Immunophenotypic characterization of leukemic stem cells in acute myeloid leukemia using single tube 10-colour panel by multiparametric flow cytometry: Deciphering the spectrum, complexity and immunophenotypic heterogeneity.

INTRODUCTION: Despite extensive research, comprehensive characterization of leukaemic stem cells (LSC) and information on their immunophenotypic differences from normal haematopoietic stem cells (HSC) is lacking. Herein, we attempted to unravel the immunophenotypic (IPT) characteristics and heterogeneity of LSC using multiparametric flow cytometry (MFC) and single-cell sequencing. MATERIALS AND METHODS: Bone marrow aspirate samples from patients with acute myeloid leukaemia (AML) were evaluated using MFC at diagnostic and post induction time points using a single tube-10-colour-panel containing LSC-associated antibodies CD123, CD45RA, CD44, CD33 and COMPOSITE (CLL-1, TIM-3, CD25, CD11b, CD22, CD7, CD56) with backbone markers that is, CD45, CD34, CD38, CD117, sCD3. Single-cell sequencing of the whole transcriptome was also done in a bone marrow sample. RESULTS: LSCs and HSCs were identified in 225/255 (88.2%) and 183/255 (71.6%) samples, respectively. Significantly higher expression was noted for COMPOSITE, CD45RA, CD123, CD33, and CD44 in LSCs than HSCs (p < 0.0001). On comparing the LSC specific antigen expressions between CD34+ (n = 184) and CD34- LSCs (n = 41), no difference was observed between the groups. More than one sub-population of LSC was demonstrated in 4.4% of cases, which further revealed high concordance between MFC and single cell transcriptomic analysis in one of the cases displaying three LSC subpopulations by both methods. CONCLUSION: A single tube-10-colour MFC panel is proposed as an easy and reproducible tool to identify and discriminate LSCs from HSCs. LSCs display both inter- and intra-sample heterogeneity in terms of antigen expressions, which opens the facets for single cell molecular analysis to elucidate the role of subpopulations of LSCs in AML progression.

Analytical Appraisal of Hematogones in B-ALL MRD Assessment Using Multidimensional Dot-Plots by Multiparametric Flow Cytometry: A Critical Review and Update.

The spectrum of benign B-cell precursors, known as hematogones (HGs), shows a significant morphological and immunophenotypic overlap with their malignant counterpart i.e. B-lymphoid blasts (BLBs). This results in a diagnostic dilemma in assessment of cases wherein there is a physiological preponderance of HGs and also poses a significant challenge in measurable residual disease assessment in B-cell acute lymphoblastic leukaemia. Consequently, expression patterns of various immunophenotypic markers are considered the most important tool in identification and delineation of HGs from BLBs. However, certain aspects of B-cell compartment evaluation by flow cytometric immunophenotyping and its relevance in clinical scenarios is yet to be defined precisely. This review summarizes current flowcytometric data on HGs and its discrimination from BLBs based on thorough review of literature and evaluation of in-house data. Furthermore, it focuses on the utility of an additional analytical tool i.e., radar plot for a comprehensive representation of various subsets of the B-cell compartment and their differentiation from BLBs. Supplementary Information: The online version contains supplementary material available at 10.1007/s12288-023-01696-5.

Therapy related AML in a case of chronic lymphocytic leukemia.

In comparison to the general population, patients with chronic lymphocytic leukemia (CLL) are at a higher risk of developing secondary malignancies. Several factors may contribute to pathogenesis, including direct effects of chemotherapy and radiation as well as the reduction of immune surveillance. Factors influencing the increased risk include the increasing age of CLL patients, chronic antigenic stimulation, and immune impairment related to CLL or chemotherapy. Compared to patients with acute myeloid leukemia (AML) that developed from de novo, therapy-related AML (t-AML) has had a poorer outcome. The range of cytogenetic abnormalities in therapy-related AML is comparable to that in de novo AML, although these patients have a significantly higher frequency of unfavourable cytogenetics, such as a complex karyotype or a deletion or loss of chromosomes 5 and/or 7. Herein, we describe a case of therapy-related AML with monocytic differentiation and t(8;16) with a residual CLL population. The aim of the present case is to highlight rare occurrence of therapy related AML with t(8;16) in CLL after fluderabine based chemotherapy (FCR: fludarabine, cyclophosphamide, and rituximab). This case also highlights flowcytometric immunophenotyping as an ideal tool to characterize secondary AML along with the identification of minimal residual disease of CLL clone, which could have ignored at t-AML diagnosis. The pathogenesis of myeloid and lymphoid malignancies as well as their co-existence can be studied by focusing on such patients. Factors predisposing to the development of t-AML should be studied further, which would help in monitoring these patients more carefully.

Acute myeloid leukemia with RAM immunophenotype: A new underdiagnosed entity.

INTRODUCTION: Acute myeloid leukemia (AML) with RAM immunophenotype is a distinct subtype of AML, as described by the Children's Oncology Group (COG), with characteristic morphological and immunophenotypic properties. It is characterized by strong CD56 expression with dim to negative CD45, HLA-DR, and CD38 expression. It is an aggressive leukemia with a poor response to induction chemotherapy and/or frequent relapses. METHODS: Seven cases with the characteristic RAM immunophenotype were identified in this retrospective analysis of newly diagnosed pediatric AML cases from January 2019 to December 2021. Herein, we have critically analyzed their clinical, morphological, cytochemical, immunophenotyping, cytogenetic, and molecular profiles. The patients were traced and followed for their current disease and treatment status. RESULTS: Of 302 cases of pediatric AML (age <18 years), seven cases (2.3%) with the distinct RAM phenotype were observed, with age ranging from 9 months to 5 years. Two patients were misdiagnosed earlier as small round cell tumor because of the strong CD56 positivity and the absence of leukocyte common antigen (LCA), but they were later correctly identified as granulocytic sarcoma. The bone marrow aspirate showed blasts with unusual cohesiveness and clumping with nuclear moulding, mimicking non-hematologic malignancies. Flow cytometry revealed blasts with low side scatter, dim to negative CD45 and CD38, negative cMPO, CD36, and CD11b; moderate to bright CD33, CD117, and bright CD56. The Mean fluorescence intensity (MFI) of CD13 expression was significantly lower as compared to the internal controls. Cytogenetic and molecular studies did not show any recurrent abnormalities. Reverse transcription polymerase chain reaction for CBFA2T3-GLIS2 fusion was performed in 5/7 cases, with one positive result. On clinical follow-up, two patients were refractory to chemotherapy. Six of the seven cases had succumbed to death (duration of survival: 3-343 days after initial diagnosis). CONCLUSION: AML with RAM immunophenotype, a distinct form of pediatric AML with a poor prognosis, may pose a diagnostic challenge if presented as a soft tissue mass. A comprehensive immunophenotypic evaluation, including stem cell and myeloid markers, is critical for an accurate diagnosis of myeloid sarcoma with the RAM-immunophenotype. Our data demonstrated weak CD13 expression as an additional immunophenotypic finding.

Cytomorphological evaluation of non-haematopoietic malignancies metastasizing to the bone marrow.

Bone marrow (BM) is one of the rare but important site of metastasis of solid tumors. The key steps of metastasis include invasion, intravasation, circulation, extravasation, and colonization. Tumor cells may express some adhesion molecules that promote the transmigration to the marrow space and link them to the marrow stroma with subsequent engraftment. It is important to detect the bone marrow metastasis for initial clinical staging, therapeutic selection, prognostic risk stratification, assessment of response to therapy and predicting relapse. Prognosis of non-hematopoietic malignancies with BM metastasis is dismal. Due to occulting and atypical clinical manifestations, bone marrow metastases can be easily missed or misdiagnosed, leading to higher mortality rates. The important factors on which the prognosis of patients with bone marrow metastases depends are primary tumor site, performance status, platelet count, and therapeutic regimens (systemic chemotherapy or palliative/supportive care). Further, in cases with BM metastasis with unknown primary sites, misdiagnosis can lead to delayed initiation of therapy and increased mortality. BM metastasis is seen in less than 10% of patients with metastatic cancer and is common in lung, breast or prostate carcinoma. Bone marrow metastasis can be presented as the initial presentation with hematological changes and may be misdiagnosed as a primary haematopoietic disorder. Leucoerythoblastic blood picture is the most common peripheral blood smear finding indicating BM metastasis, may be an indicator of associated BM fibrosis. Bone marrow aspiration and biopsy with immunohistochemistry (IHC) is an easy, cost effective and gold standard method of detection of BM metastasis. BM biopsy is superior to bone marrow aspirate for detection of metastasis. Morphology of metastatic cells is as per the primary site of tumor. Immunohistochemistry is a useful adjunct to morphology in reaching a definitive diagnosis even in case with carcinoma unknown primary (CUP) and also in diagnosing case of unsuspected malignancies. Though bone marrow is not among the most common site of involvement in CUP, which are liver, bone, lymph nodes and lung. But BM, if involved, the site of origin is determined using the immunohistochemistry panel applied to the metastatic deposits based on the morphology The aim of the review is to discuss the hematological findings of non-haematopoietic malignancies metastasizing to the bone marrow, emphasizing on histomorphology with IHC and its significance in establishing primary diagnosis in clinically unsuspected cases.

CD34 negative HLA-DR negative acute myeloid leukaemia: A higher association with NPM1 and FLT3-ITD mutations.

INTRODUCTION: CD34 and HLA-DR negativity is often used as a characteristic immunophenotypic feature of acute promyelocytic leukaemia (APL) that differentiates APL from other subtypes of acute myeloid leukaemia (AML). However, other subtypes of AML, without expression of CD34 and HLA-DR antigens, have also been reported. METHODS: We analysed the HLA-DR negative de novo non-APL AML cases by dividing HLA-DR negative non-APL group into 2 sub-groups based on CD34 expression and compared the characteristics of CD34 negative HLA-DR negative with CD34 positive HLA-DR negative non-APL AML cases with respect to morphologic, immunophenotypic, molecular and clinical parameters. RESULTS: There were 70 cases (8.54%) which were CD34 negative HLA-DR negative and 52 cases (6.34%) were CD34 positive HLA-DR negative. The median age at diagnosis was higher in CD34 negative HLA-DR negative AML than in CD34 positive HLA-DR negative AML group (38 years vs. 12 years, p < 0.001). DIC rate was higher in CD34 negative HLA-DR negative group than the other group (p < 0.001). Median total leucocyte count was higher with higher blast count in peripheral blood and bone marrow in CD34 negative HLA-DR negative AML cases than the other group (p < 0.05). CD34 negative HLA-DR negative AML was more associated with normal karyotype (96.2% vs. 38.5%; p < 0.001), NPM1 mutation (67.8% vs. 8.3%; p < 0.001) and FLT-ITD mutation (37.3% vs. 13.9%; p < 0.05). In CD34 negative HLA-DR negative group, 16 cases had co-occurrence of NPM1 and FLT3-ITD mutations, whereas no case of CD34 positive HLA-DR negative group had such dual mutation positivity. There was poor median overall survival [3.8 months (95%CI: 2.3-7.8 months) vs. 20.4 months (95% CI: 12.8-25.7 months); p = 0.0148] in CD34 positive HLA-DR negative AML than CD34 negative HLA-DR negative AML cases. CONCLUSION: We found that the CD34 negative HLADR negative non APL AML is highly associated with NPM1 and FLT3-ITD mutation, older age at diagnosis, DIC, higher total leucocyte count, higher blast counts and normal karyotype in comparison to CD34 positive HLA-DR negative AML group. Co-occurrence of NPM1 and FLT3-ITD mutation was also exclusively seen in CD34 negative HLA-DR negative group. There was poor overall survival in CD34 positive HLA-DR negative AML than CD34 negative HLA-DR negative AML cases.

Clinico-Hematological Profile of Acute Myeloid Leukemia: Experience From a Tertiary Care Cancer Center in North India.

INTRODUCTION: Complete diagnosis of acute myeloid leukemia (AML) requires knowledge of clinical information combined with morphologic evaluation, immunophenotyping, karyotyping, and molecular genetic testing. The study intends to evaluate the demographic profile, clinical workup, and investigation, including flow cytometric immunophenotyping, in adult and pediatric age groups of AML. MATERIALS AND METHODS: This is a retrospective study of AML patients treated between January 2017 and December 2021. Clinical and demographic characteristics and investigation findings were recorded from case files and the hematology database. RESULT: A total of 896 cases of AML were registered during the given period, of which 819 cases were de-novo AML. Among those 819 cases, more than two-thirds of cases, i.e., 78.9% (N = 646), received induction chemotherapy. A significantly higher male-to-female ratio was observed (1.5:1). The median age was 22 years. The median time for diagnosis was three days and the median time for treatment intervention was four days. There were significant differences in the Eastern Cooperative Oncology Group (ECOG) performance status scores between pediatric and adult AML patients. Pediatric AML patients presented with better ECOG performance scores (ECOG performance scores 0 and 1) than adult patients (74.76% vs. 43.14%, p < 0.001). Further comparing adult vs. pediatric AML patients, normal karyotype (60.56% vs. 31.93%, p < 0.001) and NPM1 (22.25% vs. 6.72%, p < 0.001) and FLT3-ITD mutations (20.28% vs. 7.98%, p<0.001) were more common in the adult group, whereas AML-ETO (40.76% vs. 16.34%, p < 0.001) was more common in the pediatric group. CONCLUSION: The study highlights the presenting age is lower than global figures. The median time for initial diagnosis and the start of treatment is within the acceptable norms. Normal karyotype and NPM1 and FLT3 mutations were common in adult AML patients, whereas AML-ETO was more common in the pediatric cohort. These findings will help plan prospective studies and see the correlation with treatment outcomes. The laboratory workup practice currently complies with the standard guidelines at our center.

Clinical and Prognostic Impact of Copy Number Alterations and Associated Risk Profiles in a Cohort of Pediatric B-cell Precursor Acute Lymphoblastic Leukemia Cases Treated Under ICiCLe Protocol.

Copy number alteration (CNA) status and CNA risk profiles of IKZF1 plus , UK-ALL CNA risk groups and MRplus scores, were evaluated for clinical and prognostic impact in a cohort of 493 B-cell acute lymphoblastic leukemia cases diagnosed and treated under the Indian Collaborative Childhood Leukemia group (ICiCLe) protocol trial. Overall CNA frequency was 59% with 60% of cases showing 2-loci deletion. CDKN2A/B deletion was most common CNA (36.3%), while IKZF1 deletion and IKZF1 plus profile were noted in 19.5% and 13.4% of cases, respectively. IKZF1 deletions and other CNA risk profiles were significantly associated with poor (PR)/high risk (HR) clinical and genetic profile parameters (P < 0.001). In addition, the 3-year OS, event-free survival (EFS) was significantly poor with high relapse rate (RR) of 38.6%, 46.5%, and 35.2% for IKZF1 deletions, IKZF1 plus profiles, and UK-ALL CNA-intermediate risk (IR)+PR risk groups, respectively (P < 0.001). Integrated evaluation of UK-ALL CNA risk profile with ICiCLe trial risk stratification groups revealed a worse overall survival, EFS, and RR of 63.3%, 43.2%, and 35.2% for CNA-IR+PR profile compared to CNA-good risk profile (81.3%, 65.0%, and 21.0%; P < 0.001). Hence, routine CNA testing in our setting is must to identify standard risk and IR cases likely to benefit from HR treatment.

Effect of the sequence of pull of bone marrow aspirates on plasma cell quantification in plasma cell proliferative disorders.

INTRODUCTION: The evaluation of plasma cell (PC) compartment is influenced by the quality of bone marrow aspirate (BMA). Herein, we evaluated the impact of sequence of pull on quality of clinical assessment in plasma cell proliferative disorders (PCPDs). METHODS: Histomorphology along with smears from first pull and second pull BMA and flow cytometric immunophenotyping (FCMI) data from second pull aspirate were evaluated for cellularity and PC%. RESULTS: Of the 484 samples, BMA smears were adequate in 87.4% of first pull (median PC = 7%; IQR = 2-25%) and 51.2% of second pull samples (median PC = 2%; IQR = 0.5-12%; p < 0.001). Recovery of PC was least on FCMI (median PC = 0.59%; IQR = 0.14-3.07%), however, sample adequacy was met in 42.6% of samples with acquisition of ≥3 million events. Second pull smears under-reported PC% in 34% of newly diagnosed multiple myeloma (NDMM) (<10% PC) and 46% of MM on therapy (<5% PC), resulting in suboptimal assessment. Bone marrow biopsy (BMBx) was evaluated in a total of 309 cases (median PC = 10.0%; IQR 4.0-40.0%) with significantly higher numbers of BMPC% on BMBx compared with first pull smears (Mean ± 2SD: 25.9% ± 30.54 vs. 20.77% ± 20.20; p = 0.001). CONCLUSION: First pull BMA smears were of superior quality but inadequate in one-tenth of samples. Second pull smears underreported PC% and recovery of PC compartment was poorest on FCMI. Concurrent bone marrow biopsy and use of the first pull sample for FCMI along with acquisition of a higher number of cells on FCMI may enhance the quality of assessment in PCPDs.

Spectrum of hematological changes in COVID-19.

Coronavirus disease 2019 (COVID-19) is caused by pathogenic and highly transmissible Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) which is a single stranded RNA virus. It rapidly emerged from an epidemic to a global pandemic form spreading in alarming levels. The pathogenesis involving spike protein which is present on the viral surface, plays a key role in host attachment and penetration. SARS-CoV-2 infection significantly affects respiratory system, but may involve other systems including haematopoietic system and homeostasis. Aim of the review article is to discuss spectrum of haematological changes in the blood counts, coagulation, peripheral blood and bone marrow in COVID-19 for complete understanding the disease process, the knowledge of which is helpful in early diagnosis and management of these patients. An extensive immune profiling of B and T cell population with analysis of spectrum of immune changes during the period of infection were also discussed. In COVID-19, changes in laboratory parameters and hematologic abnormalities have been reported and its association with early diagnosis, disease prognosis and severity has been repeatedly discussed in the literature. Changes in laboratory investigations help in risk stratification and early intervention. The most common laboratory finding in COVID-19 is lymphopenia. COVID-19 patients presented with coagulopathy is at high risk of morbidity and mortality. In severe COVID-19 patients, bone marrow aspirate shows histiocytic proliferation with hemophagocytosis. To understand the correlations between immune responses and severity of COVID-19, immune profiling of B and T cell population was compared with extensive clinical data. A deep understanding of the laboratory findings and haematological abnormalities associated with SARS-CoV-2 infection would help to raise disease suspicion in absence of Real time polymerase chain reaction or antibody results. Also the blood counts along with the morphological changes in peripheral blood would be helpful in prompt screening, diagnosis, prognosis and management of COVID-19 patients.

Acute Promyelocytic Leukemia with t(2;3): An Unusual Additional Chromosomal Abnormality.

Acute promyelocytic leukemia is a distinct subset of acute myeloid leukemia with characteristic clinical, morphological and genetic features. The gene product PML-RAR alpha resulting from reciprocal t(15;17) translocation, plays a pivotal role in the pathogenesis of acute promyelocytic leukemia and classified as favorable cytogenetic features. We are describing an unusual additional chromosomal abnormality t(2;3) in APL patient.

Seroprevalence of antibodies to SARS-CoV-2 in healthcare workers & implications of infection control practice in India.

Background & objectives: Healthcare workers (HCWs) are considered to be at a high risk of contracting COVID-19 infection. Besides, control of nosocomial infections transmitted from HCWs to the patients is also a cause of concern. This study was undertaken to investigate the seroprevalence of antibodies against the SARS-CoV-2 virus among the hospital staff of a tertiary care health facility in north India. Methods: The HCWs were tested for SARS-CoV-2 serology (IgG+IgM) using chemiluminescence immunoassay between June 22 and July 24, 2020. Venous blood (2 ml) was collected and tested for SARS-CoV-2 IgG and IgM antibodies. Results: Of the 3739 HCWs tested, 487 (13%) were positive for total SARS-CoV-2 antibodies. The highest seroprevalence was observed in administrative staff (19.6%) and least in physicians (5.4%). The staff who used public (20%) and hospital transportation (16.9%) showed higher seroprevalence compared to staff using personal transportation (12.4%). No difference was observed between HCWs posted in COVID versus non-COVID areas. All seropositive symptomatic HCWs in our study (53.6%) had mild symptoms, and the remaining 46.4 per cent were asymptomatic. The antibody positivity rate progressively increased from 7.0 per cent in the first week to 18.6 per cent in the fourth week during the study. Interpretation & conclusions: The presence of antibodies to SARS-CoV-2 in a significant number of asymptomatic HCWs, association with the use of public transport, relatively lower seroprevalence compared with the non-HCWs and rising trend during the period of the study highlight the need for serosurveillance, creating awareness for infection control practices including social distancing and study of infection dynamics in the community for effective control of an infectious pandemic.