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.

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.

Utility of CD229 as novel marker in measurable residual disease assessment in multiple myeloma-An evidence-based approach.

INTRODUCTION: CD229 has been found to be a useful plasma cell (PC) gating marker in multiple myeloma (MM). This study analyses the expression profile of CD229 on various bone marrow compartments namely, PC, non-PC and hematogones (HGs) using Multiparameter flow cytometry (MFC). Furthermore, it evaluates the ability of CD229 to delineate normal PC (NPC) from aberrant PC (APC) in measurable residual disease assessment (MRD) in MM. METHODS: Bone marrow aspirates from patients diagnosed with MM (per standard IMWG criteria) were collected in EDTA and processed for MFC using a single tube 14-color antibody panel as per standard operating procedure. RESULTS: A total of 74 patients with a diagnosis of MM (26 treatment naïve and 48 on therapy) were evaluated. The expression of CD229 was homogenous on both the PC and HG compartments as compared to CD138 and CD38. On comparing the expression of individual markers, it was found to be statistically significant between PC, HGs and non-PC for all three markers (p < 0.001). APC showed lower median expression of CD38 and higher median expression of CD138 and CD229 as compared to NPC and was found to be statistically significant for all markers (p < 0.001). In terms of differential expression on NPC and APC; CD38 was found to be the most aberrantly expressed (70%; 52/74) followed by CD229 (7%; 5/74) and CD138 (5%; 4/74). CONCLUSIONS: CD229 can be used for the identification of PC and due to relatively homogenous expression; it can be used as a suitable marker for targeted therapies. However, precise discrimination of NPC from APC cannot be reliably achieved with CD229, limiting its utility as a useful marker of diagnostic relevance and MRD assessment in MM.

Relevance of polyclonal plasma cells and post-therapy immunomodulation in measurable residual disease assessment in multiple myeloma.

BACKGROUND: Immunophenotypic profile and post-therapy alteration in antigenic expression were evaluated in normal, reactive, and aberrant plasma cells (NPC, RPC, and APC) for impact on measurable residual disease (MRD) assessment in multiple myeloma (MM). METHODS: Samples from non-MM staging marrow (n = 30), Hodgkin's lymphoma (n = 30) and MM (n = 724) were prospectively evaluated for expression profiles of NPC, RPC, and APC using antigens recommended in consensus guidelines. RESULTS: Polyclonal NPC-RPC demonstrated aberrations for all antigens evaluated with a higher frequency of aberrancies in post-therapy samples compared to treatment naïve samples (p < 0.001%). Immunomodulation in APC was observed in 79% of post-therapy samples with a change in expression of 1, 2, and ≥3 antigens in 19.9%, 15.6%, and 43.5% samples, respectively. In 13.4% of samples, APC showed no aberrancy and aberrant status was assigned based on cytoplasmic light chain restriction (cyLCR) alone. 9% samples with an admixture of NPC and APC displayed normal cytoplasmic kappa to lambda ratio (cyKLR) when the percentage of APC of total PC (neoplastic plasma cell index, NPCI), was below 25% and 50% for kappa and lambda restricted cases, respectively. CONCLUSION: The panorama and high frequency of antigenic aberrations on polyclonal PC signify the importance of MRD assay validation on a large cohort under normal and reactive conditions. Frequent Immunophenotypic shifts in APC re-confirm the redundancy of baseline immunophenotype for MRD evaluation. Small clones of APC may be missed by assessment of cyKLR alone and therefore, surface marker aberrancy supported by cyLCR is required for definitive assignment of residual APC.

Flow cytometric immunophenotyping of plasma cells across the spectrum of plasma cell proliferative disorders: A fresh insight with pattern-based recognition.

BACKGROUND: The expression pattern of common antigens including cytoplasmic kappa/lambda ratio (cyKLR) was evaluated by flow cytometric immunophenotyping (FCMI) to explore their relevance in discriminating normal and aberrant plasma cells (NPC and APC, respectively) across spectrum of plasma cell proliferative disorders (PCPD). METHODS: In this prospective analysis, 791 samples from PCPD (treatment naive = 455; partially treated = 336) were evaluated for expression of CD38, CD138, CD45, CD19, CD56, CD27, CD81, CD117, Cy-kappa, and Cy-lambda using FCMI. RESULTS: Amongst the entire cohort, 20.7% (n = 164) samples displayed only APC, 21% (n = 165) only NPC and 58% (n = 462) showed coexistence of NPC and APC. Using pattern-based recognition (PBR) for three common patterns (CD19 vs. CD56; CD27 vs. CD56 and CD19 vs. CD27), APC was separable from NPC in 93% samples. In 6.5% samples, the gating markers contributed in APC-NPC differentiation and in the remaining 0.5% CD117 and CD81 proved useful. Clonality assessment was found to be crucial to label plasma cell compartment as completely normal or aberrant in 42% cases with either all NPC or all APC. Sixty one out of 462 cases (13%) revealed cyKLR within normal reference range and in these cases; abnormal cyKLR was demonstrable only after gating APC separately based on PBR. CONCLUSION: Fair discrimination between NPC and APC is achievable in all PCPD samples using eight markers (Gating: CD38, CD138, CD45; PBR:CD19, CD56, CD27; clonality: Cy-kappa and Cy-lambda). Thus, combined assessment of clonality and immunophenotypic aberrancies is required for accurate, reliable and precise assessment of NPC and APC compartments in PCPD.

How autophagy can restore proteostasis defects in multiple diseases?

Cellular evolution develops several conserved mechanisms by which cells can tolerate various difficult conditions and overall maintain homeostasis. Autophagy is a well-developed and evolutionarily conserved mechanism of catabolism, which endorses the degradation of foreign and endogenous materials via autolysosome. To decrease the burden of the ubiquitin-proteasome system (UPS), autophagy also promotes the selective degradation of proteins in a tightly regulated way to improve the physiological balance of cellular proteostasis that may get perturbed due to the accumulation of misfolded proteins. However, the diverse as well as selective clearance of unwanted materials and regulations of several cellular mechanisms via autophagy is still a critical mystery. Also, the failure of autophagy causes an increase in the accumulation of harmful protein aggregates that may lead to neurodegeneration. Therefore, it is necessary to address this multifactorial threat for in-depth research and develop more effective therapeutic strategies against lethal autophagy alterations. In this paper, we discuss the most relevant and recent reports on autophagy modulations and their impact on neurodegeneration and other complex disorders. We have summarized various pharmacological findings linked with the induction and suppression of autophagy mechanism and their promising preclinical and clinical applications to provide therapeutic solutions against neurodegeneration. The conclusion, key questions, and future prospectives sections summarize fundamental challenges and their possible feasible solutions linked with autophagy mechanism to potentially design an impactful therapeutic niche to treat neurodegenerative diseases and imperfect aging.

Strategic Development of a Next-Generation Multi-Epitope Vaccine To Prevent Nipah Virus Zoonotic Infection.

Nipah virus (NiV) is an emerging zoonotic pathogen, reported for the recent severe outbreaks of encephalitis and respiratory illness in humans and animals, respectively. Many antiviral drugs have been discovered to inhibit this pathogen, but none of them were that much efficient. To overcome the complications associated with this severe pathogenic virus, we have designed a multi-epitope subunit vaccine using computational immunology strategies. Identification of structural and nonstructural proteins of Nipah virus assisted in the vaccine designing. The selected proteins are known to be involved in the survival of the virus. The antigenic binders (B-cell, HTL, and CTL) from the selected proteins were prognosticated. These antigenic binders will be able to generate the humoral as well as cell-mediated immunity. All the epitopes were united with the help of suitable linkers and with an adjuvant at the N-terminal of the vaccine, for the enhancement of immunogenicity. The physiological characterization, along with antigenicity and allergenicity of the designed vaccine candidates, was estimated. The 3D structure prediction and its validation were performed. The validated vaccine model was then docked and simulated with the TLR-3 receptor to check the stability of the docked complex. This next-generation approach will provide a new vision for the development of a high immunogenic vaccine against the NiV.

Combinatorial screening algorithm to engineer multiepitope subunit vaccine targeting human T-lymphotropic virus-1 infection.

Human T-lymphotropic virus (HTLV), the first human retrovirus has been discovered which is known to cause the age-old assassinating disease HTLV-1 associated myelopathy. Cancer caused by this virus is adult T cell leukemia/lymphoma which targets 10-20 million throughout the world. The effect of this virus extends to the fact that it causes chronic disease to the spinal cord resulting in loss of sensation and further causes blood cancer. So, to overcome the complications, we designed a subunit vaccine by the assimilation of B-cell, cytotoxic T-lymphocyte , and helper T-lymphocyte epitopes. The epitopes were joined together along with adjuvant and linkers and a vaccine was fabricated which was further subjected to 3D modeling. The physiochemical properties, allergenicity, and antigenicity were evaluated. Molecular docking and dynamics were performed with the obtained 3D model against toll like receptor (TLR-3) immune receptor. Lastly, in silico cloning was performed to ensure the expression of the designed vaccine in pET28a(+) expression vector. The future prospects of the study entailed the in vitro and in vivo experimental analysis for evaluating the immune response of the designed vaccine construct.

Racial disparities in prostate cancer: a molecular perspective.

Prostate cancer incidence and mortality rates are remarkably higher in African-American men as compared to their European-Americans counterparts. Despite these recognitions, precise causes underlying such prevalent racial disparities remain poorly understood. Although socioeconomic factors could account for such differences up to a certain extent, it is now being increasingly realized that such disparity has a molecular basis. Indeed, several differences, including genetic polymorphism, gene mutations, epigenetic modifications, miRNAs alterations, etc., have been reported in malignant prostate tissues from patients of diverse racial backgrounds. Here, we attempt to provide a molecular perspective on prostate cancer racial disparities by gathering available information on these associated factors and discussing their potential significance in disproportionate incidence and clinical outcomes.

Circulating MicroRNAs: Potential and Emerging Biomarkers for Diagnosis of Human Infectious Diseases.

MicroRNAs (miRNAs) are evolutionary conserved, small non-coding RNA with size ranging from 19 to 24 nucleotides. They endogenously regulate the gene expression at the post transcriptional level either through translation repression or mRNA degradation. MiRNAs have shown the potential to be used as a biomarker for the diagnosis, prognosis, and therapy of infectious diseases. Many miRNAs have shown significantly altered expression during infection. The altered expression of miRNA level in an infected human can be identified by the use of advanced diagnostic tools. In this review, we have highlighted the use of miRNA as an emerging tool for the identification of the human infectious disease. Till date, several miRNAs have been reported as a molecular biomarker in infectious diseases, such as miRNA-150 and miRNA-146b-5p in human immunodeficiency virus (HIV); miRNA-122, miRNA-21, and miRNA-34a in hepatitis; miRNA-361-5p and miRNA-29c in tuberculosis; miRNA-16 and miRNA-451 in malaria and miRNA-181 in Helicobacter pylori infection. The diagnosis of infection with the help of a biomarker is a non-invasive tool that has shown to have a key role in early diagnosis of infection. The discovery of circulating miRNA in the blood of infected patients has the potential to become a powerful non-invasive biomarker in coming future.

Arsenic exposure and outcomes of antimonial treatment in visceral leishmaniasis patients in Bihar, India: a retrospective cohort study.

BACKGROUND: In the late twentieth century, emergence of high rates of treatment failure with antimonial compounds (SSG) for visceral leishmaniasis (VL) caused a public health crisis in Bihar, India. We hypothesize that exposure to arsenic through drinking contaminated groundwater may be associated with SSG treatment failure due to the development of antimony-resistant parasites. METHODS: A retrospective cohort design was employed, as antimony treatment is no longer in routine use. The study was performed on patients treated with SSG between 2006 and 2010. Outcomes of treatment were assessed through a field questionnaire and treatment failure used as a proxy for parasite resistance. Arsenic exposure was quantified through analysis of 5 water samples from within and surrounding the patient's home. A logistic regression model was used to evaluate the association between arsenic exposure and treatment failure. In a secondary analysis survival curves and Cox regression models were applied to assess the risk of mortality in VL patients exposed to arsenic. RESULTS: One hundred and ten VL patients treated with SSG were analysed. The failure rate with SSG was 59%. Patients with high mean local arsenic level had a non-statistically significant higher risk of treatment failure (OR = 1.78, 95% CI: 0.7-4.6, p = 0.23) than patients using wells with arsenic concentration <10 µg/L. Twenty one patients died in our cohort, 16 directly as a result of VL. Arsenic levels ≥ 10 µg/L increased the risk of all-cause (HR 3.27; 95% CI: 1.4-8.1) and VL related (HR 2.65; 95% CI: 0.96-7.65) deaths. This was time dependent: 3 months post VL symptom development, elevated risks of all-cause mortality (HR 8.56; 95% CI: 2.5-29.1) and of VL related mortality (HR 9.27; 95% CI: 1.8-49.0) were detected. DISCUSSION/CONCLUSION: This study indicates a trend towards increased treatment failure in arsenic exposed patients. The limitations of the retrospective study design may have masked a strong association between arsenic exposure and selection for antimonial resistance in the field. The unanticipated strong correlation between arsenic exposure and VL mortality warrants further investigation.

Cancer "stemness"- regulating microRNAs: role, mechanisms and therapeutic potential.

Failure of conventional as well as target-based therapy against the advanced metastatic cancers is a significant clinical problem. While some cancers, such as pancreatic cancer, respond poorly to any kind of therapies, tumor relapse is often observed in many other cancer types after initial therapeutic response. Hence, significant research is being conducted to understand the mechanisms underlying therapeutic refractoriness of cancer. During the past decade, cancer stem cell (CSC) hypothesis has gained significant experimental and clinical support, and CSCs have emerged as potentially useful pharmacologic targets. MicroRNAs (miRNAs) are a group of small (~18-25 nucleotides) non-protein encoding RNAs that are now established as important regulators of gene expression. They can function as tumor promoters (oncomirs) or suppressors (anti-oncomirs) and thus hold profound implications for cancer therapy. Recent studies have identified several miRNAs to be differentially expressed in CSCs and established their role in targeting genes and pathways supporting cancer stemness properties. Here, we discuss these findings and review recent advances in miRNA-based strategies to exploit therapeutic potential of miRNAs in cancer treatment.

Efficacy of miltefosine in the treatment of visceral leishmaniasis in India after a decade of use.

BACKGROUND: Miltefosine is the only oral drug available for treatment of Indian visceral leishmaniasis (VL), which was shown to have an efficacy of 94% in a phase III trial in the Indian subcontinent. Its unrestricted use has raised concern about its continued effectiveness. This study evaluates the efficacy and safety of miltefosine for the treatment of VL after a decade of use in India. METHODS: An open-label, noncomparative study was performed in which 567 patients received oral miltefosine (50 mg for patients weighing <25 kg, 100 mg in divided doses for those weighing ≥25 kg, and 2.5 mg per kg for those aged <12 years, daily for 28 days) in a directly observed manner. Patients were followed up for 6 months to see the response to therapy. RESULTS: At the end of treatment the initial cure rate was 97.5% (intention to treat), and 6 months after the end of treatment the final cure rate was 90.3%. The overall death rate was 0.9% (5 of 567), and 2 deaths were related to drug toxicity. Gastrointestinal intolerance was frequent (64.5%). The drug was interrupted in 9 patients (1.5%) because of drug-associated adverse events. CONCLUSIONS: As compared to the phase III trial that led to registration of the drug a decade ago, there is a substantial increase in the failure rate of oral miltefosine for treatment of VL in India.