Leukemia (1997) 11, 609–618 1997 Stockton Press All rights reserved 0887-6924/97 $12.00 CORRESPONDENCE CASE REPORT A case of acute eosinophilic granulocytic leukemia with PML-RAR alpha fusion gene expression and response to all-trans retinoic acid R-Q Yu1, W Huang2, S-J Chen2, S-D Jiang1 and Z Chen2 1Division of Hematology, Department of Internal Medicine, Shanghai Chang-Zheng Hospital; and 2Laboratory of Molecular Biology, Shanghai Institute of Hematology, China A typical case of eosinophilic granulocytic leukemia with PML- RAR alpha fusion gene expression is reported. The patient achieved complete remission after oral administration of all- trans retinoic acid without any exposure to cytotoxic agents. The facts strongly suggest that the genetic event occurred at the level of pluripotent stem cells. Keywords: leukemia; eosinophilic; PML-RAR alpha; retinoic acid Introduction It has been well demonstrated that the presence of a fusion gene, PML-RAR alpha, resulting from the reciprocal translo- cation of human chromosome 15 and 17, t(15;17)(q22:q21) is a specific molecular marker of acute promyelocytic leuke- mia, and plays an important role in the pathogenesis of that disease.1–4 Until now PML-RAR alpha fusion gene has not been found in other malignant cells. Recently, we saw a typi- Figure 1 Bone marrow smear showing coarse refractile eosino- cal case of acute eosinophilic granulocytic leukemia with philic granules in leukemic cells. PML-RAR alpha fusion gene expression that achieved com- plete remission after differentiation therapy with all-trans larity with a G/E ratio of 14.1:1. The differential count showed retinoic acid (ATRA). We would like to present it for further 5.1% myeloblasts, 1.0% neutrophilic promyelocytes, 14.5% attention. eosinophilic promyelocytes, 12.5% eosinophilic myelocytes, 10.0% eosinophilic metamyelocytes, 14.0% eosinophilic band-formed granulocytes and 26.0% eosinophilic-segmented Case history granulocytes. The eosinophilic granules seen on the smear were coarse, refractile, orange-red with slight blue staining Lee XX, a 66-year-old female, was admitted to hospital in and unevenly distributed (Figure 1). In some myeloblasts and December 1991 with complaints of dizziness and chest dis- tress for 2 weeks. On physical examination the patient revealed lassitude and pallor. The superficial lymph nodes were not enlarged. There was definite tenderness on slight pressure on the lower part of the sternum. The liver and spleen were not palpable under the costal margin. No petechia and ecchymosis could be found over the surface of the body. B-ultrasonic examination showed the size of the liver and spleen was normal. The analytical test of peripheral blood showed hemoglobin 108 g/l, leukocyte count 64.3 × 109/l, platelet count 192 × 109/l. Cell morphology in peripheral blood films showed 1% eosinophilic metamyelocyte, 13% eosinophilic band-formed granulocytes and 69% eosinophilic-segmented granulocytes. On bone marrow smear there was pronounced hypercellu- Correspondence: R-Q Yu, Division of Hematology, Department of Internal Medicine, Shanghai Chang-Zheng Hospital, 415 Fong-Yang Figure 2 Electrophoresis showing the specific product of RT-PCR. Road, Shanghai 200003, PR China Lane 1, the patient; lane 2, positive specimen from patient with M3; Received 25 September 1996; accepted 11 December 1996 lane 3, normal subject; lane 4, blank control. Correspondence 610 eosinophilic promyelocytes the nuclei were twisted and twisted and folded nuclei. Auer rods could be found in their folded with a fine nuclear chromatin pattern. Auer rods could cytoplasm. On the basis of the above evidence, the diagnosis be found in their cytoplasm. Mitosis of the eosinophils could of acute eosinophilic granulocytic leukemia could be estab- easily be seen. The proliferation of erythrocytic series was lished, and according to the typically morphological feature depressed. Part of the erythroblasts underwent megaloblast- of the eosinophilic granules in the cytoplasm, acute promyelo- like transformation. Within the area of 2 × 3 cm of bone mar- cytic leukemia could also be ruled out. row smear, 65 megakaryocytes could be found; most of them PML-RAR alpha is a fusion gene resulting from reciprocal were granular and platelet-producing. There was an abundant translocation of human chromosomes by juxtaposing the PML amount of stainable iron in the histiocytes of the bone gene on chromosome 15 and the RAR alpha gene on chromo- marrow. some 17. It plays a key role in the pathogenesis of acute pro- Three days after admission ATRA therapy started 30 mg myelocytic leukemia and is also a specific molecular marker orally every 6 h. Ten days later the leukocyte count of periph- which has not been found in other malignancies. eral blood dropped from 64.3 × 109/l to 44.5 × 109/l, the plate- Here, interestingly enough, PML-RAR alpha fusion gene let count rose from 192 × 109/l to 332 × 109/l. Eosinophilic was unequivocally detected from both peripheral blood and granulocytes in the differential count decreased from 83 to bone marrow in a typical case of acute eosinophilic granulo- 64%. Twenty days after treatment tenderness of the sternum cytic leukemia that achieved complete remission after oral disappeared. Then, there was a gradual but steady decrease administration of ATRA without any exposure to cytotoxic in the leukocyte count and percentage of eosinophils in the agents. Owing to the blank control and normal control repeat- peripheral blood. Seventy days after treatment, bone marrow edly being negative, the possibility of contamination could be examination showed the cellularity had returned to normal excluded. In addition, the detection of RAR alpha-PML tran- with a G/E ratio of 3.52:1, eosinophilic promyelocytes script indicated that the abnormality of chromosomes was a decreased from 14.5 to 0.5%, eosinophilic myelocytes and reciprocal or a balanced translocation. Recently, Takatsuki et metamyelocytes fell from 12.5 and 10.0% to 1.5%, eosino- al6 found that PML-RAR alpha presented not only in CFU-GM, philic band-formed granulocytes decreased from 14.0 to but also in BFU-E. Altogether, these facts strongly suggest that 2.5%, and eosinophilic-segmented granulocytes from 26.0 to the formation of PML-RAR alpha fusion gene occurs at the 15%. Auer rods could no longer be found. One hundred and developmental stage prior to the progenitor cell for promyelo- three days after continuous oral administration of ATRA, the cyte, namely at the level of pluripotent stem cells.7 In the peripheral blood and bone marrow achieved complete main, transformed cells developed into an acute promyelo- remission. Next, three intensive courses of DAH protocol cytic leukemia, but in certain situations they could also (daunorubicin, cytosine arabinoside, harringtonine) were advance toward acute eosinophilic leukemia, although the given for consolidation. precise mechanism of distinct differentiation remains to be In June 1995, the patient was quite well with a hemoglobin elucidated. concentration of 121 g/l, leukocyte count of 4.4 × 109/l and The dosage of ATRA given to the patient was 30 mg every platelet count of 200 × 109/l. The differential count of leuko- 6 h. It corresponded to 68 mg/m2 and was much higher than cytes in the periperhal blood was normal with 1.0% eosino- the usual dose. The reason for doing so was that at the begin- philic-segmented granulocytes. Bone marrow examination ning we were really not certain if ATRA would work in that showed normal cellularity with a G/E ratio of 3.51:1. The particular case. To our surprise, that patient not only nucleated cells in all series were proportionally and morpho- responded but did not even show hyperleukocytosis at day logically normal. 10–20 after ATRA treatment. Individual discrepancy might Cytogenic study showed 46,XX. However, owing to the play a role in that situation. Another important possibility was unsatisfactory preparation of chromosome, no numerical and that most of the abnormal cells in the bone marrow and per- structural abnormality of chromosome could be found with G ipheral blood were relatively well differentiated, therefore banding analysis. they might not be prone to further division. Total RNAs were extracted from the peripheral blood and bone marrow using a one-step guanidine-thiocyanate-phenol- chloroform method. References RT-PCR was performed according to the procedures in our previous paper.5 From both peripheral blood and bone mar- 1 Kakizuka A, Miller WH Jr, Umesona K, Warrell RP Jr, Framkel SR, row samples, an L-type PML-RAR alpha transcript could be Murty VVVS, Dmitrovsky E, Evans RM. Chromosomal translo- definitely detected, which is shown in Figure 2. In the mean- cation t(15;17) in human acute promyelocytic leukemia fuses RAR alpha with a novel putative transcription factor, PML. Cell 1991; while a reverse juxtaposing product of translocation, RAR 66: 663–674. alpha-PML fusion gene could also be found. 2 de The H, Lavau C, Marchio A, Chomienne C, Degos L, Dejean A. The PML-RAR alpha fusion mRNA generated by the t(15;17) translocation in acute promyelocytic leukemia encodes a func- Discussion tionally altered RAR. Cell 1991; 66: 675–684. 3 Chen SJ, Chen Z, Chen A, Tong JH, Dong S, Wang Z, Waxman S, Zelent A. Occurrence of distinct PML-RAR alpha fusion gene The patient experienced typical manifestation of acute leuke- isoforms in patients with acute promyelocytic leukemia detected mia with rather sudden onset. There was a marked increase by reverse transcriptase-polymerase chain reaction. Oncogene of eosinophilic granulocytes in addition to the emergence of 1992; 7: 1223–1232. immature eosinophils in the peripheral blood. The bone mar- 4 Geng JP, Tong JH, Dong S, Wang ZY, Chen SJ, Chen Z, Zelent A, row was hypercellular with a pronounced augmentation of Berger R, Larsen CJ. Localization of the chromosome 15 break- eosinophilic granulocytes of all stages.