Terminal Deoxynucleotidyl Transferase Expression Research Articles

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 >/= 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.

PB1686 USE OF FLOW CYTOMETRY AND IMMUNOHISTOCHEMISTRY TO RULE OUT CNS RELAPSE OF ACUTE LYMPHOBLASTIC LEUKEMIA

Background: Increased cerebrospinal fluid (CSF) cellularity in an otherwise asymptomatic patient with acute lymphoblastic leukemia (ALL) may indicate CNS disease. Aims: To describe how flow cytometry and immunohistochemistry helped in ruling out suspicion of CNS relapse because of unexpected CSF findings in a child with acute lymphoblastic leukemia in CR during maintenance phase of chemotherapy. Methods: Examination of cytospin CSF samples with flow cytometry, fluorescent in vitro hybridization (FISH) for centromeres of chromosomes 7 and 8 on 200 cells and immunohistochemistry for CD10 and terminal deoxynucleotidyl transferase (TdT). Results: A 15-year old girl was diagnosed in 2005 with B common ALL with hyperdiploidy including trisomy 8, and no CNS involvement. She was treated according to a modified New York II protocol with good prednisone response and negative flow cytomerty minimal residual disease (MRD) on day 33, and prophylactic cranial radiation of 1200 cGy. She went on with maintenance treatment consisting of 6-mercaptopurine 300 mg/m2/d d0-3, cyclophosphamide 1200 mg/m2 d4, vincristine 1.5 mg/m2 d11, 18, 25, prednisonse 180 mg/m2/d d18-25, methotrexate 200 mg/m2 over 4 h d25, daunorubicin 20 mg/m2 d40, 41, cytosine arabinoside 100 mg/m2, thioguanine 40 mg/m2/dose 6 doses d42-44, methotrexate IT d60/0. Days 0 and 60 CSF findings were normal. However, on day 0 of the subsequent cycle, increased ceullarity was found incidentally. CSF cell number was 45/μL and increased up to 130/μL despite additional methotrexate IT injections. Examination of cytospin preparations showed 75% lymphocytes and 25% monocytes/macrophages. Morphological discrimination between lymphoblasts and mature lymphocytes was difficult. Protein level in CSF was mildly increased up to 71 mg/dL and glucose level in CSF was normal. The patient was free of symptoms and the neuroimaging was normal. Immunohistochemistry on cytospin preparation showed no TdT expression on lymphocytes. Flow cytometry of CSF using the initial immunophenotype showed normal lymphocytes and monocytes and no leukemic blasts. FISH on a cytospin preparation to detect trisomy 8, which was present at diagnosis, and using as an internal negative control chromosome 7 ploidy, was normal with two signals for chromosomes 8 and 7, respectively. Those results in addition to increasing cellularity despite intrathecal chemotherapy and progressive enrichment of CSF in monocytes and uneventful clinical course and undetactable MRD in bone marrow, led to the conclusion that high CSF high cellularity was of reactive nature. CSF cellularity returned to normal within 2 weeks. The patient completed maintenance treatment and remains in persistent complete first remission for 10 years. Summary/Conclusion: Incidental high CSF cellularity arousing suspicion of CNS relapse may be misleading. Sensitive methods of immunophenotyping and genotyping at the cellular level provide confidence in diagnosing reactive CSF pleocytosis secondary to previous intrathecal chemotherapy and avoid unnecessary treatment plan modifications.

TdT expression in germ cell tumours: a possible immunohistochemical cross-reaction and diagnostic pitfall

AimsVery recent papers proposed a possible role for the expression of terminal deoxynucleotidyl transferase (TdT) in the tumourigenesis of gonadal and extragonadal germ cell-derived tumours (GCTs). Our multicentric study evaluated...

Importance of Detection of Intracellular Myeloperoxidase, CD13, CD79a, CD22, CD3 and Terminal Deoxynucleotidyl Transferase By Flow Cytometry Diagnosis of Acute Leukemias

Background: The detection of Intracellular (IC) antigens by flow cytometry (FC) such as myeloperoxidase (MPO), cCD13, cCD79a, cCD22, cCD3 and Terminal deoxynucleotidyl Transferase (TdT) has become the useful tool in the differential diagnosis between acute myeloid leukemias (AML) and acute lymphoid leukemias (ALL). Through detection of myeloid antigens (MPO and cCD13), B cells precursors (cCD79a and cCD22) and precocity T-cells (cCD3) it has been possible to confirm the diagnosis of these acute leukemias. The detection of intracellular cell markers by FC usually requires previous permeabilization of fresh cell suspensions. TdT, also known as DNA nucleotidylexotransferase (DNTT) or terminal transferase, is a specialized DNA polymerase expressed in immature, pre-B, pre-T lymphoid cells, and acute lymphoblastic leukemia/lymphoma cells. TdT adds N-nucleotides to the V, D, and J exons of the TCR and BCR genes during antibody gene recombination, enabling the phenomenon of junctional diversity. In humans, terminal transferase is encoded by the DNTT gene. This antigen is expressed mostly in the nucleus cells from primary lymphoid organs, like the thymus and bone marrow. The TdT detection has also been shown to be useful in confirming the acute forms of B and T-lineage lymphoproliferative diseases by FC. The aim of this study was to demonstrate the importance of this cell markers' detection by FC in the differential diagnostic of acute leukemias. Methods: Bone marrow and/or peripheral blood leukemic cells from 50 cases of acute leukemia: 16 ALL and 36 AML. The cells were fixed and permeabilized in briefly exposed to Becton & Dickinson Lyse Solution at concentration of 10%, and subsequently labeled with monoclonal antibodies anti-MPO, TdT, CD3, CD13, CD22 and CD79a. Results: The MPO expression was observed in 35/36(97,22%) and cCD13 in all cases of AML and in none ALL patients. Three cases of MPO-positive ALL (FAB-L2) could be reclassified as M0-AML. These cases were CD34+;HLADR+;CD33-;CD13-;CD7+ and cCD13+. The intensity of TdT expression was observed in 15/16 (93.8%) of ALL and 5/36 (13.9%) of AML. The cCD22 and cCD79a were positive in 15/16 (93.8%) and all of pre-B ALL respectively and cCD3 was expressed in one case of Pre-T ALL that initial phenotype was CD34+/HLADR+/TdT+/CD7+ and sCD3-). Conclusions: These results indicate that monoclonal antibodies anti-MPO, cCD13, cCD79a, cCD22, cCD3 and TdT were excellent cell markers for the diagnosis and classification of acute leukemias and can be reliably detected by FC. This rapid and specific technique should be a valuable addition to routine immunophenotyping of acute leukemia. DisclosuresNo relevant conflicts of interest to declare.

The expression of terminal deoxynucleotidyl transferase and paired box gene 5 in Merkel cell carcinomas and its relation to the presence of Merkel cell polyomavirus DNA.

Merkel cell carcinoma (MCC) tumor samples frequently express B-lymphoid lineage markers. However, the reasons for expression of specific B-lymphoid lineage markers are still unclear. We studied the expression of TdT and PAX5 (two B-cell lymphoid lineage markers) in a large pool of MCC tissue microarray samples. Immunoexpression and staining intensities of TdT and Pax-5 were statistically correlated with patient, tumor, Merkel cell polyomavirus (MCV), and disease-specific parameters. In a cohort of 117 MCC patients and their corresponding tumor samples, TdT was expressed in 37 (31.6%) samples and PAX5 in 26 (22.2%). Simultaneous immunostaining for TdT and PAX5 was observed in 13 (11.1%) samples. A statistically significant relationship was observed between MCV virus copy number and positive TdT expression (P = 0.0056). Similarly, a significant relationship was also observed between positive TdT and tumor MCV virus positivity (P = 0.000495). We observed frequent TdT and PAX5 immunoexpression in MCC tumor samples. However, simultaneous immunoexpression of these markers was scarce. TdT expression was statistically significantly associated with MCV positivity. The absence of a statistically significant association between tumor parameters and disease progression markers undermines the systemic use of these markers in clinical practice.

Label-free and sensitive detection assay for terminal deoxynucleotidyl transferase via polyadenosine-coralyne fluorescence enhancement strategy

Terminal deoxynucleotidyl transferase (TdT) is a unique template-free polymerase that randomly adds multiple deoxyribonucleoside triphosphates (dNTPs) to the 3′-OH terminus of ssDNA. This characteristic makes TdT a versatile enzymatic tool in many fields. Moreover, aberrant TdT expression is a well-recognized biomarker of several leukemic diseases and is related to carcinogenesis. In this study, we developed a facile, rapid, label-free, and convenient assay for TdT detection. TdT-generated poly A tails formed a fluorescent enhancement complex in the presence of coralyne. To achieve a better signal-to-noise ratio, we used potassium thiocyanate (KSCN), instead of other halogen anions (KCl, KBr, KI, NaI) as the quenching agent of dissociate coralyne. Our results demonstrate that this assay is extremely facile, rapid, and label-free; at levels as low as 0.025 U/mL, TdT was distinctly detected within 55 min. And the determination of TdT activity in RBL-2H3 and Reh cells lysates exhibited a good sensing performance, demonstrating its potential applications in biochemical research and clinical diagnosis.

Terminal Deoxynucleotidyl Transferase Is Not Required for Antibody Response to Polysaccharide Vaccines against Streptococcus pneumoniae and Salmonella enterica Serovar Typhi.

B cell antigen receptor (BCR) diversity increases by several orders of magnitude due to the action of terminal deoxynucleotidyl transferase (TdT) during V(D)J recombination. Unlike adults, infants have limited BCR diversity, in part due to reduced expression of TdT. Since human infants and young mice respond poorly to polysaccharide vaccines, such as the pneumococcal polysaccharide vaccine Pneumovax23 and Vi polysaccharide (ViPS) of Salmonella enterica serovar Typhi, we tested the contribution of TdT-mediated BCR diversity in response to these vaccines. We found that TdT+/- and TdT-/- mice generated comparable antibody responses to Pneumovax23 and survived Streptococcus pneumoniae challenge. Moreover, passive immunization of B cell-deficient mice with serum from Pneumovax23-immunized TdT+/- or TdT-/- mice conferred protection. TdT+/- and TdT-/- mice generated comparable levels of anti-ViPS antibodies and antibody-dependent, complement-mediated bactericidal activity against S Typhi in vitro To test the protective immunity conferred by ViPS immunization in vivo, TdT+/- and TdT-/- mice were challenged with a chimeric Salmonella enterica serovar Typhimurium strain expressing ViPS, since mice are nonpermissive hosts for S Typhi infection. Compared to their unimmunized counterparts, immunized TdT+/- and TdT-/- mice challenged with ViPS-expressing S Typhimurium exhibited a significant reduction in the bacterial burden and liver pathology. These data suggest that the impaired antibody response to the Pneumovax23 and ViPS vaccines in the young is not due to limited TdT-mediated BCR diversification.

Benign TdT-positive cells in pediatric and adult lymph nodes: a potential diagnostic pitfall

Benign terminal deoxynucleotidyl transferase (TdT)-positive cells have been documented in a variety of nonhematopoietic tissues. Scant data are, however, available on their presence in nonneoplastic lymph nodes. This study is aimed to (1) characterize the presence/distribution of benign TdT-positive cells in pediatric and adult reactive lymph nodes and (2) define the phenotype and nature of such elements. This retrospective study considered 141 reactive lymph nodes from pediatric and adult patients without history of neoplastic disease. TdT-positive cells were characterized by immunohistochemical and morphometric analyses, and their presence was correlated with the clinical-pathological features. The nature of TdT-positive cells was investigated by (1) double immunostaining for early lymphoid cell markers and (2) assessment of TdT expression in fetal lymph nodes. Sparse TdT-positive cells were documented in all pediatric cases and in most (76%) adult lymph nodes. TdT-positive cell density was higher in children than adults (15.9/mm2 versus 8.6/mm2; P < .05). TdT positivity did not correlate with any clinical or histological parameter, and double immunostaining disclosed a phenotype compatible with early lymphoid precursors (positivity for CD34 and CD10, and variable expression of CD7). A very high TdT-positive cell density (802.4/mm2) was reported in all fetal lymph nodes. In conclusion, TdT-positive cells are a common finding in pediatric and adult lymph nodes. The interstitial distribution and low number of such cells allow for the differential diagnosis with precursor lymphoid neoplasms. The high density in fetal lymph nodes and the phenotype of such cells suggest their belonging to an immature lymphoid subset gradually decreasing with age.

Expression of terminal deoxynucleotidyl transferase (TdT) in classical seminoma: a potential diagnostic pitfall

Seminomas are the most frequent testicular tumors and in spite of specific markers some histological subtypes can be diagnostically challenging due to the potential overlap of morphologic features and a variant antigen expression. Terminal deoxynucleotidyl transferase (TdT) is a DNA polymerase present in hematogones, thymic T cells, lymphoblastic lymphoma/leukemia (LBL), and in some cases of acute myeloid leukemia but so far has not been described to be expressed in seminomas. After observing a reactivity of TdT in one case of seminoma, we analyzed ten additional tumors by immunohistochemistry to determine their spectrum of reactivity for TdT. In all seminoma cases investigated (10/10) as well as in two tumor-associated germ cell neoplasias in situ (2/2) the TdT staining intensity was variable but was often moderate to strong and restricted to the nucleus. We conclude that TdT expression in seminomas could represent a diagnostic pitfall in the differential diagnosis of LBL, particularly because both may lack CD45 and/or CD20 expression and-concerning relapse in long-term survivors of testicular cancer-LBL is the most frequent secondary neoplasm in the patients.

TdT expression in normal and neoplastic sebaceous cells.

Terminal deoxynucleotidyl transferase (TdT) is a DNA polymerase expressed in immature, normal and neoplastic, lymphoid or haematopoietic cells and in neuroendocrine carcinomas, such as Merkel cell carcinoma and small-cell carcinoma. It has not yet been described in cells of epithelial origin. After observing TdT immunoreactivity in normal sebaceous glands, we analysed its spectrum of expression in cases of sebaceous cell hyperplasia (SGH) and sebaceous cell neoplasm. Twelve cases of SGH and three cases of other benign lesions, namely sebaceoma, sebaceous adenoma, and sebaceous naevus, along with four archived cases of sebaceous cell carcinoma (SC) were collected and stained with TdT antibody. In addition, tissue microarrays were constructed from 11 cases of basal cell carcinoma (BCC) and 10 cases of squamous cell carcinoma (SCC), which had nine evaluable cases each, and, after carcinoma type confirmation with immunostaining for epithelial membrane antigen, TdT immunohistochemistry was performed. All cases of SGH and sebaceous cell neoplasm were positive for TdT. The staining intensity was variable, being often weak to moderate in a significant proportion of cells, apart from one case of SC and the case of sebaceous naevus, which were only focally positive. No BCCs and only one SCC showed immunoreactivity. TdT protein can be found in cells of epithelial origin and specifically sebaceous cells, both benign and malignant. It can be hypothesized that this expression is due to sebaceous cell differentiation as a prelude to apoptosis and holocrine secretion. Additional studies are needed to further elucidate its biological role.

Terminal Deoxynucleotidyl Transferase (TdT) Inhibition of Cord Blood Derived B and T Cells Expansion

Purpose: Terminal deoxynucleotidyl transferase(TdT) is a DNA polymerase that is present in immature pre-B and pre-T cells. TdT inserts N-nucleotides to the V (D) J gene segment during rearrangements of genes, therefore, it plays a vital role in the development and variation of the immune system in vertebrates. Here we evaluated the relationship between cytokines like interleukin-2 (IL-2), interleukin-7 (IL-7), and interleukin-15 (IL-15) and TdT expression in cord blood mononuclear cells and also effect of inhibition in the expansion of B and T cells derived from cord blood. Methodes: The cord blood mononuclear cells were cultured with different combination of cytokines for 21days, which they were harvested in definite days (7, 14 and 21) and evaluated by flow cytometry. Results: Our data indicated that TdT expression increased in cord blood mononuclear cells using immune cell key cytokines without being dependent on the type of cytokines. TdT inhibition reduced both the expansion of B and T cells derived from cord blood and also declined the apoptosis and proliferation. Considered together, TdT played an important role in the control of the expansion of B and T cells derived from cord blood. Conclusion: considered together, it was observed that TdT expression was increased by cytokines and TdT inhibition not only reduced B and Tcells derived from cord blood, but it also affected the rate of apoptosis and proliferation.

Survival of the fetus: fetal B and T cell receptor repertoire development.

A mature and diverse T and B cell receptor repertoire is a prerequisite for immunocompetence. In light of its increased susceptibility to infection, the human fetus has long been considered deficient in this regard. However, data accumulated since the 1990s and in earnest in the past couple of years paints a more complicated picture. As we describe in this review, mechanisms responsible for generating a diverse receptor repertoire, such as somatic recombination, class switch recombination, and somatic hypermutation, are all operational to surprising extents in the growing fetus. The composition of the fetal repertoire differs from that of adults, with preferential usage of certain variable (V), diversity (D), and joining (J) gene segments and a shorter complementarity determining (CDR3) region, primarily due to decreased terminal deoxynucleotidyl transferase (TdT) expression. Both T and B cell receptor repertoires are extremely diverse by the end of the second trimester, and in the case of T cells, are capable of responding to an invading pathogen with in utero clonal expansion. Thus, it would appear as though the T and B cell receptor repertoires are not a hindrance towards immunocompetence of the newborn. Our improved understanding of fetal receptor repertoire development is already bearing fruit in the early diagnosis of primary immunodeficiencies (PID) and may help clarify the pathogenesis of congenital infections, recurrent abortions, and autoimmune disorders in the near future.

Transformation of Follicular Lymphoma to a High Grade B-Cell Lymphoma with MYC and BCL2 Translocations and Overlapping Features of Burkitt Lymphoma and Acute Lymphoblastic Leukemia

Most commonly, histologic transformation (HT) from follicular lymphoma (FL) manifests as a diffuse large B cell lymphoma, not otherwise specified (DLBCL, NOS). Less frequently, HT may result in a high-grade B cell lymphoma with MYC and BCL2 and/or BCL6 gene rearrangements, also known as "double-hit" or "triple-hit" lymphomas. In the 2016 revision of the WHO classification of lymphoid neoplasms, the category B-cell lymphoma, unclassifiable was eliminated due to its vague criteria and limiting diagnostic benefit. Instead, the WHO introduced the high-grade B-cell lymphoma (HGBL) category, characterized by MYC and BCL2 and/or BCL6 rearrangements. Cases that present as an intermediate phenotype of DLBCL and Burkitt lymphoma (BL) will fall within this HGBL category.Very rarely, HT results in both the intermediate DLBCL and BL phenotype, and exhibits lymphoblastic features, in which case the WHO recommends that this morphological appearance should be noted. In comparison to de-novo patients with DLBCL, NOS, those with MYC and BCL2 and/or BCL6 gene rearrangements have a worse prognosis.A 63-year-old female presented with left neck adenopathy. Lab assessment including complete blood count (CBC), complete metabolic panel (CMR), serum lactate dehydrogenase (LDH) and B-2 microglobulin were all normal. A whole body computerized tomography (CT) scan revealed diffuse adenopathy above and below the diaphragm. An excisional node biopsy showed grade 3A nodular FL. The Ki-67 labeling index was 40-50%. A bone marrow biopsy showed a small focus of para-trabecular CD20+ lymphoid aggregates.She received 6 cycles of bendamustine (90 mg/m2 days +1 and + 2) and rituximab (375 mg/m2 on day+2) with each cycle delivered every 4 weeks. A follow up CT scan at completion of therapy showed a partial response with resolution of axillary adenopathy and a dramatic shrinkage of the large retroperitoneal nodes.18 months later she had crampy abdominal pain in the absence of B symptoms. Positron emission tomography with 2-deoxy-2-[fluorine-18] fluoro- D-glucose integrated with CT (18F-FDG PET/CT) scan showed widespread adenopathy, diffuse splenic involvement and substantial marrow involvement. Biopsy of a 2.4 cm right axillary node (SUVmax of 16.1) showed involvement by grade 3A FL with a predominant nodular pattern of growth. A bone marrow biopsy once again showed only a small focus of FL.She received idelalisib (150 mg twice daily), and rituximab (375 mg/m2, monthly) beginning May, 2015. After 4 cycles, a repeat CT scan showed a complete radiographic response. Idelalisib was subsequently held while she received corticosteroids for immune-mediated colitis. A month later she restarted idelalisib with a 50% dose reduction. Two weeks later she returned to clinic complaining of bilateral hip and low lumbar discomfort but no B symptoms. A restaging 18F-FDG PET/CT in January of 2016 showed dramatic marrow uptake. A bone marrow aspirate showed sheets of tumor cells representing a spectrum from intermediate sized cells with lymphoblastic features to very large atypical cells with multiple nucleoli. Two distinct histologies were present; one remained consistent with the patient's known FL with a predominant nodular pattern and the other consistent with HT (The large atypical cells expressed PAX5, CD10, BCL2, and cMYC, and were negative for CD20, MPO, CD34, CD30, and BCL6). Focal areas showed faint, heterogeneous expression of terminal deoxynucleotidyl transferase (TdT) best seen on the clot section. Ki67 proliferation index was high (4+/4). FISH analysis showed two populations with MYC amplification and/or rearrangement, and no evidence of BCL6 rearrangement; a karyotype analysis showed a complex abnormal female karyotype with t (14; 18) and multiple structural and numerical abnormalities.She started dose-adjusted rituximab, etoposide, prednisone, vincristine, cyclophosphamide, and doxorubicin (DA-R-EPOCH) with concomitant prophylactic intrathecal methotrexate and cytarabine. She had but a short lived response before dying in hospice from progressive lymphoma.Whether idelalisib could provide a microenvironment for selection of more aggressive clones needs to be addressed. Our patient's clinical course is confounded by the incorporation of idelalisib while being further complicated by the complexity of HT and the mechanisms in which first-line chemotherapy regimens impact DHL. DisclosuresNo relevant conflicts of interest to declare.

Double-strand break repair based on short-homology regions is suppressed under terminal deoxynucleotidyltransferase expression, as revealed by a novel vector system for analysing DNA repair by nonhomologous end joining.

We have constructed a novel, nonhomologous end‐joining (NHEJ) assay vector (NAV), containing mKate2, Venus and ccdB genes. Cotransfection of NAV with a construct expressing the restriction enzyme I‐SceI generated a double‐strand break (DSB) in NAV that excised mKate2 and ccdB. Repair of this DSB produced an intact vector that expressed Venus, a green fluorescent protein. Because cells bearing the repaired NAV lacked the ccdB gene which slows cell proliferation, the cultures were enriched in cells containing repaired DSBs. DNA sequence analysis of the DSB junctions indicated that the repair was carried out mainly by using the closest homology sequence. Use of the NAV yielded rapid results within 3 days after transfection. We then used the NAV to analyse NHEJ in cells overexpressing terminal deoxynucleotidyltransferase (TdT). The results indicated that TdT suppresses DNA repair that is based on short (one‐ or two‐base) homology regions, to efficiently add deoxynucleotides during VDJ recombination in lymphoid cells.

Immunophenotypic markers in adult acute lymphoblastic leukemia: the prognostic significance of CD20 and TdT expression.

BackgroundEfforts to overcome poor outcomes in patients with adult acute lymphoblastic leukemia (ALL) have focused on combining new therapeutic agents targeting immunophenotypic markers (IPMs) with classical cytotoxic agents; therefore, it is important to evaluate the clinical significance of IPMs.MethodsBaseline characteristics and clinical outcomes of patients with adult ALL were retrospectively analyzed. The percentage of blasts expressing IPMs at diagnosis was measured by multicolor flow cytometry analysis. Samples in which ≥20% of blasts expressed an IPM were considered positive.ResultsAmong the total patient population (N=230), almost all (92%) were in first or second hematological complete remission (HCR) and 54% received allogeneic hematopoietic cell transplant (allo-HCT). Five-year hematologic relapse-free survival (HRFS) and overall survival (OS) rates were 36% and 39%, respectively, and 45.6% and 80.5% of patients were positive for the IPMs CD20 and terminal deoxynucleotidyl transferase (TdT), respectively. Expression of CD20, CD13, CD34, and TdT was associated with HRFS rate, and expression of CD20 and CD13 was associated with OS rate, as was the performance of allo-HCT. In multivariate analysis, positivity for CD20 (HRFS: hazard ratio [HR], 2.21, P<0.001; OS: HR, 1.63, P=0.015) and negativity for TdT (HRFS: HR, 2.30, P=0.001) were both significantly associated with outcomes. When patients were categorized into three subgroups according to positivity for CD20 and TdT, there were significant differences in HRFS and OS among the subgroups.ConclusionPositivity for CD20 and TdT expression and clinical risk group were prognostic factors in adult ALL.

TdT+ T-Lymphoblastic Proliferation in Castleman Disease.

TdT+ T-Lymphoblastic Proliferation in Castleman Disease.

Expansion of the preimmune antibody repertoire by junctional diversity in Bos taurus.

Cattle have a limited range of immunoglobulin genes which are further diversified by antigen independent somatic hypermutation in fetuses. Junctional diversity generated during somatic recombination contributes to antibody diversity but its relative significance has not been comprehensively studied. We have investigated the importance of terminal deoxynucleotidyl transferase (TdT) -mediated junctional diversity to the bovine immunoglobulin repertoire. We also searched for new bovine heavy chain diversity (IGHD) genes as the information of the germline sequences is essential to define the junctional boundaries between gene segments. New heavy chain variable genes (IGHV) were explored to address the gene usage in the fetal recombinations. Our bioinformatics search revealed five new IGHD genes, which included the longest IGHD reported so far, 154 bp. By genomic sequencing we found 26 new IGHV sequences that represent potentially new IGHV genes or allelic variants. Sequence analysis of immunoglobulin heavy chain cDNA libraries of fetal bone marrow, ileum and spleen showed 0 to 36 nontemplated N-nucleotide additions between variable, diversity and joining genes. A maximum of 8 N nucleotides were also identified in the light chains. The junctional base profile was biased towards A and T nucleotide additions (64% in heavy chain VD, 52% in heavy chain DJ and 61% in light chain VJ junctions) in contrast to the high G/C content which is usually observed in mice. Sequence analysis also revealed extensive exonuclease activity, providing additional diversity. B-lymphocyte specific TdT expression was detected in bovine fetal bone marrow by reverse transcription-qPCR and immunofluorescence. These results suggest that TdT-mediated junctional diversity and exonuclease activity contribute significantly to the size of the cattle preimmune antibody repertoire already in the fetal period.

Unusual presentation of Precursor B Lymphoblastic Lymphoma originating from the stomach: One more case and review of the literature

Precursor B lymphoblastic lymphoma (PBL) is an aggressive neoplasm of precursor lymphocytes of B-cell lineage. Extranodal sites are often affected, however gastrointestinal involvement is rarely encountered. We report a case of PBL diagnosed in gastric biopsy of a 72-year-old female. Endoscopy showed multiple polypoid lesions in the stomach. Histologically, the tumor cells exhibited blastic morphology and immunohistochemical expression of terminal deoxynucleotidyl transferase (TdT), CD79a, CD20, CD10 and PAX-5, whereas they were CD34, MPO, and CD3 negative. The diagnosis of this rare entity in unusual extranodal sites such as the stomach is challenging, and it is based on detailed histopathological and immunohistochemical analysis combined to clinical correlations.

Absence of terminal deoxynucleotidyl transferase expression identifies a subset of high-risk adult T-lymphoblastic leukemia/lymphoma

Terminal deoxynucleotidyl transferase (TdT) can be downregulated in minimal residual disease of T-acute lymphoblastic leukemia/lymphoma (T-ALL/LBL) after chemotherapy. TdT-negative T-ALL/LBL cases are rare and have not been well characterized. We studied the clinicopathologic features of de novo T-ALL/LBL patients treated at our institution during 2003–2011, with an emphasis on immunophenotype and survival of TdT-negative versus TdT-positive cases. Absence of TdT expression was defined as <10% lymphoblasts positive. Seven (12%) TdT-negative cases were identified from a cohort of 59 de novo T-ALL/LBL. The TdT-negative and TdT-positive cases were similar with regard to gender, percentage of patients with a high leukocyte count (>100 × 109/l), central nervous system involvement, and an abnormal karyotype. However, patients with TdT-negative T-ALL/LBL had a significantly higher rate of disease progression and shorter overall survival. Although not statistically significant, TdT-negative T-ALL/LBL cases were associated with an older median age and higher percentage of ‘early T precursor’ (ETP) immunophenotype than TdT-positive cases. Absence of TdT expression identifies a subset of high-risk T-ALL/LBL that overlaps with, but is not identical to, the ETP leukemia, providing additional prognostic value.

Early B-Cell Differentiation in Merkel Cell Carcinomas: Clues to Cellular Ancestry

Merkel cell carcinoma (MCC) is a highly malignant neuroendocrine nonmelanoma skin cancer, which is associated with the Merkel cell polyoma virus (MCPyV). Recently, expression of the terminal deoxynucleotidyl transferase (TdT) and the paired box gene 5 (PAX 5) has been consistently reported in the majority of MCCs. We tested 21 MCCs for the expression of MCPyV, TdT, PAX5, IgG, IgM, IgA, kappa, and lambda by immunohistochemistry and assessed IgH and Igk rearrangement in all 21 MCCs. All of the MCCs revealed specific expression of PAX5 and 72.8% of the MCCs expressed TdT. In addition, most of the MCCs revealed specific expression of one or more Ig subclasses and kappa or lambda. One MCC did reveal monoclonal IgH and Igk rearrangement next to two other MCCs showing Igk rearrangement. As coexpression of TdT and PAX5 under physiologic circumstances is restricted to pro/pre- and pre-B cells we propose, on the basis of our results, that the cell of origin of MCCs is a pro/pre- or pre-B cell rather than the postmitotic Merkel cells. MCPyV infection and transformation of pro-/pre-B cells are likely to induce the expression of simple cytokeratins as has been shown for SV40 in other nonepithelial cells. This model of cellular ancestry of MCCs might impact therapy and possibly helps to understand why approximately 20% of MCCs are MCPyV-negative.