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.

Genomic Analysis of AZD1222 (ChAdOx1) Vaccine Breakthrough Infections in the City of Mumbai.

Background: This manuscript describes the genetic features of SARS-CoV-2 mutations, prevalent phylogenetic lineages, and the disease severity amongst COVID-19-vaccinated individuals in a tertiary cancer hospital during the second wave of the pandemic in Mumbai, India. Methods: This observational study included 159 COVID-19 patients during the second wave of the pandemic from 17th March to 1st June 2021 at a tertiary cancer care centre in Mumbai. The cohort comprised of healthcare workers, staff relatives, cancer patients, and patient relatives. For comparison, 700 SARS-CoV-2 genomes sequenced during the first wave (23rd April to 25th September 2020) at the same centre were also analysed. Patients were assigned to nonvaccinated (no vaccination or <14 days from the 1st dose, n = 92), dose 1(≥14 days from the 1st dose to <14 days from the 2nd dose, n = 29), and dose 2 (≥14 days from the 2nd dose, n = 38) groups. Primary measure was the prevalence of SARS-CoV-2 genomic lineages among different groups. In addition, severity of COVID-19 was assessed according to clinical and genomic variables. Results: Kappa B.1.1671.1 and delta B.1.617.2 variants contributed to an overwhelming majority of sequenced genomes (unvaccinated: 40/92, 43.5% kappa, 46/92, 50% delta; dose 1: 14/29, 48.3% kappa, 15/29, 51.7% delta; and dose 2: 23/38, 60.5% kappa, 14/38 36.8% delta). The proportion of the kappa and delta variants did not differ significantly across the unvaccinated, dose 1, and dose 2 groups (p = 0.27). There was no occurrence of severe COVID-19 in the dose 2 group (0/38, 0% vs. 14/121, 11.6%; p = 0.02). SARS-CoV-2 genomes from all three severe COVID-19 patients in the vaccinated group belonged to the delta lineage (3/28, 10.7% vs. 0/39, 0.0%, p = 0.04). Conclusions: Sequencing analysis of SARS-COV-2 genomes from Mumbai during the second wave of COVID-19 suggests the prevalence of the kappa B.1.617.1 and the delta B.1.627.2 variants among both vaccinated and unvaccinated individuals. Continued evaluation of genomic sequencing data from breakthrough COVID-19 is necessary for monitoring the properties of evolving variants of concern and formulating appropriate immune response boosting and therapeutic strategies.

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.

Detecting hypodiploidy with endoreduplication and masked hypodiploidy in B-cell acute lymphoblastic leukemia using multicolor flow cytometry.

BACKGROUND: Multicolor flow cytometry-based DNA-ploidy (MFC-ploidy) analysis is a simple, sensitive, and popular method for ploidy analysis in B-cell acute lymphoblastic leukemia (B-ALL). However, the utility of MFC-ploidy in the detection of B-ALL with endoreduplication or masked hypodiploidy has not been reported. Herein, we studied the patterns of MFC-ploidy assessment and its utility to detect B-ALL with hypodiploidy and endoreduplication. METHODS: MFC-ploidy analysis was performed using FxCycle Violet-dye-based method, and cytogenetic ploidy was evaluated using chromosomal-counting and FISH analysis. A total of 20 B-ALL cases with endoreduplication were studied for the patterns of MFC-ploidy analysis and compared with 250 patients with hyperdiploidy and 11 cases with pure hypodiploidy. RESULTS: All B-ALL with endoreduplication revealed two distinct peaks (populations) on MFC-ploidy analysis: the first (hypodiploid) peak (median-DNA-index [DI], 0.82; range, 0.6-0.95) and the second (hyperdiploid) peak with almost twice DI (median-DI, 1.53; range, 1.14-1.75). Cytogenetic findings were available in 19 cases and confirmed hypodiploidy with endoreduplication in 13/19 (68.4%) and only hypodiploidy in 3/19 cases. The remaining three cases showed hyperdiploid blasts in cytogenetic studies. Of these three, two cases had <10% blasts population with hypodiploidy. Thus, masked-hypodiploidy could be diagnosed correctly in 3/19 cases on MFC-ploidy analysis. CONCLUSION: MFC-ploidy analysis shows a characteristic pattern of DNA-ploidy in samples with endoreduplication. It allows the distinction between samples with masked hypodiploidy from true hyperdiploidy. An integrated approach involving cytogenetic and MFC-ploidy detection is very helpful in the risk stratification of B-ALL in routine clinical 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.

Evaluation of cytogenetic response in CML patients with variant Philadelphia translocation.

BACKGROUND AND AIM: Molecular mechanism of translocation and outcome in variant chronic myeloid leukaemia (vCML) has been a topic of debate. While several cytogenetic studies suggest a low response to Imatinib Mesylate, others demonstrate a similar disease course in both classical and vCML. Besides, many studies comprehensively also link tyrosine kinase domain (TKD) mutations with aggressive clinical outcome. Thus, we aim to study the molecular mechanism of translocation, identify the third partner chromosomes and comment on the disease course and clinical outcome. METHOD: We cytogenetically characterised 25 vCML cases to determine the third partner chromosome, mechanism of translocation and prognostic outcome. We also compared vCML cases with and without TKD mutation to most appropriately outline the clinical consequence and ascertain the potent cause of unresponsiveness to treatment. RESULTS: Third partner chromosome in variant translocation was defined by conventional and molecular cytogenetics. Although in our study most cases showed inadequate clinical response attributable to TKD mutation rather than variant translocation, we observed an inferior outcome in cases involving chromosome 5 as the third partner. CONCLUSION: Thus, we conclude that characterising and reporting new cases of variant translocations, involving various different chromosomes as third partner (with different breakpoints) by cytogenetics, will lead to a better understanding of the disease. To the best of our knowledge, this kind of delineate study has not been applied to precisely comment on the prospects of cytogenetically characterised vCML.

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.

Long term outcome of a patient with relapsed refractory early thymic precursor acute lymphoblastic leukemia treated with daratumumab.

The prognosis of patients with relapsed Early Thymic Precursor acute lymphoblastic leukemia (ETP-ALL) remains poor. Unlike B cell Precursor-ALL (BCP-ALL), there are no approved targeted therapies for ETP-ALL. Recent studies have identified a consistent expression of CD38 on the blasts of patients with T-ALL (both ETP-ALL and non ETP-ALL). Pre-clinical studies indicate that CD38 expression persists on the blasts of T-ALL even after receipt of conventional chemotherapy. These findings make CD38 an attractive targetable surface protein for patients with relapsed refractory T-ALL. We were the first to describe the clinical use of daratumumab in a patient of ETP-ALL, with relapsed disease post allogeneic transplant. We describe here the long term outcome of this patient more than 3 years after starting single agent daratumumab.

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.

Importance of conventional cytogenetics in the identification of ins(19;X)(q13.1;p11.2q28) and t(1;11)(q10;p10), both, novel cytogenetic abnormalities in a pediatric AML case.

Acute Myeloid Leukemia (AML) is a heterogeneous disease with respect to morphology, immunophenotype, chromosomal abnormalities and genetic lesions. While a majority of AML cases harbour recurrent chromosomal abnormalities, several rare, apparently unique or novel aberrations may be identified by conventional cytogenetics. In fact, with the prognostic relevance of chromosomal abnormalities, and with the advent of new-age, target-specific therapy, identifying such aberrations becomes vital. In this study, we present a case of pediatric AML with ins(19;X)(q13.1;p11.2q28) and t(1;11)(q10;p10), both, novel, previously unreported chromosomal abnormalities in AML. Post induction, both these clonal cytogenetic abnormalities persisted. The documentation of this case will help determine the significance of these cytogenetic abnormalities. Also, this case exemplifies the importance of cytogenetics in the complete characterization and risk stratification of AML patients.

Outcomes and prognostic factors in adolescents and young adults with ALL treated with a modified BFM-90 protocol.

The use of pediatrics-inspired protocols in adolescent and young adult (AYA) acute lymphoblastic leukemia (ALL) results in superior survival compared with the adult protocols. Pediatrics-inspired protocols carry an increased risk of toxicity and treatment-related mortality in low resource settings, which can offset the potential benefits. We studied the outcomes and prognostic factors in the treatment of AYA ALL with a pediatrics-inspired regimen. We retrieved data regarding demographics, investigations, treatment details, and toxicities from the electronic medical records of patients diagnosed with ALL in the 15- to 25-year-old age group who were initiated on a modified Berlin-Frankfurt-Münster 90 (BFM-90) protocol between January 2013 and December 2016 at the Tata Memorial Centre. A total of 349 patients in the 15- to 25-year-old age group were treated with a modified BFM-90 protocol. The use of this pediatrics-inspired protocol resulted in a 3-year event-free survival (EFS) and overall survival (OS) of 59.4% and 61.8%, respectively. Only 15 patients underwent an allogeneic stem cell transplant. Minimal residual disease (MRD) persistence postinduction emerged as the only factor predictive of poor outcomes. A modified BFM-90 protocol is an effective and safe regimen for AYA ALL with an OS and EFS comparable to the published literature.

Clinical impact of panel-based error-corrected next generation sequencing versus flow cytometry to detect measurable residual disease (MRD) in acute myeloid leukemia (AML).

We accrued 201 patients of adult AML treated with conventional therapy, in morphological remission, and evaluated MRD using sensitive error-corrected next generation sequencing (NGS-MRD) and multiparameter flow cytometry (FCM-MRD) at the end of induction (PI) and consolidation (PC). Nearly 71% of patients were PI NGS-MRD+ and 40.9% PC NGS-MRD+ (median VAF 0.76%). NGS-MRD+ patients had a significantly higher cumulative incidence of relapse (p = 0.003), inferior overall survival (p = 0.001) and relapse free survival (p < 0.001) as compared to NGS-MRD- patients. NGS-MRD was predictive of inferior outcome in intermediate cytogenetic risk and demonstrated potential in favorable cytogenetic risk AML. PI NGS-MRD- patients had a significantly improved survival as compared to patients who became NGS-MRD- subsequently indicating that kinetics of NGS-MRD clearance was of paramount importance. NGS-MRD identified over 80% of cases identified by flow cytometry at PI time point whereas FCM identified 49.3% identified by NGS. Only a fraction of cases were NGS-MRD- but FCM-MRD+. NGS-MRD provided additional information of the risk of relapse when compared to FCM-MRD. We demonstrate a widely applicable, scalable NGS-MRD approach that is clinically informative and synergistic to FCM-MRD in AML treated with conventional therapies. Maximum clinical utility may be leveraged by combining FCM and NGS-MRD modalities.