Aberrant Immunophenotype Research Articles

Clinical Utility of Flow Cytometry Immunophenotyping in Acute Lymphoblastic Leukemia

The acute lymphoblastic leukemia (ALL) is a malignant disease of the immune system and hematologic characterized by accumulation of neoplastic B lymphoid precursors or T (lymphoblasts) in the bone marrow and / or peripheral blood. The diagnosis of these leukemias occurs by morphological classification of French-American-British (L1, L2 or L3) associated with features of immunological profile T or B cell malignancies, based on the expression profile of monoclonal antibodies (MoAb) directed against the antigens of cell differentiation by flow cytometry (FC). Several studies have shown that blast cell immunophenotypes of cases of acute lymphoblastic leukemia does not always exhibit characteristics of lymphoid differentiation normal but exhibit aberrant immunophenotypes. Thus, blasts some cases of acute lymphoblastic leukemia of B lineage may show myeloid or T antigens. Also blasts of cases of acute lymphoblastic leukemia T cell determinants may possess B or myeloid cells.Objective:To determine the immunophenotypic profile by FC in 88 patients with ALL (B or T lineage) diagnosed in the Laboratory of Flow Cytometry Blood Center of Dalton Cunha - HEMONORTE, from State of Rio Grande do Norte, Brazil.Methods:All samples from peripheral blood and / or bone marrow were subjected to FC immunophenotyping using a panel of MoAb specific for diagnosis of acute leukemia (AL) directly conjugated to fluorochromes as fluorescein isothiocyanate (FITC), phycoerythrin (PE), peridinin-chlorophyll protein (PerCP) and allophycocyanin (APC).Results:The patients' age range was between 1 month and 84 years old, with an average of 20.3 years, with 62.5% of the patients being male. The most frequently observed strain was B and the most evident subtype was the Common Pre-B ALL. Among the cell markers evaluated, the most expressed in lineage B were CD19, CD10, HLADR and cCD79a and the antigens most frequently expressed in lineage T were cytoplasmic CD3 (cCD3) and membrane (mCD3), CD7, CD5 and CD2. A small percentage (6.8%) were doubly positive T cells.Conclusion:It is concluded that individuals with ALL in this study have demographic, clinical and immunophenotypic characteristics similar to those observed in other studies, demonstrating that CF immunophenotyping is an essential methodology in the diagnosis of follow-up of these leukemias. Disclosures No relevant conflicts of interest to declare.

EBV Negative Plasmablastic Transformation of Plasma Cell Myeloma with Aberrant Acquisition of T-Cell Associated Markers: An Aggressive Disease with Very Short Survival

Introduction: Plasma cell neoplasms can show aberrant expression of different lineages related antigens, but expression of T-cell associated markers, is exceedingly rare. Herein, we describe two patients with plasma cell Myeloma who relapsed with an aggressive disease with plasmablastic morphology, skin involvement, high Ki-67, complex karyotype (with abnormalities in chromosome 1) and interestingly both patients showed aberrant acquisition of single or multiple T-cell associated markers including CD4 and died shortly after last presentation. T-cell markers were not expressed at diagnosis. Multiple theories have been proposed to explain aberrant expression of T-cell markers on a terminally differentiated plasma cells. Due to rarity of this aberrant phenotype and scarcity of the published data, the precise causative mechanism and its clinical implications have not yet been elucidated. An extensive literature review for patients with similar findings was conducted and the relevant pathological, clinical and prognostic characteristics were summarised. Methodology: Herein we describe two patients with emergent plasmablastic morphology and aberrant acquisition of multiple T-cell markers (confirmed by both flow cytometry (FCM) and immunohistochemistry), diagnosed in national Centre for Cancer Care and Research in Qatar. In addition, we conducted a systematic literature review using PubMed, google scholar and Scopus for patients with plasma cell myeloma (PCM) and aberrant T-cell expression, using pre-defined search terms and synonyms. Results: Patients diagnosed in our centre: Case 1: A 47-year-old male presented with multiple subcutaneous swellings, skin biopsy showed plasma cell neoplasm (PCN). Serum protein electrophoresis showed two bands; IgD lambda and free lambda. The patient started on VRD followed autologous SCT. Two months later, he developed new skin nodules in the upper chest wall and right renal mass. BM at relapse, showed many pleomorphic myeloma cells with plasmablastic/ anaplastic morphology. FCM showed a large population of monotypic plasma cells expressing CD10 with aberrant expression of CD33, CD4 and CD7 (partial). By IHC: the plasma cells were positive for cMYC with aberrant expression of CD4 and CD7 (partial) and CD3 (minority of cells) with Lambda light chain restriction and negative for CD20, CD56 and CD117. KI-67 >90%. Karyotype: 47,X, Y,i(1)(q10) ,t(1;3)(p32;p13),+der(3)t(1;3)(p32;p13) ,add(4)(q35),der(8)t(8;15)(q24;q11.2) add(8)(p23),add(13)(q34),+20[20]. The patient started on second line treatment for two days, unfortunately, he progressively deteriorated and passed away. Case 2: 56-year-old male presented with solitary spinal plasmacytoma and PCM. The patient underwent local radiotherapy followed by Lenalidomide and dexamethasone for 12 cycles and achieved complete remission. 5 years later, the disease progressed with multifocal spinal lesions and right chest wall mass which was treated with Pomalidomide and Daratumumab but he progressed and developed as scalp mass lesion. BM was infiltrated by many myeloma cells with plasmablastic morphology. FCM showed a monotypic plasma cell with aberrant expression of CD4 and complex karyotype. New line of therapy include Elotuzumab-Pomalidomide-dexamethasone started and unfortunately the patient shortly succumb. Upon literature review: A total of 19 cases of PCNs (table 1) showed aberrant expression of T-cell associated markers (including the two cases reported here). 11 case at relapse & two at initial presentation. Anaplastic/plasmablastic morphology and extramedullary presentation were reported in 8/12 cases. 10/18 patients out showed aberrant expression of CD4. 9/11 cases had a very short survival. Conclusion: Here we discuss an interesting, yet a rare finding of aberrant acquisition of T-cell associated markers on PCM. This review emphasises the importance of recognising atypical and rare immunophenotypic aberrancies in PCN that could possibly lead to diagnostic pitfalls (particularly with extramedullary involvement) and provide important prognostic information. We conclude that there is an evident association between aberrant expression of T-cell associated markers on PCM and aggressive disease including plasmablastic morphology, high KI-67, extramedullary involvement and adverse outcome with short survival. Disclosures No relevant conflicts of interest to declare.

Peripheral T-cell Lymphoma, NOS With Epstein-Barr Virus Positivity in an Elderly Patient With Myelodysplastic Syndrome: An Autopsy Case Report

Abstract Casestudy Epstein-Barr virus (EBV)-associated peripheral T-cell lymphomas are a group of aggressive neoplasms with a geographic predilection for South America and Asia, but are very rare in Western populations. Results We report a case of a 74-year-old Caucasian female who presented with pancytopenia and B symptoms with EBV-IgG detected on admission. Past medical history included: ITP, chronic urticaria, and recently diagnosed myelodysplastic syndrome (MDS) on bone marrow biopsy one month prior to admission. Excisional biopsies of an enlarged right neck lymph node (repeated within 6 months) and right axillary lymph node five years ago were negative for a lymphoproliferative disorder at the time. Repeated bone marrow biopsy, performed during the current admission, confirmed the diagnosis of MDS, with scattered T-cells without aberrant immunophenotype. Despite aggressive treatment from multiple specialties, the patient deteriorated and expired four weeks later from complications of MDS. At autopsy, there was diffuse lymphadenopathy involving the mediastinum, axilla, pelvis and peripancreatic fat. Lymph node sections demonstrated nodal architecture effacement by diffuse, vaguely nodular lymphoid infiltrates. Histologically, the infiltrates were composed of medium to large lymphocytes with round to slight irregular nuclei, rare Reed-Sternberg-like multinucleated cells, clumped chromatin, and indistinct nucleoli. Individual cell necrosis was abundant with mitotic figures readily identifiable. Immunohistochemistry revealed CD2+ CD3+ neoplastic T-cells that co-express MUM1 and a subset of CD30, while negative for CD4, CD5, CD8, CD56, ALK1, and TDT. EBV-encoded RNA in-situ hybridization was focally positive. The final postmortem diagnosis was peripheral T-cell lymphoma, not otherwise specified (NOS), with focal EBV positivity. Conclusion Co-existence of a de-novo MDS and non-Hodgkin lymphoma without any prior chemotherapeutic exposure is a highly unusual finding, although MDS-like presentations can occur with EBV-associated lymphomas. Peripheral T-cell lymphoma, NOS is an aggressive lymphoma and EBV positivity has been found correlated with a poor prognosis. This case demonstrates how postmortem examination remains an important tool in clinical- pathological correlation and highlights the potential pathogenetic role EBV plays in MDS and T-cell lymphoma.

Diagnosis of gamma/delta mycosis fungoides requires longitudinal clinical observation

To the Editor: It is unclear whether epidermotropic cutaneous T cell lymphomas (CTCLs) exhibiting a T-cell receptor gamma delta (TCRγδ) phenotype initially presenting with patches and plaques should be considered a variant of mycosis fungoides (γδ-MF) or as primary cutaneous γδ T cell lymphomas (PCγδTCL).1Guitart J. Weisenburger D.D. Subtil A. et al.Cutaneous γδ T-cell lymphomas: a spectrum of presentations with overlap with other cytotoxic lymphomas.Am J Surg Pathol. 2012; 36: 1656-1665Crossref PubMed Google Scholar, 2Merrill E.D. Agbay R. Miranda R.N. et al.Primary cutaneous T-cell lymphomas showing gamma-delta (γδ) phenotype and predominantly epidermotropic pattern are clinicopathologically distinct from classic primary cutaneous γδ T-cell lymphomas.Am J Surg Pathol. 2017; 41: 204-215Crossref PubMed Scopus (40) Google Scholar, 3Kempf W. Kazakov D.V. Scheidegger P.E. Schlaak M. Tantcheva-Poor I. Two cases of primary cutaneous lymphoma with a γ/δ+ phenotype and an indolent course: further evidence of heterogeneity of cutaneous γ/δ+ T-cell lymphomas.Am J Dermatopathol. 2014; 36: 570-577Crossref PubMed Scopus (22) Google Scholar Because PCγδTCL is considered an aggressive lymphoma,4Toro J.R. Liewehr D.J. Pabby N. et al.Gamma-delta T-cell phenotype is associated with significantly decreased survival in cutaneous T-cell lymphoma.Blood. 2003; 101: 3407-3412Crossref PubMed Scopus (229) Google Scholar the misclassification of epidermotropic PCγδTCL as MF may delay the decision to use systemic treatments. Our objective was to identify unique features that differentiate γδ-MF from epidermotropic PCγδTCL. We reviewed 4 patients (2 women and 2 men 39-71 years of age) from our cutaneous lymphoma clinic who presented with features resembling early-stage MF (stages IA-IB) and who had the TCRγδ phenotype. The clinical courses and histopathologic and immunophenotypic profiles of each patient are described in detail (Supplemental Tables I and II available via Mendeley at https://doi.org/10.17632/fhww2x8yy6.2). Cutaneous signs preceded the first diagnostic biopsy specimen by a median of 20 months (range 6-48 months), with a median follow-up of 64 months. Stage progression was noted in 3 patients, and 1 patient died. Besides case 1, all patients presented with patches and plaques on the distal extremities or the face rather than on sun-protected areas typical for MF. When disease progressed to tumors, the localization was atypical for classic MF (ie, the face and plantar aspects of the feet; Fig 1 and Supplemental Fig S1). Histologically, the infiltrate was indistinguishable from classic early-stage MF (Supplemental Figs S2 and S3). Immunophenotypically, cases demonstrated cytotoxic markers (granzyme B or T-cell intracellular antigen 1), a double-negative phenotype (CD4−/CD8−), an absence of TCR-βF1, positivity for TCRγδ, and monoclonal rearrangements of TCRγ genes. In all cases, Epstein–Barr virus–encoded small RNA in situ hybridization, peripheral blood flow cytometric analysis, and positron emission tomography–computed tomography scan for systemic involvement were negative. In cases 1 and 2, lymphoma was controlled by skin-directed therapies, while a combination of skin-directed and systemic therapies failed to control the aggressive disease in cases 3 and 4. An additional 29 similar cases were identified in the published literature (Supplemental Table III). Initial cutaneous lesions developed in non–sun exposed regions in approximately half of cases with a majority demonstrating a CD4−/CD8− double negative phenotype. There was an average survival of 107 months (8.9 years) from the onset of symptoms and an approximate average survival from the date of diagnosis of ≥26 months. Stage progression was noted in approximately 50% of patients, with 2 patients dying of disease. In conclusion, CTCLs clinically and histologically presenting as early-stage MF with a cytotoxic and γδ phenotype seem to have a more aggressive disease course compared with classic MF. We suggest reserving the diagnosis of γδ-MF only after an indolent clinical course is established. Patches and plaques beyond the bathing suit area, the atypical location of early tumors, γδ-phenotype, an aberrant immunophenotype (CD4−/CD8−, activated cytotoxic molecules), and early disease progression should prompt more aggressive clinical follow-up and the consideration of early systemic therapy because these patients are better diagnosed with primary cutaneous aggressive epidermotropic cytotoxic T-cell lymphoma5Guitart J. Estela Martinez-Escala M. Subtil A. et al.Primary cutaneous aggressive epidermotropic cytotoxic T-cell lymphomas: reappraisal of a provisional entity in the 2016 WHO classification of cutaneous lymphomas.Mod Pathol. 2017; 30: 761-772Crossref PubMed Scopus (61) Google Scholar or epidermotropic PCγδTCL.

Aberrant immunophenotypes in acute lymphoblastic leukemia.

Acute lymphoblastic leukemia (ALL) is the most prevalent hematologic neoplasia worldwide. To classify leukemia, we analyzed the immunophenotypic characteristics in the neoplastic cells obtained with antibodies by cell flow cytometry or immunohistochemistry. The aberrant immunophenotypes are antigen expression patterns that differ from the normal hematopoietic maturation process, which can include some different lineage antigens such as myeloid antigens in ALL or asynchronous expression of antigens. These aberrant immunophenotypes have been studied as prognostic factors and residual disease markers. In this review, some aspects of aberrant immunophenotypes are addressed, including definition, epidemiology, and potential uses.

Determination of immunophenotypic aberrancies provides better assessment of peripheral blood involvement by mycosis fungoides/Sézary syndrome than quantification of CD26- or CD7- CD4+ T-cells.

Blood involvement by mycosis fungoides (MF)/Sézary syndrome (SS) influences prognosis and therapeutic decisions. MF/SS blood stage is currently determined by absolute CD4 + CD26- or CD4 + CD7-cell counts, which quantification method may overestimate MF/SS by including CD26- or CD7- normal CD4+ T-cells, or underestimate disease burden when MF/SS cells show incomplete loss of CD26 and/or CD7. Recently, through the standardization effort led by the International Clinical Cytometry Society (ICCS), recommendation was made to quantify MF/SS by enumerating immunophenotypically aberrant CD4+ T-cells, rather than CD26- or CD7- in isolation. We compared these two quantitation methods in 309 MF/SS patients who had blood samples analyzed by flow cytometry immunophenotyping (FCI) over a 1-year period. Using the European Organization of Research and Treatment of Cancer (EORTC)/International Society for Cutaneous Lymphomas (ISCL) criteria, 221 (71.5%) patients had a blood stage corresponding to B0, 57 (18.4%) to B1, and 31 (10%) to B2. By FCI analysis, a total of 62 patients (20.0%) were found positive for MF/SS. Among EORTC B0 patients, 11/221 (5%) were positive by FCI (false negatives), and among EORTC Stage B1 patients, 35/57 (61%) were negative by FCI (false positives). Regarding patients positive for MF/SS cells by FCI, there was an overall excellent correlation (r = .999, p < .001) between the EORTC/ISCL method and FCI method; however, four (6.5%) patients would have an altered B stage between B0 and B1. The MF/SS cell quantification method using immunophenotypic aberrancies, as recommended by the ICCS, allows to distinguish MF/SS cells from background benign T-cells and enables for more accurate staging, especially among patients currently being considered to have B0 and B1 stage diseases.

Immunophenotypic profile and aberrancies in Acute Leukaemia: A study from tertiary oncology centre in south India

Immunophenotypic profile and aberrancies in Acute Leukaemia: A study from tertiary oncology centre in south India

A Real-world Perspective of CD123 Expression in Acute Leukemia as Promising Biomarker to Predict Treatment Outcome in B-ALL and AML

IntroductionCD123 is overexpressed in many hematologic malignancies and found to be useful in characterizing leukemic blasts of both acute myeloid leukemia (AML) and B-acute lymphoblastic leukemia (B-ALL). CD123 has been recently found to be a marker of leukemic stem cells, and its utility to measure residual disease and potential role in disease relapse is under evaluation. Materials and MethodsHerein, we have evaluated the expression of CD123 in 757 samples of acute leukemia including 479 treatment-naive and 278 follow-up samples and compared with post-induction morphologic complete remission and measurable residual disease (MRD) status. Multiparametric flow cytometry was used for assessment of CD123 expression and immunophenotypic characterization of leukemic blasts at diagnostic and MRD assessment time points. ResultsUsing variable cutoffs of 5%, 10%, and 20% to define a case as CD123-positive, expression of CD123 was observed in 75.6%, 66.2%, and 50% of AML and 88.6%, 81.8%, and 75% of B-ALL, respectively. Of 11 patients, 7 (63.63%) had mixed phenotype acute leukemia, but none of the 12 patients with T-acute lymphoblastic leukemia showed positivity for CD123. CD123 expression at diagnosis was associated with post-induction MRD-positive status in both B-ALL (P < .001) and AML (P = .001). We also evaluated the utility of CD123 as a leukemia-associated aberrant immunophenotype and found it to be useful in both patients with AML (baseline, 50.6%; follow-up, 53%) and B-ALL (baseline, 75%; follow-up, 73.07%). ConclusionsIn conclusion, CD123 may be considered as a cardinal marker for residual disease assessment and response evaluation in AML and B-ALL.

Flow cytometric evaluation of TRBC1 expression in tissue specimens and body fluids is a novel and specific method for assessment of T-cell clonality and diagnosis of T-cell neoplasms.

Flow cytometric detection of T-cell clonality is challenging. The current available methodology for T-cell receptor (TCR) Vβ repertoire evaluation is a complex assay and has limited sensitivity especially for detecting low levels of disease. Therefore, there is an unmet need for a reliable, simple, and rapid assay to identify T-cell clonality. The rearrangement of the TCRB gene involves the random and mutually exclusive expression of one of two constant β chain genes (TRBC1 and TRBC2), analogous to the kappa and lambda gene utilization by B cells. Here, we used a single TRBC1 antibody, in conjunction with other T-cell associated markers, to detect T-cell clonality in tissue biopsies and body fluids. A total of 143 tissue/body fluid specimens from 46 patients with a definitive diagnosis of a T-cell neoplasm and 97 patients with no T-cell malignancy were analyzed with a cocktail of monoclonal antibodies including CD2/CD3/CD4/CD5/CD7/CD8/CD45/TCRγδ/TRBC1. We examined TRBC1 expression on neoplastic T-cell populations identified based on their immunophenotypic aberrancies, and monotypic TRBC1 expression was identified in all 46 known T-cell lymphoma cases. We applied a similar gating strategy to the 97 cases without T-cell neoplasms, and arbitrarily dissected T-cell populations into immunophenotypically distinct subsets; in this group, we found that all cases revealed an expected polytypic TRBC1 expression in all subsets. Single TRBC1 antibody detection of T-cell clonality by flow cytometry is a simple, rapid, and robust assay that could be routinely utilized in flow cytometric laboratories.

Mutant Huntingtin affects toll-like receptor 4 intracellular trafficking and cytokine production in mast cells

BackgroundHuntington’s disease (HD) is caused by the expression of a mutated variant of Huntingtin (mHtt), which results in the complex pathology characterized by a defective function of the nervous system and altered inflammatory responses. While the neuronal effects of mHtt expression have been extensively studied, its effects on the physiology of immune cells have not been fully described. Mast cells (MCs) are unique tissue-resident immune cells whose activation has been linked to protective responses against parasites and bacteria, but also to deleterious inflammatory allergic reactions and, recently, to neurodegenerative diseases.MethodsBone marrow-derived mast cells (BMMCs) were obtained from wild-type (WT-) and mHtt-expressing (R6/1) mice to evaluate the main activation parameters triggered by the high-affinity IgE receptor (FcεRI) and the Toll-like receptor (TLR) 4. Degranulation was assessed by measuring the secretion of β-hexosaminidase, MAP kinase activation was detected by Western blot, and cytokine production was determined by RT-PCR and ELISA. TLR-4 receptor and Htt vesicular trafficking was analyzed by confocal microscopy. In vivo, MC-deficient mice (c-KitWsh/Wsh) were intraperitonally reconstituted with WT or R6/1 BMMCs and the TLR4-induced production of the tumor necrosis factor (TNF) was determined by ELISA. A survival curve of mice treated with a sub-lethal dose of bacterial lipopolysaccharide (LPS) was constructed.ResultsR6/1 BMMCs showed normal β-hexosaminidase release levels in response to FcεRI, but lower cytokine production upon LPS stimulus. Impaired TLR4-induced TNF production was associated to the lack of intracellular dynamin-dependent TLR-4 receptor trafficking to perinuclear regions in BMMCs, a diminished ERK1/2 and ELK-1 phosphorylation, and a decrease in c-fos and TNF mRNA accumulation. R6/1 BMMCs also failed to produce TLR4-induced anti-inflammatory cytokines (like IL-10 and TGF-β). The detected defects were also observed in vivo, in a MCs-dependent model of endotoxemia. R6/1 and c-KitWsh/Wsh mice reconstituted with R6/1 BMMCs showed a decreased TLR4-induced TNF production and lower survival rates to LPS challenge than WT mice.ConclusionsOur data show that mHtt expression causes an impaired production of pro- and anti-inflammatory mediators triggered by TLR-4 receptor in MCs in vitro and in vivo, which could contribute to the aberrant immunophenotype observed in HD.

Clonal hematopoiesis predicts development of therapy-related myeloid neoplasms post–autologous stem cell transplantation

AbstractTherapy-related myeloid neoplasms (tMN) develop after exposure to cytotoxic and radiation therapy, and due to their adverse prognosis, it is of paramount interest to identify patients at high risk. The presence of clonal hematopoiesis has been shown to increase the risk of developing tMN. The value of analyzing hematopoietic stem cells harvested at leukapheresis before autologous stem cell transplantation (ASCT) with next-generation sequencing and immunophenotyping represents potentially informative parameters that have yet to be discovered. We performed a nested case-control study to elucidate the association between clonal hematopoiesis, mobilization potential, and aberrant immunophenotype in leukapheresis products with the development of tMN after ASCT. A total of 36 patients with nonmyeloid disease who were diagnosed with tMN after treatment with ASCT were included as case subjects. Case subjects were identified from a cohort of 1130 patients treated with ASCT and matched with 36 control subjects who did not develop tMN after ASCT. Case subjects were significantly poorer mobilizers of CD34+ cells at leukapheresis (P = .016), indicating that these patients possess inferior bone marrow function. Both clonal hematopoiesis (odds ratio, 5.9; 95% confidence interval, 1.8-19.1; P = .003) and aberrant expression of CD7 (odds ratio, 6.6; 95% confidence interval, 1.6-26.2; P = .004) at the time of ASCT were associated with an increased risk of developing tMN after ASCT. In conclusion, clonal hematopoiesis, present at low variant allele frequencies, and aberrant CD7 expression on stem cells in leukapheresis products from patients with nonmyeloid hematologic cancer hold potential for the early identification of patients at high risk of developing tMN after ASCT.

Clinicopathological spectrum of hairy-cell leukemia: A single-center study with brief review of Indian literature

The presence of specific chemotherapeutic protocols for hairy cell leukemia (HCL) makes it essential to discriminate this entity from other lymphoproliferative disorders. Hence, awareness of the variations in clinical presentations and immunophenotypic aberrancies is requisite to ensure diagnostic accuracy. A retrospective study was carried out to analyze the clinical-pathological profile of patients with HCL diagnosed over a period of 81 months (2010-September 2017) in our institute. Flow cytometry was performed in all the patients, and further, BRAFV600E mutation analysis was performed by real-time polymerase chain reaction in a limited number of samples. A total of 353 lymphoproliferative disorders were assessed during the period, of which 16 (4.5%) were diagnosed as HCL, which included 15 cases of classical HCL and single case of HCL-v. Striking male predominance was noted with a median age of 52 (range 22-90 years). 47% patients presented with pancytopenia, while 20% cases had leukocytosis. Three patients presented with bleeding diathesis in the form of melena and purpuric spots. The absence of splenomegaly was observed in 20% patients (4/15) while 2 (13.3%) cases had lymphadenopathy. Hypocellular marrow was observed in 13% cases. Bright expression of CD20/CD22 along with CD25/CD103/CD123/CD11c was noted in all the patients of classical HCL. Aberrant expression of CD23 and CD5 was seen in 33% ( n =5) and 6.7% ( n =1) cases respectively. CD200 was positive in all the 5/15 cases tested. The case of HCL-v presented with very high leukocyte count and exhibited a CD103/CD11c+ and CD123/CD25- profile. BRAFV600E, mutation was present in all the four patients tested who included patients with a hypocellular marrow and absent splenomegaly. HCL has characteristic profiles, yet it may exhibit unusual clinical and immunophenotypic presentations. Perspicacious use of flow cytometry and BRAFV600E mutation analysis will aid in the diagnosis in unprecedented cases.

Poor Prognosis Biomolecular Factors Are Highly Frequent in Childhood Acute Leukemias From Oaxaca, Mexico.

Objective:To investigate the cellular and molecular epidemiology of acute leukemias in vulnerable populations of children and adolescents in Oaxaca de Juarez, Mexico.Material and Methods:Descriptive, cross-sectional and retrospective study, conducted from 2014 to 2018 in which profiles of molecular and immunophenotypic aberrations were investigated in children and adolescents diagnosed with acute leukemia, by evaluating 28 molecular abnormalities by HemaVision-Q28 multiplex RT-PCR kit and standardized EuroFlow Immunophenotyping of bone marrow cells.Results:We included 218 patients, with 82.5% younger than 14 years and 17.5% adolescents. The median age was 9 years and a main peak of incidence was recorded at age of 4 to 5 years. B-cell acute lymphoblastic leukemia was diagnosed in 70.64% of all cases, acute myeloid leukemia was in 22.48%, T-cell acute lymphoblastic leukemia in 6.42%, and mixed lineage acute leukemia in 0.46% of cases. Overall, chromosomal translocations were positive in 29.82% of cases. While 65.31% of patients with acute myeloid leukemia reported aberrancies, only in 18.83% of B-cell acute lymphoblastic leukemia cases genetic abnormalities were obvious. Surprisingly, most prevalent translocations in B-cell acute lymphoblastic leukemia were t(9;22) in 20.7%, followed by t(4;11) in 17.2% and t(6;11) in 13.8%, whereas patients with acute myeloid leukemia showed t(15;17) in 40.6% and t(8;21) in 21.9%. In contrast, an homogeneous expression of t(3;21) and t(6;11) was recorded for T-cell acute lymphoblastic leukemia and mixed lineage acute leukemia cases, respectively. Except for t(1;19), expressed only by pre-B cells, there was no association of any of the studied translocations with differentiation stages of the B-leukemic developmental pathway.Conclusion:Our findings identify near 50% of patients with acute lymphoblastic leukemia at debut with high-risk translocations and poor prognosis in B-cell acute lymphoblastic leukemia as well as an unexpected increase of acute myeloid leukemia cases in young children, suggesting a molecular shift that support a higher incidence of poor prognosis cases in Oaxaca.

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.

Characterization of Immunophenotypic Aberrancies in Adult and Childhood Acute Lymphoblastic Leukemia: Lessons from Regional Variation.

Background & Objective:Although the antigen expression patterns of acute lymphoblastic leukemia (ALL) are well known, this study attempted to evaluate commonly used immune markers for immunophenotyping of acute leukemia to set the minimum of necessary diagnostic panels by flow cytometry.Methods:This study evaluated 89 patients referred from all over the country to the Iranian Blood Transfusion Organization (IBTO) in Tehran from 2013 to 2015. We compared the immunophenotype patterns of childhood and adult ALLs including 69(77.5%) B-cell lymphoblastic lymphoma (B-LBL), 2(2.2%) Burkitt’s lymphoma (BL), and 18(20.2%) T-cell lymphoblastic lymphoma (T-LBL) cases using flowcytometry with broad antibody panel.Results:CD19 and CD79a were the most frequent markers for B-LBL while CD7 was the most sensitive marker in T-LBL; the frequency of CD7, CD3, and CD5 antigens were 100%, 38.9%, and 88.9%, respectively. TdT+/CD34+ was significantly higher in adult B-LBLs than children, which indicates blast cells are more immature in adults. In addition, CD10 and cCD79a were significantly higher in children with B-LBL like as CD5 and CD8 in children with T-LBL. Aberrant phenotypes including CD13, CD33, CD7, and CD117 were found in 7(10.1%) cases of B-LBL. These phenotypes were CD117, HLA-DR, and CD33 in 7(38/9%) cases of T-LBL. Expression of CD117 aberrant myeloid antigen was significantly more associated with T-LBL than with B-lineage ALL.Conclusion:Significant differences were observed in antigen-expression patterns between adult and childhood ALLs. Further studies are needed to correlate specific markers with recurrent cytogenetic abnormalities and prognosis with therapeutic response.

Detection of Measurable Residual Disease (MRD) in Peripheral Blood: First Report of a Novel Microfluidic Platform in Patients with Acute Myeloid Leukemia (AML)

Background: Despite therapeutic advances, AML remains a disease in which the majority of patients relapse after attaining remission. The presence of bone marrow MRD is associated with impending AML relapse. Prediction and prevention of relapse could improve outcomes, but most current MRD tests either require bone marrow aspirate or detect individualized molecular genetic features present in a minority of AML. Furthermore, bone marrow MRD assessments are impractical if performed frequently enough to detect most early relapses. We developed and tested a novel microfluidic chip device (MCD) that can quantitate cell numbers using automated methods and explored its ability to detect low levels of peripheral blood leukemic cells with aberrant immunophenotypes. Methods: The MCD contains sinusoidal capture channels that were coated with antibodies with specificity towards one of the commonly expressed markers found on immature myeloid cells--CD33, CD34 and CD117 (capture antigens). Initial spiking experiments used fluorescently labeled leukemia cell lines HL60 (CD33+), KG1 (CD34+), Kasumi1 (CD117+) spiked into a 5000 cell/mL suspension. Cell suspensions were passed through MCDs coated with a capture antigen known to be expressed on the tested cell line. These experiments established an efficient capture using a flow rate of 1mL/sec. Then, AML patients with an aberrant immunophenotype were enrolled in a pilot study either at the start of induction chemotherapy or prior to allogeneic stem cell transplant (SCT). For patients receiving induction chemotherapy, whole blood samples were obtained monthly starting at the time of remission assessment, or monthly starting prior to SCT. Buffered whole blood was passed through MCDs coated with a capture antigen known to be expressed on patient myeloblasts. The captured cells were then released, eluted, centrifuged and plated on a glass slide. Plated cell pellets were then labeled with fluorescent antibodies targeting surface proteins known to be aberrantly (either by lineage infidelity or asynchronous expression) expressed on the patients' AML blasts. Automated fluorescence microscopy was used to identify and quantify captured cells with the known aberrant immunophenotype of the AML blasts. Descriptive statistics described serial cell counts in patients maintaining remission and relapsing patients. Results: Of 31 patients who have been enrolled in the study to date, 13 had at least 3 post-remission MCD analyses. Of these patients, 6 had either morphologic relapse or persistent/rising marrow MRD. In these patients, there was a trend towards higher initial aberrant immunophenotype cell counts, with mean initial count = 59 (95% CI 1, 108), compared to other patients with mean initial count = 15 (95% CI 5, 25). Of 5 patients who relapsed with MCD data within 1 month prior to relapse, the mean absolute rise prior to relapse above minimum MCD cell count was 54 (95% CI 2, 105), in comparison to non-relapsing patients with mean rise of 9 (95% CI 3, 15). From the initial 16 patients, 10 underwent induction therapy (the other 6 were enrolled prior to SCT). In these 10 patients there was a non-significant association between peripheral blood aberrant immunophenotype cells and remission status following induction. A total of 8 patients underwent allogeneic SCT. Two of these patients had known bone marrow MRD at the time of SCT and had a statistically significant greater number of aberrant immunophenotypic cells pre-SCT (48 and 60) compared to the 6 MRD negative patients (median = 12, range 9, 42). Conclusions: A novel MCD assay can reliably capture and detect low numbers of AML blasts from peripheral blood using immunofluorescent imaging and automated cell counts to quantify leukemia cells with aberrant immunophenotypes. Because this method uses peripheral blood, frequent sampling is feasible and of minimal risk to patients. An ongoing clinical trial will further explore the associations between MCD-based cell enumeration and clinical endpoints in AML patients that were suggested in the pilot phase of this study. Because the MCD releases trace populations of viable cells, additional experiments, such as primary cell culturing and single cell sequencing, are possible. Figure Disclosures Foster: Bellicum Pharmaceuticals, Inc: Research Funding; Daiichi Sankyo: Consultancy; MacroGenics: Research Funding; Celgene: Research Funding. Fedoriw:Alexion Pharmaceuticals: Consultancy, Speakers Bureau. Zeidner:Celgene: Consultancy, Honoraria, Research Funding; AsystBio Laboratories: Consultancy; Merck: Research Funding; Covance: Consultancy; Pfizer: Honoraria; Agios: Honoraria; Daiichi Sankyo: Honoraria; Tolero: Honoraria, Research Funding. Coombs:Covance: Consultancy; Octopharma: Honoraria; Medscape: Honoraria; Cowen &amp; Co.: Consultancy; Loxo: Honoraria; H3 Biomedicine: Honoraria; Dedham Group: Consultancy; Pharmacyclics: Honoraria; Abbvie: Consultancy. Mirkin:BioFluidica: Employment. Zomorrodi:BioFluidica: Employment. Toughiri:BioFluidica: Employment. Bartakova:BioFluidica: Employment. Carson:BioFluidica: Employment. Muller:BioFluidica: Employment.

Interim Results of Russian Acute Lymphoblastic Leukemia (RALL-2016) Study with Centralized MRD-Monitoring and Randomization for Autologous HSCT with Non-Myeloablative Conditioning in Adult Ph-Negative ALL Patients

Introduction. MRD-tailored therapy based on pediatric-inspired intensification is a back-bone of the majority of the European study groups in adult ALL. Taking in consideration the major pitfalls of the first Russian acute lymphoblastic leukemia study group trial RALL-2009 (NCT01193933) - high CR death rate, early CNS relapses in T-ALL, selection bias in auto-HSCT vs chemotherapy comparison, absence of MRD monitoring - a new RALL-2016 protocol (NCT03462095) was introduced based on the same principles as the first one - non-intensive but non-interruptive approach with low numbers of allo-HSCT, but with further deintensification of consolidation phase, centralized MRD-monitoring and randomization for autologous HSCT with non-myeloablative conditioning (CEAM). AIM. To analyze the 2,5 years efficacy and to determine significance of MRD status after induction in the new Russian ongoing prospective multicenter study RALL-2016. Materials and patients. RALL-2016 was based on the previous RALL-2009 protocol , but one day high-dose MTX and high-dose ARA-C blocks were eliminated and substituted by 2 months of non-interruptive therapy, L-asparaginase was scheduled for 1 year of treatment instead of 2,5 y, 15 intrathecal injections were increased up to 21 mostly while consolidation phase, CR T-ALL patients were brought to randomization after the informed consent: auto-HSCT vs no auto-HSCT, - with the similar further maintenance. All primary bone samples are collected and tested for cytogenetics and molecular markers, all included patients are monitored by flow cytometry by aberrant immunophenotype in a centralized lab. Results and discussion. From Dec 2016 till Jul 2019 148 Ph-negative ALL pts from 10 centers were included: median age 33 y (18-54) (BCP-ALL-80 (54%) pts, T-ALL- 64 (44%), biphenotypic- 4 (2%)). CR was achieved in 84% pts. The induction death before CR was 8% (n=12), refractory ALL was registered in 12 pts (8%). Death in CR occurred in 4%. After CR achievement 52 T-ALL patients were randomized either to chemotherapy (n=25) or to autoHSCT(n= 27). 15 of 27 T-ALL pts were transplanted at a median time of 6 months from CR (1 of 27 received alloHSCT - Neimegen Syndrom, 2 of 27 died in CR before HSCT, one pt refused the autotransplant ). OS and DFS at 2-years constituted 70,7% and 80%. 2-y OS was 65,8% for BCP-ALL, 80% for T-ALL and 66,7% for MPAL (p=0,5). 2-y DFS was 78,7% for BCP-ALL, 83,4% for T-ALL and 100% for MPAL (p=0,88). AlloHSCT in 1st CR have received only 3 (2%) pts. We have registered the differences in OS in pts who were treated in Federal Center (51 pts) or in Regional centers (97pts): 82% vs 64,6%, respectively (p=0,02). But there were no differences in DFS: 87,7% vs 77,3%, respectively (p=0,66) (Pic1). We have detected very high death rate in induction and in CR in the regional Centers despite the fact that the main pts characteristics were similar (median age, hyperleukocytosis, high risk group). MRD persistence after induction (70th day of protocol) became a significant factor of poor prognosis: 2-yeasr OS and DFS in MRD-negative (59 pts) and MRD-positive (33pts) were 91,8% vs 56,4% (p=0,017) and 88,7% vs 64,3% (p=0,16), respectively (Pic 2). Median of relapse was 7 month. Conclusion. The new RALL-2016 study pitifully continues to demonstrate high induction and CR death rate in regional centers despite of de-intensification of chemotherapy. We've observed significant differences in OS in Federal Center vs Regionals Centers, but not in DFS. MRD monitoring by FCM in ALL patients revealed that the persistence of MRD after induction (day+70) was an independent factor of poor prognosis and high relapse rate suggesting the introduction of new treatment approaches within a very short time after induction (maximum 3 months) in MRD positive patients. Figure Disclosures No relevant conflicts of interest to declare.

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.

The Sequential Flamsa-Bu Conditioning Regimen Does Not Improve Outcome in Patients Allografted for High Risk Acute Myeloid and Myelodysplasia Irrespective of Pre-Transplant MRD Status: Results of the UK NCRI Figaro Trial

INTRODUCTION: Allogeneic stem cell transplantation (allo-SCT) is an important curative strategy in adults with high risk acute myeloid leukemia (AML) and myelodysplasia (MDS). Disease relapse represents the major cause of treatment failure and whilst retrospective analyses have identified that pre-transplant measurable residual disease (MRD) is an important predictor of transplant outcome this has never been examined prospectively. The advent of reduced intensity conditioning (RIC) regimens has substantially increased the number of older adults eligible for allo-SCT but the optimal RIC regimen in high risk AML remains unknown. Registry data have demonstrated improved outcomes using a sequential transplant regimen utilizing cytosine arabinoside (araC)/amsacrine (AMSA) cytoreduction followed by a fludarabine (Flu)/busulfan (Bu) based RIC regimen (FLAMSA-Bu). However, although the FLAMSA-Bu regimen is now widely used in adults with high risk AML and MDS its benefit has not been evaluated in a randomized trial. We report the results of a randomized trial evaluating the FLAMSA-Bu regimen compared with standard RIC regimens which also represents the first prospective evaluation of the impact of pre-transplant MRD levels on transplant outcome. PATIENTS AND METHODS: 244 patients (median age 59 yrs) with high risk AML (n=164) or high risk myelodysplasia (n=80) were randomized 1:1 to a control arm determined by investigator's choice of either Flu/B2/ATG (Flu, Bu 3.2 mg/kg x 2 days, ATG 2.5 mg/kg x 2 days); Flu/Mel/Alemtuzumab (A) (Flu, Mel 140 mg/m2, A 50 mg) or Flu/Bu2/A (Flu, Bu 3.2 mg/kg x 2 days, A 50 mg) versus an experimental arm of FLAMSA-Bu (Flu, araC 2g/m2 x 4 days, AMSA 100mg/m2 x 4 days, Bu -total dose 11.2 mg/kg). Patients over the age of 60 received an adjusted FLAMSA-Bu regimen utilising a reduced dose of araC (1mg/m2 x 4 days) and a total Bu dose of 6.4 mg/kg. Patients were transplanted using either an HLA identical sibling (n=49) or matched (10/10 or 9/10) unrelated donor (n=195). All patients received cyclosporine GVHD prophylaxis. 155 patients with AML were in CR1 or CR2 at the time of transplant and 9 had primary refractory disease. MRD was monitored by flow cytometry (applying different-from-normal analysis when no diagnostic/relapse leukemic aberrant immunophenotype was available). Pre-transplant MRD levels were measured up to four weeks prior to transplantation in 201 patients (MRD positive = 80 (40%), MRD negative = 94 (47%), inadequate sample = 27 (13%)). MRD results were not reported to clinicians. The primary outcome was overall survival. RESULTS: Baseline characteristics including CR1/CR2 status, adverse cytogenetics and MRD levels were similar between regimens. Median follow up was 35 months. Transplant outcomes were comparable between patients allografted in the control and FLAMSA-Bu arms. 2 yr overall survival (OS) and cumulative incidence of relapse (CIR) were 61% and 30% respectively in the control arm vs 62% and 26% for the FLAMSA-Bu arm. Transplant related mortality at 100 days was 3.0% in patients allografted using the control regimen vs 14% in patients allografted using the FLAMSA-Bu regimen and 17% vs 21% at 1 year. In the study cohort pre-transplant MRD positivity was associated with both an increased CIR compared to patients testing MRD negative (2 yr CIR 42% vs 19%, p=0.009) and decreased OS (2 yr OS 52% vs 71%, p=0.048). The FLAMSA-Bu regimen failed to improve OS or reduce CIR in either MRD positive or MRD negative patients. CONCLUSIONS: This trial, the largest randomized trial of RIC regimens in AML to date, did not detect any benefit of intensification of the conditioning regimen in adults with high risk AML or MDS. Specifically, the FLAMSA-Bu regimen was not associated with improved transplant outcome in patients who were MRD positive pre-transplant. These data include the first demonstration in a prospective analysis that the presence of pre-transplant MRD measured in real time is associated with reduced OS consequent upon an increased risk of disease relapse. Further randomized studies of novel conditioning regimens in adult AML, crucially with integrated MRD studies, are now required but these results support exploration of alternative strategies, such as pre or post-transplant pharmacological intervention, as the most promising strategy to reduce the risk of disease relapse post allograft. Disclosures Russell: Jazz: Consultancy, Honoraria, Speakers Bureau; DSI: Consultancy, Honoraria, Speakers Bureau; Pfizer Inc: Consultancy, Honoraria, Speakers Bureau; Astellas: Consultancy, Honoraria, Speakers Bureau. Freeman:Jazz Pharmaceuticals: Speakers Bureau. OffLabel Disclosure: We report data using the combination of fludarabine, busulphan, amsacrine and cytosine arabinsoide as a conditioning regimen in patients allografted for high risk acute myeloid leukemia

Immunophenotypic Analysis of Peripheral T-Cell Lymphomas: A Single-Center Retrospective Review of Flow Cytometric Analysis

Abstract Background Peripheral T-cell lymphomas (PTCLs) are heterogenous, mature T-cell neoplasms that are a diagnostic challenge, requiring a combination of morphologic assessment and ancillary studies. Flow cytometry (FC) is a tool used routinely in lymphoma diagnosis; however, most analyses are limited to B-cell evaluation and pathologists generally lack experience evaluating for PTCL. We aimed to describe the immunophenotypic aberrancies observed by FC in PTCL. Design PTCLs with FC were collected, excluding primary leukemic processes. Four- and eight-color FC data were reanalyzed with the following antigens (when available): CD2, CD3, CD4, CD5, CD7, CD8, CD30, CD45, CD45RO, CD56, and CD57. Lymphoma cells were compared to normal T cells and an isotype control. Antigen expression was defined as &gt;20%. Results Thirty-eight cases were analyzed (29 males, 9 females, 6-86 years, median 62 years), including 29 PTCLs NOS, 4 angioimmunoblastic T-cell lymphomas (AITLs), 3 anaplastic large cell lymphomas, 1 δγ-TCL, and 1 hepatosplenic TCL from 15 bone marrows, 14 lymph nodes, 6 bloods, 2 fluids, and 1 skin. Twenty cases were CD4+, 4 were CD8+, 3 were dual +, and 10 were dual –. Thirty-seven cases (97%) showed global aberrant antigen patterns, median 4 aberrancies/case (1-8). Lymphoma cells accounted for 0.07% to 68% (median 2.6%) of total events. Aberrant CD7 expression was present in 34 of 38 (89%) and was underexpressed in 22 of 34 (65%). CD3 and CD5 were aberrant in 79% of cases each, with two-thirds showing underexpression. CD2 and CD45RO were aberrant in two-thirds of PTCLs, with overexpression in 61% and 92% of those cases, respectively. One AITL showed no aberrancies. Conclusions Nearly all PTCLs show immunophenotypic aberrancy compared to normal T cells. Most commonly, PTCL showed aberrant underexpression of CD7, CD3, and CD5 and overexpression of CD2 and CD45RO. Our data support FC panels with CD2, CD3, CD4, CD5, CD7, CD8, and CD45RO to optimize recovery of aberrant T cells.