Aberrant Marker Research Articles

Standardized Approach for Monitoring Plasma Cell Disorders

Abstract Multi-color flow cytometry using consensus surface markers (CD138, CD38, CD19, CD45, CD27, CD56, CD81, and CD117), intracellular kappa and lambda, as well as other auxiliary markers is routinely used for the identification and characterization of plasma cells. Here, we describe the development of a modular 10-color plasma cell panel for immunophenotyping of the plasma cell compartment. The panel was developed as an 8-color dried backbone, manufactured in a single test-per-tube, ready to use format. The dried backbone can be supplemented with liquid (drop in) reagents, which provides the flexibility of using intracellular kappa and lambda to examine clonality or using CD38-multi-epitope and/or anti-p63 (VS38c) reagent(s). The single test-per-tube format is ideally suited for MRD applications, where sample size is limited and acquisition of millions of events is needed for data analysis. In addition, the dried reagent tubes have a long shelf life (12 months at 20–25°C vs. 24 hours for liquid cocktails with multiple BV dyes in BSB buffer at 2–8°C). Furthermore, utilization of multiple high-performing polymer dyes in the panel affords high resolution of the relevant cell populations. The dried plasma cell tube(s) were tested on bone marrow samples from healthy and diseased subjects in conjunction with intracellular staining for kappa and lambda. Our results demonstrate that the 10-color plasma cell panel can be used for identification of the plasma cell compartment (based CD38 and CD138 expression), assessment of aberrant marker expression (CD19, CD27, CD81, CD117, CD45, and CD56) for the identification of normal and abnormal plasma cells, and examining clonality in multiple patient samples (Kappa and Lambda).

Clinical and hematological correlates of aberrant immunophenotypic profiles in adult and pediatric acute myeloid leukemia at presentation.

Aberrant phenotypes in acute leukemia have been reported with varying frequencies in independent studies and their association with prognostic factors is still a matter of debate. This study aims to identify the frequency of aberrant immunophenotypes in de novo acute myeloid leukemia (AML) and to evaluate their association with initial clinical and hematological features. A total of 181 patients of de novo AML were included during the time (July 2010-June 2012). The immunophenotype of all cases of AML was studied by using flow cytometry. Aberrant lymphoid antigen expression was seen in 43.1% cases. Most frequent aberrant lymphoid antigen was CD7, seen in 26.5% cases. All French-American-British (FAB) subtypes except AML-M3 expressed aberrant lymphoid antigens. The expression was most common in AML-M4 in the current study. CD34 expression in AMLs was significantly associated with the expression of aberrant lymphoid antigens. Lymphoid antigen expression in adult AML was significantly associated with higher white blood cell (WBC) count (>50,000/mm3) and higher number of peripheral blasts (>70%). In summary, CD7 is the most common aberrant lymphoid antigen expressed in AML. FAB subtype AML-M3 is usually not associated with aberrant lymphoid antigen expression. AML cases with CD34 positivity are more likely to express aberrant lymphoid markers. The current study also supports that aberrant lymphoid antigen expression in adult AML is associated with adverse presenting hematological features (WBC count >50,000/mm3, peripheral blasts >70%). Pediatric Ly + AML cases are not associated with adverse presenting clinical and biological features.

Rhabdomyosarcomas: structural distribution and analysis of an immunohistochemical profile

To qualitatively and quantitatively assess the expression of myogenic transcription factors on a large sample, to identify potential phenotypic differences, and to estimate the distribution and frequency of aberrant markers, such as ALK, PAX5, WT1, PCK, CAM5.2, SIX1, and Synaptophysin. The investigation included 202 tumor tissue samples. Five tissue microarrays were assembled from the obtained material for subsequent histological and immunohistochemical studies. Embryonal RMS (ERMS) was diagnosed in 103 cases; alveolar RMS (ARMS) was detected in 80; spindle-cell/sclerosing RMS (SRMS) was found in 16 cases; epithelioid RMS (EpiRMS) was diagnosed in 2 patients. The expression of Myogenin and MyoD1 was detected in all the examined RMS tissue samples. ARMS was more characterized by staining at 1+ and 2+ intensities; at the same time, more than 50% of ERMS, SRMS, and EpiRMS cases showed staining at 1+ intensity. ALK expression was investigated using the D5F3 and p80 clones. The D5F3 clone displayed a higher staining intensity than the p80 clone (p<0.05). The expression of PAX5 was observed in 13 of 75 ARMS cases. That of WT1 and SIX1 was found in all RMS groups. The morphological diagnosis of RMS requires a careful assessment of all of the above factors, especially taking into account the variability in the expression of myogenic transcription factors and the high level of phenotypic aberration.

Verification of aberrant expression of CD7 in acute myeloid leukemia

Introduction Acute myeloid leukemia (AML) is an aggressive disorder characterized by accumulation of blast cells in bone marrow. The heterogeneous phenotype of AML is based on cytogenetic mutations and molecular aberrations. Immunophenotyping is a convenient method for quick and reproducible diagnosis of most hematological malignancies. The higher frequency of aberrant expression of lymphoid markers in AML may be related to environmental changes and accumulation of biological defects. The CD7, a T-cell antigen, is expressed in a minority of patients with AML, and it is the most common aberrant marker found in AML in most studies. Aim We aimed to determine the frequency of CD7 expression in AML and to verify if this aberrant expression is true or false. The study included 32 newly diagnosed patients with AML. Detection of CD7 in AML was done by using an independent method to check the gene expression, namely, reverse transcriptase-PCR, besides the usually used monoclonal antibody-based flow cytometric measurements. Patients and methods The study revealed the frequency of CD7 expression in newly diagnosed AML cases to be 18.1% by flow cytometry. The AML cases with positive CD7 expression by flow cytometry were subjected to reverse transcriptase-PCR to determine gene expression. All cases with positive CD7 expression by flow cytometry were found to be negative for CD7 gene expression by PCR. Conclusion This study raises a reasonable possibility of a false-positive detection of aberrant CD7 expression in AML using immunophenotyping by flow cytometry.

Enestpath Leukemic Stem Cell (LSC) Sub-Study: Analyzing Characteristics of LSC-Positive Patients and Impact of Switch from Imatinib to Nilotinib Therapy on LSCs in Patients with Chronic Myeloid Leukemia

Enestpath Leukemic Stem Cell (LSC) Sub-Study: Analyzing Characteristics of LSC-Positive Patients and Impact of Switch from Imatinib to Nilotinib Therapy on LSCs in Patients with Chronic Myeloid Leukemia

Evaluation of Clinical, Hematological and Flow Cytometry Dates in Elderly Patients with Acute Myeloid Leukemia: Impact on Clinical Outcome

BACKGROUND: Acute Myeloid Leukemia (AML) is the most frequent acute leukemia in older patients (over 60 years of age) with more than 50% of the cases, accounting for 15 to 20% of childhood leukemia and 80% of adult leukemias, with poor prognosis, especially in elderly patients. Myelodysplastic syndromes (MDS), another spectrum of clonal myeloproliferative disorders that also involve mainly older age groups and are characterized by ineffective hematopoiesis, peripheral cytopenia's, chromosomal abnormalities and predisposition for progression to AML (AML/MDS). Elderly patients with AML are part of a biological and clinically distinct group with presence of high-risk cytogenetic abnormalities, hematological abnormalities and immunophenotypic aberrations. OBJECTIVE: Evaluation of clinical, demographic, hematological and immunophenotypic parameters in elderly patients with AML and determination of correlation these parameters with disease status. METHODS: Bone marrow (BM) and peripheral blood (PB) samples from 80 elderly patients diagnosed with AML were evaluated for hematological, immunophenotypic and karyotype parameters. The hematological parameters evaluated were: count of leukocytes, platelets and blastic cells and also determination of hemoglobin levels, cytomorphologic analysis of bonne marrow, immunophenotyping by flow cytometry for immunologic AML classification, determination of aberrant lymphoid cell markers, and detection of antigens related to multidrug resistance (MDR) such us P-glycoprotein (Pgp) and multidrug resistance proteins type 1 (MRP1), p53 protein and also karyotype analysis for confirmation of AML/MDS cases. At the same time, demographic and clinical data were obtained from all patients. RESULTS: Data analysis showed that 56 patients had "de novo" AML, 6 recurrent disease, 15 transformed MDS and 3 refractory AML. In relation to clinical aspects, there was a predominance of splenomegaly (91.2%), followed by hepatomegaly (76.2%). Laboratory findings showed a predominance of hyperleukocytosis (91.3%), thrombocytopenia (85%) and anemia (86.2%), with cytopenias being more pronounced in cases of AML/MDS. Most cases were classified as FAB M1 (36.6%), M2 (17.5%) and M4 (23.7%). Our data analysis was statistically significant (p &lt; 0.05) and showed a correlation with aberrant T-lymphoid antigens (CD3 and CD7), Pgp, MRP1, p53 protein and transferrin receptor (CD71) with the increase in the unfavorable status of the disease, contributing to a worse prognosis in these patients. CONCLUSIONS: Our results demonstrate the importance of clinical and laboratory investigations of these patients in order to obtain further information on these cancers. Disclosures No relevant conflicts of interest to declare.

Perinatal protein restriction with postnatal catch-up growth leads to elevated p66Shc and mitochondrial dysfunction in the adult rat liver.

Epidemiological data suggest an inverse relationship between birth weight and long-term metabolic deficits, which is exacerbated by postnatal catch-up growth. We have previously demonstrated that rat offspring subject to maternal protein restriction (MPR) followed by catch-up growth exhibit impaired hepatic function and ER stress. Given that mitochondrial dysfunction is associated with various metabolic pathologies, we hypothesized that altered expression of p66Shc, a gatekeeper of oxidative stress and mitochondrial function, contributes to the hepatic defects observed in MPR offspring. To test this hypothesis, pregnant Wistar rats were fed a control (20% protein) diet or an isocaloric low protein (8%; LP) diet throughout gestation. Offspring born to control dams received a control diet in postnatal life, while MPR offspring remained on a LP diet (LP1) or received a control diet post weaning (LP2) or at birth (LP3). At four months, LP2 offspring exhibited increased protein abundance of both p66Shc and the cis-trans isomerase PIN1. This was further associated with aberrant markers of oxidative stress (i.e. elevated 4-HNE, SOD1 and SOD2, decreased catalase) and aerobic metabolism (i.e., increased phospho-PDH and LDHa, decreased complex II, citrate synthase and TFAM). We further demonstrated that tunicamycin-induced ER stress in HepG2 cells led to increased p66Shc protein abundance, suggesting that ER stress may underlie the programmed effects observed in vivo. In summary, because these defects are exclusive to adult LP2 offspring, it is possible that a low protein diet during perinatal life, a period of liver plasticity, followed by catch-up growth is detrimental to long-term mitochondrial function.

Could Immunophenotype Guide Molecular Analysis in Patients with Myeloid Malignancies

Objective: Immunophenotype has been correlated with molecular aberrations in several studies. The aim of this study was the discovery of immunophenotypic features related to mutations in AML and MDS patients connected to prognostic factors. Moreover, an effort to evaluate a method for the detection of the most common NPM1 mutations of exon12 and Internal Tandem Duplications (ITD) mutations of FLT3 gene by flow cytometry was performed. Method: Patients with de novo myeloid neoplasms [ AML and MDS (AML-M3 patients were excluded)] were included. FLT3/ITD/TKD and NPM1 mutations were detected by PCR and fragment analysis. The immunophenotypic analysis was performed by multi-dimensional flow cytometry (FC) with a standardized panel of monoclonal antibodies on peripheral blood or bone marrow samples. Nucleophosmin Antibody and CD135 were used for the mutations immunophenotypic detection. Results: NPM1 and/or FLT3 mutations correlated with low or no expression of more immature cells markers such as CD34, CD117, HLADR, as well as higher expression of more mature markers such as CD11b. The higher expression of CD33 should be mentioned as well. The presence of NPM1mut and FLT3/ITD does not seem to be detectable by FC at least using these two monoclonal antibodies. The presence of CD7 aberrant lymphoid marker’s expression was associated with FLT3mut, NPM1wt genotype. CD56 or CD2 positivity was found only in patients’ samples negative for NPM1 and/or FLT3 mutations. Conclusions: Certain immunophenotype findings including the presence of aberrant lymphoid markers may be indicative of the presence of mutations in NPM1 and FLT3 linked to prognosis.

EZH2, new diagnosis and prognosis marker in acute myeloid leukemia patients

PurposeAcute myeloid leukemia (AML) is a heterogeneous disease. The discovery of novel discriminative biomarkers remains of utmost value for improving outcome predictions. Enhancer of zeste homolog 2 (EZH2) is a histone methyltransferase of H3K27me3. It is frequently up-regulated in human cancers and associated with silencing of differentiation genes. We aimed herein to investigate the prevalence and prognosis impact of somatic EZH2 mutations and their potential associations with other prognostic markers FLT3, NPM1, DNMT3A and IDH2. Materials and methodsOur study population was composed of 211 Tunisian patients with de novo AML and 14 healthy donors. The 11 last exons coding the set domain of EZH2 were investigated by PCR and Sanger sequencing. ResultsEZH2 mutations were identified in 66/211 (31%) patients with a sex ratio of 1.06. The presence of EZH2 mutations was statistically significantly associated with failure consolidation therapy (p = 0.004). There were no differences in the incidence of EZH2 mutations and FLT3-ITD, NPM1, DNMT3A and IDH2 mutations. When EZH2 mutations were associated with those of FLT3 or IDH2, a short duration of progression free survival was observed (p < 0.05). Moreover, CD7 aberrant markers conferred a poor prognosis in EZH2 mutated patients (p < 0.05). ConclusionsGiven these data we conclude that EZH2 mutations are frequent in our patients, and can be used as a prognosis marker in combination with FLT3, IDH2 mutations and CD7 marker, to stratify AML patients and to guide therapeutic decisions.

Proteomic analysis of peripheral blood polymorphonuclear cells (PBMCs) reveals alteration of neutrophil extracellular trap (NET) components in uncontrolled diabetes

Neutrophils have been thought to play a major role in inflammation and diabetic complications especially in poor glycemic control patients as demonstrated by their aberrant inflammatory markers. The aim of the present study was to compare neutrophil proteome profiles between diabetic patients with good glycemic control and those with poor glycemic control to see whether there might be any differences that could be related to the cause of complications which are found more commonly in the latter. Using 2-dimensional gel electrophoresis (2-DE) followed by quadrupole time of flight mass spectrometry (Q-TOF MS) and/or tandem mass spectrometry (MS/MS), we identified 35 differentially expressed proteins, some of which were protein components of neutrophil extracellular traps (NETs), in the poor glycemic control group compared to the good glycemic control group. The observed alterations of protein components of NETs included downregulation of myeloperoxidase, azurocidin (CAP37), and S100A9; and upregulation of the glycolytic enzymes transketolase and alpha-enolase. Manganese superoxide dismutase (MnSOD), functioning in cellular response and defense, was also found downregulated in the poor control group. Most of the glycolysis-related proteins were downregulated in the good control group but upregulated in the poor control group, including phosphoglycerate kinase 1 (PGK1) and L-lactate dehydrogenase B chain (LDHB). The findings of this study demonstrate the dysregulation of protein components of NETs in neutrophils in patients with poorly controlled diabetes. More specifically, these findings suggest association between NETs and inflammation in diabetes and provide further insights into the role of neutrophils in the complications of poorly controlled diabetes.

PB1702 EXPRESSION OF ANGIOTENSIN‐CONVERTING ENZYME IN PATIENTS WITH ACUTE LEUKEMIAS

Background:Blast cells are characterized by aberrant expression normal markers of early hematopoietic progenitor cells (HPC). Angiotensin‐converting enzyme (ACE/CD143), a key regulator of renin‐angiotensin system, was shown to be expressed in blood‐forming tissues of the human embryo and primitive human HPC. Recent studies indicated that CD143 is over‐expressed in leukemic myeloid blast cells.Aims:To evaluate the surface expression of CD143 in blast cells in patients with newly diagnosed acute leukemia (AL).Methods:The expression of CD143 was estimated in patients with newly diagnosed untreated acute leukemia, using eight‐color flow cytometry. The blast gate was defined on the basis of CD45dim expression and side‐scatter characteristics. Anti‐CD143 monoclonal antibody (clone BB9, BD) conjugated with phycoerythrin was used for analysis of CD143 expression. The percentage of positive cells (PPC) and the mean fluorescence intensity (MFI) was applied to describe expression. The expression of CD143 was devided into three groups according range of PPC ≤1%, 1 – 20 %, and ≥20 %.Results:A total of untreated 68 patients with newly diagnosed acute leukemia were included in the study group. They were diagnosed with non‐promyelocytic acute myeloid leukemia (non‐APL AML) – 35 (51.5 %), of them 3 (8.5 %) patients were related to favorable genetic group, 9 (25.7 %) – intermediate, 23 (65.8 %) – adverse; promyelocytic leukemia (APL) – 10 (14.7 %); B acute lymphoblastic leukemia/lymphoma (B‐ALL) – 13 (19.1 %), including 2 patients with Ph‐positive B‐ALL; T acute lymphoblastic leukemia/lymphoma (T‐ALL) – 10 (14.7 %), including 2 cases of early T‐precursor ALL (ETP). Median age of patients was 35.5 years, 29 male, 39 female.In total group with CD143 expression with PPC≥20% was observed in 7% (5/68) of all cases AL. CD143 expression in non‐APL AML with a range of PPC 1 – 20 % was detected in 12/35 cases (34.2 %), PPC ≥20 % was observed in 2/35 cases with complex karyotype (5.7 %). In group with APL 2/10 (20 %) patients had 1 – 20%, and 2/10 (20 %) patients had &gt;20 %. Among T‐ALL only 1/10 (10 %) had a CD143 expression. ACE expression in B‐ALL was absent (defined as ≤1 %). Median MFI of ACE expression was higher in patients with APL – 477 (range 159 – 4796) than in non‐APL AML ‐ 175 (range 43 – 3253). CD143 expression with PPC≥1% was significantly higher in patients with AML (non‐APL AML and APL), than in ALL (B‐ and T‐ALL), 40 % (18/45) vs. 4.3 % (1/23) (p = 0.0016).Summary/Conclusion:Thus, CD143 expression with PPC≥20% was observed only in 7% (5/68) of all cases AL, 2 was observed in AML with PML‐RARA, 2 ‐ AML with complex karyotype and 1 in ETP‐ALL. No expression of CD143 was detected in B‐ALL. CD143 expression was significantly higher in myeloid blast cells than lymphoid. No correlation was found between the expression of ACE and age, sex, neuroleukemia, leucocytes count, bone marrow blasts, lactic acid dehydrogenase activity and cytogenetic abnormalities in different subgroups. The significance of CD143 expression in AL is not completely clear and has to be studied.

Clinical and Prognostic Significance of Cell Sensitivity to Chemotherapy Detected in vitro on Treatment Response and Survival of Leukemia Patients.

Multidrug resistance (MDR) is a major challenge in leukemia treatment. The objective of this study was to identity predictors of MDR to allow for rapid and economical assessment of the efficacy of planned antitumor therapy for leukemia patients. The study included 113 patients with acute and chronic leukemias. Prior to antitumor therapy, we measured the sensitivity of tumor cells of patients to the panel of chemotherapeutic drugs, together with MDR1 mRNA and P-glycoprotein (P-gp) expression as one of the mechanisms of MDR, and compared these data with the response to therapy. The scales for leukemia patients according to therapy response, drug sensitivity of tumor cells, MDR1 mRNA and P-gp levels, and the presence of unfavorable immunological and cytogenetic markers were introduced for subsequent correlation analysis. We show that the drug resistance of tumor cells of leukemia patients estimated in vitro at diagnosis correlates with a poor response to chemotherapy and is usually combined with aberrant and immature immunological markers, cytogenetic abnormalities, and a high expression of MDR1 mRNA and P-gp. All together, these factors indicate unfavorable prognosis and low survival of leukemia patients. Thus, the sensitivity of tumor cells to chemotherapeutic drugs measured in vitro at diagnosis may have prognostic value for individual types of leukemia.

Single-cell analysis reveals the KIT D816V mutation in haematopoietic stem and progenitor cells in systemic mastocytosis.

BackgroundSystemic mastocytosis (SM) is a haematological disease characterised by organ infiltration by neoplastic mast cells. Almost all SM patients have a mutation in the gene encoding the tyrosine kinase receptor KIT causing a D816V substitution and autoactivation of the receptor. Mast cells and CD34+ haematopoietic progenitors can carry the mutation; however, in which progenitor cell subset the mutation arises is unknown. We aimed to investigate the distribution of the D816V mutation in single mast cells and single haematopoietic stem and progenitor cells.MethodsFluorescence-activated single-cell index sorting and KIT D816V mutation assessment were applied to analyse mast cells and >10,000 CD34+ bone marrow progenitors across 10 haematopoietic progenitor subsets. In vitro assays verified cell-forming potential.FindingsWe found that in SM 60–99% of the mast cells harboured the KIT D816V mutation. Despite increased frequencies of mast cells in SM patients compared with control subjects, the haematopoietic progenitor subset frequencies were comparable. Nevertheless, the mutation could be detected throughout the haematopoietic landscape of SM patients, from haematopoietic stem cells to more lineage-primed progenitors. In addition, we demonstrate that FcεRI+ bone marrow progenitors exhibit mast cell-forming potential, and we describe aberrant CD45RA expression on SM mast cells for the first time.InterpretationThe KIT D816V mutation arises in early haematopoietic stem and progenitor cells and the mutation frequency is approaching 100% in mature mast cells, which express the aberrant marker CD45RA.

Contribution of Altered Endocannabinoid System to Overactive mTORC1 Signaling in Focal Cortical Dysplasia

Alterations of the PI3K/Akt/mammalian target of rapamycin complex 1 (mTORC1) signaling pathway are causally involved in a subset of malformations of cortical development (MCDs) ranging from focal cortical dysplasia (FCD) to hemimegalencephaly and megalencephaly. These MCDs represent a frequent cause of refractory pediatric epilepsy. The endocannabinoid system -especially cannabinoid CB1 receptor- exerts a neurodevelopmental regulatory role at least in part via activation of mTORC1 signaling. Therefore, we sought to characterize the possible contribution of endocannabinoid system signaling to FCD. Confocal microscopy characterization of the CB1 receptor expression and mTORC1 activation was conducted in FCD Type II resection samples. FCD samples were subjected to single nucleotide polymorphism screening for endocannabinoid system elements, as well as CB1 receptor gene sequencing. Cannabinoid CB1 receptor levels were increased in FCD with overactive mTORC1 signaling. CB1 receptors were enriched in phospho-S6-positive cells including balloon cells (BCs) that co-express aberrant markers of undifferentiated cells and dysplastic neurons. Pharmacological regulation of CB1 receptors and the mTORC1 pathway was performed in fresh FCD-derived organotypic cultures. HU-210-evoked activation of CB1 receptors was unable to further activate mTORC1 signaling, whereas CB1 receptor blockade with rimonabant attenuated mTORC1 overactivation. Alterations of the endocannabinoid system may thus contribute to FCD pathological features, and blockade of cannabinoid signaling might be a new therapeutic intervention in FCD.

Gene Expression Profiling Reveals Aberrant T-cell Marker Expression on Tumor Cells of Waldenström's Macroglobulinemia.

That the malignant clone of Waldenström's macroglobulinemia (WM) demonstrates significant intraclonal heterogeneity with respect to plasmacytoid differentiation indicates the mechanistic complexity of tumorigenesis and progression. Identification of WM genes by comparing different stages of B cells may provide novel druggable targets. The gene expression signatures of CD19+ B cells (BC) and CD138+ plasma cells (PC) from 19 patients with WM were compared with those of BCs from peripheral blood and tonsil and to those of PCs from the marrow of healthy (N-PC) and multiple myeloma donors (MM-PC), as well as tonsil (T-PC). Flow cytometry and immunofluorescence were used to examine T-cell marker expression on WM tumor cells. Consistent with defective differentiation, both BCs and PCs from WM cases expressed abnormal differentiation markers. Sets of 55 and 46 genes were differentially expressed in WM-BC and WM-PC, respectively; and 40 genes uniquely dysregulated in WM samples were identified. Dysregulated genes included cytokines, growth factor receptors, and oncogenes not previously implicated in WM or other plasma cell dyscrasias. Interestingly, strong upregulation of both IL6 and IL6R was confirmed. Supervised cluster analysis of PC revealed that marrow-derived WM-PC was either MM-PC-like or T-PC-like, but not N-PC-like. The aberrant expression of T-cell markers was confirmed at the protein level in WM-BC. We showed that comparative microarray profiles allowed gaining more comprehensive insights into the biology of WM. The data presented here have implications for the development of novel therapies, such as targeting aberrant T-cell markers in WM.

Uncommon CD markers in acute myeloid leukemia

This study was done to assess the unusual CD expression in 100 cases of acute myeloid leukemia from October 2016 to April 2018. The age limit was from 3 to 50 years. Four color flow cytometry was used to diagnose the fresh aspirated bone marrow or peripheral blood sample of acute leukemia. The unusual lymphoid CD expression on myeloblasts was analyzed. Among the cases, 44% were acute myeloid leukemia, 52% of patients were of acute lymphoblastic leukemia and mixed phenotype acute leukemia was 4%. Aberrant CD expression was observed in 58% acute myeloid leukemia patients. Both aberrant CD5 and CD7 lymphoid markers expressed in acute myeloid leukemia patients were 4.2%. Aberrant CD7, CD5, cCD79a and cCD3 were in 45.8%, 33.3%, 8.3%, 8.3% of acute myeloid leukemia patients respectively. In acute myeloid leukemia, the frequency of aberrant CD expression was compared with recent international data.

Evaluation of TIM-3+ LSCs Is a Highly Sensitive MRD Monitoring Strategy to Predict AML Relapse after Allogenic Stem Cell Transplantation

Relapse of acute myeloid leukemia (AML) is a critical problem in clinics. Particularly, the prognosis of patients who relapsed after allogeneic stem cell transplantation (allo-SCT) is still poor. Self-renewing leukemic stem cells (LSCs), which are mainly enriched within CD34+CD38- fraction, cause re-growth of leukemia and eventually lead to recurrence. Therefore, evaluation of residual LSCs is crucial to estimate the efficacy of therapies including allo-SCT. As we previously described, T-cell immunoglobulin mucin-3 (TIM-3) is a useful marker for discrimination of normal and malignant HSCs. We, therefore, prospectively evaluated the frequencies of LSCs at multiple time points and assessed the validity of TIM-3 as a minimal residual disease (MRD) marker in the setting of allo-SCT.50 AML patients who had been detected CD34+CD38-TIM-3+ LSCs in BM sample at least once before SCT were analyzed. All 50 patients underwent allo-SCT at Fukuoka Blood and Marrow Transplantation Group (FBMTG)-related hospitals from July 2015 to May 2018. 43 out of 50 patients achieved complete donor chimerism confirmed by short tandem repeat PCR (STR-PCR) at the time of engraftment (day 15-36), and these 43 patients was prospectively evaluated the frequencies of residual CD34+CD38-TIM-3+ LSCs using multi-color flow cytometry. Median age of the evaluated 43 AML patients at SCT was 44.8 years (28-67). 26 patients underwent SCT on disease and 17 patients at hematological CR. Relapse-free survival (RFS) was calculated from the time of SCT until last follow-up or documentation of relapse.In each timing (at diagnosis, relapse, engraftment, etc.), we tracked the frequencies of TIM-3+ cells within CD34+CD38- fraction in BM. Of note, the proportion of CD34+CD38- fraction was 0.016 [0.0056 - 0.0267] % of BMMNC at engraftment. Then, according to the frequency of TIM-3+ cells within CD34+CD38- fraction, we classified patients into 3 groups; ‘high’ (> 90 % of CD34+CD38- cells were positive for TIM-3, n=5), ‘intermediate’ (61-90 % of CD34+CD38- cells were positive for TIM-3, n=11), and ‘low’ (< 60 % of CD34+CD38- cells were positive for TIM-3, n=27). This classification revealed that the recurrence rate within the observation period (median 334.4 days) was 100 % (5 of 5) in ‘high’ group, 45.4 % (5 of 11) in ‘intermediate’ group and 14.8 % (4 of 27) in ‘low’ group. Relapse-free survival was 83 (72-92) days in ‘high’ group, 368 (56-869) days in ‘intermediate’ group and 482.6 (45-1029) days in ‘low’ group, respectively (p<0.01) (Figure 1). Of note, all of 5 patients in ‘high’ group exhibited hematological relapse within 100 days. Thus, our new classification using TIM-3 as LSC-specific marker could isolate the patients at high risk for early relapse in allo-SCT settings even if they were considered as hematological CR and maintained complete donor chimerism assessed by clinically available methods (three-color FACS, STR-PCR, FLT3-ITD status and fusion gene specific qPCR) at the same timepoint. It suggests that monitoring of LSCs using TIM-3 should be a more sensitive and versatile strategy to predict early relapse of AML than aberrant surface markers monitoring by multi-color FACS because conventional FACS did not detect MRD population at engraftment while our strategy detected (we could detect LSCs (CD34+CD38-TIM-3+ fraction) within BMMNC in 0.0053 [0 - 0.012] %), and, the cases whose aberrant markers could be traced were only 28 cases of 43 in this study.We also confirmed that the identical driver mutations were detected at both the initial diagnosis and relapse using whole exome sequencing (WES) of purified LSCs. As a typical example, WES detected CEBPA mutation (c.11dupG, 72.7 % and 50.0 %) and WT1 mutation (c.1091_1092insTTGTACGGTC, 41.9 % and 39.5 %) in a single patient. Additionally, we also validated that purified TIM-3+ LSCs at engraftment harbored the identical mutations by amplicon sequencing. It indicates that CD34+CD38-TIM-3+ cells should represent LSCs throughout the clinical course, from diagnosis to relapse.In summary, TIM-3 expression represents the clones of functional LSCs are involved in relapse. Evaluation of TIM-3+ LSCs by multi-color FACS should be a highly sensitive strategy to predict relapse in clinical allo-SCT settings, and might enable us to appropriately intervene to overcome the poor clinical outcomes of AML. [Display omitted] DisclosuresAkashi:Taiho Pharmaceutical: Research Funding; Novartis pharma: Research Funding; Pfizer: Research Funding; Kyowa Hakko Kirin: Research Funding, Speakers Bureau; Eisai: Research Funding; Celgene: Research Funding, Speakers Bureau; Astellas Pharma: Research Funding; sanofi: Research Funding; MSD: Research Funding; Bristol-Myers Squibb: Research Funding, Speakers Bureau; Chugai Pharma: Research Funding; Eli Lilly Japan: Research Funding; Ono Pharmaceutical: Research Funding; Otsuka Pharmaceutical: Research Funding; Asahi-kasei: Research Funding.

Clinical Significance of Sex Chromosome Loss in Sex-Mismatched Bone Marrow Transplantations: A Potential Candidate for Predicting Adverse Prognosis

Background: Sex chromosome loss (SCL) has long been deemed a physiological age-related phenomenon, but due to its increasingly common appearance in pathogenic settings, there has been renewed debate as to its clinical significance. In hematologic patients receiving sex-mismatched bone marrow transplantations (BMT), depending on the donor/patient sex combination, the appearance and/or disappearance of the Y chromosome can have various implications such as engraftment, relapse, or even donor cell leukemia. This diversity, especially when coupled with deletion/duplication of an X chromosome, can be extremely misleading when determining post-BMT chimerism and may even cause misdiagnosis. We investigated the incidence and clinical implications of SCL in patients who received sex-mismatched BMT.Methods: The present study includes 476 patients (262 females and 214 males) that underwent sex-mismatched BMT over a 10-year period (2005-2015) at Seoul National University Hospital. Retrospective review of electronic medical records was performed to obtain relevant clinical data. SCL was determined by X/Y Fluoresence in situ Hybridization (FISH) and its clinical significance was evaluated in conjunction with other available cytogenetic (karyotyping, FISH), molecular (short-tandem repeat (STR) analysis), and bone marrow (BM) aspiration/biopsy results. Correlation between clinical factors were analyzed via Kendall's rank correlation coefficients. Kaplan-Meier survival analysis was utilized to obtain and compare survival curves of the patients according to age, sex, recipient STR percentage, SCL percentage, and post-BMT time interval till appearance of SCL. Two types of survival was analyzed: the first being the survival from initiation of BMT, and the second being the survival from the onset of SCL.Results: From a total of 476 patients who received sex-mismatched BMT, 77 patients (16.2%) showed post-BMT SCL. Interestingly, the number of female recipients with SCL was significantly higher than males (67 vs 10), but the overall degree of SCL (%) was higher in male patients. Acute myeloid leukemia (AML) was the most common primary diagnoses in all patients, but the distribution of diagnoses was not different between sexes. Females had a lower average age compared to males (33.8 vs 50.9 years). Correlation analysis showed that SCL positively correlated with both STR and age, however, there were a few cases with discrepancies between the SCL percentage and recipient STR percentage. These discrepancies usually occurred at low SCL and STR percentages (<5%), and required clinical interpretation via co-reviews of other available cytogenetic and/or molecular results. According to Kaplan-Meier survival analysis, the mortality rates measured from both initiation of BMT and onset of SCL were all significantly higher in patients >32 years (p=0.003, Figure 1 a), males (p<0.001, Figure 1 b), recipient STR >8.25% (p<0.001, Figure 1 c), SCL >3.8% (p = 0.006, Figure 1 d), and time interval from BMT to onset of SCL ≤10.9 months (p = 0.002, Figure 1 e). Comprehensive analyses of X/Y FISH combined with aberrant FISH markers, G-banding, and BM blast counts confirmed both higher relapse and mortality rates in SCL patients.Conclusions: Vigilance is required when interpreting X/Y FISH results, especially when relatively low percentages (<5%) of SCL is present. In ambiguous cases, it is recommended to co-review cytogenetic and molecular results. Furthermore, based on the positive correlation of SCL with relapse and poor survival, we propose that SCL could have significant prognostic implications, and patients with SCL could be classified as high-risk and more carefully monitored in the post-BMT setting. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Expression of TCF3-ZNF384 in Human Hematopoietic Cells Induces Lineage Disruption in Vitro and Acute Leukemia in Vivo

Introduction: Rearrangement of ZNF384, a transcription factor of poorly characterized function, defines a subtype of acute leukemia that may manifest as either B-ALL with aberrant myeloid marker expression or B/myeloid mixed phenotype acute leukemia (MPAL). Such leukemias are characterized by chromosomal rearrangements that result in the fusion of a diverse group of partners, often transcription factors or epigenetic modifiers, to ZNF384. Our prior studies have shown that ZNF384 B-ALL and B/myeloid MPAL are genomically indistinguishable, and that the fusion may be identified in a subset of hematopoietic stem cells, suggesting that the acquisition of a ZNF384 fusion in a primitive progenitor directly perturbs hematopoietic differentiation. The goals of this study were to determine the effect of expression of ZNF384 rearrangements on human hematopoietic stem and progenitor cell differentiation in vitro and in vivo, using TCF3-ZNF384 as a commonly observed exemplar of this form of leukemia.Methods: For in vitro experiments, human CD34+ cord blood cells were sorted into stem and progenitor populations (hematopoietic stem cell (HSC), multipotent progenitor (MPP), common myeloid progenitor (CMP), granulocyte-macrophage progenitor (GMP), and megakaryocyte-erythroid progenitor (MEP)) and lentivirally infected with wild type ZNF384, TCF3-ZNF384, or vector control. Single cells were sorted onto an MS-5 stromal layer and the immunophenotype of colonies was determined 15 days later by flow cytometry. In vivo studies were performed by sorting human CD34+ cord blood cells into stem-enriched (CD34+CD38-) or progenitor-enriched (CD34+CD38+) populations and lentivirally infecting with wild type ZNF384, TCF3-ZNF384, or vector control. Cells were transplanted into sub-lethally irradiated NOD.Cg-Prkdcscid Il2rgtm1Wjl Tg(CMV-IL3,CSF2,KITLG)1Eav/MloySzJ (NSG-SGM3) mice.Results: Single-cell MS-5 stromal experiments revealed that expression of the fusion protein perturbed hematopoietic differentiation. In all stem and progenitor populations, cells expressing TCF3-ZNF384 lost the ability to differentiate into erythroid colonies. HSC, MPP, and CMP cells expressing the fusion most commonly form undifferentiated, CD45+, CD33+ colonies. Additionally, GMP and MEP cells expressing the fusion lost their ability to form colonies. Human CD34+ cells expressing TCF3-ZNF384 successfully initiate leukemia in NSG-SGM3 mice with a median latency of 123 days. Mice presented with anemia and pathological analysis using hematoxylin and eosin staining showed infiltration of leukemic cells into the bone marrow, spleen, liver, central nervous system, and ovary. Additionally, CD33, myeloperoxidase, and major basic protein staining confirmed myeloid leukemia with a subset of eosinophil differentiation.Conclusion: Our results demonstrate that hematopoietic lineage determination is altered by the expression of TCF3-ZNF384 in human stem and progenitor cell populations. Additionally, we have created the first model of TCF3-ZNF384 leukemia which mimics the complexity of lineage deregulation in ZNF384-rearranged leukemia. DisclosuresMullighan:Cancer Prevention and Research Institute of Texas: Consultancy; Amgen: Honoraria, Speakers Bureau; Loxo Oncology: Research Funding; Pfizer: Honoraria, Research Funding, Speakers Bureau; Abbvie: Research Funding.

Primary Effusion Lymphoma with Extracavitary Presentation and A T-Cell Immunophenotype: A Potential Diagnostic Pitfall

<p class=MsoListParagraph style=margin:0in;margin-bottom:.0001pt;mso-add-space: auto;text-align:justify;text-indent:0in;line-height:normal;mso-list:l0 level1 lfo1; mso-mirror-indents:yes> 1. Abstract <p class=MsoNormalCxSpMiddle style=margin-bottom:0in;margin-bottom:.0001pt; mso-add-space:auto;text-align:justify;line-height:normal;mso-mirror-indents: yes> Primary Effusion Lymphoma (PEL) is a unique immunodeficiency-associated malignancy that requires infection with Kaposi sarcoma herpesvirus/human herpesvirus 8 (KSHV/HHV8). Although thought to arise from mature B-lymphocytes, PEL typically has a null Immunophenotype but can very rarely show lineage specific aberrancies, including aberrant T-cell markers. Here we present one such case of PEL where aberrant T-cell markers led to a misdiagnosis of peripheral T-cell lymphoma. We performed a review of the literature to identify similar cases of PEL with strong T-cell antigen expression. Most cases occurred in men who were HIV positive or otherwise immunodeficient. Many were EBV positive. Rare cases demonstrated T-cell receptor gene rearrangements. Postulated theories for the cause of disordered differentiation include immunodeficiency and/or EBV infection. The expression of T-cell antigens on PELs may be a source for error in diagnosis and awareness of this correlation is of practical diagnostic relevance. <p class=MsoListParagraph style=margin:0in;margin-bottom:.0001pt;mso-add-space: auto;text-align:justify;text-indent:0in;line-height:normal;mso-list:l0 level1 lfo1; mso-mirror-indents:yes> 2. Keywords: <span style=font-size:10.0pt;font-family: times= new= roman,serif;color:black;mso-themecolor:text1=> Body Cavity Lymphoma; Effusion Lymphoma; Extracavitary Primary Effusion Lymphoma; KSHV/HHV8; PEL; Primary Effusion Lymphoma; T-Cell Primary Effusion Lymphoma