-NHL in real-world practice are scarce. Methods: We included T-cell non-Hodgkin's lymphoma (T-NHL) patients evaluated for the diagnosis and/or staging of T/NK-NHL using FCI between 2014 and 2020. We studied the utility of FCI in the diagnosis and subtyping of T/NK-NHL and correlated the FCI findings with the results of histopathology/immunohistochemistry. For correlation purposes, patients were categorized under definitive diagnosis and subtyping, inadequate subtyping, inadequate diagnosis, and misdiagnosis based on the findings of each technique. Results: A total of 232 patients were diagnosed with T/NK-NHL. FCI findings provided definitive diagnoses in 198 patients and subtyping in 187/198 (95.45%) patients. The correlation between FCI and histopathological/immunohistochemistry results (n = 150) demonstrated an agreement on the diagnosis and subtyping in 69/150 (46%) patients. Of the remaining cases, the diagnosis and subtyping were inadequate in 64/150 (42.7%), and 14/150 (9.33%) were misdiagnosed on histopathology/immunohistochemistry results. FCI provided definitive diagnosis and subtyping in 51/64 (79.7%) patients. Among these, 13 patients diagnosed with peripheral T-cell lymphoma not-otherwise-specified were reclassified (angioimmunoblastic T-cell lymphoma (AITL)-11 and prolymphocytic leukemia-2) on FCI. It corrected the diagnosis in 14 patients that were misdiagnosed (6 B-cell NHL (B-NHL), 3 Hodgkin's lymphoma, 1 acute leukemia, and 1 subcutaneous panniculitis-like T-cell lymphoma) and misclassified (3 T-NHL) on histopathological results. AITL was the commonest T-NHL misclassified on histopathological results. FCI also confirmed the definite involvement in 7/83 (8.4%) and 27/83 (32.5%) bone marrow (BM) samples reported as suspicious and uninvolved, respectively, on histopathological evaluation. Conclusion: AITL was the most frequently diagnosed T/NK-NHL in this study. FCI provided a distinct advantage in detecting BM involvement by T/NK-NHL, especially in patients with low-level involvement. Overall, our study concluded that FCI plays a critical role in the diagnosis, subtyping, and staging of T/NK-NHL in real-world practice.

Mimics and artefacts of measurable residual disease in a highly sensitive multicolour flow cytometry assay for B-lymphoblastic leukaemia/lymphoma: critical consideration for analysis of measurable residual disease.

High-sensitivity multicolour flow cytometry (MFC)-based B-lymphoblastic leukaemia (B-ALL) measurable residual disease (BMRD) assay is increasingly being used in clinical practice. Herein, we describe six consistently present low-level populations immunophenotypically mimicking abnormal B-ALL blasts in 441 BMRD samples from 301 children. These included CD19+ CD123+ plasmacytoid dendritic cells differentiating from lymphoid precursors, CD10+ transitional B cells with CD10+ /CD38dim-to-negative/CD20bright/CD45bright phenotype, CD19+ natural killer (NK) cells, CD73bright/CD10+ mesenchymal stromal/stem cells, CD73bright/CD34+ endothelial cells, and a CD34+ CD38dim-to-negative/CD10- /CD20bright/CD45bright subset of mature B cells. We provide the proportions, comprehensive immunophenotype, and practical clues for proper identification of these low-level populations. Knowledge regarding the presence and immunophenotype of these mimics is essential for accurate interpretation in high-sensitivity MFC-BMRD analysis.

Comprehensive immune cell profiling depicts an early immune response associated with severe coronavirus disease 2019 in cancer patients.

Recent studies have highlighted multiple immune perturbations related to severe acute respiratory syndrome coronavirus 2 infection-associated respiratory disease [coronavirus disease 2019 (COVID-19)]. Some of them were associated with immunopathogenesis of severe COVID-19. However, reports on immunological indicators of severe COVID-19 in the early phase of infection in patients with comorbidities such as cancer are scarce. We prospectively studied about 200 immune response parameters, including a comprehensive immune-cell profile, inflammatory cytokines and other parameters, in 95 patients with COVID-19 (37 cancer patients without active disease and intensive chemo/immunotherapy, 58 patients without cancer) and 21 healthy donors. Of 95 patients, 41 had severe disease, and the remaining 54 were categorized as having a nonsevere disease. We evaluated the association of immune response parameters with severe COVID-19. By principal component analysis, three immune signatures defining characteristic immune responses in COVID-19 patients were found. Immune cell perturbations, in particular, decreased levels of circulating dendritic cells (DCs) along with reduced levels of CD4 T-cell subsets such as regulatory T cells (Tregs ), type 1 T helper (Th1) and Th9; additionally, relative expansion of effector natural killer (NK) cells were significantly associated with severe COVID-19. Compared with patients without cancer, the levels of terminal effector CD4 T cells, Tregs , Th9, effector NK cells, B cells, intermediate-type monocytes and myeloid DCs were significantly lower in cancer patients with mild and severe COVID-19. We concluded that severely depleted circulating myeloid DCs and helper T subsets in the initial phase of infection were strongly associated with severe COVID-19 independent of age, type of comorbidity and other parameters. Thus, our study describes the early immune response associated with severe COVID-19 in cancer patients without intensive chemo/immunotherapy.

Improved protocol for plasma microRNA extraction and comparison of commercial kits.

INTRODUCTION: MicroRNAs are small, non-coding RNA molecules that are becoming popular biomarkers in several diseases. However, their low abundance in serum/plasma poses a challenge in exploiting their potential in clinics. Several commercial kits are available for rapid isolation of microRNA from plasma. However, reports guiding the selection of appropriate kits to study downstream assays are scarce. Hence, we compared four commercial kits to evaluate microRNA-extraction from plasma and provided a modified protocol that further improved the superior kit's performance. MATERIALS AND METHODS: We compared four kits (miRNeasy Serum/Plasma, miRNeasy Mini Kit from Qiagen; RNA-isolation, and Absolutely-RNA MicroRNA Kit from Agilent technologies) for quality and quantity of microRNA isolated, extraction efficiency, and cost-effectiveness. Bioanalyzer-based Agilent Small RNA kit was used to evaluate quality and quantity of microRNA. Extraction efficiency was evaluated by detection of four endogenous control microRNA using real-time-PCR. Further, we modified the manufacturer's protocol for miRNeasy Serum/Plasma kit to improve yield. RESULTS: miRNeasy Serum/Plasma kit outperformed the other three kits in microRNA-quality (P < 0.005) and yielded maximum microRNA-quantity. Recovery of endogenous control microRNA i.e. hsa-miR-24-3p, hsa-miR-191-5p, hsa-miR-423-5p and hsa-miR-484 was higher as well. Modification with the inclusion of a double elution step enhanced yield of microRNA extracted with miRNeasy Serum/Plasma kit significantly (P < 0.001). CONCLUSION: We demonstrated that miRNeasy Serum/Plasma kit outperforms other kits and can be reliably used with a limited plasma quantity. We have provided a modified microRNA-extraction protocol with improved microRNA output for downstream analyses.

Expression of CD304/neuropilin-1 in adult b-cell lymphoblastic leukemia/lymphoma and its utility for the measurable residual disease assessment.

INTRODUCTION: Many new markers are being evaluated to increase the sensitivity and applicability of multicolor flow cytometry (MFC)-based measurable residual disease (MRD) monitoring. However, most of the studies are limited to childhood B-cell lymphoblastic leukemia/lymphoma (B-ALL), and reports in adult B-ALL are extremely scarce and limited to small cohorts. We studied the expression of CD304/neuropilin-1 in a large cohort of adult B-ALL patients and evaluated its practical utility in MFC-based MRD analysis. METHODS: CD304 was studied in blasts from adult B-ALL patients and normal precursor B cells (NPBC) from non-B-ALL bone marrow samples using MFC. CD304 expression intensity and pattern were studied with normalized-mean fluorescent intensity (nMFI) and coefficient of variation of immunofluorescence (CVIF), respectively. MFC-based MRD was performed at end of induction (EOI; day-35), end of consolidation (EOC; day 78-80), and subsequent follow-up (SFU) time points. RESULTS: CD304 was positive in 120/214(56.07%) and was significantly associated with BCR-ABL1 fusion (P = .001). EOI-MRD and EOC-MRD were positive in 129/214(60.3%) and 50/81(61.72%), respectively. CD304 was positive in a significant percentage of EOI (48%, 62/129) and EOC (52%, 26/50) MRD-positive B-ALL samples. Its expression was retained, lost, and gained in 73.7%, 26.3%, and 11.3% of EOI-MRD and 85.7%, 14.3%, and none of EOC-MRD samples, respectively. Low-level MRD (<0.01%) was detectable in 34 of all (EOI + EOC + SFU = 189) MRD-positive samples, and CD304 was found useful in 50% of these samples. CONCLUSION: CD304 is commonly expressed in adult B-ALL and clearly distinguish B-ALL blasts from normal precursor B cells. It is a stable MRD marker and distinctly useful in the detection of MFC-based MRD monitoring, especially in high-sensitivity MRD assay.

Sudden blast phase in pediatric chronic myeloid leukemia-chronic phase with abnormal lymphoid blasts detected by flow cytometry at diagnosis: Can it be considered a warning sign?

BACKGROUND: Inconclusive knowledge persists regarding the course of chronic myeloid leukemia-chronic phase (CML-CP) patients with detectable abnormal blasts by flow-cytometry at diagnosis. The 2016 WHO classification is not specific regarding sub-classification of CML with <10% abnormal B-lymphoid blasts (ABLB), and suggests these patients often show rapid progression. We report the clinical course of pediatric CML-CP patients who had detectable abnormal blasts by flow-cytometry at baseline. METHODS: Retrospective audit of all pediatric CML patients between January 2013 and December 2017 were included. Their clinical presentation, demographic profile, and treatment outcomes were extracted from electronic medical records. Some of these patients got flow-cytometry done by default, though it was not a routine part of diagnostic CML marrow studies. RESULTS: Amongst 65 pediatric CML patients, flow-cytometry at initial diagnosis was available in 15 (CP-12; AP-3). Of the 12 CML-CP patients, 10 (83%) had abnormal flow-cytometric findings-5 (50%) with mixed lineage blasts (4-B/Myeloid, 1-B/T/Myeloid), and myeloid lineage blasts in the remaining 5 (50%). At a median follow-up of 26 months (range: 9-34 months), 3/5 patients with ABLB at diagnosis progressed to frank blast crisis (2 B-cell; 1 Mixed lineage). None among the five patients with diagnostic myeloid-alone aberrant blasts progressed to blast crisis. Imatinib resistant mutation was also found in 3/5 (60%) CML-CP patients with these ABLB at baseline. CONCLUSIONS: Although a retrospective study with limited sample size, presence of ABLB detected on flow-cytometry in CML-CP patients, had a noticeable early conversion to CML-BC in our cohort. Incorporation of flow-cytometry in diagnostic work-up can provide useful insight regarding the behavior of pediatric CML-CP patients and guide therapy.

Mast cell differentiation of leukemic blasts in diverse myeloid neoplasms: A potential pre-myelomastocytic leukemia condition.

INTRODUCTION: Myeloid neoplasm with blasts showing mast cell (MC)-differentiation and MC-component less than 10% of all nucleated cells but not fulfilling the criteria for systemic mastocytosis with associated hematological neoplasm (SM-AHN) or myelomastocytic leukemia (MML) has not been described in the literature. Herein, we report a study of diverse myeloid malignancies with blasts showing MC-differentiation but not meeting the criteria for SM-AHN or MML. We also evaluated the utility of flow-cytometric immunophenotyping (FCI) in the characterization of immature-MCs (iMCs). METHODS: We identified nine patients of myeloid neoplasms and studied their morphological, FCI, immunohistochemistry, cytogenetic and molecular characteristics. We also compared the immunophenotypic features of MCs from patient samples with control samples. RESULTS: The study included patients with newly-diagnosed acute myeloid leukemia (n = 4), chronic myelomonocytic leukemia (n = 1), and chronic myeloid leukemia on follow-up (n = 4) showing MC differentiation in leukemic-blasts. These patients had mildly increased MCs (range, 0.5%-3%) in bone-marrow morphology, including immature-forms and did not meet the criteria for either SM-AHN or MML. On FCI, iMCs were positive for bright-CD117, heterogeneous-CD34, dim-to-negative-HLADR, and moderate-CD203c expression. Expression-levels of CD123 and CD38 were higher (p < 0.001) but CD33 and CD45 were lower in iMCs compared to mature-MC from control samples (p = 0.019 and p = 0.0037). CONCLUSION: We reported a rare finding of MC differentiation of leukemic blasts in diverse myeloid neoplasms and proposed it as a potential pre-myelomastocytic leukemia condition. We described the distinct immunophenotypic signature of immature-MCs using commonly used markers and highlighted the utility of FCI for the diagnosis of this entity.

Immunophenotypic shift in the B-cell precursors from regenerating bone marrow samples: A critical consideration for measurable residual disease assessment in B-lymphoblastic leukemia.

Accurate knowledge of expression patterns/levels of commonly used MRD markers in regenerative normal-B-cell-precursors (BCP) is highly desirable to distinguish leukemic-blasts from regenerative-BCP for multicolor flow cytometry (MFC)-based measurable residual disease (MRD) assessment in B-lymphoblastic leukemia (B-ALL). However, the data highlighting therapy-related immunophenotypic-shift in regenerative-BCPs is scarce and limited to small cohort. Herein, we report the in-depth evaluation of immunophenotypic shift in regenerative-BCPs from a large cohort of BALL-MRD samples. Ten-color MFC-MRD analysis was performed in pediatric-BALL at the end-of-induction (EOI), end-of-consolidation (EOC), and subsequent-follow-up (SFU) time-points. We studied normalized-mean fluorescent intensity (nMFI) and coefficient-of-variation of immunofluorescence (CVIF) of CD10, CD19, CD20, CD34, CD38, and CD45 expression in regenerative-BCP (early, BCP1 and late, BCP2) from 200 BALL-MRD samples, and compared them with BCP from 15 regenerating control (RC) TALL-MRD samples and 20 treatment-naïve bone-marrow control (TNSC) samples. Regenerative-BCP1 showed downregulation in CD10 and CD34 expression with increased CVIF and reduced nMFI (p < 0.001), upregulation of CD20 with increased nMFI (p = 0.014) and heterogeneous CD45 expression with increased CVIF (p < 0.001). Immunophenotypic shift was less pronounced in the BCP2 compared to BCP1 compartment with increased CVIF in all but CD45 (p < 0.05) and reduced nMFI only in CD45 expression (p = 0.005). Downregulation of CD10/CD34 and upregulation of CD20 was higher at EOI than EOC and SFU time-points (p < 0.001). Regenerative-BCPs are characterized by the significant immunophenotypic shift in commonly used B-ALL-MRD markers, especially CD10 and CD34 expression, as compared to treatment-naïve BCPs. Therefore, the templates/database for BMRD analysis must be developed using regenerative-BCP.

Utility of CD36 as a novel addition to the immunophenotypic signature of RAM-phenotype acute myeloid leukemia and study of its clinicopathological characteristics.

INTRODUCTION: In 2016, Children Oncology Group (COG) described a new high-risk subtype of acute myeloid leukemia (AML) with a distinct immunophenotypic-signature, RAM-phenotype (RAM-AML). Data on clinical and laboratory features of RAM-AML are still limited to COG report only. Herein, we report the clinicopathological characteristics and detailed immunophenotypic features of RAM-AML patients. In COG report, 38% of RAM-AML belonged to acute megakaryoblastic leukemia (AMKL)-subtype. Hence, we further compared the immunophenotypic features RAM-AML with non-RAM-AMKL diagnosed during the same study period. METHODS: We included RAM-AML and non-RAM AMKL patients diagnosed between January 2017 and December 2019. We studied their morphological, cytochemical, immunophenotyping, cytogenetic, and molecular characteristics. Mean fluorescent intensity (MFI) and expression-pattern of immunophenotypic markers of RAM-AML were compared with non-RAM AMKLs patients. RESULTS: We identified 11 RAM-AML (1%) and 21 non-RAM AMKL (1.9%) patients in 1102 pediatric-AML patients. Seven of 11 (63.64%) patients belonged to FAB-M7-subtype. CD56, CD117, and CD33 demonstrated overexpression, whereas CD45 and CD38 showed under-expression in RAM-AML patients. CD36 was consistently negative in RAM-AML, whereas moderate-bright positive in non-RAM AMKLs patients (p < 0.0001). On principle component analysis, addition of CD36 enhanced the visual-separation between RAM-AML and non-RAM AMKL clusters. Cytogenetic and molecular studies did not show any recurrent abnormality; however, RNA-sequencing study revealed CBFA2T3-GLIS2-fusion in three of seven (42.8%) RAM-AML patients. CONCLUSION: We report the clinicopathological characteristics and the detailed immunophenotypic profile in the world's second series of RAM-AML patients. We further report a novel finding of CD36-negative expression as an additional parameter to the multidimensional immunophenotypic signature of this entity.

Eleven-marker 10-color flow cytometric assessment of measurable residual disease for T-cell acute lymphoblastic leukemia using an approach of exclusion.

Measurable/minimal residual disease (MRD) status has been suggested as a powerful indicator of clinical-outcome in T-cell lymphoblastic leukemia/lymphoma (T-ALL). Multicolor flow cytometric (MFC)-based T-ALL MRD reports are limited and traditionally based on the utilization of markers-of-immaturity like TdT and CD99. Moreover, studies demonstrating the multicolor flow cytometric (MFC) approach for the assessment of T-ALL MRD are sparse. Herein, we describe an 11-marker, 10-color MFC-based T-ALL MRD method using an "approach of exclusion." METHODS: The study included 269 childhood T-ALL patients treated with a modified-MCP841 protocol. An 11-marker, 10-color MFC-based MRD was performed in bone marrow (BM) samples at the end-of-induction (EOI) and end-of-consolidation (EOC) time-points using Kaluza-version-1.3 software. RESULTS: We studied EOI-MRD in 269 and EOC-MRD in 105 childhood T-ALL patients. EOI-MRD was detectable in 125 (46.5%) samples (median, 0.3%; range, 0.0007-66.3%), and EOC-MRD was detectable in 34/105 (32.4%) samples (median, 0.055%; range, 0.0008-27.6%). Leukemia-associated immunophenotypes (LAIPs) found useful for MRD assessment were dual-negative CD4/CD8 (40.9%), dual-positive CD4/CD8 (23.3%) and only CD4 or CD8 expression (35.8%); dim/subset/dim-negative surface-CD3 (39%), dim/subset/dim-negative/negative CD5 (28.3%), dim/dim-negative/negative/heterogeneous CD45 (44.7%) and co-expression of CD5/CD56 (7.5%). EOI-MRD-positive status was found to be the most-relevant independent factor in the prediction of inferior relapse-free and overall survival. CONCLUSION: We described an 11-marker 10-color MFC-based highly sensitive MRD assay in T-ALL using an approach of exclusion. The addition of CD4 and CD8 to the pan-T-cell markers in a 10-color assay is highly useful in T-ALL MRD assessment and extends its applicability to almost all T-ALL patients.

Post-induction Measurable Residual Disease Using Multicolor Flow Cytometry Is Strongly Predictive of Inferior Clinical Outcome in the Real-Life Management of Childhood T-Cell Acute Lymphoblastic Leukemia: A Study of 256 Patients.

Background: Measurable/minimal residual disease (MRD) status is suggested as a powerful indicator of clinical-outcome in T-cell lymphoblastic leukemia/lymphoma (T-ALL). Contrary to B-cell ALL, reports on T-ALL MRD are limited and mostly based on molecular methods, mainly from developed countries. Multicolor flow cytometry (MFC)-based T-ALL studies are very few. Clinically relevant cut-off levels and ideal time-point for MRD assessment are still inconclusive. In view of lack of T-ALL MRD data from the developing world, we evaluated the prognostic value of MFC-based post-induction (PI)-MRD assessment in T-ALL in the context of standard practice. Methods: We included 256 childhood-T-ALL patients (age < 15 years) treated with a modified-MCP841 protocol, which uses high-dose cytarabine during consolidation, as a part of standard hospital practice. MRD was studied using 10-color 11-antibody MFC with any level of detectable disease being considered positive. Post-induction (PI)-MRD was available in all patients, and post-consolidation (PC) MRD was available mostly in PI-MRD-positive patients (n = 88). Results: Three years cumulative-incidence-of-relapse (3years-CIR) in PI-MRD-positive patients was inferior to negative patients (46.3% vs. 18.4%). The median relapse-free-survival (RFS), event-free-survival (EFS) and overall-survival (OS) with hazard ratio (HR) of PI-MRD-positive patients were 21.4 months vs not reached (p < 0.0001, HR-4.7), 21.6 months vs. not-reached (p = 0.0003, HR-2.01) and 37.3 months vs. not reached (p = 0.026, HR-1.64) respectively. RFS, EFS and OS of patients with PI-MRD<0.01% (n = 17) were as inferior as PI-MRD ≥ 0.01% in comparison with MRD-negative patients with HR of 4.7 (p < 0.0001), 2.45 (p = 0.0003), and 2.5 (p = 0.029), respectively. Three-years-CIR of patients with hyperleukocytosis (≥100 × 109/L) was also higher (50.5 vs. 27.6%) with inferior RFS, EFS, and OS. Among PI-MRD-positive patients, 3years-CIR, RFS, EFS, and OS of PC-MRD-positive were also inferior to that of negative patients. On multivariate analysis any-level detectable PI-MRD and hyperleukocytosis remained independently associated with inferior RFS, EFS, and OS. A combination of PI-MRD-positive status and hyperleukocytosis identified the patients with the worst clinical outcomes. Conclusion: Detectable PI-MRD using MFC was found to be the strong predictive factor of inferior clinical outcome in T-ALL patients. The combination of PI-MRD status and hyperleukocytosis provides the most influential tool for the management of T-ALL in resource constrained settings from developing world.