I kemia (CML), the blast cell type is usually of neutrophi1

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1 Promyelocytic Blast Crisis of Chronic Myelocytic Leukemia With Both t(9;22) and t(l5; 17) in M3 Cells SYLVIE CASTAIGNE, MD,' ROLAND BERGER, MD,t VERONIQUE JOLLY, MD,' MARIE THERESE DANIEL, MD,* ALAIN BERNHEIM, MD,t MICHEL MARTY, MD,' LAURENT DEGOS, MD," AND GEORGES FLANDRIN, MDS A blast crisis with the features of promyelocytic leukemia (M3) occurred during the evolution of chronic myelocytic leukemia (CML) with the t(9;22) translocation. This rare form of transformation was confirmed by means of cytologic and electron microscopic examination. Cytogenetic studies showed two simultaneous translocations t(15;17) and t(9;22) in the promyelocytes. After intensive chemotherapy, a complete remission was obtained and only karyotypes with t(9;22) translocation were present. These data confirm the specificity of the t(15;17) translocation in malignant promyelocytic proliferation and provide evidence for a second genetic event in the genesis of blast crisis occurring in a committed cell belonging to the abnormal population defined by the Phl chromosome. Cancer 54: , N THE TRANSFORMATION Of chronic myelocytic leu- I kemia (CML), the blast cell type is usually of neutrophi1 origin, and less frequently of lymphoid, erythroid, or megakaryocytoid origin. We have had the opportunity to observe a case of typical acute promyelocytic leukemia (FAB M3) supervening during the course of CML and to perform precise morphologic and cytogenetic analyses of both the CML and M3 cell populations. This study provides evidence for a two-step event in the genesis of blast crisis of CML. Case Report A 37-year-old white man was referred to the department of Hematology of Saint-Louis Hospital, Paris, in April, with a 4-month history of weakness, nocturnal sweats, and weight loss. He had a 20 year history of schizophrenia controlled with neuroleptics. Physical examination showed splenomegaly 12 cm below the costal margin. A full blood count showed a hemoglobin level of 13.5 g/dl, leukocyte count of 218,000/mm3 with a * Institut de Recherches sur les Maladies du Sang (Directeur: Professor Boiron), Hopital Saint-Louis, Pans, France. t Laboratoire de Cytog&n&tique, Hopital Saint-Louis, Pans, France. $ Laboratoire d'h&matologie, Hopital Saint-Louis, Pans, France. Address for reprints: Sylvie Castaigne, MD, Institut de Recherches sur les Maladies du Sang, Hopital Saint-Louis, 2 Place du Dr. A. Fournier, Pans, France. Accepted for publication October 18, differential of 35% segmented neutrophils, 2% eosinophils, 3% basophils, 27% metamyelocytes, 24% myelocytes, 2% promyelocytes, 1 % myeloblasts and 2 nucleated/ 100 leukocytes. The platelet count was 255,000/mm3. A bone marrow aspirate was hypercellular with myeloid hyperplasia. Megacaryocytes were increased. A diagnosis of chronic myelocytic leukemia was made, and chemotherapy was started with busulfan. After 8 months of treatment, the leukocyte count was 34,000 and the spleen had diminished to 4 cm below the costal margin. In January, 1982, the patient was referred with fever and hematuria. The hemoglobin level was 6.6 gjdl and the leukocyte count was 52,900/mm3 with a differential of 14% segmented neutrophils, 1% lymphocytes, I % eosinophils, I % metamyelocytes, and 83% blasts. The blast cells were identified cytologically as promyelocytes from the variant form of M3 in the FAB classification.' The bone marrow aspirate showed increased cellularity with 50% abnormal promyelocytes with numerous bundles of Auer rods (faggot cells). No megakaryocytes were seen. The platelet count was 4 1,000. Coagulation studies showed disseminated intravascular coagulation with prothrombin consumption 44%, factor V 51%, fibrinogen 0.70 g/l, and fibrinogen degradation products 100 mmg/l. Therapy was started with heparin (1 mg/kg), platelet transfusions, and high-dose chemotherapy using daunorubicin ( 100 mg/m2/day) for 3 days and cytosine arabinoside (200 mg/m2/ day) for 5 days. By the 12th day after initiation of therapy, hemostasis was normal and bone marrow was h ypocellular. 2409

2 2410 CANCER December I 1984 VOl. 54 therapy, cutaneous lesions increased, and the patient died of sepsis with pancytopenia in July, Results FIG. I. Peripheral blood. Characteristic faggot cells, with bundles of Auer rods (Wright-Giemsa, X640). One month later, the bone marrow was poorly cellular with granulocytic differentiation and few megakaryocytes, but without a blast cell component. In May, 1982, the patient received a consolidation course of daunorubicin and cytosine arabinoside. In June, some papules appeared on the limbs and the trunk. Examination of a skin biopsy specimen demonstrated dermal infiltration by abnormal promyelocytes. At this time, the bone marrow aspirate showed relapse with an excess af abnormal promyelocytes and absence of megakaryocytes. The patient was treated with 6-mercaptopunne (6-MP) and VP-16 (etoposide). Despite FIG. 2. Peripheral blood. Abnormal blast cell with folded nucleus (M3 variant blast cell type) (Wright-Giemsa, X640). Cytologic Findings At the time of blast crisis, the peripheral blood blast cells were large, with irregular, bilobed nucleus. They frequently had a clearly visible nucleolus and abundant cytoplasm with few fine, dust-like azurophilic granules. A few cells contained bundles of faggot cells (Fig. 1). In the peripheral blood films, these features were compatible with the appearance of the M3 variant (Fig. 2), whereas the bone marrow blast cells were more closely related to the classic M3 features.2 Neutrophilic cells at various stage of maturation and few erythroblasts were seen; megakaryocytes were absent (Fig. 3). Myeloperoxidase and chloracetate esterase reactions were strongly positive in the blasts of the peripheral blood and the bone marrow. Ultrastructural studies showed hypergranulated cells typical of M3 with large, folded nuclei and abundant primary granules of variable size scattered throughout the cytoplasm (Fig. 4). The characteristic crystalized structure of faggot cells was found (Fig. 4, Znset). Cytogenetic Studies Chromosome studies were performed on direct preparation samples and/or on cultures (24 and 48 hours) of blood and bone marrow. RHG and CBG banding techniques were applied (Table 1). A first chromosome analysis was performed at diagnosis, before any treatment, from unstimulated blood cell cultures incubated for 24 and 48 hours. A11 37 mitoses examined had a common Philadelphia (Ph ) chromosome resulting from a t(9;22) translocation without other anomalies (Figs. 5A and 5B). A second chromosomal study was performed in the blast crisis of the disease and before chemotherapy for acute leukemia (February 2, 1982). Three types of metaphases were observed: 46, XY, t(9;22); hyperdiploid with Phl and 46, XY, t(9;22), and t(15;17). The last clone thus had the two translocations for chronic granulocytic leukemia (CGL) and for acute promyelocytic leukemia (Figs. 5A and 5B). Cytologic study of bone marrow mitoses (Fig. 6) from the same samples as those used for cytogenetic study revealed the nature of the dividing cells; in the direct sample, 38 promyelocytes, 34 erythrocytes, and 3 neutrophilic cells were present, whereas in cultured bone marrow the only cells in mitoses were promyelocytes ( 16 cells).

3 No. 11 M3 PROMYELOCYTIC BLAST CRISIS - Castaigne et al FIG. 3. Bone marrow. Hypergranular promyelocytes and maturing neutrophilic cells (Wright- Giemsa, X640). A third chromosomal study was performed during the remission (March 22, 1982), on bone marrow cells after 48-hour culture. The t(9;22) translocation was present in all 23 metaphases studied, and chromosomes 15 and 17 were normal.3a During relapse of disease, two chromosomal studies were performed: one showed only metaphases with both translocations, and the other showed both translocations and two extra chromosomes, 8 and?f. myelocytic leukemia (APL), since the blast crisis was associated with disseminated intravascular coagulation. However, an unusual feature was the cutaneous relapse, histologically proved by the demonstration of abnormal promyelocytes in the dermis (Fig. 7). Particularly interesting is the correlation between the cytologic and cytogenetic findings. An additional chromosome abnormality was found Discussion The heterogeneity of the blast cells in the transformation of CML is well known. Morphologic appearances resembling almost every type of blast cell found in hemopoietic malignancies have been described. Myeloblastic and lymphoblastic cells are the most common types of blast cells. Promyelocytes with large azurophilic granules in the cytoplasm and/or faggot cells have been ~bserved.~-~ However, promyelocytic transformation seems extremely rare. Alimena et al. in a series of 69 cases of blast crisis of CML found only one case classified as M3. In this case, t( 15; 17) was not reported. Brodsky et af.* reported one patient with M3 features and t(15;17). In the case described here, besides the persistence of erythroblasts and granulocytes at various stages of maturation, the main feature was the proliferation of typical abnormal promyelocytes with the appearance of M3. The clinical features were also typical of acute pro- FIG. 4. Electron microscopic study of the ultrastructural feature of a hypergranular blast cell. Many granules and sections of Auer rods (arrow) (X6750). Inset: Detail of transversal section of Auer rod with characteristic tubular pattern.

4 2412 CANCER December Vol. 54 TABLE 1. Karyotype and Mitose Numbers 46, t(9;22) Hyperdiploid 48, t(9;22) Date Material 46, t(9;22) t(15;17) + Ph 1 t(15;17), Blood (unstimulated) I982 Bone marrow direct Bone marrow 48 hr culture hr unstimulated blood culture I982 Bone marrow 48 hr culture hr unstimulated blood culture hr unstimulated blood culture hr unstimulated blood culture 5 3 during the blast crisis, a t(15;17) translocation that is thought to be specific to acute promyelocytic leukemia and has never been reported in other types of acute le~kemia.~ Comparison of cytogenetic and cytologic features of dividing cells either in direct or in cultured bone marrow samples suggest that metaphases having both translocations t( 15; 17) and t(9;22) were those of promyelocytes (M3 cells). Metaphases having only Ph' could be those of immature neutrophils and erythroblast^.^,'^ When the remission was obtained, the t( 15; 17) disappeared, although the Ph' persisted in bone marrow cultured cells. When the acute phase recurred, t( 15; 17) was again found in blood cells, in addition to a new abnormality, probably a trisomy 8. The existence of a t(15;17) and t(9;22) in a patient with M3 type of acute transformation of CML confirms the specificity of this translocation. As with de novo M3, the t( 15; 17) was mainly observed in abnormal promyelocytes, excluding erythrobla~ts~~'~; this translocation was not observed during remission, although it reappeared at the time of relapse. The t( 15; 17) was evidently a secondary chromosomal event, the primary one being the Ph', since only promyelocytes had both translocations, whereas all other dividing cells had only the common Ph' chromosome. These data reinforce the hypothesis that CML blast crisis may result from a second genetic event occurring in a committed cell belonging to the monoclonal abnormal population defined by the Ph' chromosome.'' FIG. 5A AND 5B. Partial karyotypes (RHG bands). (A) Chronic phase: t(9;22), and normal 15 and 17. Rearranged chromosomes are shown by triangles. (B) Blastic phase: t(9;22), t( 15;17). Rearranged chromosomes are shown by triangles.

5 No. I1 M3 PROMYELOCYTIC BLAST CRISIS - Castaigne et al FIG. 6. Bone marrow. Mitosis of abnormal promyelocytes (arrow) and mitosis of erythroblasts (double arrow) (Wright-Giemsa, X640). FIG. 7. Plastic-embedded section of a skin biopsy specimen. Notice the large blast cells with folded nucleus (H & E, X400). REFERENCES 1. Bennett JM, Catovsky D, Daniel MT et a1 A variant form of hypergranular promyelocytic leukemia (M3). Br J Huematol 1980; 44: Bennett JM, Catovsky D, Daniel MT et al. Proposals for the classification of the acute leukemia. Br J Huematol 1976; 33: Berger R, Bernheim A, Daniel MT, Valensi F, Flandrin G. Cytological types of mitoses and chromosome abnormalities in acute leukemia. Leukemia Res 1983; 7: a. Berger R, Bernheim A, Daniel MT, Flandrin G. t(15:17) in a promyelocytic form of chronic granulocytic leukemia blastic crisis. Cancer Genet Cytoget 1983: 8: Ben-Zeev D, Scwartz SO, Friedman IA. Promyelmytic-myelmytic leukemia as a terminal manifestation of chronic granulocytic leukemia: Report of a case. Blood 1966; 27: Mayer S, Falkenrodt A, Albert A er a[. Corps d Auer et chromosome Ph I : pseudo leuctmies myeloides chroniques? Nouv Rev Fr Hematol 1982; 24: Shaw MT, Bottomley RH, Grozea PN. Heterogeneity of morphological, cytological and cytogenetics features in the blastic phase of chronic granulocytic leukemia. Cancer 1975; 35: Alimena G, Dallapicolla B, Castaldi R et al. Chromosomal, morphological and clinical correlations in blastic crisis of chronic myeloid leukemia: A study of 69 cases. Scand J Haematol 1982; 28: Brodsky I, Fuscaldo KE, Strayer DR, Kahn SB, Contoy JF. Management of chronic myelocytic leukemia: Aggressive therapy (Abstr). Blood 1981; 58(Suppl. 1):135a. 9. Rowley JD, Golomb HM, Dougherty C Translocation: A consistent chromosome change in acute promyelmytic leukemia. Lancet 1977; i: Berger R, Bernheim A, Flandrin G. Absence d anomalies chromosomiques et leucemie aigue: relations avec les cellules mtdullaires normales. C R Acad Sci Paris : Bernheim A, Berger R, Preud homme JL, Labaume S, Bussel A, Barot-Ciorbaru R. Philadelphia chromosome positive blood B lymphocytes in chronic myelocytic leukemia. Leukemia Res 198 1; 5: