Terminal Deoxynucleotidyl Transferase Expression Research Articles

Terminal deoxynucleotidyl transferase expression in different subtypes of childhood B-cell acute lymphoblastic leukemia

The lack of expression of terminal deoxynucleotidyl transferase (TdT) is frequently associated with KMT2A-rearranged subtype of pediatric acute lymphoblastic leukemia (ALL). However, this association has not been investigated extensively in the Asian population. A retrospective analysis of TdT expression in pediatric B-cell ALL (B-ALL) was performed in patients treated using the Taiwan Pediatric Oncology Group (TPOG) ALL 2002 and 2013 protocols. Among the 331 patients with B-ALL, 12 patients showed TdT negativity at initial diagnosis. Among these, eight patients showed KMT2A rearrangement (66.7%). Other patients showing negative TdT expression had ETV6::RUNX1, MEF2D-rearranged, and other B-ALL subtypes. However, in the context of KMT2A-rearranged B-ALL (n = 20), only eight patients showed TdT negativity. The 5-year event-free survival and overall survival of patients with and without TdT expression were 83.8% versus 46.8% (P <0.001) and 86.3% versus 55.4% (P = 0.004), respectively. Moreover, several aberrant markers, such as CD2, CD56, CD7, and CD117, were rarely expressed in the B-ALL samples, and if expressed, they were enriched in specific genetic subtypes. The results of this study indicate that immunophenotypic features are correlated with specific genetic subtypes of childhood B-ALL.

Increased Incidence of TdT-negative Pre-B Acute Lymphoblastic Leukemia Associated With Poor Prognostic Features Among Mexican Children in Central Mexico.

Mexican and Hispanic children in Mexico and the United States, respectively, have the highest incidence and worst outcomes of pre-B acute lymphoblastic leukemia (ALL) compared with other racial/ethnic groups. Terminal deoxynucleotidyl transferase (TdT) is an intranuclear DNA polymerase normally present on immature lymphocytes (TdT-positive) and distinguishes ALL from mature lymphoid malignancies. We performed a multisite retrospective study to determine the incidence of TdT-negative precursor B-cell acute lymphoblastic leukemia (pre-B ALL) among Mexican, Caucasian, and US-born Hispanic children to correlate TdT expression with patient characteristics and known prognostic factors. Fisher exact test was performed for categorical variables and the Wilcoxon rank-sum test was used for continuous variables. TdT-negative pre-B ALL was most frequently identified in patients with National Cancer Institute high-risk disease ( P =0.014). TdT-negative expression was also most frequently associated with hypodiploid pre-B ALL ( P =0.001) and KMT2A gene rearrangement ( P =0.0012). Mexican children had the highest incidence of TdT-negative ALL compared with Caucasians and US Hispanics ( P <0.001), with an increased incidence of poor prognostic features as well. This study demonstrates significant differences in TdT-negative expression, genomic alterations, and leukemic ploidy based on race and ethnicity.

Absence of terminal deoxynucleotidyl transferase expression in T-ALL/LBL accumulates chromosomal abnormalities to induce drug resistance.

T-acute lymphoblastic leukemia/lymphoma (T-ALL/LBL) is a malignant neoplasm of immature lymphoblasts. Terminal deoxynucleotidyl transferase (TDT) is a template-independent DNA polymerase that plays an essential role in generating diversity for immunoglobulin genes. T-ALL/LBL patients with TDT- have a worse prognosis. However, how TDT- promotes the disease progression of T-ALL/LBL remains unknown. Here we analyzed the prognosis of T-ALL/LBL patients in Shanghai Children's Medical Center (SCMC) and confirmed that TDT- patients had a higher rate of recurrence and remission failure and worse outcomes. Cellular experiments demonstrated that TDT was involved in DNA damage repair. TDT knockout delayed DNA repair, arrested the cell cycle and decreased apoptosis to induce the accumulation of chromosomal abnormalities and tolerance to abnormal karyotypes. Our study demonstrated that the poor outcomes in TDT- T-ALL/LBL might be due to the drug resistance (VP16 and MTX) induced by chromosomal abnormalities. Our findings revealed novel functions and mechanisms of TDT in T-ALL/LBL and supported that hematopoietic stem cell transplantation (HSCT) might be a better choice for these patients.

Humanized V(D)J-rearranging and TdT-expressing mouse vaccine models with physiological HIV-1 broadly neutralizing antibody precursors

Antibody heavy chain (HC) and light chain (LC) variable region exons are assembled by V(D)J recombination. V(D)J junctional regions encode complementarity-determining-region 3 (CDR3), an antigen-contact region immensely diversified through nontemplated nucleotide additions ("N-regions") by terminal deoxynucleotidyl transferase (TdT). HIV-1 vaccine strategies seek to elicit human HIV-1 broadly neutralizing antibodies (bnAbs), such as the potent CD4-binding site VRC01-class bnAbs. Mice with primary B cells that express receptors (BCRs) representing bnAb precursors are used as vaccination models. VRC01-class bnAbs uniformly use human HC VH1-2 and commonly use human LCs Vκ3-20 or Vκ1-33 associated with an exceptionally short 5-amino-acid (5-aa) CDR3. Prior VRC01-class models had nonphysiological precursor levels and/or limited precursor diversity. Here, we describe VRC01-class rearranging mice that generate more physiological primary VRC01-class BCR repertoires via rearrangement of VH1-2, as well as Vκ1-33 and/or Vκ3-20 in association with diverse CDR3s. Human-like TdT expression in mouse precursor B cells increased LC CDR3 length and diversity and also promoted the generation of shorter LC CDR3s via N-region suppression of dominant microhomology-mediated Vκ-to-Jκ joins. Priming immunization with eOD-GT8 60mer, which strongly engages VRC01 precursors, induced robust VRC01-class germinal center B cell responses. Vκ3-20-based responses were enhanced by N-region addition, which generates Vκ3-20-to-Jκ junctional sequence combinations that encode VRC01-class 5-aa CDR3s with a critical E residue. VRC01-class-rearranging models should facilitate further evaluation of VRC01-class prime and boost immunogens. These new VRC01-class mouse models establish a prototype for the generation of vaccine-testing mouse models for other HIV-1 bnAb lineages that employ different HC or LC Vs.

The presence of Auer rods in neutrophils and monocytes besides blasts in a case of acute myeloid leukemia with CSF3R mutation

An 81-year-old male with a history of diabetes, hypertension, hypercholesterolemia, previous DVT not on anticoagulation, and arthritis presented to the emergency department with a chief complaint of generalized weakness. Complete blood counts revealed white blood cells (WBC) 165.3 × 109/L, hemoglobin (Hb) 4.4 g/dl, and platelet (PLT) < 10 × 109/L, with 62% blasts. Review of peripheral blood showed blasts of variable sizes with a moderate amount of basophilic cytoplasm (Figure 1A–D; peripheral blood; Leishman stain; 100× objective), and pale-salmon-pink-colored cytoplasmic granules in a subset (Figure 1A, see arrows). Auer rods are easily identified in blasts (Figure 1A,B, see asterisk), occasionally seen in monocytes (Figure 1C, see asterisk) and neutrophils (Figure 1d, see asterisk). Bone marrow was examined to establish diagnosis, showing marked hypercellularity with 72% blasts on aspirate smears with morphologic features similar to those in peripheral blood (Figure 1E [bone marrow biopsy; hematoxylin and eosin, 40× objective] and F [bone marrow aspirate; Leishman stain; 50× objective]). Granulocytic dysplasia with pelgeroid morphology and abnormal nuclear chromatin clumping was seen in both peripheral blood and bone marrow (> 10% and < 50%). Concurrent flow cytometry showed increased myeloblasts with expression of CD34, CD13, CD 117, CD33, HLA-DR, CD19 (dim; minor subset), CD11b (dim; subset), CD14 (dim; minor subset), CD15 (dim; minor subset), myeloperoxidase (dim; subset), and terminal deoxynucleotidyl transferase (dim; minor subset). Conventional cytogenetics showed normal male karyotype. Concurrent FISH studies using acute myeloid leukemia (AML) and myelodysplastic syndrome panels of probes are negative for all loci studied, including 8q22 and 21q22 probes for RUNX1-RUNX1T1 rearrangement. Next generation sequencing showed mutation on the CSF3R gene (p.T640N, variant allele frequency 48.4%). An integrated diagnosis of AML with maturation was established (French–American–British Classification Subtype M2). Auer rods are a morphologic feature in leukemic blasts defining myeloid differentiation. The presence of Auer rods in neutrophils and monocytes are rarely reported, most commonly in association with acute promyelocytic leukemia (APL) with PML-RARA and AML with RUNX1-RUNX1T1, occasionally seen in other subtypes of AML and myelodysplastic syndrome (MDS) in the literature as well [1]. We presented a case of AML with Auer rods in non-blast cells as well as morphologic and immunophenotypic features resembling AML with RUNX1-RUNX1T1: blasts with basophilic cytoplasm, salmon-pink (pale) cytoplasmic granules and perinuclear Hof besides Auer rods as well as granulocytic dysplasia in the background. However, no recurrent cytogenetic and molecular genetic abnormalities as defined by World Health Organization (WHO) classification are detected, while a CSF3R mutation seen. Although mutations in the colony stimulating factor 3 receptor (CSF3R) occurs rarely in AML, comprising only about 1% of adults with AML, it is suggested that CSF3R can be a potential therapeutic target in AML [2]. In summary, we presented for the first time a case of AML with a few rare morphologic features including the presence of Auer rods in non-blast cells coinciding with a potential therapeutic target of CSF3R mutation. Whether the association between the morphologic features and the CSF3R mutation can be established warrants additional studies. In addition, a potential diagnostic pitfall to be taken into consideration in this case is that no reverse transcription-polymerase chain reaction (RT-PCR) was done to further exclude RUNX1-RUNX1T1 rearrangement, although the likelihood of having undetected rearrangement by karyotyping and FISH studies is extremely low. What is the new aspect of your work? Here, we for the first time presented a case of AML with morphologic features resembling AML with RUNX1-RUNX1T1, including Auer rods in non-blast cells, coinciding with CSF3R mutation, which is a rare mutation in AML while representing a potential novel therapeutic target. What is the central finding of your work? A rare case of CSF3R-mutated AML with no recurrent cytogenetic abnormality, showing unusual morphologic features resembling AML with RUNX1-RUNX1T1, including Auer rods in non-blast cells. What is (or could be) the specific clinical relevance of your work? The rare morphological features present in this AML could potentially serve as a trigger to identify cases of AML with CSF3R mutation for targeted therapy. None. The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported. The authors received no specific funding for this work. Not applicable. Not applicable. Not applicable. The data that support the findings of this study are available from the corresponding author upon reasonable request.

Burkitt leukaemia with B-cell precursor immunophenotype.

A 64-year-old male presented with malaise and B symptoms. An automated full blood count showed haemoglobin 124 g/L, white blood cells 40 × 109/L, platelet 51 × 109/L. Positron emission tomography/computed tomography indicated widespread activity above and below the diaphragm with bone marrow, peritoneal, pleural, hepatic, renal, small bowel and possible adrenal involvement. The blood film (Figure 1A, May-Grünwald-Giemsa stain ×100 objective) showed numerous large cells with basophilic cytoplasm and cytoplasmic vacuolation. By flow cytometry (Beckman Coulter Duraclone) (Figure 1E–H) these cells (red colour population) were CD45 low (Figure 1E). They were positive for surface CD19, CD10, HLADR, CD38 and cytoplasmic CD79a, terminal deoxynucleotidyl transferase (TdT) (Figure 1H); negative for surface CD34, CD20 (panel F), CD22, light chains (Figure 1G) and cytoplasmic CD3, myeloperoxidase (MPO). Bone marrow aspirate (Figure 1B, May-Grünwald-Giemsa stain x100 objective) was packed with medium to large-sized cells with basophilic cytoplasm, and frequent cytoplasmic vacuolation. No abnormalities were detected by reverse transcription-polymerase chain reaction screening for common leukaemic fusion genes using the Q30 leukaemia assay (QuanDX). Fluorescence in situ hybridization (FISH) analysis of the liquid sample detected immunoglobulin heavy chain/MYC translocation and gain of 1q. Next-generation sequencing (Archer VariantPlex) identified NRAS p.Gln61Arg variant (VAF 36%). Bone marrow histology (Figure 1C, haematoxylin and eosin stain x40 objective) revealed effacement by large cells with finely dispersed chromatin, with frequent mitoses and some with multiple small nucleoli. A classical ‘starry sky’ pattern was not seen. Immunohistochemical staining was strongly positive for CD45, CD20 (most positive) and TdT (Figure 1D, x40 objective); positive for paired box 5 (PAX5), CD19, CD79a, CD10, multiple myeloma 1; and negative for B-cell lymphoma 2 (BCL2), BCL6, CD5, cyclin D1, CD1a, CD99, CD117, CD34, MPO and light chains. Staining for Epstein-Barr virus ribonucleic acid was negative. The MIB1 proliferation factor was near 100%. FISH confirmed the MYC translocation but neither BCL6 nor BCL2 translocations were detected. A diagnosis of “Burkitt leukaemia (BL) with a B-cell precursor immunophenotyped”. The patient's medical comorbidities made him unfit for chemotherapy and he received palliative corticosteroid treatment. BL with precursor B cell immunophenotype is rare in adults. The WHO Classification of Tumours of Haematopoietic and Lymphoid Tissue (revised 4th edition) notes approximately 2% of paediatric cases of BL that “have a phenotype of precursor” B-cells, with an expression of TdT, and sometimes CD34, and absence of CD20 and surface immunoglobulin expression. The reason for this aberrant phenotype remains unknown [1]. A small study reported IG-MYC+ neoplasms with precursor B cell immunophenotype to resemble precursor B-cell acute lymphoblastic leukaemia/ lymphoblastic lymphoma rather than BL in genomic, epigenomic profiling, the mutational landscape, and the DNA methylation pattern, with frequent activation of the RAS pathway [2]. Further studies are needed for a better understanding of this rare subgroup of disease (see Figure 1). Ke Xu wrote the manuscript. Ke Xu, Anna Childerhouse and Rajeev Gupta critically revised the final version of the manuscript.

Dntt expression reveals developmental hierarchy and lineage specification of hematopoietic progenitors.

Abstract Intrinsic and extrinsic cues determine developmental trajectories of hematopoietic stem cells (HSCs) towards erythroid, myeloid and lymphoid lineages. To dissect early lymphoid specification events, we generated two transgenic mouse models reporting and tracing the expression of terminal deoxynucleotidyl transferase (TdT). Surprisingly, transient induction of TdT was detected on a newly identified multipotent progenitor (MPP) subset which lacked self-renewal capacity but maintained multilineage differentiation potential. TdT induction by MPPs reflected a transcriptionally dynamic but uncommitted stage characterized by low expression of lineage-associated genes. Further, analysis using single-cell CITE-Seq indicated that multipotency is associated with expression of Endothelial Cell-Selective Adhesion Molecule (ESAM). Developmental progression along all lineages is defined by downregulation of ESAM and by stable TdT expression within the lymphoid trajectory. Collectively, we propose a new developmental hierarchy of early hematopoietic progenitors for which we provide their transcriptional profile as well as their phenotypic and functional properties at single cell resolution. Supported by NIH intramural funding

Dntt expression reveals developmental hierarchy and lineage specification of hematopoietic progenitors.

Intrinsic and extrinsic cues determine developmental trajectories of hematopoietic stem cells (HSCs) towards erythroid, myeloid and lymphoid lineages. Using two newly generated transgenic mice that report and trace the expression of terminal deoxynucleotidyl transferase (TdT), transient induction of TdT was detected on a newly identified multipotent progenitor (MPP) subset that lacked self-renewal capacity but maintained multilineage differentiation potential. TdT induction on MPPs reflected a transcriptionally dynamic, but uncommitted stage, characterized by low expression of lineage-associated genes. Single-cell CITE-Seq indicated that multipotency in the TdT+ MPP is associated with expression of the endothelial cell adhesion molecule ESAM. Stable and progressive upregulation of TdT defined the lymphoid developmental trajectory. Collectively, we here identify a new multipotent progenitor within the MPP4 compartment. Specification and commitment are defined by downregulation of ESAM which marks the progressive loss of alternative fates along all lineages.

Diagnostically misleading aberrant terminal deoxynucleotidyl transferase expression in germ cell tumors.

Terminal deoxynucleotidyl transferase (TdT) is aunique type of DNA polymerase predominantly expressed in precursor lymphoid cells and acute lymphoblastic leukemia. It participates in the junctional diversity of T-cell receptors and immunoglobulins. Recently, aberrant TdT expression was found in seminomas. Here, we evaluated the expression of TdT in our cohort of germ cell tumors (GCTs) with two anti-TdT antibody clones. We included 173 cases of testicular GCTs, 5 ovarian dysgerminomas, and one gonadoblastoma in the study. Tissue microarrays containing representative tumor samples were constructed and subsequently stained with anti-TdT monoclonal rabbit antibody EP266 (Dako) and TdT rabbit polyclonal antibody (Cell Marque). Expression was assessed with the H-score. No specific nuclear reaction was observed for the polyclonal anti-TdT antibody. The H-score values varied between the histological subtypes for the EP266 antibody. Positive nuclear staining was consistently seen in germ cell neoplasia in situ , seminoma, dysgerminoma, and embryonal carcinoma. Pure tumors had higher TdT H-scores than the mixed ones. Teratomas, yolk sac tumors, and choriocarcinomas were almost uniformly negative. Our study confirms that aberrant expression of TdT by testicular and ovarian GCTs exemplifies apotential diagnostic pitfall in histopathological diagnostics.

Clinicopathologic and prognostic features of TdT-negative pediatric B-lymphoblastic leukemia

Little is known about B-lymphoblastic leukemia (B-ALL) that lacks expression of terminal deoxynucleotidyl transferase (TdT). To address this, we performed the largest study to date of TdT-negative B-ALL using data from St. Jude Total XV and XVI clinical trials. Compared to TdT-positive B-ALL (n = 896), TdT-negative B-ALL (n = 21) was associated with younger age (median, 1.4 versus 6.8 years, P < 0.001), higher white blood cell count (median, 52.8 versus 9.9 × 109/L, P < 0.001), absence of hyperdiploidy (0 versus 27.8%, P = 0.002), KMT2A rearrangement (100 versus 1.9%, P < 0.001), and inferior 5-year event-free survival (EFS) (76.2 versus 90.3%, P = 0.047). In the context of KMT2A-rearranged B-ALL (n = 38), TdT-negativity was significantly associated with the MLLT1 rearrangement partner (P = 0.026) but was not independently predictive of survival, suggesting that the high-risk features of TdT-negative B-ALL are secondary to underlying KMT2A rearrangements. Finally, we compared the sensitivity of TdT-negativity to neuron-glial antigen 2 (NG.2) expression for the detection of KMT2A rearrangements and found that 63% of KMT2A-rearranged B-ALL cases not identified by NG.2 were TdT-negative. The results of this study expand the spectrum of immunophenotypic features that are specific for high-risk KMT2A rearrangements in pediatric B-ALL and can be readily implemented using existing standard acute leukemia flow cytometry panels.

Incremental Value of FDG PET/CT in Aggressive High Grade B Cell lymphoma with TdT Expression

High-grade B-cell lymphoma, an aggressive form of Non-Hodgkin's Lymphoma, is known as a double or triple hit lymphoma based on the presence of MYC and BCL2 without or with BCL6 genetic rearrangements, respectively. It carries a poorer prognosis, compared to other variants of B-cell lymphoma, and its management also differs which requires more intensive chemotherapy in contrast to the routine regimen. Terminal deoxynucleotidyl transferase (TdT), a marker of immaturity is commonly expressed in B-cell lymphoblastic leukemia or lymphoma (B cell ALL) which is absent in mature forms of B-cell lymphoma. The TdT is expressed in high-grade B-cell lymphoma; therefore, it poses a classification and management dilemma, which should be accurately differentiated from B-cell ALL and mandates molecular analysis. Herein, we report a case of a 52-year-old female with biopsy reported as high-grade B-cell lymphoma with TdT expression. She was referred for Fluor-deoxyglucose (FDG) Positron Emission Tomography-Computed Tomography (PET/CT) scan for staging in the absence of molecular analysis for B-cell ALL. It was diagnosed as lymphoma on FDG PET/CT based on its characteristic findings of extensive extranodal involvement of multiple organs with no significant lymphadenopathy establishing the incremental value of FDG PET/CT scan, which helped the clinician to arrive at a conclusion.

Terminal Deoxynucleotidyl Transferase Commonly Expresses in Germ Cell Tumors: Evaluation on a Large Series from Multiple Centers.

AimThe concrete features of expression of terminal deoxynucleotidyl transferase (TdT) are needed to be revealed in male and female germ cell tumors (GCTs).MethodsTdT immunostaining was performed in 195 GCTs, and the tumor and/or tumorous components included seminomas, germ cell neoplasias in situ (GCNISs), dysgerminomas, embryonal carcinomas (ECs), extragonadal germinomas, yolk sac tumors (YSTs), teratomas, and spermatocytic tumors. Twenty-one sex cord-stromal tumors were also added. Expression of the classical germ cell tumor markers (PLAP, OCT4, SALL4, CD117, and D2-40) was compared to that of TDT.ResultsNearly all (tumors or tumorous components) seminomas (99%, 107/108), GCNISs (98%, 51/52), dysgerminomas (94%, 17/18), ECs (100%, 15/15), and extragonadal germinomas (100%, 11/11) were positive for TdT. None of the cells in YSTs (0/38), teratomas (0/19), spermatocytic tumors (0/1), or sex cord-stromal tumors (0/21) were immunoreactive for TdT staining. The normal testicular and ovarian gonadal tissues were also negative for TdT. However, TdT presented with significant loss of antigen immunoreactivity in the paraffin-embedded tissues older than 3 years, giving rise to weak or moderate staining in a subset of cases. The expressions of TdT showed no significances with PLAP, OCT4, SALL4, CD117, and D2-40 during the diagnosis of the most GCTs (P>0.05), except for with PLAP, SALL4, or CD117 in YST (P=0.000 each), and D117 (P=0.000) or D2-40 (P=0.006) in ECs.ConclusionOur findings further verify that TdT can serve as a new GCT marker for seminomas, GCNISs, dysgerminomas, ECs, and extragonadal germinomas, with a highly positive rate. Awareness of TdT positivity in GCTs contributes to the prevention of erroneous diagnoses, particularly in the setting of core needle biopsies. To determine the properties where TdT staining may not be apparent in some old archived paraffin-embedded tissues, one could circumvent the potential misinterpretations of false-negative immunohistochemistry results.

Expression of terminal deoxynucleotidyl transferase in the pharyngeal and palatine tonsils in local infectious and inflammatory diseases of the upper respiratory tract and pharynx in childhood

Along with central immune organs, the peripheral lymphoepithelial organs of the pharynx are actively involved in protecting the body from infections. Adaptive, or induced, immunity occurs during the postnatal ontogenesis of immunocompetent lymphocytes, which includes the secondary somatic recombination of the V genes with the participation of recombination-activating gene (RAG) and terminal deoxynucleotidyl transferase (Tdt) proteins. This publication discusses the results of detection of Tdt-positive cells in the pharyngeal and palatine tonsils of children of different ages, who had been operated on for adenoid vegetations and chronic tonsillitis. Attention is drawn to the localization of Tdt+ cells, the level of Tdt expression, an attempt to clarify the phenotype, destination, and place in the diagnostic arrays of functional markers when an adaptive immunity is generated in children.

Large nuclear size correlated with better overall survival, Merkel cell polyomavirus positivity, and terminal deoxynucleotidyl transferase expression in Merkel cell carcinoma

To the Editor: The histologic appearance of Merkel cell polyomavirus (MCPyV)-positive Merkel cell carcinoma (MCC) has been reported to be distinctive: more uniform, round nuclei and less cytoplasm than cells in MCPyV-negative tumors.1,2 In most of these studies, however, the described morphologic differences were identified by light microscopy, and morphometric analysis was done on a limited number of cases.1,3 Extraction of nuclear features by computational methods from high-resolution whole-slide digital scans of histologic slides has been shown to improve accuracy and consistency.

Terminal deoxynucleotidyl transferase (TdT) expression is associated with FLT3-ITD mutations in Acute Myeloid Leukemia

The terminal deoxynucleotidyl transferase (TdT) is a DNA polymerase expressed in acute myeloid leukemias (AMLs), where it may be involved in the generation of NPM1 and FLT3-ITD mutations. We studied the correlations between TdT expression and FLT3-ITD or NPM1 mutations in primary AML samples, and the impact on patients’ survival. TdT expression was analyzed in 143 adult AML patients by flow cytometry as percentage of positivity and mean fluorescence intensity (MFI) on blasts. TdT was positive in 49 samples (34.2%), with a median of 48% TdT-positivity (range 7-98) and a median MFI of 2.70 (range 1.23-30.54). FLT3-ITD and NPM1 mutations were present in 24 (16.7%) and 34 (23.7%) cases, respectively. Median TdT expression on blasts was significantly higher in FLT3-ITD+, as compared with FLT3-ITD- AMLs (median 8% vs 0% respectively, p = 0.035). NPM1 mutational status, FLT3-ITD allelic ratio, karyotype, and ELN risk groups, did not correlate with TdT expression or MFI on blasts. TdT + patients had poorer survival as compared to TdT-, but this result was not confirmed by the multivariable analysis, where ELN risk stratification as well as age and type of treatment remained independent prognostic factors for OS. In summary, our results support the possible implication of TdT enzyme in the generation of FLT3-ITD mutations in AML.

Transformation of a low-grade follicular lymphoma into a composite lymphoma combining a high-grade B-cell lymphoma and a lymphoblastic neoplasm expressing Terminal deoxynucleotidyl Transferase: a case report

BackgroundHigh-grade B-cell lymphoma with rearrangements of MYC and BCL2 and/or BCL6 is an aggressive mature B-cell neoplasm, whereas B-lymphoblastic lymphoma is immature cell proliferation, with a frequent positivity for terminal deoxynucleotidyl transferase. The transformation of a low-grade follicular lymphoma into a lymphoblastic neoplasm expressing terminal deoxynucleotidyl transferase is a very rare event.Case presentationA 55-year-old Caucasian man was followed for a grade 1–2 follicular lymphoma carrying a t(14;18) IGH/BCL2+ and was initially treated with R-CHOP. The follicular lymphoma presented two relapses. In the third relapse, the patient had multiple lymphadenopathy and ascites, which motivated a retroperitoneal biopsy and an ascitic tap. These samples were analyzed by histological, cytological, flow cytometric, cytogenetic, and molecular assessments. The patient died of a multiple organ dysfunction syndrome 2 weeks after his third relapse. The biopsy revealed a diffuse proliferation made up of two types of tumor cells: centroblasts (Bcl-6-positive) and immature cells (terminal deoxynucleotidyl transferase-positive). Flow cytometric analysis confirmed the immature phenotype, with an expression of terminal deoxynucleotidyl transferase, combined with a loss of membrane immunoglobulins. The cytogenetic analysis performed on the ascites revealed a clonal evolution characterized by a t(8;22)(q24;q11) MYC+ translocation not previously detected in follicular lymphoma. Fluorescence in situ hybridization confirmed the double rearrangement of the BCL2 and MYC genes. Polymerase chain reactions and sequencing were used to study the clonal relationship between follicular lymphoma and the secondary tumors. The IGVH gene rearrangement revealed a unique clonal rearrangement involving an IGVH4–59 subset in all three specimens.ConclusionThese findings suggest a clonal relationship between the two types of lymphoma cells. Furthermore, they support the transformation of an acute follicular lymphoma into a composite lymphoma combining a high-grade B-cell lymphoma and a lymphoblastic neoplasm expressing terminal deoxynucleotidyl transferase. This case report highlights the possible transformation of follicular lymphoma into a highly aggressive and immature proliferation.

Aggressive B‐cell lymphomas with a primary bone marrow presentation

Aggressive B-cell lymphomas present as a heterogeneous spectrum of disease. A primary diagnosis in the bone marrow (BM) may be challenging in terms of diagnostic classification and clinical handling, owing to limited architectural information. Aggressive B-cell lymphomas can be subdivided into entities that typically present primarily in the BM, and cases with BM involvement in which the bulk of disease is present in other organs. One main topic at the 2018 BM workshop of the European Association of Haematopathology/Society of Hematopathology was therefore aggressive B-cell lymphomas with a primary BM presentation. The spectrum of cases submitted to this topic gave a good overview of commonly encountered problems, as well as unusual manifestations, and highlighted areas of imprecise disease definitions and diagnostic grey zones. The categories submitted to the workshop included cases of Burkitt lymphoma (BL) with unusual features, high-grade B-cell lymphomas (HG-BCLs) with and without so-called double/triple-hit, and diffuse large B-cell lymphomas (DLBCLs) with a primary BM presentation. Areas of difficulties included the morphological boundaries of HG-BCL not otherwise specified, cases with MYC and bcl-2 or bcl-6 translocations and terminal deoxynucleotidyl transferase (TdT) expression, which were categorised as B-cell lymphoblastic leukaemia/lymphoma if most cells showed TdT positivity, and the clinicopathological overlap between intravascular large B-cell lymphoma, CD5-positive DLBCL, and DLBCL with primary presentations in the BM, spleen, and liver. This review summarises our understanding of the main aggressive B-cell lymphoma categories with a common primary BM presentation and potential problem areas, and makes suggestions for the immunophenotypic and genetic work-up, illustrated by the interesting and challenging cases submitted to the workshop.

Expression of TdT in Myoepithelial Cells: Investigation in Breasts, Sweat Glands, and Salivary Lesions Emphasizing the Never-Documented Immunohistochemical Findings.

Background. The expression of terminal deoxynucleotidyl transferase (TdT) in myoepithelial cells (MECs) within the breast was recently incidentally observed in our routine practice. This study aimed to elucidate the expression of TdT in MECs. Methods. TdT immunostaining was performed on 180 mammary, 89 cutaneous, and 94 salivary tissues or lesions. Other myoepithelial markers, including P63, calponin, and SMA as well as double staining for TdT and calponin, were also evaluated in some cases. Selected lesions with basal or myoid differentiation were also included in the investigation. Results. MECs were positive for TdT in mammary lesions that contained MECs (132/135) but negative when they did not contain MECs (45/45). MECs in sweat glands (24/30) and their neoplastic counterparts, including those in hidradenoma papilliferum (2/9), spiradenoma (6/6), and cutaneous mixed tumor (9/9), showed weak to moderate TdT positivity. MECs were variably immunolabeled for TdT in salivary or salivary gland-type tumors with myoepithelial differentiation (pleomorphic adenoma, 24/25; basal cell adenoma, 6/7; adenoid cystic carcinoma, 7/7; Warthin tumor, 0/6; mucoepidermoid carcinoma, 0/8; acinic cell carcinoma, 0/4), but MECs in normal salivary gland barely stained for TdT (30/32). Conclusions. Our findings indicate that TdT may be eligible as an additional auxiliary immunohistochemical marker as P63, but not a surrogate, to identify the MECs in the breast with limited cross-reactivity, particularly in lesions with a prominent proportion of MECs. Positivity for TdT, along with other relevant markers, in a subset of sweat gland lesions and salivary tumors may contribute to their diagnosis.

Prognostic implications of TdT expression in acute myeloid leukemia with an intermediate-risk karyotype.

Terminal deoxynucleotidyl transferase (TdT) is expressed on precursor lymphoblastic neoplasms and some acute myeloid leukemia (AML) cells. The clinical impact of TdT expression on AML outcomes remains unclear. Here, we conducted a retrospective analysis to identify prognostic implications of TdT expression in AML with an intermediate-risk karyotype. Forty-eight cases of intermediate-risk AML were enrolled. TdT positivity was defined as expression on ≥ 10% of the gated cells. Of 48 cases, 12 (25%) were positive for TdT [median expression rate of TdT 0.9% (range 0-86.9%)]. No significant differences in patient characteristics or complete remission rate were observed between TdT-positive and TdT-negative cases. The probability of overall survival (OS) and event-free survival (EFS) at 1year was not significantly different between TdT-positive and TdT-negative cases (OS: 58.3% vs. 65.2%, p = 0.32; EFS: 33.3% vs. 57.1%, p = 0.06). Relapse-free survival (RFS) probability at 1year was significantly lower for TdT-positive than TdT-negative cases (10% vs. 71.3%, p = 0.002). Multivariate analyses revealed that TdT positivity was an independent significant adverse factor for RFS [hazard ratio: 3.309, 95% confidence interval: 1.334-8.209, p = 0.009]. Our results suggest that TdT expression is associated with increased risk of relapse in patients with intermediate-risk AML.

The Grey Zone: Characterization of Aggressive High-Grade B-Cell Malignancy with TdT Expression and IGH/BCL2 and MYC Rearrangement

Accurately distinguishing between mature and immature B-cell neoplasms is crucial for determining therapy. However, overlapping morphologic, cytogenetic and immunophenotypic characteristics can have diagnostic and therapeutic implications. Morphologically, high grade B-cell lymphoma with double/triple hit (HGBL) may have a "starry sky" or blastoid appearance that can resemble B-lymphoblastic leukemia (B-LBL), Burkitt's Lymphoma (BL) or diffuse large B cell lymphomas (DLBCL), but differ from immature B-cell neoplasms with expression of CD20, CD10, MUM1, and surface immunoglobulin light chains (sIG). Conversely, B-LBL and B-ALL lymphoblasts have dim or lack expression of CD20 and sIG and express markers of immaturity such as CD34 and terminal deoxynucleotidyl transferase (TdT). In this study, we present our institutional experience of 8 patients with a "grey zone" leukemia/lymphoma: 4 patients with aggressive TdT-positive HGBL and 4 patients with B-LBL or B-ALL with a "double-hit" karyotype. We hypothesize these cases may represent a separate entity between HGBL and B-LBL/B-ALL with activating Myc rearrangements. Our cohort was compiled by reviewing cases encountered in our clinical practice from July 2015 to July 2019 that could be identified as follows: (1) de novo HGBL with MYC, BCL2, and/or BCL6 rearrangements or amplifications with concurrent aberrant TdT expression at diagnosis or in subsequent biopsies and (2) de novo B-LBL or B-ALL with a "double‐hit" karyotype. Clinical and diagnostic data were obtained by review of the medical records. The Moffitt Cancer Center Institutional Review Board approved this retrospective study. Of patients with HGBL with aberrant TdT expression, 3 were HGBL with triple hit and 1 with a Bcl6 rearrangement and Bcl2 and Myc amplifications. These patients presented with aggressive features including elevated LDH at diagnosis, high IPI score, and Ki-67 &gt;/= 80%, however without bone marrow or CNS involvement. They express a typical GCB immunophenotype, positive for CD10, CD20, and MUM1 and negative for CD34 (Table 1). These patients were treated with DA-EPOCH-R (3) and R-CHOP (1), of which 1 achieved a partial response and 3 developed progressive disease (Table 2). Three patients went on to consolidative therapy with CD19-directed CAR-T (66%) or autologous transplant (33%). The median overall survival (mOS) was 12.1 months. In the second group, 2 were considered B-ALL based on immature morphology and lack of mature B-cell markers (CD20 negative or variable and MUM1 negative), although they did not express TdT. They both showed IGH-Bcl2 and Myc rearrangements. They were treated with DA-EPOCH-R, of which 1 experienced progressive disease and 1 was unable to complete therapy. The other 2 patients were diagnosed with de novo B-LBL that was CD10 and TdT positive. Of these, 1 showed MUM1 positivity and genomic testing revealed IGH-Bcl2 and Myc rearrangements accompanied by a TP53 mutation. The other showed IGH-Bcl2 rearrangement and Myc activating mutation. One was treated with HyperCVAD with an interval CR and the other with modified DFCI died 5.1 months after diagnosis. Prognosis in this second subset was poor with a mOS of 5.3 months. Within the entire cohort, the majority (75%) were treated as DLBCL with R-CHOP-based therapy, while the remaining (25%) were treated with HyperCVAD. 25% experienced a PR or interval CR, while the remainder experienced progressive disease (PD). Overall, we observed a median PFS of 6.6 months and mOS 5.6 months with 0% undergoing allogeneic transplant in first remission, 1 (12.5%) undergoing autogeneic transplant in first remission and 2 (25%) undergoing CD19-directed CAR-T following salvage therapy. HGBL that has acquired TdT expression and B-LBL/B-ALL associated with IGH/BCL2 translocation and activating MYC rearrangements or mutations are uncommon, but represent a biologically unique group with a poor clinical outcome, likely due to accumulated somatic hypermutations. The high percentage of MUM1 expression in these cases suggests a role of dysregulated MUM1 in cooperation with Myc dysregulation. The recognition of this group may be under-appreciated due to the lack of awareness. Inclusion of evaluation of TDT in "mature" HGBL and identification of genetic alterations including BCL-2, BCL-6 and Myc in "immature" B-LBL/B-ALL will be helpful. Consolidation upon first remission with transplant or CAR-T may be of benefit. Disclosures Chavez: Genentech: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Kite: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Karyopharm: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; Bayer: Membership on an entity's Board of Directors or advisory committees. Bello:Celgene: Speakers Bureau. Bunting:Transenterix: Equity Ownership; Tilray: Equity Ownership; Crispre: Equity Ownership; Celgene: Equity Ownership. Shah:Kite/Gilead: Honoraria; Adaptive Biotechnologies: Honoraria; AstraZeneca: Honoraria; Novartis: Honoraria; Spectrum/Astrotech: Honoraria; Incyte: Research Funding; Jazz Pharmaceuticals: Research Funding; Pharmacyclics: Honoraria; Celgene/Juno: Honoraria.