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.

KIT exon 17 mutations are predictive of inferior outcome in pediatric acute myeloid leukemia with RUNX1::RUNX1T1.

BACKGROUND: Pediatric core binding factor acute myeloid leukemia (CBF-AML), although considered a favorable risk subtype, exhibits variable outcomes primarily driven by additional genetic abnormalities, such as KIT mutations. PROCEDURE: In this study, we examined the prognostic impact of KIT mutations in 130 pediatric patients with CBF-AML, treated uniformly at a single center over 4 years (2017-2021). KIT mutations were detected via next-generation sequencing using a myeloid panel comprising 52 genes for most patients. RESULTS: Our findings revealed that KIT mutations were present in 31% of CBF-AML cases. Exon 17 KIT mutation was most commonly (72%) seen with notable occurrences at the D816 and N822 residue in 48% and 39% of cases, respectively. The 3-year cumulative incidence of relapse (CIR) and overall survival (OS) for patients with exon 17 KIT mutation were 36% and 40%, respectively, and was significantly worse in comparison to other site KIT mutations (3-year CIR: 11%; OS: 64%) and without KIT mutation (3-year CIR: 13%; OS:71%). Notably, the prognostic impact of KIT mutations was prominent in patients with RUNX1::RUNX1T1, but not in those with CBFB::MYH11 fusion. Additionally, a high KIT variant-allele frequency (VAF) (>33%) predicted for a higher disease relapse; 3-year CIR of 40% for VAF greater than 33% versus 7% for VAF less than 33%. When adjusted for site of KIT mutation and end-of-induction measurable residual disease, VAF greater than 33% correlated with poor OS (hazard ratio [HR]: 4.4 [95% CI: 1.2-17.2], p = .034). CONCLUSION: Exon 17 KIT mutations serve as an important predictor of relapse in RUNX1::RUNX1T1 pediatric AML. In addition, a high KIT VAF may predict poor outcomes in these patients. These results emphasize the need to incorporate KIT mutational analysis into risk stratification for pediatric CBF-AML.

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.

Low levels of CD26 on certain cellular subtypes of donor harvest is associated with better clinical outcomes post allogeneic stem cell transplantation through regulation of NF-κB pathway and pro-inflammatory cytokines.

BACKGROUND: We had previously reported significant association of immunoectoenzyme CD26 expression on donor harvest with acute Graft-versus-Host-Disease (aGVHD) in allogeneic stem cell transplantation (ASCT) patients. The current study was aimed at analysing CD26 signaling pathway molecules and understanding their impact on immune reconstitution and clinical outcomes post-ASCT. SUBJECTS AND METHODOLOGY: The study cohort included 26 transplant donors/patients who underwent reduced intensity (n = 21), myeloablative (n = 4) and non-myeloablative (n = 1) ASCT for hematological malignancies. Donors were matched related donors (n = 19) and haploidentical donors (n = 7). Surface expression of CD26, CD73 and ADA, and various immune cell subtypes were assessed by multicolour-flow cytometry. Soluble CD26 (sCD26) and cytokine levels were measured in plasma samples by ELISA and Multiplex Luminex assay, respectively. Immune cells from healthy individuals were stimulated with phytohemagglutinin (PHA) in the presence or absence of CD26 inhibitor. Effect of CD26 inhibition on NF-κB localization in PHA stimulated cells was analysed by immunofluorescence and confocal microscopy. Pro-inflammatory cytokines from the culture supernatants were detected with Cytometric bead array flow cytometry. Association of all measured markers with clinical outcomes was evaluated using appropriate statistical tests. RESULTS: CD26 surface expression on PBSC donor harvest cells showed increased risk of chronic GVHD (cGVHD, p = 0.055). Amongst the various immune cell subtypes, decreased B cells in harvest showed significant association with aGVHD (p = 0.022) whereas increased myeloid dendritic cells and CD3+T cells at Day100 in peripheral blood of transplant recipients correlated with cGVHD (p = 0.046) and aGVHD (p = 0.035), respectively. Further, high sCD26 in transplant recipients at Day100 exhibited association with reduced event-free survival (EFS) (p = 0.011). Higher CD26 expression on more & less mature NK cells, naïve & post-switched memory B cells and Treg cells in the donor harvest (p < 0.05) led to lower EFS in transplant recipients. Mechanistically, CD26 inhibitor caused dose-dependent reduction in CD26 enzyme activity and in pro-inflammatory cytokine production in post mitogen-stimulated T cell cultures. CONCLUSION: Our study has implicated that lower CD26 expression on immune cell subtypes of the donor stem cell harvest is associated with reduced risk of GVHD and better survival. The underlying mechanism was found to be through NF-κB pathway and pro-inflammatory cytokines. Based on these observations, chemically designed or natural resources-based CD26 inhibitors can be explored further in clinical trials for improving ASCT outcomes.

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.

Novel covalent CDK7 inhibitor potently induces apoptosis in acute myeloid leukemia and synergizes with Venetoclax.

INTRODUCTION: The emergence of resistance to the highly successful BCL2-directed therapy is a major unmet need in acute myeloid leukemia (AML), an aggressive malignancy with poor survival rates. Towards identifying therapeutic options for AML patients who progress on BCL2-directed therapy, we studied a clinical-stage CDK7 inhibitor XL102, which is being evaluated in solid tumors (NCT04726332). MATERIALS AND METHODS: To determine the anti-proliferative effects of XL102, we performed experiments including time-resolved fluorescence resonance energy transfer, target occupancy, cell cycle and apoptosis-based assays. We also included genetically characterized primary myeloid blasts from de novo and relapsed/refractory AML patients. For mechanistic studies, CRISPR/Cas9 mediated knockout of CDK7 and c-Myc and immunoblotting were performed. NOD/SCID orthotropic and subcutaneous AML xenografts were used to determine anti-leukemic effects. To assess the synergistic effects of XL102 with Venetoclax, we performed RNA sequencing and gene set enrichment analysis using Venetoclax sensitive and resistant model systems. RESULTS: XL102, a highly specific, orally bioavailable covalent inhibitor of CDK7. Inhibitory effect on CDK7 by XL102 in primary myeloid blasts (n = 54) was in nanomolar range (mean = 300 nM; range = 4.0-952 nM). XL102 treated AML cells showed a reduction in phosphorylation levels of Serine 2/5/7 at carboxy-terminal domain of RNA polymerase II. T-loop phosphorylation of CDK1(Thr161) and CDK2(Thr160) was inhibited by XL102 in dose-dependent manner leading to cell-cycle arrest. c-Myc downregulation and enhanced levels of p53 and p21 in XL102 treated cells were observed. Increased levels of p21 and activation of p53 by XL102 were mimicked by genetic ablation of CDK7, which supports that the observed effects of XL102 are due to CDK7 inhibition. XL102 treated AML xenografts showed remarkable reduction in hCD45 + marrow cells (mean = 0.60%; range = 0.04%-3.53%) compared to vehicle control (mean = 38.2%; range = 10.1%-78%), with corresponding increase in p53, p21 and decrease in c-Myc levels. The data suggests XL102 induces apoptosis in AML cells via CDK7/c-Myc/p53 axis. RNA-sequencing from paired Venetoclax-sensitive and Venetoclax-resistant cells treated with XL102 showed downregulation of genes involved in proliferation and apoptosis. CONCLUSION: Taken together, XL102 with Venetoclax led to synergistic effects in overcoming resistance and provided a strong rationale for clinical evaluation of XL102 as a single agent and in combination with Venetoclax.

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.

Targeting transcriptional kinase of CDK7 halts proliferation of multiple myeloma cells by modulating the function of canonical NF-kB pathway and cell cycle regulatory proteins.

Multiple myeloma (MM) is an incurable plasma cell neoplasm. Despite several effective frontline therapeutic regimens, including Bortezomib (BTZ), relapse is almost inevitable; therefore, better therapeutic modalities to improve the outcomes are needed. Cyclin-dependent kinases (CDKs) are an essential constituent of the cellular transcriptional machinery and tumors including MM are critically dependent on transcription to maintain their oncogenic state. In the present study, we explored the efficacy of THZ1, a covalent CDK7 inhibitor in MM treatment using Bortezomib resistant (H929BTZR) cells and zebrafish xenografts. THZ1 showed anti-myeloma activity in the models of MM but had no effect on healthy CD34+ cells. THZ1 suppresses phosphorylation of carboxy-terminal domain of RNA polymerase II and downregulates the transcription of BCL2 family of proteins both in H929BTZS and H929BTZR cells leading to G1/S arrest and apoptosis. THZ1 mediates inhibition of bone marrow stromal cells-induced proliferation and activation of NF-kB signaling. The data derived from zebrafish xenografts of MM demonstrate that THZ1 combined with BTZ synergistically reduces tumor growth in zebrafish embryos. Collectively, our results reveal that THZ1 alone as well as in combination with BTZ has effective anti-myeloma activity.

Treatment of Pediatric Acute Promyelocytic Leukemia with Retinoic Acid and Arsenic Trioxide along with Chemotherapy.

OBJECTIVES: Outcomes of childhood acute promyelocytic leukemia (APL) have exceeded 90% in the era of differentiating agents. In resource-limited settings, early mortality secondary to coagulopathy remains a significant challenge. Differentiation syndrome is a unique complication of APL therapy that requires a high degree of suspicion for timely initiation of therapy. METHODS: A retrospective study of children ≤15 y of age with APL diagnosed between January-2013 and June-2019 treated at a tertiary cancer centre was conducted. Patients with a total leukocyte count ≥10,000/µL were risk stratified as high-risk. Treatment included differentiating agents, all-trans retinoic acid and arsenic trioxide along with chemotherapy. Baseline demographics, clinical complications and outcomes were analysed. RESULTS: Out of 90 patients treated, 48 (53%) had high-risk APL and 25 (28%) presented with significant bleeding manifestations. Response to therapy was excellent with 96% of evaluable patients achieving molecular remission by the end of consolidation phase. Differentiation syndrome occurred in 23 (25%) patients of which two expired. Early mortality rate was 5.5% and was due to severe hemorrhage most often at the time of presentation. The 3-y overall survival of the entire cohort was 91% (95% CI: 85-97%). Two of 4 patients with relapse of disease could be salvaged with only differentiating agents followed by autologous transplantation. CONCLUSIONS: Long-term outcomes of Indian children with APL are excellent. Timely management of coagulopathy and prompt initiation of differentiating agents along with appropriate cytoreductive measures is critical. Efforts to build academic-community partnerships to ensure timely diagnosis and emergency care in order to reduce early mortality are needed.

Withaferin-A alleviates acute graft versus host disease without compromising graft versus leukemia effect.

Acute graft versus host disease (aGvHD) contributes to a significant proportion of non-relapse mortality and morbidity in patients undergoing allogeneic hematopoietic stem cell transplantation (alloHSCT). Withaferin-A (WA), a phytomolecule obtained from Withania somnifera (Ashwagandha), is known to have anti-inflammatory, anti-proliferative and immunomodulatory properties. The efficacy of WA for the prevention and treatment of aGvHD was evaluated using a murine model of alloHSCT. Prophylactic administration of WA to mice mitigated the clinical symptoms of aGvHD and improved survival significantly compared to the GvHD control [HR = 0.07 (0.01-0.35); P < 0.001]. Furthermore, WA group had better overall survival compared to standard prophylactic regimen of CSA + MTX [HR = 0.19 (0.03-1.1), P < 0.05]. At the same time, WA did not compromise the beneficial GvL effect. In addition, WA administered to animals after the onset of aGvHD could reverse the clinical severity and improved survival, thus establishing its therapeutic potential. Our findings suggest that WA reduced the systemic levels of Th1, Th2 and Th17 inflammatory cytokine and increased the anti-inflammatory cytokine IL-10 levels significantly (P < 0.05). WA also inhibited lymphocytes migration to gut, liver, skin and lung and protected these organs from damage. Ex-vivo, WA inhibited proliferation of human peripheral blood mononuclear cells (hPBMCs), modulated immune cell phenotype and decreased cytokine release. In addition, WA inhibited pJAK2 and pSTAT3 protein levels in mouse splenocytes and hPBMCs. In conclusion, our study demonstrates the utility of WA for the prevention and treatment of aGvHD, which should be further evaluated in a clinical setting.

Demographics, Pattern of Care & Outcomes of Primary CNS Lymphoma- Experience from a Tertiary Care Cancer Center in India.

Primary CNS lymphoma (PCNSL) is a rare subtype of non-Hodgkin lymphoma with the worst outcomes amongst all extranodal lymphomas. There is a scarcity of data on real-world outcomes of primary CNS lymphoma (PCNSL) owing to the rarity of the disease. This study analyzed the demographic patterns, risk stratification, treatment regimens used, & outcomes of patients treated at Tata Memorial Center Mumbai, India. This is a retrospective analysis of newly diagnosed primary CNS lymphoma patients treated at our centre over seven years from January 2013 to December 2019. A total of 142 patients with PCNSL were diagnosed during this period. Thirty (21.1%) patients were deemed ineligible for any systemic or local therapies,ten patients were referred to other hospitals, two patients had relapsed disease, and one was excluded because age less than 18 years. Finally 99 patients were included in the final analysis. Among these 99 patients,72 patients (72.7%) were < 60 years,70 (70.7%) patients had Eastern cooperative oncology group (ECOG) performance status (PS) less than equal to 2. DLBCL was the most common histology (86.4%) while rests were high grade B cell NHL NOS (11.4%),Burkitt's Lymphoma(1%),Peripheral T-cell Lymphoma NOS (1.2%). Only one of 99 patients was positive for HIV serology. Multiple intracranial lesions were found in 59.5%. Surgical resection was performed in 28.4% of patients. Out of 63 patients in whom the International extranodal lymphoma study group (IELSG) score is available, 34(54%) were IELSG high-risk groups. As per Memorial Sloan Kettering Cancer Center (MSKCC) risk grouping, patients were almost equally distributed in all the risk groups, with 32(32.3%) patients in risk group 1 (age < 50 years), 36(36.4%) patients in risk group 2 (age > 50 years, KPS > = 70), and 31(31.3%) patients in risk group 3 age > 50 years, KPS < 70). First-line treatment with high dose methotrexate (HD-MTX) based regimens was administered to 92 (92.9%) patients, and 72.8% of these patients received rituximab. Of these 92 patients, 59 (64.1%) patients could complete induction, and 52 patients received consolidation. Thirty-one patients received high dose cytarabine based chemo consolidation, one patient underwent high dose chemotherapy followed by autologous stem cell transplantation (ACST), and 19 patients received whole-brain radiotherapy (WBRT) and 1 patient received temozolomide as consolidation regimen. Thus only 52 patients completed the entire course of induction with consolidation therapy. The response to treatment was assessed using International PCNSL Collaborative Group Criteria. Post completion of consolidation, 49(94.2%) patients had a complete response. With a median follow-up duration of 39.2 months, the median progression-free survival (PFS) and the median overall survival (OS) of the patients taken into the analysis (N = 99) were 21 and 37 months respectively. On multivariate analysis, age < 60 yrs, > = 5 HD-MTX cycles received & the use of rituximab predicted better OS.Outcomes of patients with PCNSL treated with HD-MTX based therapy are comparable to reported literature however a large proportion of patients do not undergo required treatment despite the curable nature of disease. Supplementary Information: The online version supplementary material available at 10.1007/s12288-022-01557-7.

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.

Evaluation of multiple myeloma measurable residual disease by high sensitivity flow cytometry: An international harmonized approach for data analysis.

BACKGROUND: Multiple myeloma (MM) measurable residual disease (MRD) evaluated by flow cytometry is a surrogate for progression-free and overall survival in clinical trials. However, analysis and reporting between centers lack uniformity. We designed and evaluated a consensus protocol for MM MRD analysis to reduce inter-laboratory variation in MM MRD reporting. METHODS: Seventeen participants from 13 countries performed blinded analysis of the same eight de-identified flow cytometry files from patients with/without MRD using their own method (Stage 1). A consensus gating protocol was then designed following survey and discussions, and the data re-analyzed for MRD and other bone marrow cells (Stage 2). Inter-laboratory variation using the consensus strategy was reassessed for another 10 cases and compared with earlier results (Stage 3). RESULTS: In Stage 1, participants agreed on MRD+/MRD- status 89% and 68% of the time respectively. Inter-observer variation was high for total numbers of analyzed cells, total and normal plasma cells (PCs), limit of detection, lower limit of quantification, and enumeration of cell populations that determine sample adequacy. The identification of abnormal PCs remained relatively consistent. By consensus method, average agreement on MRD- status improved to 74%. Better consistency enumerating all parameters among operators resulted in near-unanimous agreement on sample adequacy. CONCLUSION: Uniform flow cytometry data analysis substantially reduced inter-laboratory variation in reporting multiple components of the MM MRD assay. Adoption of a harmonized approach would meet an important need for conformity in reporting MM MRD for clinical trials, and wider acceptance of MM MRD as a surrogate clinical endpoint.

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.