Acute lymphoblastic leukemia (ALL) represents approximately

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1 Terminal Deoxynucleotidyl Transferase Negative Acute Lymphoblastic Leukemia Joana Faber, MD; Hagop Kantarjian, MD; W. Mark oberts, MD; Michael Keating, MD; Emil Freireich, MD; Maher Albitar, MD Objective. Terminal deoxynucleotidyl transferase (TdT) is a useful marker in the diagnosis of acute lymphoblastic leukemia (ALL) (French-American-British [FAB] L and L) and is most useful in distinguishing ALL from mature B-lymphoid neoplasms, such as Burkitt lymphoma (FAB L3) and other lymphoid malignancies. The frequency of TdT-negative ALL is not known. Here we report 3 TdT-negative ALL cases that met the criteria for T- cell ALL. Design. We reviewed approximately 00 cases of ALL retrieved from the database at our institution. All cases were evaluated using Wright-iemsa, myeloperoxidase, butyrate, and TdT staining; immunophenotyped using flow cytometry; and studied using Southern blot analyses for T-cell receptors and immunoglobulin gene rearrangement. esults. All ALL cases (L and L) were TdT-positive, except for 3 cases that were of early T-cell lineage. None of the 3 cases demonstrated positivity for TdT in immunofluorescence staining with polyclonal antibodies or flow cytometry with monoclonal antibodies. Flow cytometric analysis confirmed a pre T-cell immunophenotype in all 3 cases. One of the cases showed rearrangement of a T-cell antigen receptor and immunoglobulin heavy chain (J H ). A second case showed germline configuration of T-cell receptors, but also showed rearrangement of the J H, despite the expression of T-cell markers only. (Arch Pathol Lab Med. 000;4:9 97) Acute lymphoblastic leukemia (ALL) represents approximately 80% of all acute leukemias in children and 0% of the cases seen in adults. The vast majority of ALLs (80%) are of a B-cell lineage. Acute lymphoblastic leukemia with a T-cell lineage characteristically presents with a mediastinal mass (50% of T-cell cases). According to the French-American-British (FAB) morphologic criteria, 3 ALL can be classified as L, L, or L3. Morphologically, the major features distinguishing L blasts from L blasts are a high nuclear-cytoplasmic ratio (L) and the presence of nucleoli (L). 4 These blasts, whether of a B-cell or T-cell immunophenotype, are negative for myeloperoxidase and are almost always positive for terminal deoxynucleotidyl transferase (TdT). L3 ALL (Burkitt lymphoma) represents a lymphoid population of more mature B-cell lineage and usually shows a distinct morphology with a deep basophilic and vacuolated cytoplasm. L3 blasts also lack myeloperoxidase, but they differ from L and L blasts in that they do not express TdT, but do express surface immunoglobulins. A small number of acute myeloblastic leukemias and chronic myelogenous leukemias in lymphoid blastic phase also express TdT. During the past 4 years, we diagnosed 3 cases of TdT- Accepted for publication July, 999. From the Division of Pathology and Laboratory Medicine, Section of Hematopathology (Drs Faber and Albitar), the Division of Medicine, Department of Leukemia (Drs Kantarjian and Freireich), and the Department of Pediatrics (Dr oberts), The University of Texas M. D. Anderson Cancer Center, Houston, Tex. eprints: Maher Albitar, MD, Division of Pathology and Laboratory Medicine, The University of Texas M. D. Anderson Cancer Center, 55 Holcombe Blvd, Box 7, Houston, TX negative ALL out of approximately 00 ALL cases. The 3 cases did not meet the criteria for L3 ALL and appeared to be of pre T-cell or early T-cell lineage. Cases and presented with increased blasts in the peripheral blood and bone marrow. Immunochemical stains revealed the blasts were negative for myeloperoxidase and TdT, and we confirmed that they were of the T-cell immunophenotype using flow cytometric analysis. Molecular studies demonstrated clonal T-cell receptor (TC) rearrangement in one case but no TC gene rearrangement in a second case, despite the demonstration of the primitive T-cell immunophenotype. In this article, we present the clinical data and discuss the diagnosis, therapy, and outcomes in these 3 cases. EPOT OF CASES Case An 8-month-old male infant presented with fever and rhinorrhea. His initial white blood cell count (WBC) was /L with 65% blasts. Computed tomographic scans showed a large anterior mediastinal mass. Upon referral to The University of Texas M. D. Anderson Cancer Center (Houston, Tex), the patient s initial hemoglobin concentration was 50 g/l and his platelet count was /L. Physical examination was remarkable for mild cervical lymphadenopathy and hepatomegaly. A bone marrow aspiration was performed, and only peripheral blood and aspirate smears were available for evaluation. The blasts showed some variation in cell size and moderately abundant cytoplasm. There was some degree of irregularity of the nuclear contour, and the nuclei had coarsely reticular chromatin and inconspicuous nucleoli. The flow cytometric studies (Table) showed the following values: CD, %; CD3, 9%; CD4, 6%; CD8, 4%; cytoplasmic CD3, 40%; CD5, 8%; CD7, 97%; CD9, %; CD0, 0.%; CD0, 96%; cytoplas- 9 Arch Pathol Lab Med Vol 4, January 000 TdT-Negative ALL Faber et al

2 Table. Immunophenotype CD, % CCD3, % SCD3, % CD4, % CD5, % CD7, % CD8, % CD0, % CD9, % CD0, % CCD, % CD3, % CD33, % CD34, % HLA-D, % c-kit, % TdT, % Molecular studies TC TC TC J H Immunophenotype and Molecular Studies* Case Case Case * CCD3, cytoplasmic CD3; SCD3, surface CD3;, not done; CCD, cytoplasmic CD;, rearranged; and, germline. (vincristine and steroids) was initiated but produced no response. He was referred to M. D. Anderson Cancer Center, where physical examination was remarkable for splenomegaly ( cm below the costal margin), and radiography showed an anterior mediastinal mass and lymphadenopathy. His WBC was /L with 94% blasts, his platelet count was / L, and his hemoglobin concentration was 08 g/l. Pathologic examination of the bone marrow showed 9% blasts with oval to round nuclei, reticular chromatin, and moderately abundant cytoplasm (Figure, A); a trephine biopsy showed sheets of blasts (Figure, B). Myeloperoxidase and TdT stains were negative. Flow cytometry studies (Table) showed CD, 70.; CD3, %; cytoplasmic CD3, 99% (Figure ); CD7, 99%; CD4, %; CD8, %; CD9, %; CD0, 66%; CD3, %; CD33, 7%; CD4, 0.%; CD64, 0.%; CD34, 96%; HLA-D, 86%; c-kit, %; and TdT, %. Flow cytometric analysis suggested the blasts were of a pre T-cell immunophenotype, based on the expression of CD, cytoplasmic CD3, CD7, and CD0. Expression of TdT was negative. Molecular studies showed J H rearrangement but no rearrangement for the light chain (Figure ). There was no evidence of rearrangement for the TC, TC, ortc genes, and no evidence of p5 and p6 deletions. Likewise, rearrangement of the c-myc oncogene was not detected. The MLL gene was rearranged at chromosome q3, which was consistent with the cytogenetic finding of the pseudodiploid clone 46XY, del()(p3), del()(q3). The patient received the compound 506-U (araguanosine prodrug) but did not have a complete remission after courses of therapy. mic CD, 0.3%; CD3, 56%; CD33, %; CD34, %; c-kit, 0.8%; CD4, %; glycophorin A, 40%; and TdT, 9%. Flow cytometric analysis suggested the blasts were of a T-cell immunophenotype, expressing cytoplasmic CD3, CD5, and CD7. CD0 was also expressed. The only positive myeloid marker was CD3; c-kit and TdT were negative. The lack of expression of more mature markers (CD4 or CD8), as well as the coexpression of CD4/CD8 and lack of surface CD3 expression, excluded a postthymic origin, such as that seen in T-cell lymphomas. Samples of the patient s cerebrospinal fluid showed 9% blasts with a WBC of 0 9 /L. Cytogenetic studies showed a diploid male karyotype, 46XY. Molecular studies showed no rearrangement of the TEL gene, but the TC, TC, and TC chains were rearranged. earrangement of the immunoglobulin heavy chain (J H ) was also detected, but rearrangement of the light chain was not. On the basis of these findings, a diagnosis of T-cell ALL (FAB L) was made, and chemotherapy consisting of dexamethasone, vincristine, L-asparaginase, daunomycin, and intrathecal methotrexate was initiated, according to an infant treatment protocol. Bone marrow examination on days 7 and 4 of the chemotherapy regimen revealed % and 9% blasts, respectively. The patient was discharged from the hospital during week 3 of treatment with a WBC of /L, hemoglobin concentration of 85 g/l, and platelet count of /L. Three months later, the patient underwent reinduction with daunomycin, vincristine, L-asparaginase, and cyclophosphamide. The patient has been in complete remission for more than months and as of this report was receiving intensified maintenance course number 3, according to the CC-953 protocol. Case A 9-year-old man had a diagnosis of T-cell ALL in 99. He received chemotherapy consisting of doxorubicin hydrochloride, vincristine, teniposide, L-asparaginase, cyclophosphamide, 6- mercaptopurine, and intrathechal chemotherapy, after which he experienced complete remission. The patient did well until June 997, when he started experiencing weakness, fatigue, and weight loss. A relapse of ALL was diagnosed. Chemotherapy Case 3 A 39-year-old woman presented with shortness of breath, weakness, and chest discomfort. She was found to be cytopenic. Upon referral to M. D. Anderson Cancer Center in September 997, physical examination revealed no adenopathy or hepatosplenomegaly, and radiography of the chest was normal. Her initial WBC was /L with 7% neutrophils, her hemoglobin concentration was 75 g/l, and her platelet count was / L. Bone marrow examination showed diffuse interstitial infiltration by blasts (Figure, D). The cytoplasm was relatively scant, and the nuclei had slightly coarse chromatin with inconspicuous nucleoli (Figure, C). Tests for myeloperoxidase and TdT were negative. The flow cytometry studies (Table) showed the following values: CD, %; CD, %; CD3, 3%; cytoplasmic CD3, 6%; CD5, 96%; CD7, 97%; CD9, %; CD0, 0.5%; CD33, %; CD3, 5%; CD4, 7%; CD64, %; c-kit,, 80%; CD34, 0.4%; HLA-D, 6%; and TdT, 7%. Flow cytometric analysis showed the blasts were of a pre Tcell immunophenotype, expressing cytoplasmic CD3, CD5, and CD7, and negative for TdT. Expression of c-kit was positive; however, there was no expression of other myeloid markers. Cytogenetic studies showed a diploid female karyotype, 46XX. Electron microscopic studies were negative for myeloperoxidase. No samples were available for molecular analysis, but based on the findings cited here, a diagnosis of ALL was made. The patient experienced a complete remission while receiving hyperfractionated cyclophosphamide, vincristine, adriamycin, dexamethasone chemotherapy, and she has remained in complete remission for more than 7 months. METHODS Blood and Bone Marrow Aspirate Smears Approximately 00 cases of ALL (excluding Burkitt lymphoma) were reviewed from our database. They consisted of cases of early pre B-cell type (%), common (65%), pre B-cell type (0%), and T-cell type (3%) ALL. Of these cases, only 3 were negative for TdT. Wright- iemsa stained peripheral blood and bone marrow aspirate smears were obtained from all 3 of these cases at Arch Pathol Lab Med Vol 4, January 000 TdT-Negative ALL Faber et al 93

3 Figure. shown. epresentative morphology from case (A and B) and case 3 (C and D). Both bone marrow aspirate smears and biopsy samples are M. D. Anderson Cancer Center. In cases, a bone marrow trephine biopsy was also available for examination. Staining was performed using standard procedures. Cytochemical and Cytoimmunochemical Staining Cytochemical and cytoimmunochemical stainings were performed according to routine protocols and included Wright-iemsa, periodic acid Schiff reaction, Sudan black B, myeloperoxidase, -naphthyl butyrate esterase, tartrateresistant acid phosphatase, and TdT staining. Terminal deoxynucleotidyl transferase staining by immunofluorescence was performed using rabbit anti-tdt Ig and fluorescein-conjugated goat anti-rabbit Ig (Supertechs, Inc, Bethesda, Md). A positive control (cell line provided by Supertechs) and negative sample were used with every TdT staining in addition to the patient sample without primary antibody. Immunophenotyping Immunophenotyping was performed on fresh bone marrow samples using a standard flow cytometric analysis technique as described previously. 5 Briefly, tricolor flow cytometric analysis was performed on samples after using a standard whole blood lysis technique. First, 0.5 to 0 6 cells were chilled for 0 minutes at C to8 C. After incubation, the erythrocytes were lysed in ammonium chloride for 5 minutes. The cells were then washed twice with a phosphate-buffered saline solution. Next, the stained cells were fixed with % paraformaldehyde. Flow cytometric analysis was performed using a Becton Dickinson FACScan (Becton Dickinson, Mountain View, Calif), and the data were evaluated with the LYSIS II software program (Becton Dickinson). Blasts were gated based on side-scatter and CD45 antigen expression. The results were expressed as the percentage of blast cells expressing the antigen. Isotype control was used with staining to establish the background staining. Positivity was defined as a staining that was discrete and separate from the background isotype control in more than 0% of the gated cells. Monoclonal antibodies included CD, CD3 (surface and cytoplasmic), CD4, CD5, CD8, CD0, CD, CD3, CD33, CD34, c-kit, CD4, and TdT (Becton Dickinson Immunocytometry Systems, San Jose, Calif). 94 Arch Pathol Lab Med Vol 4, January 000 TdT-Negative ALL Faber et al

4 Figure. Molecular studies for case show a germline configuration for T-cell receptors (TCs) and rearrangement of J H. Three restriction enzymes were used for TC studies, but only EcoI and HindIII were used for J H.BHI indicates BamHI; M, molecular weight marker (lambda/hindiii); P, patient; N, normal; and C, control. Arch Pathol Lab Med Vol 4, January 000 TdT-Negative ALL Faber et al 95

5 Figure 3. Example of histograms used in evaluating cytoplasmic CD3 expression. A, Distribution of a side-scattered pattern versus CD45 showing the gated blast region. B, Histogram showing the background using isotype controls. C, Histogram of blast cells showing that most of the CD34- positive cells were also positive for cytoplasmic CD3. Molecular Studies High-molecular-weight DNA was extracted from the leukemic cells according to previously published protocols. 6 Southern blot analyses were also performed. Briefly, 0 g of digested DNA (EcoI, HindIII, and BamHI) was loaded onto a 0.7% agarose gel. The electrophoresed DNA was transported to a nylon membrane, and the blots were probed with TC, TC, and TC, as well as for the J H and light-chain gene fragments (Oncor, aithersburg, Md). For J H rearrangement analysis, we routinely performed digestion with only EcoI and HindIII. BamHI digestion was used only when questionable results were obtained. ESULTS Clinical Characteristics Of the 00 cases of L and L ALL in our database, we found only 3 that were negative for TdT. These ALL cases were a mixture of early pre B-cell (%), common (65%), pre B-cell (0%), and T-cell (3%) ALL. Two patients (cases and ) presented with a high leukocyte count, and the third presented with cytopenia. A mediastinal mass was detected in cases. Case involved a patient experiencing relapsed ALL. Peripheral Blood and Bone Marrow Examination Morphologically, the neoplastic cells were classified as blasts (Figure, A and C). They showed some variation in size, ranging from medium to large. Their chromatin pattern was open but more condensed when compared with that of L and L lymphoblasts. Nuclear convolution was more prominent in case. The pattern of bone marrow infiltration was interstitial and diffuse, and was similar to that seen in acute leukemias (Figure, B and D). Immunophenotyping Cases and 3 were best classified as early T-cell ALL, which is characterized by the expression of cytoplasmic CD3, CD5, and CD7, and the lack of expression of CD, CD, and surface CD3. 7 Case was best classified as pre T-cell ALL, which is characterized by the expression of CD7, cytoplasmic CD3, CD34, and HLA-D. Appropriate controls and gating on the blast cells were documented (Figure 3). Terminal deoxynucleotidyl transferase was negative in flow cytometric and cytoimmunochemical analysis. Molecular Studies Southern blot analyses for B- and T-cell lineages were performed in cases and. ene rearrangement of the TC, TC, and TC chains was detected in case. Case demonstrated a very immature state of T-cell differentiation in flow cytometric analysis, but rearrangement of the TC, TC, and TC chains was not detected. J H rearrangement was detected in both cases (Figure ). 96 Arch Pathol Lab Med Vol 4, January 000 TdT-Negative ALL Faber et al

6 COMMENT Terminal deoxynucleotidyl transferase is an intranuclear protein that catalyzes the polymerization of deoxynucleoside triphosphate and differs from the DNA polymerases by not requiring template information for polymerization. In the normal lymphoid ontogeny, genomic recombinations occur in a tightly regulated sequence and require an enzyme called recombinase. Although not necessary for recombination, TdT provides additional nucleotides to junctional sequences, which gives it an important function in the rearrangement of immunoglobulins and TC chains. 6 Terminal deoxynucleotidyl transferase is expressed in 90% of cortical thymocytes and in a minor population of bone marrow lymphocytes ( %). 5,8 The level of TdT expression, in conjunction with other immunologic markers and morphologies, is still the major criterion for distinguishing ALL from acute myeloblastic leukemia. Terminal deoxynucleotidyl transferase is present in virtually all ALL cases (L and L), but in only 5% to % of acute myeloblastic leukemia cases. 9 To our knowledge, this is the first article to describe TdT-negative ALL of L or L morphology confirmed using molecular and immunophenotypic studies. None of our 3 cases of T-cell ALL demonstrated positivity for TdT using current techniques of immunofluorescence staining with polyclonal antibodies or flow cytometry with monoclonal antibodies. Flow cytometric analysis confirmed a pre T-cell immunophenotype in all 3 cases, even though case had a very primitive subset. The authors of a previous study of 9 cases with a similar pre T-cell ALL immunophenotype suggested that expression of surface and cytoplasmic CD3 occurs at a very immature state of T-cell differentiation before rearrangement of the TC -, TC -, and TC -chain genes in a third of the cases. 0 In the same study, a germline configuration of TC, TC, and TC was also confirmed in cases. Previous studies also showed that very early pre T-cell ALL may fail to show rearrangement of any TC gene; none of these reports documented clonal rearrangement of the heavy chain in such cases. Furthermore, the presence of the J H rearrangement has been reported in 7% of T-cell neoplasms with TC gene rearrangement.,3 J H rearrangement in T-cell tumors seems limited to the heavy-chain gene locus and has not been reported for the light chains. The best explanation for these bigenotypic B- or T-lymphoid neoplasms is attributed to their similar structural configuration and to a common recombinase enzyme system. 3 5 Both T- and B-cell lineages were involved in the leukemic process in molecular studies, but the immunophenotype showed commitment to one lineage. Case differed from previously described cases because of the presence of clonal heavy-chain rearrangement and a germline configuration for the TC, TC, and TC chains (Figure ). Because of the strong flow cytometry data supporting a T-cell immunophenotype, we can speculate that in very primitive T-cell ALL, J H rearrangement precedes TC gene rearrangement in the ontogeny of lymphoid neoplasms. Case also showed bigenotypic rearrangement with a T-cell immunophenotype, despite the lack of TdT expression, which raises questions as to whether TdT is necessary for gene rearrangement. It is possible that TdT was expressed at a very low level that was undetectable using current immunologic methods but can be detected at the level of mna or more sensitive immunoassays. 8 Studies using knockout mice showed that TdT is necessary for appropriate TC gene rearrangement and diversity. In summary, our data indicate that a lack of TdT, as indicated using flow cytometric or immunohistochemical analysis, does not rule out the diagnosis of L or L ALL morphology. 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