Enzymatic Preparation of DNA with an Expanded Genetic Alphabet Using Terminal Deoxynucleotidyl Transferase and Its Applications.

Efficient preparation of DNA oligonucleotides containing unnatural nucleobases (UBs) that can pair with their cognates to form unnatural base pairs (UBPs) is an essential prerequisite for the application of UBPs in vitro and in vivo. Traditional preparation of oligonucleotides containing unnatural nucleobases largely relies on solid-phase synthesis, which needs to use unstable nucleoside phosphoramidites and a DNA synthesizer, and is environmentally unfriendly and limited in product length. To overcome these limitations of solid-phase synthesis, we developed enzymatic methods for daily laboratory preparation of DNA oligonucleotides containing unnatural nucleobase dNaM, dTPT3, or one of the functionalized dTPT3 derivatives, which can be used for orthogonal DNA labeling or the preparation of DNAs containing UBP dNaM-dTPT3, one of the most successful UBPs to date, based on the template-independent polymerase terminal deoxynucleotidyl transferase (TdT). Here, we first provide a detailed procedure for the TdT-based preparation of DNA oligonucleotides containing 3'-nucleotides of dNaM, dTPT3, or one of dTPT3 derivatives. We then present the procedures for enzyme-linked oligonucleotide assay (ELONA) and imaging of bacterial cells using DNA oligonucleotides containing 3'-nucleotides of dTPT3 derivatives with different functional groups. The procedure for enzymatic synthesis of DNAs containing an internal UBP dNaM-dTPT3 is also described. Hopefully, these methods will greatly facilitate the application of UBPs and the construction of semi-synthetic organisms with an expanded genetic alphabet.

Recording Binding Information Directly into DNA-Encoded Libraries Using Terminal Deoxynucleotidyl Transferase.

Terminal deoxynucleotidyl transferase (TdT) is an unusual DNA polymerase that adds untemplated dNTPs to 3'-ends of DNA. If a target protein is expressed as a TdT fusion and incubated with a DNA-encoded library (DEL) in the presence of dATP, the binders of the target induce proximity between TdT and the DNA, promoting the synthesis of a poly-adenine (polyA) tail. The polyA tail length is proportional to the binding affinity, effectively serving as a stable molecular record of binding events. The polyA tail is also a convenient handle to enrich binders with magnetic poly(dT)25 beads before sequencing. In a benchmarking system, we show that ligands spanning nanomolar to double-digit micromolar binding can be cleanly identified by TdT extension, whereas only the tightest binding ligands are identified by classical affinity selection. The method is simple to implement and can function on any DEL that bears a free 3'-end.

Multiplex enzymatic synthesis of DNA with single-base resolution.

Enzymatic DNA synthesis (EDS) is a promising benchtop and user-friendly method of nucleic acid synthesis that, instead of solvents and phosphoramidites, uses mild aqueous conditions and enzymes. For applications such as protein engineering and spatial transcriptomics that require either oligo pools or arrays with high sequence diversity, the EDS method needs to be adapted and certain steps in the synthesis process spatially decoupled. Here, we have used a synthesis cycle comprising a first step of site-specific silicon microelectromechanical system inkjet dispensing of terminal deoxynucleotidyl transferase enzyme and 3' blocked nucleotide, and a second step of bulk slide washing to remove the 3' blocking group. By repeating the cycle on a substrate with an immobilized DNA primer, we show that microscale spatial control of nucleic acid sequence and length is possible, which, here, are assayed by hybridization and gel electrophoresis. This work is distinctive for enzymatically synthesizing DNA in a highly parallel manner with single base control.

Spatially Selective Electrochemical Cleavage of a Polymerase-Nucleotide Conjugate.

Novel enzymatic methods are poised to become the dominant processes for de novo synthesis of DNA, promising functional, economic, and environmental advantages over the longstanding approach of phosphoramidite synthesis. Before this can occur, however, enzymatic synthesis methods must be parallelized to enable production of multiple DNA sequences simultaneously. As a means to this parallelization, we report a polymerase-nucleotide conjugate that is cleaved using electrochemical oxidation on a microelectrode array. The developed conjugate maintains polymerase activity toward surface-bound substrates with single-base control and detaches from the surface at mild oxidative voltages, leaving an extendable oligonucleotide behind. Our approach readies the way for enzymatic DNA synthesis on the scale necessary for DNA-intensive applications such as DNA data storage or gene synthesis.

Novel sperm chromatin dispersion test with artificial intelligence-aided halo evaluation: A comparison study with existing modalities.

BACKGROUND: Sperm chromatin dispersion test is a common and inexpensive technique to assess sperm DNA fragmentation, but its subjectivity in assessing a small number of spermatozoa is a disadvantage. OBJECTIVES: To study the efficacy of a new sperm chromatin dispersion test kit (R10) combined with an artificial intelligence-aided halo-evaluation platform (X12) and compare the results to those of existing sperm DNA fragmentation testing methods. MATERIALS AND METHODS: Semen samples from normozoospermic donors (n = 10) and infertile men with abnormal semen parameters (n = 10) were enrolled. DNA fragmentation indices were examined by multiple assays, including R10, Halosperm G2 (G2), sperm chromatin structure assay, and terminal deoxynucleotidyl transferase deoxynucleotidyl transferase nick end labeling. In R10 assay, the DNA fragmentation indices were obtained both manually (manual R10) and by X12 (AI-R10). The obtained DNA fragmentation indices were analyzed by agreement analyses. RESULTS: The DNA fragmentation indices obtained by manual R10 and those obtained by AI-R10 showed a strong significant correlation (r = 0.97, p < 0.001) and agreement. The number of spermatozoa evaluated by AI-R10 was 2078 (680-5831). The DNA fragmentation indices obtained by manual R10 and AI-R10 both correlated with those of G2 (r = 0.90, p < 0.001; r = 0.88, p < 0.001). Between the AI-R10 and G2 results, Passing-Bablok regression showed no systematic or proportional difference, and Bland-Altman plots revealed overall agreement and a mean bias of 6.3% with an SD of 6.9% (95% limit of agreement: -7.2% to 19.9%). AI-R10 and sperm chromatin structure assays showed systematic differences with a mean bias of -1.9%, while AI-R10 and terminal deoxynucleotidyl transferase deoxynucleotidyl transferase nick end labeling revealed proportional differences with a mean bias of -10.7%. CONCLUSIONS: The novel sperm chromatin dispersion kit and artificial intelligence-aided platform demonstrated significant correlation and agreement with existing sperm chromatin dispersion methods by assessing greater number of spermatozoa. This technique has the potential to provide a rapid and accurate assessment of sperm DNA fragmentation without technical expertise or flow cytometry.

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.

DNTT activation, TdT-aided gene length mutation, and better prognosis in ATG-based regimen allo-HSCT in AML.

This study aimed to investigate the relationship between anomalous DNA nucleotidylexotransferase (DNTT) activation and the mutagenesis of gene length mutations (LMs) in acute myeloid leukemia (AML), and the relevance of their prognosis in antithymocyte globulin (ATG)-based regimen allogeneic hematopoietic stem cell transplantation (allo-HSCT). A cohort of 578 AML cases was enrolled. Next-generation sequencing was performed to screen mutations of 86 leukemia driver genes. RNA-seq was used to analyze gene expression. Prognostic analysis was investigated in 239 AML cases who underwent ATG-based regimen allo-HSCT. We report a refined subtyping algorithm of LMs (type I-IV) based on sequence anatomy considering the TdT-aided mutagenesis mechanism. GC content adjacent to LM junctions, inserted nontemplate nucleotide bases, and DNTT expression analysis supported the DNTT activation and TdT-aided mutagenesis in type II/III LMs in the total AML cohort. Both single-variate and multivariate analyses showed a better overall survival of FLT3 type III compared to type I in a subset of ATG-based regimen allo-HSCT cases. The novel LM subtyping algorithm not only deciphers the etiology of the mutagenesis of LMs but also helps to fine-tune prognosis differentiation in AML. The possible prognostic versatility of this novel LM subtyping algorithm in terms of chemotherapy, targeted therapy, and allo-HSCT merits further investigation.

Detection of apoptotic cells based on in situ hybridization chain reaction using specific hairpins.

There are an increasing number of experiments to study programmed cell death/apoptosis, one of the characteristics of which is DNA fragmentation. The only current method for in situ detection of DNA fragmentation is Terminal deoxynucleotidyl transferase mediated-dUTP Nick End Labeling, TUNEL. In this study, a new method for in situ detection of apoptotic DNA fragments, namely In Situ Hybridization Chain Reaction, isHCR, was established. The principle of the assay is that the sticky end sequence of the apoptotic cell DNA fragment non-specifically initiates a hybridization chain reaction that specifically detects the apoptotic cell. The results of the combined TUNEL and isHCR method demonstrated that the majority of isHCR-positive cells were also labeled by TUNEL. In situ HCR often detect DNA fragments in the cytoplasm that the classical TUNEL method couldnot, and these cells may be in the early stages of apoptosis. It also indicates that DNA fragments are transferred to the cytoplasm during apoptosis. Because the staining process does not require terminal deoxynucleotidyl transferase as TUNEL staining does, isHCR staining cost low and can be performed on a large number of tissue specimens. It is believed that isHCR has the potential to detect DNA fragmentation of apoptotic cells in situ.

Enzymatic Synthesis of DNA with an Expanded Genetic Alphabet Using Terminal Deoxynucleotidyl Transferase.

Development of unnatural base pairs (UBPs) has significantly expanded the genetic alphabet both in vitro and in vivo and led to numerous potential applications in the biotechnology and biopharmaceutical industry. Efficient synthesis of oligonucleotides containing unnatural nucleobases is undoubtedly an essential prerequisite for making full use of the UBPs, and de novo synthesis of oligonucleotides with terminal deoxynucleotidyl transferases (TdTs) has emerged as a method of great potential to overcome limitations of traditional solid-phase synthesis. Herein, we report the efficient template-independent incorporation of nucleotides of unnatural nucleobases dTPT3 and dNaM, which have been designed to make one of the most successful UBPs to date, dTPT3-dNaM, into DNA oligonucleotides with a TdT enzyme under optimized conditions. We also demonstrate the efficient TdT incorporation of dTPT3 derivatives with different functional linkers into oligonucleotides for orthogonal labeling of nucleic acids and applications thereof. The development of a method for the daily laboratory preparation of DNAs with UBPs at arbitrary sites with the assistance of TdT is also described.

Long Non-Coding RNA DUXAP8 Acts as an Oncogene in Sinonasal Squamous Cell Carcinoma Through miR-584-5p/FNDC3B Pathway.

Sinonasal squamous cell carcinoma (SNSCC) is one of the least frequent carcinomas in the head and neck and accounts for 60% to 75% of sinonasal malignancies. The role of long non-coding RNAs (lncRNAs) in cancer development has drawn great attention over the years. The current study intended to assess the role and specific mechanism of lncRNA double homeobox A pseudogene 8 (DUXAP8) in SNSCC. Quantitative real-time PCR (qRT-PCR) analysis was implemented to assess the expression level of DUXAP8, microRNA-584-5p (miR-584-5p), and fibronectin type III domain containing 3B (FNDC3B). Proliferation assays included colony formation assay, Cell Counting Kit-8 (CCK-8) assay, and 5-ethynyl-2'-deoxyuridine (EdU) assay. Transwell assays were implemented to monitor cell migration and invasion. Cell apoptosis was evaluated via terminal deoxynucleotidyl transferase (TdT) dUTP nick-end labeling (TUNEL) and JC-1 experiments. Mechanism experiments included RNA pull-down assay, RNA binding protein immunoprecipitation (RIP) assay, and luciferase reporter assay. DUXAP8 is overexpressed in SNSCC cells. Functionally, DUXAP8 silencing suppresses the malignant progression of SNSCC. Furthermore, DUXAP8 up-regulates the expression of FNDC3B via sponging miR-584-5p. Rescue experiments demonstrated that DUXAP8 mediates the progression of SNSCC via up-regulating FNDC3B expression. In conclusion, DUXAP8 acts as an oncogene in SNSCC, which may be a new molecular marker for SNSCC.

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+ MPPs 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.

Mutational Landscape of TdT+ Large B-cell Lymphomas Supports Their Distinction From B-lymphoblastic Neoplasms: A Multiparameter Study of a Rare and Aggressive Entity.

In the current World Health Organization classification, terminal deoxynucleotidyl transferase (TdT) expression in a high grade/large cell B-cell lymphoma (LBCL) indicates a B-lymphoblastic lymphoma/leukemia (B-LBL), although TdT expression in what appear to be mature LBCL or following mature B-cell neoplasms is reported. The frequency of TdT+ LBCL, how to best categorize these cases, and their clinicopathologic features, molecular landscape, and relationship to classic B-LBL remain to be better defined. TdT expression was therefore assessed in 258 LBCL and the results correlated with the cytologic, phenotypic, and cytogenetic findings. Targeted mutational analysis, review of prior biopsies, and assessment of clinical associations was performed in the 6 cases with >10% TdT+ cells. All 6 TdT+ LBCL were blastoid-appearing, CD34-, MYC+, BCL2+, and had MYC rearrangements (R) (5/6 with BCL2 and/or BCL6-R). 5/6 had a prior TdT- LBCL and/or follicular lymphoma and all had an aggressive course. Fifteen nonsynonymous variants in 11 genes were seen in the 4/5 tested cases with mutations. TdT+ and TdT- areas in 1 case showed identical mutations. The mutational profiles were more like those reported in germinal center B-cell type-diffuse LBCL rather than B-LBL. Evolution from preceding TdT- lymphomas was nondivergent in 1/3 tested cases and partially divergent in 2. The clinicopathologic and cytogenetic features of these 6 cases were similar to those found in a meta-analysis that included additional cases of TdT+ LBCL or B-LBL following follicular lymphoma. Thus, TdT+, CD34- large B-cell neoplasms with MYC rearrangements and often a "double hit" are rare, frequently a transformational event and aggressive but are distinct from classic B-LBL.

[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]. / Ekspressiya terminal'noi dezoksinukleotidiltransferazy v glotochnoi i nebnykh mindalinakh pri mestnykh infektsionno-vospalitel'nykh zabolevaniyakh verkhnikh dykhatel'nykh putei i glotki v detskom vozraste.

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.

Labeling the 3' Termini of Oligonucleotides Using Terminal Deoxynucleotidyl Transferase.

Terminal deoxynucleotidyl transferase (TdT, also simply called terminal transferase) is a template-independent polymerase that catalyzes the addition of deoxynucleotides and dideoxynucleotides to the 3'-hydroxyl terminus of a DNA molecule. Cobalt (Co2+) is a necessary cofactor for the activity of this enzyme. Incorporation at the 3' terminus can be limited to just 1 nt by using [α-32P]ddATP or biotin-, digoxigenin (DIG)-, or fluorescein-ddUTP. Because none of these molecules carries a 3'-hydroxyl group, no additional molecules can be incorporated. Alternatively, the enzyme is capable of adding several (2-100) nt to 3' ends in a so-called homopolymeric "tailing" reaction. A tailing reaction is performed in the presence of a mixture of labeled and unlabeled dNTPs. The rate of addition of dNTPs, and thus the length of the tail, is a function of the ratio of 3' DNA ends to dNTP concentration and, in addition, the specific dNTP that is used.

Attenuated asthma phenotype in mice with a fetal-like antigen receptor repertoire.

We hypothesized that the scarcity of N-nucleotides might contribute to the inability of the neonate to mount a robust allergic immune response. To test this, we used terminal deoxyribunucleotidyl Transferase deficient (TdT-/-) mice, which express "fetal-like" T cell receptor and immunoglobulin repertoires with largely germline-encoded CDR3 regions. Intraperitoneal sensitization was followed by aerosol provocation with either PBS or the allergen OVA in both TdT-/- mice and wild-type mice to develop allergic respiratory inflammation. The effects of this procedure were investigated by lung function test, immunological analysis of serum and brochoalveolar lavage. The local TH2 cytokine milieu was significantly attenuated in TdT-/- mice. Within this group, the induction of total IgE levels was also significantly reduced after sensitization. TdT-/- mice showed a tendency toward reduced eosinophilic inflow into the bronchial tubes, which was associated with the elimination of respiratory hyperreactivity. In conclusion, in a murine model of allergic airway inflammation, the expression of fetal-like antigen receptors was associated with potent indications of a reduced ability to mount an asthma phenotype. This underlines the importance of somatically-generated antigen-receptor repertoire diversity in type one allergic immune responses and suggests that the fetus may be protected from allergic responses, at least in part, by controlling N addition.

T Cells Expressing Receptor Recombination/Revision Machinery Are Detected in the Tumor Microenvironment and Expanded in Genomically Over-unstable Models.

Tumors undergo dynamic immunoediting as part of a process that balances immunologic sensing of emerging neoantigens and evasion from immune responses. Tumor-infiltrating lymphocytes (TIL) comprise heterogeneous subsets of peripheral T cells characterized by diverse functional differentiation states and dependence on T-cell receptor (TCR) specificity gained through recombination events during their development. We hypothesized that within the tumor microenvironment (TME), an antigenic milieu and immunologic interface, tumor-infiltrating peripheral T cells could reexpress key elements of the TCR recombination machinery, namely, Rag1 and Rag2 recombinases and Tdt polymerase, as a potential mechanism involved in the revision of TCR specificity. Using two syngeneic invasive breast cancer transplantable models, 4T1 and TS/A, we observed that Rag1, Rag2, and Dntt in situ mRNA expression characterized rare tumor-infiltrating T cells. In situ expression of the transcripts was increased in coisogenic Mlh1-deficient tumors, characterized by genomic overinstability, and was also modulated by PD-1 immune-checkpoint blockade. Through immunolocalization and mRNA hybridization analyses, we detected the presence of rare TDT+RAG1/2+ cells populating primary tumors and draining lymph nodes in human invasive breast cancer. Analysis of harmonized single-cell RNA-sequencing data sets of human cancers identified a very small fraction of tumor-associated T cells, characterized by the expression of recombination/revision machinery transcripts, which on pseudotemporal ordering corresponded to differentiated effector T cells. We offer thought-provoking evidence of a TIL microniche marked by rare transcripts involved in TCR shaping.

A case of primary cutaneous B-cell lymphoma with immature features in an old man. Diffuse large B-cell lymphoma with immature features or B-cell lymphoblastic lymphoma?

Primary cutaneous B-cell lymphomas are a heterogeneous group of lymphoid neoplasms primarily occurring in the skin. Although most cases are represented by primary cutaneous follicle center cell lymphoma, primary cutaneous marginal zone lymphoma and leg-type diffuse large B-cell lymphoma, other diffuse large B-cell lymphomas and B-cell lymphoblastic lymphoma may rarely present primarily in the skin. In this setting, the presence of histopathologic and immunohistochemical features of cellular immaturity is exceedingly rare and may represent a diagnostic challenge. We present the first case of a primary cutaneous diffuse large B-cell lymphoma characterized by diminished expression of CD45, expression of TdT and rearrangement of MYC gene. The differential diagnosis mainly included B-cell lymphoblastic lymphoma, and required the genetic analysis of heavy chain (IGH) gene rearrangements.

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.

Targeting TdT gene expression in Molt-4 cells by PNA-octaarginine conjugates.

Peptide nucleic acid (PNA) is an amide based structural nucleic acid mimic with potential applications in gene therapeutic drug discovery. In the present study, we evaluated and compared the effects on gene expression, cell viability and apoptosis of two antisense PNA-d-octaarginine conjugates, targeting sequences at the AUG translation start site or the 5'-UTR of the TdT (terminal deoxynucleotidyl transferase) gene, as well as a sense oligomer corresponding to the 5'-UTR-antisense, in Molt-4 cells. The protein level of TdT was determined by flow cytometry, and qPCR was used for mRNA expression analysis. Mismatch PNAs were used as control to address the sequence/target spcifity of the biological effects. The results showed that treatment with the AUG- and to slightly lesser extent with the 5'-UTR-antisense PNAs reduced the TdT mRNA as wel as the protein level, whereas only very low effect was observed for the 5'-UTR-sense PNA. A parallel effect was observed on reduced cell survival and increased rate of apoptosis. Our findings suggest that antisense PNAs can inhibit expression of the TdT gene and induce apoptosis in Molt-4 cells.

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.

BACKGROUND: High-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 PRESENTATION: A 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. CONCLUSION: These 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.