Association of Mortality with Lymphocyte Subset in Patients with COVID-19-associated Acute Respiratory Failure: A Subgroup Analysis.

Background: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes pneumonia and lymphopenia. We investigated the predictive value of T-lymphocyte subset absolute counts for outcomes following coronavirus disease-2019 (COVID-19)-associated acute respiratory failure (C-ARF). Patients and methods: A retrospective chart review of adult patients with C-ARF was undertaken from 23 March 2020 to 20 November 2021 to obtain relevant data. Patients were divided into two groups based on survival. The T-lymphocyte subsets were determined by flow cytometric analysis. A binomial logistic regression was performed to ascertain factors affecting survival. Cut-off values to differentiate between survivors and non-survivors were identified with the receiver operating characteristic (ROC) analysis. Results: A total of 379 patients were analyzed. Age was negatively correlated with survival. Non-survivors had significantly lower T-lymphocyte subset absolute counts than survivors. Serum ferritin levels were significantly higher in non-survivors. Baseline lymphocyte (%) and a subset were predictive of survival in patients [lymphocyte (%) <5.65%, CD3+ <321 cells/µL, CD4+ <205 cells/µL, CD8+ <103 cells/µL]. Conclusions: Lower T-lymphocyte subsets were associated with higher mortality in patients with C-ARF. Monitoring trends may help in identifying patients at increased risk of poor outcomes. How to cite this article: Vadi S, Pednekar A, Suthar D, Sanwalka N, Rabade N, Ghodke K. Association of Mortality with Lymphocyte Subset in Patients with COVID-19-associated Acute Respiratory Failure: A Subgroup Analysis. Indian J Crit Care Med 2023;27(1):52-56.

In Response to Author: COVID-19 and T Cells: Do T Cells Really Matter?

How to cite this article: Vadi S, Pednekar A, Suthar D, Sanwalka N, Ghodke K, Rabade N. In Response to Author: COVID-19 and T Cells: Do T Cells Really Matter? Indian J Crit Care Med 2023;27(1):76.

Characteristics and Predictive Value of T-lymphocyte Subset Absolute Counts in Patients with COVID-19-associated Acute Respiratory Failure: A Retrospective Study.

Background: Of the factors influencing severity and outcomes following coronavirus disease-2019 (COVID-19), cellular immune response has a strong impact. The spectrum of response varies from over-activation to hypo-functioning. The severe infection leads to reduction in numbers and dysfunction of T-lymphocytes/subsets. Patients and methods: This retrospective, single-center study aimed to analyze the expression of T-lymphocyte/subsets by flow cytometry and inflammation-related biomarker, serum ferritin in real-time polymerase chain reaction (RT-PCR) positive patients. According to oxygen requirements, patients were stratified into nonsevere (room air, nasal prongs, and face mask) and severe [nonrebreather mask (NRBM), noninvasive ventilation (NIV), high-flow nasal oxygen (HFNO), and invasive mechanical ventilation (IMV)] subgroups for analysis. Patients were classified into survivors and nonsurvivors. Mann-Whitney U test was used to analyze differences in T-lymphocyte and subset values when classified according to gender, the severity of COVID, outcome, and prevalence of diabetes mellitus (DM). Cross tabulations were computed for categorical data and compared using Fisher's exact test. Spearman correlation was used to analyze the correlation of T-lymphocyte and subset values with age or serum ferritin levels. p <0.05 values were considered to be statistically significant. Results: A total of 379 patients were analyzed. Significantly higher percentage of patients with DM were aged ≥61 years in both nonsevere and severe COVID groups. A significant negative correlation of CD3+, CD4+, and CD8+ was found with age. CD3+ and CD4+ absolute counts were significantly higher in females as compared to males. Patients with severe COVID had significantly lesser total lymphocyte (%), CD3+, CD4+, and CD8+ counts as compared to those with nonsevere COVID (p <0.05). T-lymphocyte subsets were reduced in patients with severe disease. A significant negative correlation of total lymphocyte (%), CD3+, CD4+, and CD8+ counts was found with serum ferritin levels. Conclusions: T-lymphocyte/subset trends are an independent risk factor for clinical prognosis. Monitoring may help in intervening in patients with disease progression. How to cite this article: Vadi S, Pednekar A, Suthar D, Sanwalka N, Ghodke K, Rabade N. Characteristics and Predictive Value of T-lymphocyte Subset Absolute Counts in Patients with COVID-19-associated Acute Respiratory Failure: A Retrospective Study. Indian J Crit Care Med 2022;26(11):1198-1203.

BRAFV600E mutation in hairy cell leukemia: A single-center experience.

BACKGROUND: BRAFV600E mutation has been reported as a unique genetic lesion of hairy cell leukemia (HCL), a subset of which lacks this lesion and shows adverse outcomes. AIMS: To determine the prevalence of BRAFV600E in HCL from our center and derive clinicopathological correlation, if any. MATERIALS AND METHODS: A 9-year retrospective analysis of 46 consecutive cases of HCL diagnosed on morphology and immunophenotyping was done. Stained smears were used as samples for amplification refractory mutation system polymerase-chain reaction using fluorescent primers for mutation detection. RESULTS: BRAFV600E mutation was detected in 41/46 patients (89.1%) while absent in control samples of chronic lymphocytic leukemia. Cases mimicking HCL-variant clinically or immunophenotypically too showed the presence of this mutation. HCL with mutated BRAF presented at a younger age. No statistical difference in blood counts, tumor load, and immunophenotype patterns existed among BRAF mutated and unmutated group. Nine patients (45%) with mutated BRAF had residual disease following treatment with cladribine. CONCLUSION: BRAFV600E mutation analysis has a definitive role in the diagnosis of HCL.

Molecular genetics of BCR-ABL1 negative myeloproliferative neoplasms in India.

INTRODUCTION: Over the past decade, we have moved on from a predominantly morphological and clinical classification of myeloproliferative neoplasms (MPN) to a more evolved classification that accounts for the molecular heterogeneity that is unique to this subgroup of hematological malignancies. This usually incorporates mutations in Janus kinase 2 (JAK2), MPL, and calreticulin (CALR) genes. In this manuscript, we report the frequency of these mutations in a cohort of Indian patients at a tertiary cancer center. MATERIALS AND METHODS: One hundred and thirty cases of MPN were included in this study. These cases were diagnosed and classified based on the World Health Organization 2008 criteria. JAK2 and MPL mutations were detected using high sensitivity allele-specific polymerase chain reaction using fluorescent labeled primers followed by capillary electrophoresis. A subset of JAK2 and CALR mutations were assessed using a fragment length assay. RESULTS: Among the MPN, we had 20 cases of polycythemia vera (PV), 34 cases of essential thrombocythemia (ET), and 59 of myelofibrosis (MF). JAK2, MPL, and CALR mutations were mutually exclusive of each other. Seventeen cases were categorized as MPN unclassifiable (MPN-U). JAK2p.V617F and MPL mutations were present in 60% (78 of 130) and 5.3% (7 of 130) of all MPN. All the PV cases harbored the JAK2 p.V617F mutation. A total of 23.8% (31 of 130) of patients harbored CALR mutations. CALR exon 9 mutations were detected in 60.8% (14 of 23) and 50% (5 of 10) of JAK2 and MPL negative MF and ET cases, respectively. MPN-U cases included three JAK2 p.V617F positive, two MPL p.W515 L, and 12 CALR positive cases. Ten different types of CALR indels (8 deletions and 2 insertions) were detected of which Type I and Type II mutations were the most common, occurring at a frequency of 45.1% (14 of 31) and 22.5% (7 of 31), respectively. DISCUSSION AND CONCLUSION: We report frequencies of JAK2 p. V617F, MPL exon 10 and CALR mutations in 130 patients similar to those reported in western literature. These mutations carry not only diagnostic but also prognostic relevance.

Molecular Heterogeneity in Acute Promyelocytic Leukemia - a Single Center Experience from India.

Atypical breakpoints and variant APL cases involving alternative chromosomal aberrations are seen in a small subset of acute promyelocytic leukemia (APL) patients. Over seven different partner genes for RARA have been described. Although rare, these variants prove to be a diagnostic challenge and require a combination of advanced cytogenetic and molecular techniques for accurate characterization. Heterogeneity occurs not only at the molecular level but also at clinico-pathological level influencing treatment response and outcome. In this case series, we describe the molecular heterogeneity of APL with a focus on seven variant APL cases from a single tertiary cancer center in India over a period of two and a half years. We discuss five cases with ZBTB16-RARA fusion and two novel PML-RARA variants, including a Bcr3 variant involving fusion of PML exon4 and RARA exon3 with an additional 40 nucleotides originating from RARA intron2, another involving exon 6 of PML and exon 3 of RARA with addition of 126 nucleotides, which mapped to the central portion of RARA intron 2. To the best of our knowledge, this is the first case series of this kind from India.

An integrated genomic profile that includes copy number alterations is highly predictive of minimal residual disease status in childhood precursor B-lineage acute lymphoblastic leukemia.

INTRODUCTION: Copy number alterations (CNA) have been described in childhood precursor B-lineage acute lymphoblastic leukemia (B-ALL) which in conjunction with chromosomal abnormalities drive leukemogenesis. There is no consensus on the clinical incorporation of CNA in B-ALL. An integrated genomic classification (IGC) has been proposed which includes CNA and cytogenetics. METHODS: We correlated this IGC with immunophenotypic minimal residual disease (MRD) as well as other standard criteria for 245 patients of B-ALL such as National Cancer Institute (NCI) risk, D+8 prednisolone response, cytogenetics, and ploidy status. RESULTS: MRD was detectable in 81 patients (33.1%). The most common abnormalities were seen in CDKN2A/B (25.7%) followed by PAX5(20%), ETV6(16.7%), IKZF1(15.5%), Rb1(5.3%), BTG (3.3%), EBF1(2.0%), and PAR1(0.8%). On integrating CNA into the IGC, 170 patients (69.4%) were classified into good genomic risk (GEN-GR) whereas 75 (30.6%) belonged to the poor genomic risk (GEN-PR) category. The IGC showed a significant correlation with MRD and NCI risk. The presence of CNA predicted MRD clearance in intermediate cytogenetics group. CONCLUSION: These data seem to indicate that in addition to cytogenetics, CNA should be incorporated into routine clinical testing and risk algorithms for B-ALL. The IGC is of prognostic relevance and offers an additional avenue for prognostication and risk-adapted therapy.

Immunogenetics of chronic lymphocytic leukemia.

INTRODUCTION: Cytogenetic aberrations as well as presence of IGVH mutations are the underlying reason for clinical heterogeneity in Chronic Lymphocytic Leukemia (CLL). The presence of IGVH mutations as well as the predominant gene usage shows geographical variations. However, there is no study from India addressing immunogenetics of CLL. In a first Indian study we document the immunogenetics of CLL in a large tertiary hospital. METHODS: We analyzed IGVH mutation status, VH gene usage, cytogenetic abnormalities using FISH, immunophenotyping data and correlated them with standard clinical variables in 84 patients of CLL. RESULTS: Advanced Rai stage (Stage 3/4) was seen in 45% of our patients, where as 13q deletion was the commonest clonal cytogenetic abnormality detected in 48.4% of the cases. IGVH unmutated cases (55.2%) showed higher proportion expressing CD38 and CD49d, a preferential usage for VH1 and VH3 families (55.2%), presentation at an advanced Rai stage (52.8%) as well as more frequent presence of p53 deletions. As compared to the IGVH mutated cases greater proportion of IGVH unmutated patients (70%) required treatment. However, there was no significant difference in the time to treatment between mutated and unmutated cases which can be attributed to relatively short median follow up of 10 months. CONCLUSION: To summarize, we have seen a higher proportion of IGVH unmutated patients in our cohort (55.2%). The commonly used VH genes in the Indian population are IGVH 2-5, IGVH 1-2 and IGVH 1-69. Longer clinical follow up and a larger cohort is necessary to confirm the prognostic value of IGVH mutation analysis in Indian Patients with CLL.

CD19 negative precursor B acute lymphoblastic leukemia (B-ALL)-Immunophenotypic challenges in diagnosis and monitoring: A study of three cases.

BACKGROUND: CD19 is a B-cell specific marker, expressed on all stages of B-lymphocytes including plasma cells. It is widely used in the flow cytometric immunophenotyping (FCI) of B-cell and plasma cell malignancies. The analysis approach of FCI for the diagnosis and monitoring of B-cell acute lymphoblastic leukemia (B-ALL) is totally based on the CD19-based primary gating strategy and it would be challenging to study B-ALL without CD19 expression. Since CD19 negative B-ALL are extremely rare, we report three cases of B-ALL with negative expression of CD19 and discussed its implication in the diagnosis, residual disease monitoring and future targeted therapy. METHODS: Peripheral blood (PB) and bone marrow (BM) samples from three cases suspicious of acute leukemia were studied for morphology, cytochemistry, immunophenotyping and cytogenetics. FCI was performed using a comprehensive six to eight-color multiparametric assay. The cytogenetic studies for standard recurrent genetic translocations were performed by FISH & Karyotyping. RESULTS: The three cases studied were diagnosed as B-ALL based on the expression of CD10, CD20, CD22, CD34, and CD79a by leukemic blasts. CD19 was studied using two different clones (i.e. J3-119 & HIB-19) and found to be severely down regulated in all three cases. There were no significant differentiating features in morphology. Cytogenetic studies were negative for recurrent translocations. CONCLUSION: We report three cases of extremely rare CD19 negative B-ALL. Lack of awareness of negative CD19 expression in B-ALL can leads to incorrect immunophenotypic diagnosis and monitoring of B-ALL, especially in laboratories using limited markers. © 2016 International Clinical Cytometry Society.

'Childhood systemic mastocytosis associated with t (8; 21) (q22; q22) acute myeloid leukemia'.

Systemic mastocytosis (SM) with associated clonal nonmast cell lineage disease is seen in up to 20% cases of SM. SM is uncommon in the pediatric population. T (8; 21) (q22; q22) is a good prognostic factor in acute myeloid leukemia (AML). However, the presence of SM confers poor prognosis in t (8; 21) (q22; q22) associated AML. We report the case of a child with t (8; 21) (q22; q22) associated AML with SM and her minimal residual disease status over the course of her treatment. In our case, the abnormal mast cells, showing co-expression of CD25 and CD2, persisted even after the marrow showed no evidence of residual AML.

Lymphoblastic leukemia with surface light chain restriction: A diagnostic dilemma.

Surface light chain expression is a feature of mature B-cell neoplasms. Light chain restriction in precursor B acute lymphoblastic leukemia is infrequently seen. We report a case of a 28-year-old female with non-FAB L3 morphology blasts and immunophenotypic features showing overlap between a precursor and mature B-cell neoplasm.

Unusual immunophenotype of T-cell large granular lymphocytic leukemia: report of two cases.

Large granular lymphocytes (LGL) leukemias are commonly of the T-cell or NK-cell type. T-cell LGL leukemia is typically a disorder of mature CD3, CD8 and T-cell receptor TCR (TCR - T cell receptor)-αß positive cytotoxic T-cells. Rare variants include TCRγδ+ variants and CD4 + TCRαß+ cases. We report a case of each of these rare variants. An 83-year-old female presented with anemia and lymphocytosis with LGLs on peripheral smear. Six-color multiparametric flowcytometric analysis showed expression of CD3, heterogeneous CD7, dim CD2 and TCRγδ and lacked expression of CD5, TCRαß, CD56, CD4 and CD8. A final diagnosis of TCRγδ+ T-cell LGL leukemia was made. Differentiation between TCRγδ+ T-cell LGL leukemia and other γδ+ T-cell malignancies is of utmost importance due to the indolent nature of the former as compared to the highly aggressive behavior of the latter. An 85-year-old male diagnosed with liposarcoma was identified to have lymphocytosis during preoperative evaluation. Peripheral smear showed presence of LGLs. Flowcytometric immunophenotyping showed expression of TCRαß, CD3, CD2, CD5, CD4, dim CD8, CD56 with aberrant loss of CD7 expression. Vß repertoire analysis by flowcytometry showed 97% cells with Vß14 clonality. A final diagnosis of TCRαß+ CD4 + T-cell LGL leukemia was made. CD4 + T-cell large granular lymphocytic leukemias have an indolent, less aggressive course when compared to their CD8 + counterparts and are not necessarily associated with cytopenias. However, their association with secondary neoplasia (29% of the cases) warrants a high degree of suspicion in the diagnosis as also noted in the index case. Use of a wide panel of antibodies and newer modalities such as Vß repertoire analysis helps in accurate subtyping of LGL leukemia.

Clear cell myoepithelial carcinoma ex pleomorphic adenoma.

Pleomorphic adenoma is the most common epithelial neoplasm of lacrimal gland. A clear cell myoepithelial carcinoma arising in the background of pleomorphic adenoma is common in the salivary glands but very rare in the lacrimal glands. We report the case of a 27 year old man whose lacrimal gland pleomorphic adenoma recurred several times over a period of four years and ultimately evolved into a clear cell myoepithelial carcinoma ex pleomorphic adenoma.

Clival dedifferentiated chordoma: a case report.

BACKGROUND: Chordoma is a rare, slowly growing tumor arising from notochordal rests and occurring in several anatomical locations with different clinical patterns of presentation. Dedifferentiation or sarcomatous transformation in a chordoma is a known but rarely recorded event. CASE: We report the case of a 58-year-old man diagnosed with chordoma of the clivus who, over the course of 5 years, showed histological and immunohistochemical evidence of progressive dedifferentiation in the tumor. CONCLUSION: Sarcomatous transformation in chordomas is seen less frequently in the clival region. A high degree of suspicion and extensive sampling are essential for diagnosis, especially in recurrent tumors.