| Japanese Journal of Clinical Oncology | Pages |
Dual Rearrangement of Immunoglobulin and T-cell Receptor Genes in a Case of Philadelphia Chromosome-positive Acute Leukemia
Introduction
Case report
Discussion
References
Dual Rearrangement of Immunoglobulin and T-cell Receptor Genes in a Case of Philadelphia Chromosome-positive Acute Leukemia
INTRODUCTION
Acute leukemias sometimes provide good models to study the differentiation pathway of hematopoietic cells at early stages of maturation. Rearrangement of antigen receptor genes, as well as cell surface antigen expression, has generally been regarded as evidence of lymphoid differentiation. However, it has been reported that immunoglobulin and T-cell receptor (TCR) genes are rearranged in many cases of acute myeloid leukemia (AML) (1-3). Although these studies were performed in short series of cases, dual rearrangement of immunoglobulin and TCR genes in AML was quite rare.
On the other hand, the appearance of the Philadelphia chromosome (Ph1) in AML is a rare phenomenon that occurs in only 2-3% of these patients (4-6). With regard to analysis of immunophenotype, most Ph1-positive AML tend to have B-cell-associated antigens and some characteristics of acute mixed lineage leukemia (AMLL) (7). Recently, CD10 expression has been considered to be characteristic of blast cells with Ph1-positive AML in some reports (8-10), but its clinical implication is unclear.
Here we report a patient with Ph1-positive acute leukemia who showed myeloid lineage commitment, promiscuous CD10 expression, and dual rearrangement of immunoglobulin and TCR genes. The clinical implication of immunophenotype, gene rearrangement and Ph1 positivity is discussed.
CASE REPORT
A 48-year-old Japanese man was admitted to our hospital because of elevation of white blood cell count at the regular check-up on 17 February 1992. The liver was palpable 2 cm below the right costal margin, but the spleen was not palpable. Laboratory data on admission were: red blood cell count 3.97 × 1012/l, hematocrit 38.1%, hemoglobin 12.9 g/dl, platelet count 151 × 109/l and white blood cell count 76.7 × 109/l with a differential of 52% blasts, 7% promyelocytes, 3.0% myelocytes, 1% metamyelocytes, 9% bands, 9% segmented neutrophils, 1% eosinophils, 2% monocytes and 16% lymphocytes. Bone marrow aspiration revealed myeloid hyperplasia with 80.4% of blasts. Most of the blasts had myeloid features with poor azurophil granules (Fig. 1). By cytochemistry, 3.4% of the blasts were positive for myeloperoxidase (Fig. 1), 7.0% for Sudan black B, but all of the blasts were negative for Periodic acid-Schiff and alpha-naphthyl butyrate esterase. The bone marrow cells expressed CD10, CD13, CD33 and HLA-DR, but did not express CD3, CD7, CD19 and CD20 by flow cytometry of conventional single-color method (11) (Table 1). Chromosomal analysis revealed Ph1 with no additional abnormalities in all 20 cells analyzed. By the reverse transcriptase-polymerase chain reaction (RT-PCR) method, which has been previously described (12), a 446 bp fragment corresponding to a major bcr-abl fusion gene (b3a2) was observed. Minor bcr-abl fusion gene was not detected (Fig. 2). According to the French-American-British (FAB) criteria, the patient was tentatively diagnosed as AML-M2, but CD10 expression of blast cells raised a possibility of B-cell lineage commitment. Southern blot analysis using probes recommended in previous reports (13-15) disclosed rearrangement of both TCR beta chain (Fig. 3) and immunoglobulin heavy chain genes (Fig. 4). Rearrangement of immunoglobulin light chain (kappa) gene was not detected (Fig. 4). To clarify whether blast cells express CD10, two-color analysis of surface antigens of bone marrow cells was performed. It was revealed that most CD13- and CD33-positive cells did not express CD10 (Fig. 5a and b).
Figure 1. May-Grunwald-Giemsa-stained bone marrow smear from the patient at diagnosis shows that most of the blasts have poor azure granules (a). Myeloperoxidase stain reveals that >3% of the blasts are positive (b). Figure 2. Analysis of major and minor bcr-abl fusion mRNA expression by the RT-PCR method. The 446 bp band of major bcr-abl (b3a2) is observed in lane 2, but no 472 bp band of minor bcr-abl in lane 3. Abl mRNA is shown as an internal control. Lane 1 (M), size marker ([phis]X174/HaeIII digestion); lane 2 (1), major bcr-abl; lane 3 (2), minor bcr-abl; lane 4 (3), internal control (253 bp). Figure 3. TCR C[beta]1 gene rearrangement by Southern hybridization analysis. After DNA was extracted from the patient's mononuclear cells, it was digested by BamHI and EcoRV restriction enzymes, and electrophoresed. The 24 kb band in the left lane and the 7 kb band in the right lane are germ line. Rearranged band (R) is noted by BamHI digestion, while the 12 kb germ line band disappeared with EcoRV digestion. Figure 4. Immunoglobulin gene rearrangement by Southern hybridization analysis. The patient's DNA was digested by HindIII with or without BamHI for the analysis of the IgH J region. Rearranged bands are observed in both lanes (a). Digestion by HindIII and BamHI for the analysis of IgL kappa shows no rearrangement (b). Figure 5. Two-color analysis of cell surface antigens by flow cytometry. Cytograms of CD10 versus CD13 (a) and CD10 versus CD33 (b) are shown. The percentage of each cell population is shown under the cytogram. Most of the CD13-positive and CD33-positive cells are negative for CD10. Since blast cells were thought to be mainly in myeloid lineage, a course of combined chemotherapy consisting of 300 mg of behenoyl cytosine arabinoside, 60 mg of daunorubicin and 100 mg of 6-mercaptopurine was begun on the day of admission. Although the white blood cell count decreased in the course of combined chemotherapy, blasts remained, comprising 40.4% of the nucleated cell count in the marrow aspiration examination on the sixth day of hospitalization. Initial chemotherapy was discontinued and lymphoid lineage-targeted chemotherapy consisting of 2 mg of vincristine, 60 mg of prednisolone and 30 mg of adriamycin was started on the seventh day, but little improvement was observed. Serial studies of marrow cell components showed persistent existence of blasts, over 10% of whole nucleated cells. Although normal clones without Ph1 appeared in up to 25% of total cells analyzed on day 15, they decreased to 5% on day 43. On the 64th day of hospitalization, the patient died of pulmonary bleeding. Permission for autopsy was not obtained. Table 1. 
CD1 0.5%
CD10 32.1%
CD2 0.4%
CD13 39.6%
CD3 1.5%
CD14 1.8%
CD4 0.2%
CD19 5.9%
CD5 1.3%
CD20 0.3%
CD7 1.0%
CD33 45.8%
CD8 1.1%
HLA-DR 92.0%
DISCUSSION
Investigation of rearrangement of immunoglobulin and TCR genes is useful to determine the lineage, stage of differentiation and clonality of the proliferating lymphoid cells. Moreover, its clinical application has contributed to the classification of lymphoid malignancies, elucidation of disease etiology, and decision making in therapy. However, during these several years, immunoglobulin heavy chain genes have been found to be rearranged in T-cell leukemias (16), and TCR [alpha], [beta], [gamma] and [delta] gene rearrangements have been reported in B-precursor acute lymphoid leukemia (ALL) (17). It is speculated that these phenomena occur from the fact that both immunoglobulin genes and TCR genes have similar recombinational sequence, and common recombinase (18,19). However, in most cases, immunoglobulin gene rearrangement in T-cell malignancies and TCR gene rearrangement in B-cell malignancies will stop in the early stage of D-J binding not forming V-D-J recombination. The pathognomonic significance of this abortive recombination is unclear, but it is now accepted that gene rearrangements are not an absolute indicator of lymphoid lineage.
The presence of immunoglobulin and TCR gene rearrangement has also been shown to occur in AML. Recent reports show that the incidence of gene rearrangement in AML is higher than previously suspected, and that it is not associated with early stages of cell differentiation nor with the expression of lymphoid markers (20-22). The vast majority of them are thought to be AMLL with myeloid morphology and cytochemistry. In this case, myeloperoxidase and Sudan black B staining confirmed myeloid lineage, but CD10 positivity may raise a possibility of B/myeloid lineage-associated AMLL. However, many investigators now require expression of more than one unexpected lineage-associated antigen for a diagnosis of AMLL. According to the scoring system for biphenotypic acute leukemia considered by Buccheri et al. (23), the lack of B-lineage antigens other than CD10 denied AMLL in this case.
CD10 (also called common ALL antigen: CALLA) is considered as a lymphocyte-associated antigen and is very rarely found in AML. Some investigators reported that CD10 expression was quite common in Ph1-positive AML and AMLL (8,9,24-27). Moscinski and his colleagues (10) considered that CD10 positivity is characteristic of blasts with Ph1-positive AML, but these studies were performed in a short series of cases and cell surface antigens were analyzed by single labeling experiments. Our result from two-color analysis of surface antigens disclosed promiscuity or infidelity of CD10 expression. It may be possible that most of them are reactive cells, but not blasts themselves. Another possibility is that there were two clones in the leukemic cells, namely a CD10-positive leukemic clone and a CD10-negative one. To clarify this, accumulation of cases concerning CD10 expression in AML is necessary.
The existence of Ph1 is the other specific point in this case. Hamaguchi and colleagues (28) reported that a certain population of Ph1-positive acute leukemia, such as one with monosomy 7, may originate from the pluripotent stem cell level. Moreover, the existence of Ph1 in AML may have a close relationship with the expression of B-cell-associated surface antigens (7). These findings led to speculation that B-cell lineage commitment may occur in the very early stage of differentiation, probably near the pluripotent stem cell level.
Major bcr-abl fusion gene observed by RT-PCR raised the possibility that the patient was in myeloid crisis of chronic myeloid leukemia (CML). Whether it is AML or blast crisis of CML is still a big problem when we encounter Ph1-positive leukemias, but no definite answer has been given until now. Some reports suggest that the following findings may contribute to distinguish them: (1) marked splenomegaly; (2) increase in basophils; (3) occasional disappearance of Ph1-positive cells during remission; (4) mosaic consisting of Ph1-positive and Ph1-negative cells (29-34). In this case, he was tentatively diagnosed as AML-M2 because he had no splenomegaly, no increase in basophils nor eosinophils in the peripheral blood and bone marrow, and no preceding history of hematological disorders. Moreover, normal clones without Ph1 which appeared during the combined chemotherapy support, to some extent, that this case is AML. Yet we still have to be careful to give a definite diagnosis because few cases which had a reduction in Ph1-positive cells in the blast crisis of CML during chemotherapy have been reported (35,36).
Although the association of Ph1 with multilineage differentiation is unclear, our case has significant implication for further investigation of the relationship between Ph1-positive cells and lineage selection.
References
For reprints and all correspondence: Kazuo Motoyoshi, The Third Department of Internal Medicine, National Defense Medical College, 3-2, Namiki, Tokorozawa, Saitama 359, Japan
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Last modification: 24 Jul 1998
Copyright©Japanese Journal of Clinical Oncology, 1998.
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