Precursor B Lymphoblastic Leukemia With Surface Light Chain Immunoglobulin Restriction A Report of 15 Patients

Hematopathology / PRECURSOR B LYMPHOBLASTIC LEUKEMIA Precursor B Lymphoblastic Leukemia With Surface Light Chain Immunoglobulin Restriction A Report of 15 Patients Rina Kansal, MD, 1 George Deeb, MD, 1 Maurice Barcos, MD, PhD, 2 Meir Wetzler, MD, 3 Martin L. Brecher, MD, 4 AnneMarie W. Block, PhD, 5 and Carleton C. Stewart, PhD 6 Key Words: Precursor B cell; Acute lymphoblastic leukemia; Surface immunoglobulin positive acute leukemia; Flow cytometry; WHO classification; Immunophenotyping Abstract We describe 15 patients (9 children) with precursor B-cell (pb) acute lymphoblastic leukemia (ALL) with surface immunoglobulin (sig) light chain restriction revealed by flow cytometric immunophenotyping (FCI). The same sig+ immunophenotype was present at diagnosis and in 3 relapses in 1 patient. In 15 patients, blasts were CD19+CD10+ (bright coexpression) in 14, CD34+ in 12, surface κ+ in 12, surface λ+ in 3; in 8 of 8, terminal deoxyribonucleotidyl transferase (TdT)+; and in 4, surface IgD+ in 2 and surface IgM+ in 1. The 3 CD34 cases included 1 TdT+ case, 1 with t(1;19)(q23;p13), and 1 infant with 70% marrow blasts. One adult had CD10 CD19+CD20 CD22+CD34+ TdT+sIg+ blasts with t(2;11)(p21;q23). Blasts were L1 or L2 in all cases (French-American-British classification). Karyotypic analysis in 12 of 12 analyzable cases was negative for 8q24 (myc) translocation. Karyotypic abnormalities, confirmed by fluorescence in situ hybridization in 6 cases, included hyperdiploidy, t(1;19)(q23;p13), t(12;21)(p13;q22), t(9;22)(q34;q11), t(2;11)(p21;q23), and trisomy 12. The sig light chain restriction in pb ALL might be present in neoplasms arising from the early, intermediate, and late stages of precursor B-cell maturation; sig light chain restriction revealed by FCI does not necessarily indicate a mature B-cell phenotype, further emphasizing the importance of a multidisciplinary approach to diagnosing B-lymphoid neoplasms. Neoplasms of the B-lymphoid cell may have a precursor B-cell phenotype or a mature B-cell phenotype, as described in the World Health Organization (WHO) classification of hematopoietic malignant neoplasms. 1 The latter category includes Burkitt leukemia/lymphoma and other neoplasms of the mature B-cell, which may have a leukemic manifestation. In the diagnostic evaluation of these B-cell neoplasms, flow cytometric immunophenotypic (FCI) analysis has a critical role in the differentiation of a precursor B-cell phenotype from a mature B-cell phenotype. 2 While a mature B-cell phenotype shows the presence of surface immunoglobulins by FCI analysis, this expression typically is absent in a precursor B-cell phenotype. Precursor B-cell (pb) acute lymphoblastic leukemia (ALL) is a neoplasm of B lymphoblasts that characteristically are negative for surface light chain immunoglobulins by FCI analysis and express markers related to the degree of B-cell differentiation. 1-3 Cases of pb ALL with the expression of immunoglobulins include those described as transitional pre-b ALL, which express cytoplasmic and surface IgM but no surface light chain immunoglobulins. 4 The expression of surface light chain immunoglobulins in pb ALL seems to be rare in the pediatric age group. 5 In adults, we found 7 published cases of ALL with surface light chain immunoglobulin restriction 6,7 that would be classified as pb ALL by the WHO classification. Previous authors have suggested that the lymphoblasts in these pb ALL cases might represent a B-cell stage intermediate between the transitional pre B-cell stage and the mature B cell. 6,7 We present findings from 15 patients with pb ALL with surface light chain immunoglobulin restriction, diagnosed by a multidisciplinary approach in a single institution, which demonstrate 512 Am J Clin Pathol 2004;121:512-525 Downloaded 512 from https://academic.oup.com/ajcp/article-abstract/121/4/512/1759325

Hematopathology / ORIGINAL ARTICLE for the first time that surface light chain immunoglobulin restriction in precursor B lymphoblasts is not confined to any particular stage of B-lymphoblast differentiation and, instead, may be seen in a heterogeneous group of pb ALLs showing different degrees of differentiation of pb lymphoblasts. To the best of our knowledge, this is the largest reported group of cases with this unusual immunophenotypic expression in pb ALL. Materials and Methods This single-institution retrospective study was designed at Roswell Park Cancer Institute (RPCI; Buffalo, NY) following an index case of pb ALL with surface light chain immunoglobulin restriction, which was observed in the clinical practice of one of us (R.K.) at Buffalo General Hospital, Buffalo, NY. The flow cytometry database at RPCI was searched for the period January 1995 to December 2001 for all cases of acute lymphoblastic leukemia and for all Blineage malignancies with surface immunoglobulin (sig) expression. The pathology database also was searched to identify cases of pb ALL. At least 198 cases of ALL were identified; however, the cases retrieved by the specific key word searches represented only a subset of the total number of cases of ALL analyzed by flow cytometry at RPCI, precluding an estimate of the incidence of sig+ pb ALL in our cases. The immunophenotypic and relevant pathologic data were reviewed, and 19 cases of pb ALL were identified in 15 patients, showing sig light chain restricted neoplastic cells by FCI analysis. All cases were classified according to the WHO criteria. 1 Cases of Burkitt lymphoma/leukemia were excluded. The clinical, morphologic, cytogenetic, and flow cytometric immunophenotypic findings were reviewed in all 19 cases. Clinical information, including age, sex, sites of involvement by disease, leukocyte counts at diagnosis, and treatment and clinical follow-up data were recorded by review of medical records. Flow Cytometry Multiparameter flow cytometry was performed in all cases in the Laboratory of Flow Cytometry, RPCI, as previously described. 8,9 All cases were analyzed by 4-color (15 cases) or 3-color flow cytometry (1995-1997; 4 cases in 3 patients) using a FACScan Flow Cytometer (Becton Dickinson, San Jose, CA). An acute leukemia panel of antibodies was used in all cases. At RPCI, this panel routinely includes surface κ and λ, HLA-DR, CD2, CD3, CD4, CD5, CD7, CD8, CD10, CD11b, CD11c, CD13, CD14, CD15, CD16, CD19, CD20, CD22, CD23, CD25, CD32, CD33, CD34, CD38, CD41, CD45, CD56, CD57, CD64, CD71, and CD79b. Surface IgD and IgM were analyzed in 4 cases using 3-color FCI analysis. The 4-color fluorochrome combinations included fluorescein isothiocyanate (FITC)/phycoerythrin (PE)/peridinium chlorophyll protein complex (PerCP) or PE-cyanin-5 (PECY5 or Tricolor (TC, Caltag, Burlingame, CA)/allophycocyanin (APC). The 3-color fluorochrome combinations included FITC/PE/PerCP or PECY5 or TC. The corresponding antibody combinations included CD3/CD14/PerCP-HLA-DR/CD45, CD3/CD4/TC-CD8/CD45, CD7/CD13/TC-CD2/CD19, CD5/λ/PECY5-CD19/κ, CD20/CD11c/TC-CD22/CD25, CD5/CD19/PECY5-CD10/CD34, CD11b/CD13/PECY5- CD33/CD34, CD15/CD56/PECY5-CD19/CD34, and IgD/IgM/PECY5-CD19. The surface κ and λ antibodies used were APC-κ (clone TB28-2, BD Biosciences, San Jose, CA) and PE-λ (clone 1-155-2, BD Biosciences). All other antibodies were obtained from the following sources: Caltag (CD2, CD8, CD14, CD15, CD16, CD19, CD22, CD23, CD32, CD41, CD57, CD71, CD79b, IgD, and IgM), BD Biosciences (CD3, CD4, CD5, CD11c, CD13, CD14, CD20, CD25, CD34, CD45, CD64, and HLA-DR), and Immunotech-Coulter, Hialeah, FL (CD7, CD10, CD11b, CD19, CD33, CD38, CD56, and CD71). The samples analyzed included 18 bone marrow aspirates and 1 peripheral blood sample. Cell viability was determined by using ethidium monoazide fluorescence, as previously described. 10 All specimens exhibited more than 98% viable cells after processing. All data were obtained in list mode and displayed as 2-parameter dot plots using WinList multiparameter analysis software (Verity Software House, Topsham, ME). The antibody panel that best resolved leukemia cells was chosen to identify the cell population of interest, and the data were reanalyzed ( back gated ) to produce bivariate displays in the forward vs the side scatter plot. New regions were drawn around the cells of interest in this display ( leukemia gate ), and these scatter regions then were used to analyze all other panels. Cells stained with isotype-matched antibody-fluorochrome combinations and biologically negative control cells within each tube were used as negative controls. The cell clusters of interest were called positive for a surface antigen if they showed a distinct shift in fluorescence intensity in comparison with the negative controls. The intensity of fluorescence was reported as dim if the mean fluorescence intensity was within the second logarithmic decade (the first logarithmic decade being negative). Morphologic Examination and Terminal Deoxyribonucleotidyl Transferase Immunohistochemical Analysis The corresponding peripheral blood smears, bone marrow aspirate smears (Wright-Giemsa stained and cytochemically stained), and H&E-stained bone marrow aspirate clot sections and core biopsy sections were reviewed. The Downloaded from https://academic.oup.com/ajcp/article-abstract/121/4/512/1759325 Am J Clin Pathol 2004;121:512-525 513 513 513

Kansal et al / PRECURSOR B LYMPHOBLASTIC LEUKEMIA French-American-British (FAB) morphologic type of the lymphoblasts 11 and bone marrow blast percentages were recorded. In patients with lymph node and bone marrow involvement, more than 25% bone marrow blasts were required for the diagnosis of pb ALL, as opposed to fewer than 25% marrow blasts and fewer than 10% circulating blasts that would be classified as lymphoblastic lymphoma according to the Pediatric Oncology Group criteria. 12 Retrospective assessment for nuclear terminal deoxyribonucleotidyl transferase (TdT) enzyme could not be performed by flow cytometry because additional cryopreserved cell samples were not available for study. Therefore, an immunohistochemical stain for TdT (TdT, DAKO, Carpinteria, CA) was performed in all cases with available paraffin-embedded sections (cases 7, 10-15) or unstained air-dried marrow aspirate smears, using the DAKO automated staining system. 13 The aspirate smear slides were fixed in cold methanol (4 C, 15 minutes) before antigen retrieval. In all cases, a microwave oven (100 C, 14 minutes, Target Retrieval Solution [DAKO], ph 8.9-9.9) was used for antigen retrieval. Conventional Cytogenetics and Fluorescence In Situ Hybridization Conventional cytogenetic analyses were performed on trypsin Wright stain banded bone marrow. 14 Cells were examined from 24- or 48-hour unstimulated cultures or from methotrexate-synchronized unstimulated cells cultured for 24 hours. At least 20 cells per case were examined whenever possible. Chromosomal abnormalities were described according to the International System for Human Cytogenetic Nomenclature (1995). 15 Fluorescence in situ hybridization (FISH) analyses were performed using commercially available (Vysis, Downers Grove, IL) α satellite centromeric and locus-specific rearrangement probes in 6 cases. At least 500 interphase nuclei were examined for FISH analysis. Results Clinical and Morphologic Features for 15 Patients The clinical and morphologic features for all patients are given in Table 1. The 15 patients included 9 children (cases 1-9, Table 1) with an age range of 11 months to 13 years (median, 5 years) and 6 adults (cases 10-15, Table 1) with an age range of 20 to 73 years (median, 60 years). All 15 patients were given a diagnosis of pb ALL by a multidisciplinary approach on referral to RPCI, with the unusual finding of surface light chain immunoglobulin restricted lymphoblasts by FCI analysis. The white blood cell counts at diagnosis ranged from 840 to 169,100/µL (0.8-169.1 10 9 /L; median, 4,200/µL [4.2 10 9 /L]). The corresponding Wright-Giemsa stained bone marrow aspirate smears were available for review in all cases. By FAB morphologic type, the blasts were L1 or L2 in all cases Image 1. Bone marrow blast percentages ranged from 38.5% to 97.4% (median, 91%) in the smear preparations. Enzyme cytochemical stains on smear preparations were Table 1 Clinical and Pathologic Features of 15 Patients With Precursor B Lymphoblastic Leukemia Sites Blasts Outcome Case No./Sex/ Spleen/ Mediastinum/ WBC FAB Time of CR/Duration Age (y) * LN Liver CNS Count (/µl ) BM (%) Type FCI Diagnosis (mo) Survival 1/F/3 No No/2-3 No/No 1,400 61.5 L1 Initial Yes/49 Alive; NED 2/M/3 Yes 2/2 No/No 59,100 95.8 L1 Initial Yes/17 Alive; NED 3/M/3 No No/No No/No 4,200 96.4 L2 Initial Yes/27 Alive; NED 4/F/5 No No/No No/No 4,300 96.2 L1 Initial Yes/21 Alive; NED 5/M/7 Yes No/No No/No 2,800 59.6 L2 Initial Yes/72 Alive; NED 6/M/9 No No/No No/No 2,300 97.0 L1 Initial Yes/25 Alive; NED 7/M/12 Yes No/4 No/No 169,100 97.4 L2 Initial No Died; 5 mo 8/M/11 mo Yes Tip/No No/Yes 22,900 71.5 L1 Initial Yes/57 Alive; NED 9/F/13 Yes No/2-3 No/No 13,200 91.4 L2 Initial Yes/19 Alive; NED 10/M/20 No No/No No/No 2,600 40.2 L1 Initial Yes/4 Died 11/M/34 Yes No/No No/No 1,900 97.0 L2 Initial Yes/1 Died 12/M/56 No No/No No/No 10,980 92.0 L2 Initial Yes/22 Alive; NED 13/F/62 No No/No No/No 7,300 63.2 L2 Relapse No Died 14/F/67 No No/No No/No 3,200 59.4 L2 Initial No Died 15/F/73 No No/No No/No 840 38.5 L2 Relapse Yes/6 Died BM, bone marrow; CNS, central nervous system; CR, complete remission; FAB, French-American-British co-operative group 11 ; FCI, flow cytometric immunophenotypic analysis; LN, lymph nodes; NED, no evidence of disease. * Unless otherwise indicated. Numeric values are centimeters. Values are given in conventional units; to convert to Système International units ( 10 9 /L), multiply by 0.001. The time of FCI (at initial diagnosis or relapse) that showed surface light chain immunoglobulin restriction on lymphoblasts. FCI at primary diagnosis and at persistent/relapsed leukemia in these cases showed surface light chain immunoglobulin restriction on lymphoblasts. 514 Am J Clin Pathol 2004;121:512-525 Downloaded 514 from https://academic.oup.com/ajcp/article-abstract/121/4/512/1759325

Hematopathology / ORIGINAL ARTICLE available for review for 10 patients (cases 1-6, 8, 9, 11, 12). By these cytochemical stains, the leukemic cells were negative for myeloperoxidase and Sudan black in smears from all 10 patients, negative for chloroacetate esterase in 9 of 9, and showed granular periodic acid Schiff positivity in 2% to 83% of blasts (median, 35%) in 9 of 9. Bone marrow trephine biopsies were performed in all adults, revealing effacement of marrow architecture by leukemic cells in all cases. Peripheral blood smears at the time of diagnostic bone marrow biopsies were available for review for 1 patient (case 12). One adult (case 11) underwent a concurrent lymph node biopsy at a local referring hospital at the time of diagnosis, which was reviewed (R.K. and M.B.) and showed architectural effacement by lymphoblasts that were revealed as CD10+TdT+ by paraffin section immunohistochemical stains. Fresh tissue from this lymph node was not submitted for FCI analysis. Bone marrow trephine biopsy sections in this case, however, showed extensive bilateral effacement of marrow architecture by lymphoblasts, consistent with pb ALL as opposed to precursor B lymphoblastic lymphoma by the Pediatric Oncology Group criteria. 12 FCI Analysis and TdT Immunohistochemical Findings for 15 Patients The flow cytometric immunophenotype for all patients is given in Table 2. The FCI analyses for all except 2 patients were performed at the time of initial diagnosis. For 1 adult (case 12), the initial FCI analysis was done at Buffalo General Hospital at diagnosis and was followed by repeated diagnostic FCI analysis at RPCI. The scatter plots of both FCI analyses for case 12 were reviewed (R.K. and C.C.S.) and showed the same surface light chain restriction on the lymphoblasts and additional dim surface IgM expression on the lymphoblasts in the initial analysis. For 1 adult (case 14), FCI analyses were done on 2 separate bone marrow aspirate samples at diagnosis and at the time of 5 subsequent analyses, all of which were performed at the RPCI flow cytometry laboratory and showed persistent leukemia. The same immunophenotype and surface light chain restriction were present in the first 5 of the 7 FCI analyses; the sixth and the seventh consecutive analyses did not show surface light chain restriction in this patient with persistent lymphoblastic leukemia. For 2 patients, the results of FCI analysis at the time of relapse showed surface light chain immunoglobulin restriction. Review of the FCI analysis results at the time of primary diagnosis in these 2 cases did not show surface light chain restriction on the lymphoblasts. The blasts were CD19+ in all cases and showed bright coexpression of CD19 and CD10 in samples from 14 of 15 patients. CD45 was dim or absent, with HLA-DR positivity in all cases. The leukemic cells were CD34+ by FCI analysis in 12 of 15 patients and TdT+ by immunohistochemical analysis in 8 of 8 Image 2. The 3 CD34 cases included 1 TdT+ case, 1 with t(1;19)(q23;p13), and 1 infant with 71.5% bone marrow lymphoblasts. Surface CD22 expression was present on the neoplastic cells in all cases and showed less fluorescence intensity than the residual benign B cells in the sample. CD20 expression was dim in 2 cases and absent in 13. Dim CD5 expression was noted in the sample from 1 infant with CD10+CD19+CD34+ blasts; all other cases were CD5. The surface light chain immunoglobulin expressed was κ in the samples from 12 of 15 patients and λ in 3 of 15, with dim fluorescence intensity in all cases. Samples from 4 patients were analyzed for surface heavy chain immunoglobulins (cases 5, 8, 12, and 14); 2 showed IgD dim +IgM lymphoblasts (cases 5 and 8); case 12 showed IgM+IgD lymphoblasts; and the neoplastic cells in case 14 were negative for IgD and IgM. Aberrant dim expression of a myeloid marker CD13 or CD33 was detected on the leukemic cells in 6 patients, with CD13+CD33+ lymphoblasts in the samples from 3 patients, CD13+CD33 lymphoblasts in 2, and CD13 CD33+ leukemic cells in 1. Examples of flow cytometry plots from pediatric and adult cases of surface light chain positive pb ALL are shown in Image 3, Image 4, Image 5, Image 6, Image 7, and Image 8. Conventional Cytogenetics and FISH Findings for 15 Patients Cytogenetic findings are summarized in Table 3. Karyotypic analyses were performed on samples from 12 patients, Image 1 Several lymphoblasts are present in this bone marrow smear from a 62-year-old woman (Wright-Giemsa, 1,000). Downloaded from https://academic.oup.com/ajcp/article-abstract/121/4/512/1759325 Am J Clin Pathol 2004;121:512-525 515 515 515

Kansal et al / PRECURSOR B LYMPHOBLASTIC LEUKEMIA Table 2 Flow Cytometric Immunophenotypic Features for 15 Patients With Precursor B Lymphoblastic Leukemia * Case No. TdT CD34 HLA-DR CD45 CD19 CD10 CD22 CD20 sig CD13 CD33 CD5 1 NA + + +(d) + + + +(d) λ(d) +(d) 2 NA + + +(d) + + +(d) κ(d) 3 NA + +(d) + + + κ(d) +(d) 4 + + +(d) +(d) + +(d) +(d) κ(d) +(d) +(d) 5 NA + + + + + +(d) κ(d) 6 NA + + +(d) + + +(d) κ(d) +(d) +(d) 7 + + + +(d) + + +(d) κ(d) +(d) +(d) 8 NA + +(d) + + + κ(d) 9 NA + +(d) + + + κ(d) 10 + +(d) +(d) + + + λ(d) 11 + +(d) +(d) +(d) + + +(d) κ(d) 12 + + + +(d) + + + λ(d) +(d) 13 + + +(d) +(d) + + +(d) κ(d) 14 + + + +(d) + + κ(d) +(d) 15 + + + +(d) + + + κ(d) NA, not available; sig, surface immunoglobulin; TdT, nuclear terminal deoxyribonucleotidyl transferase (by immunohistochemical stains); +, positive; (d), dim;, negative. * The samples for flow cytometric immunophenotypic analysis were bone marrow aspirates in all cases except case 12, for which a peripheral blood sample was used. Samples were diagnostic in all cases except 13 and 15, which were samples at relapse of sig precursor B-cell ALL. Flow cytometric immunophenotypic analysis was 4-color in all cases except 5, 8, and 14, in which it was 3-color. Surface IgD and IgM were analyzed in cases 5, 8, 12, and 14; see the text. abnormalities for 2 (cases 3 and 6) without an analyzable karyotype. FISH studies confirmed trisomies 4 and 10 in case 1, trisomies 4 and 21 in case 2, trisomy 12 in case 4, and bcr/abl fusion in case 7. In case 3, FISH analysis showed 2 clonal populations: one with trisomy 4 and the other with trisomies 4 and 10. A cryptic t(12;21)(p13;q22) was revealed by FISH in case 6 by using the TEL/AML1 locus-specific fusion probe. Image 2 Terminal deoxyribonucleotidyl transferase (TdT)+ lymphoblasts in paraffin-embedded core biopsy section (TdT, immunohistochemical stain, 400). but samples were not analyzable for the remaining 3 patients (cases 3, 6, and 15) owing to inadequate mitoses or a hypocellular specimen. The karyotypes in the 12 analyzable cases were negative for an 8q24 (myc) rearrangement. The dividing cells showed t(1;19)(q23;p13) in 3 patients, t(9;22)(q34;q11) in 1 patient, t(2;11)(p21;q23) in 1 patient, trisomy 12 in 1 patient, hyperdiploidy in 3 patients, and a normal karyotype in 3 patients. FISH studies were performed on the samples from 6 patients. These studies confirmed karyotypic findings for 4 patients and provided evidence of specific cytogenetic Multiparametric Correlation in 9 Pediatric Cases The 9 children in our study (cases 1-9) included 6 boys and 3 girls, with ages ranging from 11 months to 13 years (median, 5 years). The total leukocyte counts at diagnosis ranged from 1,400 to 169,100/µL (1.4-169.1 10 9 /L; median, 4,200/µL [4.2 10 9 /L]). The diagnostic bone marrow aspirate samples from all pediatric cases were obtained at diagnosis. The lymphoblasts in all cases showed FAB L1 or L2 morphologic features on Wright-Giemsa stained bone marrow aspirate smears. Bone marrow blast percentages ranged from 59.6% to 97.4% (median, 95.8%). Bone marrow trephine core biopsies were not performed on any of the children. Regrettably, marrow aspirate clots or evaluable TdT-stained aspirate smears were available for only 2 children; immunohistochemical analysis showed TdT+ lymphoblasts in these 2 cases. FCI analysis of the samples from all patients revealed lymphoblasts with bright coexpression of CD19 and CD10, with surface κ light chain restriction for all except 1 patient with λ light chain restriction (case 1). This patient was a 3- year-old boy with CD5 dim +CD10+CD19+CD20 dim +CD34+, surface λ+ lymphoblasts, and this seems to be the first 516 Am J Clin Pathol 2004;121:512-525 Downloaded 516 from https://academic.oup.com/ajcp/article-abstract/121/4/512/1759325

Hematopathology / ORIGINAL ARTICLE 120 Side Scatter 100 80 60 40 20 0 0 20 40 60 80 100 120 0 20 40 60 80 100 120 Forward Scatter Side Scatter Anti HLA-DR PerCP CD19PE CD19 PE CD22 TC CD34 APC CD10 PC CD20 FITC κ APC CD19 PC CD19 PC κ APC Image 3 (Case 2) Surface immunoglobulin positive precursor B lymphoblastic leukemia in a 3-year-old boy with CD45 dim + HLA-DR+CD10+CD19+CD22 dim +CD34+ surface κ dim + leukemic cells (arrows) at diagnosis (4-color flow cytometric immunophenotypic analysis). Sparse residual benign B cells are present in this case (arrowheads). Note that the color assigned to the neoplastic cells varies in different tubes. APC, allophycocyanin; FITC, fluorescein isothiocyanate; PC, PE-cyanin-5; PE, phycoerythrin; PerCP, peridinium chlorophyll protein complex; TC, Tricolor (Caltag, Burlingame, CA). reported pediatric case of a CD5+ pb ALL with sig light chain restriction revealed by FCI analysis. Owing to the coexpression of CD5 and CD19 with surface λ, which is more common than κ in mantle cell lymphoma, 2 the unlikely possibility of the blastoid variant of mantle cell lymphoma in a child was considered but rejected owing to the unequivocal presence of CD34 in FCI analysis and a karyotype characteristic of pb ALL without the t(11;14) translocation, in addition to the dim expression of CD45, CD20, and surface immunoglobulin by FCI analysis that argue against a diagnosis of mantle cell lymphoma. For 1 child (case 4) with CD10+CD19+CD34+HLA- DR+, surface κ+ blasts, trisomy 12 was revealed as the sole karyotypic abnormality. Our pediatric cases also included cases with a cryptic t(12;21) translocation, TEL/AML1 fusion 16 (n = 1), t(9;22)(q34;q11), bcr/abl fusion 17 (n = 1), and a hyperdiploid karyotype (n = 3). 18 Two children in our study had the t(1;19)(q23;p13) translocation, which often is associated with a late stage of precursor B-cell differentiation that shows cytoplasmic IgM expression with or without surface IgM but no surface light chain expression. 19,20 The lymphoblasts in samples from 1 of 2 patients with the t(1;19) showed dim surface IgD κ expression, in the absence of surface IgM expression by FCI analysis. Dim surface IgD without surface IgM by FCI analysis also was present in 1 infant with CD10+CD19+CD20 CD22+CD34 HLA-DR+ blasts that correspond to an intermediate stage of B- lymphoblast differentiation. The 9 children were treated according to the Pediatric Oncology Group chemotherapy protocols. All patients Downloaded from https://academic.oup.com/ajcp/article-abstract/121/4/512/1759325 Am J Clin Pathol 2004;121:512-525 517 517 517

Hematopathology / ORIGINAL ARTICLE 120 Side Scatter 100 80 60 40 20 0 0 20 40 60 80 100 120 0 20 40 60 80 100 120 Forward Scatter Side Scatter Anti HLA-DR PerCP CD19PE CD19 PE CD22 TC CD34 APC CD10 PC CD20 FITC κ APC CD19 PC CD19 PC κ APC Image 5 (Case 6) Surface immunoglobulin positive precursor B lymphoblastic leukemia in a 9-year-old boy with CD45 dim +HLA- DR+CD10+CD19+CD22 dim +CD34+ surface κ dim + leukemic cells (arrows) at diagnosis (4-color flow cytometric immunophenotypic analysis). Residual benign B cells (arrowheads) show brighter CD22 expression in comparison with the leukemic cells. Note that the color assigned to the neoplastic cells varies in different tubes. APC, allophycocyanin; FITC, fluorescein isothiocyanate; PC, PE-cyanin-5; PE, phycoerythrin; PerCP, peridinium chlorophyll protein complex; TC, Tricolor (Caltag, Burlingame, CA). (median, 59 years). The total leukocyte counts at diagnosis ranged from 840 to 10,980/µL (0.8-11.0 10 9 /L; median, 2,630/µL [2.6 10 9 /L]). The leukemic samples with surface light chain immunoglobulin restriction were obtained at diagnosis from 4 patients and at relapse from 2 patients. The lymphoblasts in samples from all patients showed FAB L1 or L2 morphologic features on Wright-Giemsa stained marrow aspirate smears. Bone marrow blast percentages ranged from 38.5% to 97.0% (median, 61.3%). Immunoperoxidase stains performed on paraffin-embedded trephine core biopsy or marrow aspirate clot sections revealed that the leukemic cells were TdT+ in all cases. By FCI analysis, the leukemic cells coexpressed CD10 and CD19 in samples from all patients except 1 (case 14) with an CD10 CD15 CD19+CD22+CD20 CD34+TdT+ immunophenotype, which corresponded to an early stage of precursor B-cell differentiation. Surface IgM and IgD were not detectable by FCI analysis in this case, which showed the t(2;11)(p21;q23) translocation by karyotypic analysis. Surface IgM was present in 1 patient (case 12) with sig+ results but without a karyotypic abnormality. Surface heavy chain immunoglobulins were not analyzed in the sample from the patient (case 10) with the t(1;19)(q23;p13). In the 2 patients with surface light chain expressing lymphoblasts at relapse, review of the initial FCI analysis did not show surface light chain expression on the leukemic cells at primary diagnosis. Two patients (cases Downloaded from https://academic.oup.com/ajcp/article-abstract/121/4/512/1759325 Am J Clin Pathol 2004;121:512-525 519 519 519

Kansal et al / PRECURSOR B LYMPHOBLASTIC LEUKEMIA 120 Side Scatter 100 80 60 40 20 0 0 20 40 60 80 100 120 0 20 40 60 80 100 120 Forward Scatter Side Scatter Anti HLA-DR PerCP CD19PE CD19 PE CD22 TC CD34 APC CD10 PC CD20 FITC κ APC CD19 PC CD19 PC κ APC CD13 PE CD13 PE CD34 APC CD19 APC CD34 APC CD33 PC Image 6 (Case 7) Surface immunoglobulin positive precursor B lymphoblastic leukemia in a 12-year-old boy with CD45 dim +HLA-DR+CD10+CD19+CD22 dim +CD34+ surface κ dim + leukemic cells (arrows) at diagnosis (4-color flow cytometric immunophenotypic analysis). Also shown is the dim aberrant expression of the myeloid markers, CD13 and CD33, on the neoplastic cells in this case. Note that the color assigned to the neoplastic cells varies in different tubes. APC, allophycocyanin; FITC, fluorescein isothiocyanate; PC, PE-cyanin-5; PE, phycoerythrin; PerCP, peridinium chlorophyll protein complex; TC, Tricolor (Caltag, Burlingame, CA). 10 and 14) showed surface light chain restricted lymphoblasts at initial diagnosis and at subsequent FCI analyses to detect residual or relapsed leukemia. In case 14, this surface light chain immunoglobulin expression contributed to the immunophenotypic fingerprint of the leukemic cells in 3 FCI analyses performed for residual leukemia and was no longer detectable in the fourth and fifth consecutive FCI analyses for persistent disease. All adults received chemotherapy according to the Cancer and Leukemia Group B protocols. Of the 6 patients, 4 achieved complete remission, with a duration ranging from 41 days to 666 days; however, 5 of 6 did not survive. 520 Am J Clin Pathol 2004;121:512-525 Downloaded 520 from https://academic.oup.com/ajcp/article-abstract/121/4/512/1759325

Hematopathology / ORIGINAL ARTICLE 120 Side Scatter 100 80 60 40 20 0 0 20 40 60 80 100 120 0 20 40 60 80 100 120 Forward Scatter Side Scatter Anti HLA-DR PerCP CD19PE CD19 PE 101 CD34 APC CD10 PC κ APC CD19 PC CD19 PC κ APC Image 7 (Case 10) Surface immunoglobulin positive precursor B lymphoblastic leukemia in a 20-year-old man with CD45 dim +HLA-DR+CD10+CD19+CD34 surface λ dim + leukemic cells (arrows) at diagnosis (4-color flow cytometric immunophenotypic analysis). Note that the color assigned to the neoplastic cells varies in different tubes. APC, allophycocyanin; PC, PE-cyanin-5; PE, phycoerythrin; PerCP, peridinium chlorophyll protein complex. Discussion Accurate diagnostic distinction between pb ALL and mature B-cell leukemia (Burkitt leukemia) is critical for disease management. Burkitt leukemia typically shows FAB L3 morphologic features, has a mature B-cell phenotype (CD34 TdT, surface light chain+) by FCI analysis and the c-myc gene rearrangement by cytogenetics, and manifests clinically with bulky disease that requires more intensive chemotherapy than standard ALL therapy, with central nervous system prophylaxis. 21 However, discrepancies in the morphologic features and immunophenotype of Burkitt leukemia have been described. Hammami et al 22 reported 9 cases of mature B-cell ALL with FAB L1 or L2 morphologic features. Navid et al 23 reported 5 pediatric cases of Burkitt leukemia, which originally were misinterpreted as pb ALL owing to the absence of sig by FCI analysis. Coexpression of precursor cell markers CD34 and TdT also has been reported in Burkitt leukemia. 24-26 In all of the aforementioned cases with an aberrant immunophenotype or morphologic features, the critical feature that determined the course of the disease as Burkitt leukemia was the c-myc gene translocation. All patients in our study were given a diagnosis of pb ALL by a multidisciplinary approach, and they represent pb ALL cases with morphologic and cytogenetic features typical of this entity but with unexpected surface light chain immunoglobulin restriction revealed by FCI analysis. Our pediatric cases included cases with a cryptic t(12;21) translocation, TEL/AML1 fusion, 16 t(9;22)(q34;q11), bcr/abl fusion, 17 and a hyperdiploid karyotype. 18 These cytogenetic abnormalities are different from those reported in the 4 cases of sig+ pb ALL identified in a large study of pediatric pb ALLs. 5 One of our pediatric cases also showed trisomy 12, which reportedly is uncommon in precursor B-cell neoplasms, 27,28 although it often is associated with B-cell Downloaded from https://academic.oup.com/ajcp/article-abstract/121/4/512/1759325 Am J Clin Pathol 2004;121:512-525 521 521 521

Kansal et al / PRECURSOR B LYMPHOBLASTIC LEUKEMIA 120 Side Scatter 100 80 60 40 20 0 0 20 40 60 80 100 120 0 20 40 60 80 100 120 Forward Scatter Side Scatter Anti HLA-DR PerCP CD19PE CD19 PE CD22 TC CD34 APC CD10 PC CD20 FITC κ APC CD19 PC CD19 PC κ APC Image 8 (Case 13) Surface immunoglobulin positive precursor B lymphoblastic leukemia in a 62-year-old woman with CD45 dim +HLA-DR+CD10+CD19+CD22 dim +CD34+ surface κ dim + leukemic cells (arrows) at relapse (4-color flow cytometric immunophenotypic analysis). Note that the color assigned to the neoplastic cells varies in different tubes. APC, allophycocyanin; FITC, fluorescein isothiocyanate; PC, PE-cyanin-5; PE, phycoerythrin; PerCP, peridinium chlorophyll protein complex; TC, Tricolor (Caltag, Burlingame, CA). chronic lymphocytic leukemia, 29 a neoplasm of mature B cells. Of interest, trisomy 12 was reported previously in 2 adult cases of sig+ pb ALL. In 1 case, it was part of a complex karyotype in a 23-year-old man with CD9+CD10+CD24+HLA-DR+TdT+, surface IgMλ+ lymphoblasts with non FAB L3 morphologic features. 6 In our case with trisomy 12, dim aberrant expression of CD13 and CD33 also was present; these markers were not analyzed in the case reported by Michiels and colleagues. 6 Vasef and colleagues 7 reported the second adult case of sig+ pb ALL with trisomy 12. The immunophenotype in their case, however, was CD5+CD10 CD19+CD20+CD34 TdT+, surface λ+, in contrast with our case that was CD5 CD10+CD19+CD20 CD22+CD34+, surface κ+. The immunologic classification of ALL of B-cell lineage is based on the stages of maturation of a B cell, with pb ALL arising from the early, intermediate, and late precursor stages of B-cell differentiation. 1-3,21 In addition to nuclear TdT and HLA-DR that are expressed by B lymphoblasts at all precursor stages of maturation, the earlystage blasts express CD19 and cytoplasmic CD22 and are CD10. 1,21 The intermediate-stage B lymphoblasts also express CD10, and the late precursor stage is characterized by the expression of cytoplasmic heavy chain immunoglobulin IgM. 1,21 All 6 adult pb ALL cases with surface light chain expression described by Vasef et al 7 seemed to have an immunophenotype consistent with the most mature precursor B-cell stage, and it was suggested that the immunophenotype in these sig+ pb ALL cases was intermediate between that of pre B cells, which are TdT+ and cytoplasmic Ig+, and mature B cells, which express surface heavy and light chain immunoglobulins. 6,7 Our study cohort of pb ALL included 522 Am J Clin Pathol 2004;121:512-525 Downloaded 522 from https://academic.oup.com/ajcp/article-abstract/121/4/512/1759325

Hematopathology / ORIGINAL ARTICLE Table 3 Cytogenetic Findings for 15 Patients With Precursor B Lymphoblastic Leukemia Case No. Karyotype Comments 1 51 57,inc [2 cells]/46,xx [1 cell] FISH +4,+10 2 52,XY,+X,+4,+6,+14,+21,+21 [20 cells] FISH +4,+21 3 Not analyzable FISH +4/+4,+10 4 47,XX,?+12,inc [2 cells]/46,xx [15 cells] FISH +12 5 60,XY,t(1;19)(q23;p13),i(7)(q10),inc [3 cells]/46,xy [17 cells] 6 Not analyzable FISH t(12;21)(p13;q22),tel/aml1 fusion 7 46,XY,ider(9)(q10)t(9;22)(q34;q11),der(22)t(9;22) [19 cells]/48,xy,t(9;22)(q34;q11), FISH bcr/abl fusion +21,+der(22)t(9;22) [1 cell] 8 46,XY [4 cells] Hypocellular specimen 9 46,XX,t(1;19)(q23;p13) [15 cells] / 46,XX [5 cells] 7,XY,+5,der(19)t(1;19)(q23;p13) [19 cells]/47,idem, 3,+8 [1 cell] 11 45,XY,?inv(10), 20 [14 cells]/46,xy [6 cells] 12 46,XY [20 cells] 13 46,XX [20 cells] 14 46,XX,t(2;11)(p21;q23) [20 cells] 15 Not analyzable Hypocellular specimen FISH, Fluorescence in situ hybridization. cases in the early, intermediate, and late stages of precursor B-cell differentiation and demonstrated, for the first time, that surface light chain immunoglobulin restriction in pb ALL might be present in neoplasms arising from all (early, intermediate, and late) stages of a precursor B-cell, which otherwise have typical morphologic and cytogenetic features of pb ALL. Neoplastic pb cells (lymphoblasts) show aberrant surface marker expression and deviation from the normal sequence of maturation that can help to differentiate them from normal marrow progenitor B cells (hematogones), 30,31 which also can coexpress TdT and CD34. 32 We have not systematically assessed the expression of surface light chain immunoglobulins on normal hematogones in our study. Nevertheless, the neoplastic cells in our cases provide an additional example of deviation from the normal sequence of B-cell maturation by showing unexpected surface light chain immunoglobulin expression, which likely would not be present in normal hematogones. Studies of normal B- lymphocyte development 32 have suggested subtle differences between the developmental stages identified in models of normal bone marrow compared with those deduced from B- lineage leukemias. 33 The expression of surface light chains on precursor B lymphoblasts in our study further suggests that it is not always possible to correlate leukemic B cells with normal B-cell developmental stages. One possible mechanism that would explain the occurrence of sig in this heterogeneous group of pb ALL cases is uncoupling of proliferation and maturation in B cells, which has been reported to occur in this group of neoplasms. 34 The expression of surface IgD in 2 (50%) of 4 of our cases, instead of surface IgM that might be present in transitional pre B-cell ALL, 4,20 also is likely an example of aberrant surface antigen expression in neoplastic B-lineage cells. The acquisition of surface CD22 often is considered to indicate a more mature precursor B-cell stage. This may lead to the argument that the presence of surface CD22 on neoplastic cells in all our cases suggests that these actually are examples of a more mature or transitional pb ALL. There are at least 2 reasons that do not support this view. First, the detection of surface CD22 on B-lineage lymphoblasts most likely is fluorochrome-dependent and has been reported in virtually all pb ALLs in at least 3 studies. 35-37 Furthermore, in the flow cytometry laboratory at RPCI, all sig pb ALL cases also show surface CD22 positivity, similar to our sig+ pb ALL cases (unpublished observation, C.C.S.), indicating that the surface CD22 positivity in our cases cannot be interpreted as evidence of a more mature stage of precursor B-cell maturation. Second, the interpretation that these cases represent a specific precursor stage of B-cell development is not supported by the cytogenetic findings in our cases, which include findings considered to be typically associated with the early-, intermediate-, and late-stage precursor B-cell ALL groups. Instead, it is possible that these sig+ ALL cases arise when a subset of leukemic cells within a typical sig ALL, whether early, intermediate, or late pre B-cell stage, progress toward greater maturational status and eventually become the predominant clone. Such a mechanism might also explain the 2 sig+ cases in our series that were detected only at relapse. Nevertheless, regardless of the underlying mechanism, these cases show that if surface light chain restriction is detected in a neoplastic B-cell population, it does not necessarily indicate a classification of a mature B-cell lymphoma/leukemia. In other words, a precursor B neoplasm cannot be ruled out solely by the presence of surface light chain immunoglobulin restriction on neoplastic cells by using FCI analysis. Downloaded from https://academic.oup.com/ajcp/article-abstract/121/4/512/1759325 Am J Clin Pathol 2004;121:512-525 523 523 523

Kansal et al / PRECURSOR B LYMPHOBLASTIC LEUKEMIA Information from systematic studies of pb ALL that have assessed surface light chain immunoglobulin expression on B lymphoblasts is limited. Behm and colleagues 5 reported 4 (0.7%) of 551 pediatric pb ALL cases with weak surface IgM and λ positivity. To the best of our knowledge, the published reports of sig+ pb ALL in adults include a case report 6 and retrospectively identified random cases. 7 Besides, the expression of surface light chain immunoglobulins on pb cells has not been evaluated in most recent large studies with focus on FCI evaluation of benign and neoplastic pb cells. 31,38,39 Although the incidence of sig+ pb ALL in our cases cannot be ascertained from our present data, this unusual expression of sig on precursor B lymphoblasts seems to be more frequent in our cases than currently reported in pb ALL. Furthermore, within a period of 8 months after we closed our study, we identified 3 additional patients given a diagnosis of sig+ pb ALL at RPCI (not included in this report). However, we do not have any further explanation for the apparently increased occurrence of sig+ pb ALL in our cases compared with the cases at most other tertiary institutions. From a diagnostic standpoint, our cases demonstrate that the expression of surface light chain immunoglobulins on neoplastic B cells does not necessarily indicate a mature B-cell phenotype, further emphasizing the need for a multiparametric approach in the differential diagnosis of B-cell neoplasms, including Burkitt leukemia vs precursor B lymphoblastic leukemia, and leukemic mantle cell lymphoma vs pb ALL. While the c-myc gene rearrangement would point to Burkitt leukemia in the differential diagnosis of pb ALL from Burkitt, the differential diagnosis of sig+ pb ALL from a leukemic manifestation of a mature B-cell lymphoma such as the blastoid variant of mantle cell lymphoma would be facilitated by the absence of the progenitor cell markers CD34 and TdT in the latter 40 ; the typically bright expression of CD20, CD45, and surface light chain immunoglobulin in FCI analysis in the latter; and the presence of the t(11;14) translocation by cytogenetic analysis or cyclin D1 expression by immunohistochemical analysis in the latter. 40 Furthermore, the expression of surface light chain immunoglobulins on B-lineage lymphoblasts might be of particular relevance in diagnosis when a limited antibody panel must be used for FCI analysis and indicates the need for caution in the classification of B-cell neoplasms by FCI analysis in such instances. The significance of this immunophenotypic finding in the clinical course of pb ALL is not clearly determined from our study. Nevertheless, in cases with sig+ lymphoblasts, the expression of sig light chains might contribute to the diagnostic immunophenotypic fingerprint of the neoplastic cells, as illustrated in 2 of our cases. This immunophenotypic finding also might appear for the first time at relapse in a typical pb ALL, as we noted in 2 cases. We draw the following conclusions from our study: (1) Surface light chain immunoglobulin restriction in pb ALL might be more common than currently recognized. (2) When present in pb ALL, it is not confined to a late precursor stage of B-cell differentiation, as previously suggested. Instead, it might be present in neoplasms arising from the early, intermediate, and late stages of a precursor B cell, which otherwise have typical morphologic and cytogenetic features of pb ALL. (3) The significance of this finding in the clinical course of pb ALL remains to be determined. (4) From a diagnostic standpoint, the presence of surface light chain restriction on neoplastic B cells by FCI analysis does not necessarily indicate a mature B-cell phenotype, emphasizing the need for caution in evaluating limited FCI antibody panels and the importance of multiparametric correlation in the immunophenotypic diagnosis and classification of B- lineage neoplasms. From the 1 Department of Pathology, Buffalo General Hospital, the State University of New York at Buffalo; Departments of 2 Pathology, 3 Medicine, and 4 Pediatrics, the 5 Clinical Cytogenetics Laboratory, and the 6 Laboratory of Flow Cytometry, Roswell Park Cancer Institute, Buffalo, NY. Address reprint requests to Dr Kansal: Dept of Pathology, Medical College of Wisconsin, 9200 W Watertown Plank Rd, Milwaukee, WI 53226. Acknowledgments: We thank Sally Bialy for assistance with retrieval of the flow cytometry data, Mary Beth Dell for assistance with the flow cytometry illustrations, and Bertram Schnitzer, MD, for reviewing the manuscript. References 1. Jaffe ES, Harris NL, Stein H, et al, eds. Pathology and Genetics of Tumours of Haematopoietic and Lymphoid Tissue. Lyon, France: IARC Press; 2001:109-187. World Health Organization Classification of Tumours. 2. Jennings CD, Foon KA. Recent advances in flow cytometry: application to the diagnosis of hematologic malignancy. Blood. 1997;90:2863-2892. 3. Foon KA, Todd RF. Immunologic classification of leukemia and lymphoma. Blood. 1986;68:1-31. 4. Koehler M, Behm FG, Shuster J, et al. Transitional pre B-cell acute lymphoblastic leukemia of childhood is associated with favorable prognostic clinical features and an excellent outcome: a Pediatric Oncology Group study. Leukemia. 1993;7:2064-2068. 5. Behm FG, Head DR, Pui C-H, et al. B-precursor ALL with unexpected expression of surface immunoglobulin (sig) mu and lambda [abstract]. Lab Invest. 1995;72:106A. 6. Michiels JJ, Adriaansen HJ, Hagemeijer A, et al. TdT positive B-cell acute lymphoblastic leukemia (B-ALL) without Burkitt characteristics. Br J Haematol. 1988;68:423-426. 7. Vasef MA, Brynes RK, Murata-Collins JL, et al. Surface immunoglobulin light chain positive acute lymphoblastic leukemia of FAB L1 or L2 type: a report of 6 cases in adults. Am J Clin Pathol. 1998;110:143-149. 524 Am J Clin Pathol 2004;121:512-525 Downloaded 524 from https://academic.oup.com/ajcp/article-abstract/121/4/512/1759325

Hematopathology / ORIGINAL ARTICLE 8. Stewart CC, Stewart SJ. Cell preparation for the identification of leukocytes. In: Darzynkiewicz Z, Crissman H, Robinson JP, eds. Methods in Cell Biology. New York, NY: Academic Press; 2001:218-270. 9. Stewart CC, Stewart SJ. Multiparameter data acquisition and analysis of leukocytes by flow cytometry. In: Darzynkiewicz Z, Crissman H, Robinson JP, eds. Methods in Cell Biology. New York, NY: Academic Press; 2001:289-312. 10. Riedy MC, Muirhead KA, Jensen CP, et al. Use of a photolabeling technique to identify nonviable cells in fixed homologous or heterologous cell populations. Cytometry. 1991;12:133-139. 11. Bennett JM, Catovsky D, Daniel M-T, et al, for the French- American-British (FAB) co-operative group. Proposals for the classification of acute leukemias. Br J Haematol. 1976;33:451-458. 12. Griffith RC, Kelly DR, Nathwani BN, et al. A morphologic study of childhood lymphoma of the lymphoblastic type: the Pediatric Oncology Group experience. Cancer. 1987;59:1126-1131. 13. Chilosi M, Pizzolo G. Review of terminal deoxynucleotidyl transferase: biological aspects, methods of detection, and selected diagnostic applications. Appl Immunohistochem. 1995;3:209-221. 14. Yunis JJ. New chromosome techniques in the study of human neoplasia. Hum Pathol. 1981;12:540-549. 15. Mitelman F, ed. ISCN 1995: An International System for Human Cytogenetic Nomenclature 1995. Basel, Switzerland: S Karger; 1995. 16. Rubnitz JE, Downing JR, Pui C-H, et al. TEL gene rearrangement in acute lymphoblastic leukemia: a new genetic marker with prognostic significance. J Clin Oncol. 1997;15:1150-1157. 17. Crist W, Carroll A, Shuster J, et al. Philadelphia chromosome positive acute lymphoblastic leukemia: clinical and cytogenetic characteristics and treatment outcome: a Pediatric Oncology Group study. Blood. 1990;76:489-494. 18. Pui C-H, Crist WM, Look AT. Biology and clinical significance of cytogenetic abnormalities in childhood acute lymphoblastic leukemia. Blood. 1990;76:1449-1463. 19. Raimondi SC, Behm FG, Roberson PK, et al. Cytogenetics of pre B-cell acute lymphoblastic leukemia with emphasis on prognostic implications of the t(1;19). J Clin Oncol. 1990;8:1380-1388. 20. Borowitz MJ, Hunger SP, Carroll AJ, et al. Predictability of the t(1;19)(q23;p13) from surface antigen phenotype: implications for screening cases of childhood acute lymphoblastic leukemia for molecular analysis: a Pediatric Oncology Group study. Blood. 1993;82;1086-1091. 21. Silverman LB, Sallan SE. Acute lymphoblastic leukemia. In: Handin RI, Lux SE, Thomas P, eds. Blood: Principles and Practice of Hematology. 2nd ed. Philadelphia, PA: Lippincott Williams & Wilkins; 2003:779-804. 22. Hammami A, Chan WC, Michels SD, et al. Mature B-cell acute leukemia: a clinical morphological, immunological, and cytogenetic study of nine cases. Hematol Pathol. 1991;5:109-118. 23. Navid F, Mosijczuk AD, Head DR, et al. Acute lymphoblastic leukemia with the (8;14)(q24;q23) translocation and FAB L3 morphology associated with a precursor immunophenotype: the Pediatric Oncology Group experience. Leukemia. 1999;13:135-141. 24. Drexler HG, Messmore HL, Menon M, et al. A case of TdTpositive B-cell acute lymphoblastic leukemia. Am J Clin Pathol. 1986;85:735-738. 25. Secker-Walker L, Stewart E, Norton J, et al. Multiple chromosome abnormalities in a drug resistant TdT positive B- cell leukemia. Leuk Res. 1987;11:155-161. 26. Shende A, Festa RS, Wedgwood JF, et al. A paediatric case of a TdT positive B-cell acute lymphoblastic leukaemia (B-ALL) without Burkitt characteristics [letter]. Br J Haematol. 1988;70:129-130. 27. Oshimura M, Freeman AI, Sandberg AA. Chromosomes and causation of human cancer and leukemia, XXVI: banding studies in acute lymphoblastic leukemia (ALL). Cancer. 1977;40:1161-1172. 28. Palka G, Fioritoni G, Lambardo M, et al. A cytogenetic survey of 13 patients with acute lymphocytic leukemia (ALL). Hematologica. 1987;72:511-514. 29. Matutes E, Oscier D, Garcia-Marco J, et al. Trisomy 12 defines a group of CLL with atypical morphology: correlation between cytogenetic, clinical and laboratory features in 544 patients. Br J Haematol. 1996;92:382-388. 30. Rimsza LM, Larson RS, Winter SS, et al. Benign hematogone-rich lymphoid proliferations can be distinguished from B-lineage acute lymphoblastic leukemia by integration of morphology, immunophenotype, adhesion molecule expression, and architectural features. Am J Clin Pathol. 2000;114:66-75. 31. McKenna RW, Washington LT, Aquino DB, et al. Immunophenotypic analysis of hematogones (B-lymphocyte precursors) in 662 consecutive bone marrow specimens by 4- color flow cytometry. Blood. 2001;98:2498-2507. 32. Loken MR, Shah VO, Dattilio KL, et al. Flow cytometric analysis of human bone marrow, II: normal B lymphocyte development. Blood. 1987;70:1316-1324. 33. Anderson KC, Bates MP, Slaughenhoupt BL, et al. Expression of human B-cell associated antigens on leukemias and lymphomas: a model of human B cell differentiation. Blood. 1984;63:1424-1433. 34. Hurwitz C, Loken MR, Graham ML, et al. Asynchronous antigen expression in B lineage acute lymphoblastic leukemia. Blood. 1988;72:299-307. 35. Boue DR, LeBien TW. Expression and structure of CD22 in acute leukemia. Blood. 1988;71:1480-1486. 36. Dorvault CC, Jones PA, Swerdlow SH, et al. Frequent cell surface expression of CD22 on B-lineage acute lymphoblastic leukemias (ALL) [abstract]. Lab Invest. 1999;79:135A. 37. Siebert JD, Katzke KA, Copp MV, et al. Cell surface CD22 is commonly expressed in precursor B-lymphoblastic leukemia [abstract]. Am J Clin Pathol. 2001;116:606. 38. Ciudad J, San Miguel JF, Lopez-Berges MC, et al. Detection of abnormalities in B-cell differentiation pattern is a useful tool to predict relapse in precursor-b-all. Br J Haematol. 1999;104:695-705. 39. Weir EG, Cowan K, LeBeau P, et al. A limited antibody panel can distinguish B-precursor acute lymphoblastic leukemia from normal B precursors with four-color flow cytometry: implications for residual disease detection. Leukemia. 1999;13:558-567. 40. Soslow RA, Zukerberg LR, Harris NL, et al. bcl-1 (PRAD- 1/cyclin D-1) overexpression distinguishes the blastoid variant of mantle cell lymphoma from B-lineage lymphoblastic lymphoma. Mod Pathol. 1997;10:810-817. Downloaded from https://academic.oup.com/ajcp/article-abstract/121/4/512/1759325 Am J Clin Pathol 2004;121:512-525 525 525 525