American Journal of Hematology 77:156–160 (2004)
Acute Lymphoblastic Leukemia With the Phenotype of a Putative B-Cell/T-Cell Bipotential Precursor Lee Gong Lau,1* Lip Kun Tan,2 Evelyn S.C. Koay,3 Melvin H.L. Ee,4 Suat Hoon Tan,4 and Te Chih Liu2 1
Department of Hematology-Oncology, National University Hospital, Singapore Department of Laboratory Medicine, National University Hospital, Singapore 3 Molecular Diagnosis Center, National University Hospital, Singapore 4 National Skin Center, Singapore
2
Biphenotypic acute leukemias (BALs) are uncommon. Most are of myeloid-B-cell or myeloid-T-cell lineage. We report herein a 70-year-old man with an unusual acute leukemia where the blasts expressed both B- and T-lymphoid markers. He presented to us with an enlarging cutaneous tumor. The presenting peripheral blood and bone marrow aspirate showed 40% and 90% blasts, respectively, which were negative for the usual cytochemical stains. The flow cytometric analysis revealed that the blasts were positive for CD19, CD20, CD22, cytoplasmic (Cyt) CD79a, CD10, Cyt CD3, CD5, CD7, CD4, HLA-DR, TdT, and were negative for myeloid markers. According to the scoring system from the European Group for the Immunological Characterization of Acute Leukaemias (EGIL), this case was an unequivocal B-cell/T-cell BAL. Conventional cytogenetic analysis revealed 46XY [t(4;11)(q31;q13), add(8)(q24), der(9)del(9)(p21)del(9)(q32q34), –13, +mar] in all 25 metaphases analyzed. Fluorescence in situ hybridization (FISH) and polymerase chain reaction (PCR) for 11q23 rearrangements as well as t(9;22) were negative. PCR for both TCR-g and IgH gene analyses revealed polyclonal rearrangements. We postulate that this case of BAL might have arisen from the putative common lymphoid progenitor cell. Am. J. Hematol. 77:156–160, 2004. ª 2004 Wiley-Liss, Inc. Key words: acute lymphoblastic leukemia; immunophenotyping; lymphoid progenitor cell
INTRODUCTION
Most acute leukemias can be assigned a specific lineage as either acute lymphoblastic leukemia (ALL) or acute myeloid leukemia (AML) according to the morphologic, cytochemical, and immunophenotypic characteristics of the blast cells. With the widespread availability of flow cytometry, acute leukemias that express cross-lineage antigens are increasingly being recognized [1,2]. These can either be leukemias with two separate populations of blasts, each from a different lineage, or alternatively, a single blast population expressing multiple lineage markers. The two groups have been referred to as bi-lineal acute leukemia and biphenotypic acute leukemia (BAL), respectively. All these are categorized as ‘‘acute leukemias of ambiguous lineage’’ in the new World Health Organization (WHO) classification system and are believed to have arisen from the multipotent hematopoietic stem cells (HSC) ª 2004 Wiley-Liss, Inc.
[3]. In an effort to unify the definition of BAL, the European Group for the Immunological Classification of Leukaemias (EGIL) proposed guidelines for the scoring of lineage-specific antigens commonly used in the assignment of lineage to acute leukemia [4]. Using this scoring system, BALs are uncommon, generally accounting for less than 5% of all acute leukemias [5–9]. Most of these are of combined myeloid/B-cell or myeloid/T-cell lineage, whereas B-cell/ T-cell BALs are exceedingly rare. Recognition of *Correspondence to: Dr. Lee Gong Lau, Department of HematologyOncology, National University Hospital, 5 Lower Kent Ridge Road, Singapore 119074. E-mail:
[email protected] Received for publication 4 August 2003; Accepted 27 March 2004 Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/ajh.20163
Case Report: ALL and Common Lymphoid Progenitor
BAL is important as they are uniformly associated with poorer prognosis with conventional treatment regimes [3,8,10]. We describe and discuss the morphologic, immunophenotypic, chromosomal as well as molecular features of a B-cell/T-cell BAL.
CASE REPORT
A previously healthy, 70-year-old man presented to the dermatologist with a 5 cm 3 cm rapidly enlarging cutaneous tumor over the right chest wall. A skin biopsy was suggestive of leukemic infiltrates, and he was transferred to the Department of Hematology/ Oncology for further management. Physical examination revealed a fit and well man with no lymphadenopathy or hepatosplenomegaly. The blood count on admission showed the following: white blood cell count, 14.92 109/L (40% blasts); hemoglobin, 14.7 g/dL; and platelet count, 122 109/L. A bone marrow (BM) aspirate and trephine biopsy were performed. The sample was submitted for morphologic, cytochemical, cytogenetic, molecular and flow cytometric evaluations. A diagnosis of B-cell/T-cell BAL was established, and the patient was commenced on induction chemotherapy as for acute lymphoblastic leukemia (prednisolone, daunorubicin, vincristine, and L-asparaginase). The cutaneous tumor responded and resolved rapidly. In the 4th week of treatment, he was pancytopenic and developed fulminant Pseudomonas aeruginosa pneumonia and septicemia. Peripheral blood films at that time revealed no circulating blasts. Despite aggressive management, the patient succumbed.
MATERIALS AND METHODS Histomorphology, Cytochemistry, and Immunohistochemistry
Standard Romanowsky-stained blood film and BM smears were prepared for morphologic analysis. In addition, the aspirate was also stained with myeloperoxidase (MPO), Sudan black B (SBB), periodic acidSchiff (PAS), and acid phosphatase (AP). The histology of the skin and BM biopsy was analyzed in hematoxylin–eosin (HE)-stained sections. Immunoperoxidase staining on paraffin-embedded tissue sections included CD3, CD4, CD5, CD8, CD20, CD30, CD43, CD45, CD56, CD79a, and MPO for the skin and CD3, CD10, CD20, CD79a, and TdT (all from Dako Corporation, Carpinteria, CA, except CD4, CD56, and TdT, which were from Novocastra Laboratory Ltd, Newcastle-upon-Tyne, U.K.) for the BM.
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Flow Cytometry
Four-color immunophenotyping of the leukemic cells was performed on filtered fresh whole BM aspirate after red cell lysis. The following antibodies were used: CD2, CD3, CD4, CD5, CD7, CD8, CD10, CD11c, CD13, CD14, CD19, CD20, CD22, CD33, CD34, CD38, CD45, CD56, CD64, CD71, CD117, HLA-DR, Cyt CD3, Cyt CD79a, Cyt MPO, Cyt IgM, surface IgM, and nuclear TdT (all from BectonDickinson, San Jose, CA, except MPO, IgM, and TdT, from DakoCytomation, Glostrup, Denmark). Blasts were identified, gated, and analyzed based on their CD45 dim/side-scatter low pattern. Metaphase and Interphase Cytogenetics
Metaphase cytogenetic studies were performed on the BM aspirate and analyzed using standard trypsin– Giemsa-banding techniques. In addition, fluorescence in situ hybridization (FISH) evaluation of chromosome 11q rearrangements was performed according the manufacturer’s instructions (Vysis, Downers Grove, IL). Polymerase Chain Reaction for IgH Gene and T-Cell Receptor g (TCR-g ) Gene Rearrangements and t(4;11)(q21;q23) and t(9;22)(q34;q11) Analyses
Genomic DNA was extracted from the mononuclear cell fraction of the BM aspirate. Polymerase chain reaction (PCR) amplification of the IgH gene was performed on a conserved VH framework III region and J region, while that for the TCR- gene was carried out on gene fragments at the Ja and Jp loci of the g chain. The amplified PCR products were detected by capillary electrophoresis (CE) on the ABI Prism 310 Genetic Analyser, using GeneScan software (both from Applied Biosystems Inc., Foster City, CA). Messenger RNA was similarly isolated from BM mononuclear cells and tested using the HemaVisionÒ-7 kit (DNA Technology A/S, Aarhus, Denmark) for commonly reported translocations including t(4;11)(q21;23). For t(9;22)(q34;q11) analysis, two additional amplifications were carried out to detect fusion products involving the major and minor breakpoint cluster regions of the BCR gene.
RESULTS Morphology
The peripheral blood and BM smears revealed 40% and 90% blast cells, respectively. These cells were large and heterogenous with irregular nuclei, lacy chromatin, prominent nucleoli, and moderately abundant agranular cytoplasm resembling L2 lymphoblasts (Fig. 1). They were negative for all cytochemical stains.
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Fig. 1. Peripheral blood film showing blast cells with prominent nucleoli, open chromatin and moderately abundant agranular cytoplasm (original magnification 400·). [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]
Fig. 3. Skin biopsy showing infiltration by neoplastic cells with round to irregularly shaped nuclei with dispersed chromatin and distinct nucleoli (original magnification 400·). [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]
Fig. 2. Bone marrow trephine biopsy showing extensive replacement by blast cells (original magnification 100·). [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]
Histology and Immunohistochemistry
Marrow spaces on the BM trephine biopsy showed almost complete replacement by blasts (Fig. 2), the majority of which stained positively for CD10 and TdT. Occasional cells were positive for CD3, CD20, and CD79a. The skin biopsy revealed a non-epidermotropic diffuse infiltrate of basophilic neoplastic cells replacing the reticular dermis and extending through the subcutaneous tissue (Fig. 3). The cells were mediumsized with a high nuclear cytoplasmic ratio, dispersed nuclear chromatin, and prominent nucleoli. Brisk mitotic activity was evident. The majority of these
Fig. 4. Flow cytometric antigenic profiling of BM blasts (R1 in red) showing positive expression of HLA-DR, Cyt CD79a, Cyt CD3, CD19, and TdT (B–D) but negative expression of CD34 (B). R2 (green) is the normal lymphocyte population (A). [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]
cells expressed only CD45 and CD43 with scattered CD4 positivity. Immunophenotyping
Flow cytometric immunophenotypic analysis revealed a single blast population with distinct
Case Report: ALL and Common Lymphoid Progenitor
positivity for numerous B-cell and T-cell markers. In brief, the blasts were positive for HLA-DR, TdT, Cyt CD79a, CD19, CD20, CD22, CD10, Cyt CD3, CD5, CD7, CD4, CD38, and CD71 (Fig. 4). They were negative for CD34, CD2, CD3, CD8, CD11c, CD56, IgM, and all other myeloid markers. Aberrant positivity for CD33 was noted. According to the EGIL criteria, this case scored 7.5 points for B-lymphoid markers and 5 points for T-lymphoid markers, making it a B-cell/T-cell BAL. As described above, immunoperoxidase staining was performed on paraffin-embedded skin tissue sections while flow cytometric analysis was done on freshly aspirated marrow tissues. This could have accounted for the apparent discrepancy noted in immunophenotypic expressions between the cutaneous and marrow blasts cells. Cytogenetics
Conventional cytogenetic analysis revealed 46XY [t(4;11)(q31;q13), add(8)(q24), der(9)del(9)(p21)del(9) (q32q34), 13, +mar] in all 25 metaphases analyzed. Studies by both FISH as well as PCR analysis did not indicate a MLL gene rearrangement. Molecular Analysis
The PCR for both TCR- and IgH genes revealed polyclonal rearrangements. Specific RT-PCR for detection of t(9;22)(q34;q11) was negative. DISCUSSION
We describe and characterize a case of acute leukemia in which the blasts showed clear expression of both B-lymphoid and T-lymphoid markers. Using the EGIL criteria [4], the blasts scored 7.5 points for B-lymphoid markers (Cyt CD79a, 2; CD22, 2; CD19, 1; CD20, 1; CD10, 1; TdT, 0.5) and 5 points for T-lymphoid markers (Cyt CD3, 2; CD5, 1; CD10, 1; CD7, 0.5; TdT, 0.5), establishing it as a B-cell/T-cell BAL. To our knowledge, this is the first reported case of a well-characterized de novo B-cell/T-cell BAL, although the existence of this leukemic entity has been alluded to in case series [5–8]. The lack of monoclonal TCR- and IgH gene rearrangements may appear contradictory. However, present experience with the use of gene rearrangement studies for the determination of clonality and/or assignment of lineage involvement would suggest that these tests are lacking in both sensitivity and specificity [11]. Leukemic cells are generally thought to have resulted from perturbation of a normal precursor cell population [12]. Based on this hypothesis, it is tempting to speculate that the leukemic population in our patient
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could have originated from the putative common lymphoid progenitor (CLP) cell with the capacity to differentiate into committed cells of either B-lymphoid or T-lymphoid lineage. An orderly T- and B-lymphoid antigen maturation sequence is definable on flow cytometric analysis of precursor cells from normal BM. The aberrant and disordered expression of antigens in our patient is consistent with a malignant process [12]. Based on mouse experiments, the phenotype of the normal mouse CLP is that of a lineage-marker-negative, immature precursor cell. In our patient, a block in the development of the cell at the CLP stage is likely with accompanying dysregulation of the genes controlling the sequential appearance of lineage-associated antigens. The existence of CLP in human BM and cord blood as well as murine BM has long been the subject of intense investigations [13–15]. The cells are of particular interest to clinicians as a potential tool for the augmentation of immune reconstitution following stem cell transplantation [16,17]. Our patient may provide further corroborative evidence for the existence of the CLP in humans. Our patient exhibited a few unique cytogenetic abnormalities. Involvement of the MLL gene at 11q23 and the bcr-abl t(9;22)(q34;q11) proto-oncogene have been frequently reported in BAL [3,6–8]. These two changes were not present in our patient. He had two other abnormalities not previously associated with acute leukemias: t(4;11)(q31;q13) and monosomy 13. The multiple abnormalities present, however, make it difficult to guess at the pathogenetic role played by each karyotypic abnormality. In summary, we present a well-characterized de novo case of B-cell/T-cell BAL with distinct clinical, immunophenotypic, and cytogenetic features. We postulate that this acute leukemia might have arisen from a CLP cell. This corroborative evidence for the existence of the human counterpart to the mouse CLP provides greater impetus for the experimental studies using CLP as a therapeutic transplantation tool. ACKNOWLEDGMENT
We thank Miss Jenny B. Li and Miss Jean Y.C. Chen for helping with the immunophenotyping and cytogenetic studies. REFERENCES 1. Khalidi HS, Medeiros LJ, Chang KL, Brynes RK, Slovak ML, Arber DA. The immunophenotype of adult acute myeloid leukemia: high frequency of lymphoid antigen expression and comparison of immunophenotype, French-American-British classification, and karyotypic abnormalities. Am J Clin Pathol 1998;109:211–220.
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