Measurable/minimal residual disease (MRD) status has been suggested as a powerful indicator of clinical-outcome in T-cell lymphoblastic leukemia/lymphoma (T-ALL). Multicolor flow cytometric (MFC)-based T-ALL MRD reports are limited and traditionally based on the utilization of markers-of-immaturity like TdT and CD99. Moreover, studies demonstrating the multicolor flow cytometric (MFC) approach for the assessment of T-ALL MRD are sparse. Herein, we describe an 11-marker, 10-color MFC-based T-ALL MRD method using an "approach of exclusion." Methods: The study included 269 childhood T-ALL patients treated with a modified-MCP841 protocol. An 11-marker, 10-color MFC-based MRD was performed in bone marrow (BM) samples at the end-of-induction (EOI) and end-of-consolidation (EOC) time-points using Kaluza-version-1.3 software.Results: We studied EOI-MRD in 269 and EOC-MRD in 105 childhood T-ALL patients. EOI-MRD was detectable in 125 (46.5%) samples (median, 0.3%; range, 0.0007-66.3%), and EOC-MRD was detectable in 34/105 (32.4%) samples (median, 0.055%; range, 0.0008-27.6%). Leukemia-associated immunophenotypes (LAIPs) found useful for MRD assessment were dual-negative CD4/CD8 (40.9%), dualpositive CD4/CD8 (23.3%) and only CD4 or CD8 expression (35.8%); dim/subset/ dim-negative surface-CD3 (39%), dim/subset/dim-negative/negative CD5 (28.3%), dim/dim-negative/negative/heterogeneous CD45 (44.7%) and co-expression of CD5/CD56 (7.5%). EOI-MRD-positive status was found to be the most-relevant independent factor in the prediction of inferior relapse-free and overall survival. Conclusion:We described an 11-marker 10-color MFC-based highly sensitive MRD

BackgroundRecently, anti-CD38 monoclonal antibody (Mab) therapy has become a focus of attention as an additional/alternative option for many hematological neoplasms including T-cell acute lymphoblastic leukemia (T-ALL). It has been shown that antitumor efficacy of anti-CD38-Mab depends on the level of CD38 expression on tumor cells. Reports on CD38 expression in T-ALL are scarce, and data on the effect of cytotoxic chemotherapy on CD38 expression are limited to very few samples. Moreover, it lacks entirely in refractory disease and in adult T-ALL. We report the flow cytometric evaluation of CD38 expression in T-ALL blasts at diagnosis and the effect of cytotoxic chemotherapy on its expression in measurable residual disease (MRD), refractory disease (MRD≥5%), and relapsed disease in a large cohort of T-ALL.MethodsThe study included 347 samples (188 diagnostic, 100 MRD, 24 refractory and 35 relapse samples) from 196 (children: 85; adolescents/adults: 111) patients with T-ALL. CD38-positive blasts percentages (CD38-PBPs) and expression-intensity (mean fluorescent intensity, CD38-MFI) were studied using multicolor flow cytometry (MFC). MFC-based MRD was performed at the end-of-induction (EOI-MRD, day 30–35) and end-of-consolidation (EOC-MRD, day 78–85) subsequent follow-up (SFU-MRD) points.ResultsPatients were classified into early thymic precursor subtype of T-ALL (ETPALL, 54/188, 28.7%), and non-ETPALL (134/188, 71.3%). Of 188, EOI-MRD assessment was available in 152, EOC-MRD was available in 96 and SFU-MRD was available in 14 patients. CD38 was found positive in 97.9% (184/188) of diagnostic, 88.7% (110/124) MRD (including 24-refractory) and 82.9% (29/35) relapsed samples. Median (95% CI) of CD38-PBPs/MFI in diagnostic, MRD, refractory, and relapsed T-ALL samples were, respectively, 85.9% (82.10%–89.91%)/4.2 (3.88–4.47), 74.0% (58.87%–83.88%)/4.6 (3.67–6.81), 79.6% (65.25%–96.11%)/4.6 (3.33–8.47) and 85.2% (74.48%–93.01%)/5.6 (4.14–8.99). No significant difference was noted in CD38 expression between pediatric versus adult and patients with ETPALL versus non-ETPALL. No change was observed in CD38-MFI between diagnostic versus MRD and diagnostic versus relapsed paired samples. However, we noticed a mild drop in the CD38-PBPs in MRD samples compared with the diagnostic samples (p=0.016).ConclusionWe report an in-depth analysis of CD38 expression in a large cohort of T-ALL at diagnosis, during chemotherapy, and at relapse. Our data demonstrated that CD38 is robustly expressed in T-ALL blasts with a little effect of cytotoxic chemotherapy making it a potentially effective target for antiCD38-Mab therapy.

Accurate knowledge of expression patterns/levels of commonly used MRD markers in regenerative normal-B-cell-precursors (BCP) is highly desirable to distinguish leukemicblasts from regenerative-BCP for multicolor flow cytometry (MFC)-based measurable residual disease (MRD) assessment in B-lymphoblastic leukemia (B-ALL). However, the data highlighting therapy-related immunophenotypic-shift in regenerative-BCPs is scarce and limited to small cohort. Herein, we report the in-depth evaluation of immunophenotypic shift in regenerative-BCPs from a large cohort of BALL-MRD samples. Ten-color MFC-MRD analysis was performed in pediatric-BALL at the end-of-induction (EOI), end-of-consolidation (EOC), and subsequent-follow-up (SFU) time-points. We studied normalized-mean fluorescent intensity (nMFI) and coefficientof-variation of immunofluorescence (CVIF) of CD10, CD19, CD20, CD34, CD38, and CD45 expression in regenerative-BCP (early, BCP1 and late, BCP2) from 200 BALL-MRD samples, and compared them with BCP from 15 regenerating control (RC) TALL-MRD samples and 20 treatment-naïve bone-marrow control (TNSC) samples.Regenerative-BCP1 showed downregulation in CD10 and CD34 expression with increased CVIF and reduced nMFI (p < 0.001), upregulation of CD20 with increased nMFI (p = 0.014) and heterogeneous CD45 expression with increased CVIF (p < 0.001). Immunophenotypic shift was less pronounced in the BCP2 compared to BCP1 compartment with increased CVIF in all but CD45 (p < 0.05) and reduced nMFI only in CD45 expression (p = 0.005). Downregulation of CD10/CD34 and upregulation of CD20 was higher at EOI than EOC and SFU time-points (p < 0.001). Regenerative-BCPs are characterized by the significant immunophenotypic shift in commonly used B-ALL-MRD markers, especially CD10 and CD34 expression, as compared to treatment-naïve BCPs. Therefore, the templates/database for BMRD analysis must be developed using regenerative-BCP.

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 À /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.

Background Eradicating cancer stem cells (CSCs) that are termed as the “beating heart” of various malignant tumors, including cervical cancer, holds great importance in cancer therapeutics. CSCs not only confer chemo‐radio resistance but also play an important role in tumor metastasis and thereby pose a potential barrier for the cure of cervical cancer. Cervical cancer, a common malignancy among females, is associated with high morbidity and mortality rates, and the study on CSCs residing in the niche is promising. Recent findings Biomarker approach to screen the cervical CSCs has gained impetus since the past decade. Progress in identification and characterization of the stem cell biomarkers has led to many insights. For the diagnostic purpose, several biomarkers like viral (HPV16), stem cell markers, transcription factors (viz, SOX2, OCT 4, and c‐Myc), and CSC surface markers (viz, ALDH1 and CD44) have been identified. The research so far has been directed to study the CSC stemness and demonstrates various gene expression signatures in cervical CSCs. Such studies hold a potential to improve diagnostic accuracy and predict therapeutic response and clinical outcome in patients. Conclusions Stem cell biomarkers have been validated and their therapeutic targets are being developed as “strategies to improve therapeutic ratio in personalized medicine.” This review gives a brief overview of the cervical CSC biomarkers, their current and future diagnostic, prognostic, and therapeutic potential.

Introduction MicroRNAs are small, non-coding RNA molecules that are becoming popular biomarkers in several diseases. However, their low abundance in serum/plasma poses a challenge in exploiting their potential in clinics. Several commercial kits are available for rapid isolation of microRNA from plasma. However, reports guiding the selection of appropriate kits to study downstream assays are scarce. Hence, we compared four commercial kits to evaluate microRNA-extraction from plasma and provided a modified protocol that further improved the superior kit’s performance. Materials and methods We compared four kits (miRNeasy Serum/Plasma, miRNeasy Mini Kit from Qiagen; RNA-isolation, and Absolutely-RNA MicroRNA Kit from Agilent technologies) for quality and quantity of microRNA isolated, extraction efficiency, and cost-effectiveness. Bioanalyzer-based Agilent Small RNA kit was used to evaluate quality and quantity of microRNA. Extraction efficiency was evaluated by detection of four endogenous control microRNA using real-time-PCR. Further, we modified the manufacturer’s protocol for miRNeasy Serum/Plasma kit to improve yield. Results miRNeasy Serum/Plasma kit outperformed the other three kits in microRNA-quality (P < 0.005) and yielded maximum microRNA-quantity. Recovery of endogenous control microRNA i.e. hsa-miR-24-3p, hsa-miR-191-5p, hsa-miR-423-5p and hsa-miR-484 was higher as well. Modification with the inclusion of a double elution step enhanced yield of microRNA extracted with miRNeasy Serum/Plasma kit significantly (P < 0.001). Conclusion We demonstrated that miRNeasy Serum/Plasma kit outperforms other kits and can be reliably used with a limited plasma quantity. We have provided a modified microRNA-extraction protocol with improved microRNA output for downstream analyses.

The goal of this study was to isolate cancer stem-like cells marked by high expression of CD44, a putative cancer stem cell marker, from primary oral squamous cell carcinomas and identify distinctive gene expression patterns in these cells. From 1 October 2013 to 4 September 2015, 76 stage III-IV primary oral squamous cell carcinoma of the gingivobuccal sulcus were resected. In all, 13 tumours were analysed by immunohistochemistry to visualise CD44-expressing cells. Expression of CD44 within The Cancer Genome Atlas-Head and Neck Squamous Cell Carcinoma RNA-sequencing data was also assessed. Seventy resected tumours were dissociated into single cells and stained with antibodies to CD44 as well as CD45 and CD31 (together referred as Lineage/Lin). From 45 of these, CD44Lin and CD44Lin subpopulations were successfully isolated using fluorescence-activated cell sorting, and good-quality RNA was obtained from 14 such sorted pairs. Libraries from five pairs were sequenced and the results analysed using bioinformatics tools. Reverse transcription quantitative polymerase chain reaction was performed to experimentally validate the differential expression of selected candidate genes identified from the transcriptome sequencing in the same 5 and an additional 9 tumours. CD44 was expressed on the surface of poorly differentiated tumour cells, and within the The Cancer Genome Atlas-Head and Neck Squamous Cell Carcinoma samples, its messenger RNA levels were higher in tumours compared to normal. Transcriptomics revealed that 102 genes were upregulated and 85 genes were downregulated in CD44Lin compared to CD44Lin cells in at least 3 of the 5 tumours sequenced. The upregulated genes included those involved in immune regulation, while the downregulated genes were enriched for genes involved in cell adhesion. Decreased expression of PCDH18, MGP, SPARCL1 and KRTDAP was confirmed by reverse transcription quantitative polymerase chain reaction. Lower expression of the cell-cell adhesion molecule PCDH18 correlated with poorer overall survival in the The Cancer Genome Atlas-Head and Neck Squamous Cell Carcinoma data highlighting it as a potential negative prognostic factor in this cancer.

Recent studies have highlighted multiple immune perturbations related to severe acute respiratory syndrome coronavirus 2 infection–associated respiratory disease [coronavirus disease 2019 (COVID‐19)]. Some of them were associated with immunopathogenesis of severe COVID‐19. However, reports on immunological indicators of severe COVID‐19 in the early phase of infection in patients with comorbidities such as cancer are scarce. We prospectively studied about 200 immune response parameters, including a comprehensive immune‐cell profile, inflammatory cytokines and other parameters, in 95 patients with COVID‐19 (37 cancer patients without active disease and intensive chemo/immunotherapy, 58 patients without cancer) and 21 healthy donors. Of 95 patients, 41 had severe disease, and the remaining 54 were categorized as having a nonsevere disease. We evaluated the association of immune response parameters with severe COVID‐19. By principal component analysis, three immune signatures defining characteristic immune responses in COVID‐19 patients were found. Immune cell perturbations, in particular, decreased levels of circulating dendritic cells (DCs) along with reduced levels of CD4 T‐cell subsets such as regulatory T cells (T regs ), type 1 T helper (Th1) and Th9; additionally, relative expansion of effector natural killer (NK) cells were significantly associated with severe COVID‐19. Compared with patients without cancer, the levels of terminal effector CD4 T cells, T regs , Th9, effector NK cells, B cells, intermediate‐type monocytes and myeloid DCs were significantly lower in cancer patients with mild and severe COVID‐19. We concluded that severely depleted circulating myeloid DCs and helper T subsets in the initial phase of infection were strongly associated with severe COVID‐19 independent of age, type of comorbidity and other parameters. Thus, our study describes the early immune response associated with severe COVID‐19 in cancer patients without intensive chemo/immunotherapy.