* The Philadelphia (Ph) chromosome [der(22) t(9;22) (q34;q11)] is the characteristic chromosomal abnormality found in chronic myelogenous leukemia (CML). This chromosome has been reported in patients with other chromosomal abnormalities. In this study, we describe a patient with hematologically typical chronic-phase CML with an unusual and complex translocation involving chromosomes 9, 11, and 22. These complex translocations were identified by G-banded conventional cytogenetics and confirmed by fluorescence in situ hybridization (FISH) using whole chromosome painting probes (wcp). To the best of our knowledge, these are unique translocations involving the short and the long arms of chromosome 9 in 4 different translocations with the short arm of chromosome 11 and the long arm of chromosome 22.
(Arch Pathol Lab Med. 2001;125:437-439)
The Ph chromosome [der(22) t(9;22)(q34;q11)] is the characteristic chromosomal aberration in CML and was first described by Nowell and Hungerford in 1960.1 Later, Rowley2 demonstrated the precise translocation between chromosome 9 and 22. The c-abl proto-oncogene is translocated from chromosome 9 to the breakpoint cluster region on chromosome 22.(3,4) The c-abl and breakpoint cluster genes are juxtaposed in a head-to-tail configuration, creating a chimeric gene that is transcribed to an 8.5-- kilobase mRNA and resulting in up-regulated tyrosine kinase activity.5 Additional chromosomal abnormalities of clonal evolution precede the development of the blastic or acute phase in 70% to 80% of CML cases. Cytogenetic abnormalities that occur in addition to the Ph chromosome at the time of initial diagnosis have not been associated with a poor prognosis.6 We describe a case of typical CML with unusual and, to the best of our knowledge, unique complex translocations involving chromosomes 9,11, and 22.
CASE REPORT
In January 2000, a 39-year-old asymptomatic man was found to have an elevated white blood cell count of 35 x 10^sup 9^/L on routine blood examination. A bone marrow aspiration and biopsy were performed, followed by conventional cytogenetics. A diagnosis of CML was made. The patient received hydroxyurea to reduce his white blood cell count. Three months after the initial diagnosis, the patient was referred to the M. D. Anderson Cancer Center for additional treatment options. Complete blood count, bone marrow aspiration, and biopsy with cytogenetic analysis were repeated. The cytogenetic and fluorescence in situ hybridization (FISH) studies are reported in detail.
MATERIALS AND METHODS
Cytogenetic analysis was done on a 24-hour culture of a bone marrow specimen. The cells were cultured by conventional methods and processed by standard techniques. The chromosomes were banded with a trypsin-Giemsa technique (GTG or G banding).7
The FISH chromosomal studies were performed according to the manufacturer's directions. To delineate the nature of the complex translocations, the FISH technique using whole chromosome painting probes (wcp) for chromosomes 9 (wcp9, SO/DAPI [spectrum orange/4',6-diamidino-2-phenylindole dihydrochloride], Vysis), 22 (wcp22, SO/DAPI, Vysis), and 11 (wcp11, fluorescein isothiocyanate/propidium iodide, American Laboratory Technology, Inc) was applied. Metaphases were photographed using a Zeiss fluorescence microscope (Zeiss, Germany) equipped with the appropriate filter sets.
RESULTS
The complete blood count revealed a hemoglobin concentration of 136 g/L, a hematocrit of 0.43, a platelet count of 150 x 10^sup 9^/L, and a white blood cell count of 11.1 x 10^sup 9^/L. The white blood cell differential revealed the following values: 0.63 segmented neutrophils; 0.02 band neutrophils; 0.13 lymphocytes; 0.15 monocytes; 0.02 basophils; 0.03 myelocytes; and 0.02 metamyelocytes. No blasts were seen in the peripheral blood.
The bone marrow aspirate was hypercellular with granulocytic hyperplasia. The megakaryocytes were normal. The differential on 500 cells revealed the following values: 0.01 myeloblasts; 0.01 promyelocytes; 0.15 myelocytes; 0.28 metamyelocytes; 0.40 granulocytes; 0.02 eosinophils; 0.01 basophils; 0.01 lymphocytes; 0.01 monocytes; and 0.09 normoblasts. The myeloid-erythroid ratio was 9.8:1. The clinicopathologic findings were diagnostic of CML in chronic phase.
Cytogenetic analysis of 20 metaphases derived from an unstimulated culture of bone marrow cells using GTG banding revealed the modal number of chromosomes to be 46 per cell, with a male sex constitution and apparent complex translocations involving chromosomes 9, 11, and 22. The cytogenetic diagnosis from the GTG banding study was as follows: 46,XY,der(9)t(9;11)(p13;p13)t(9;11)(q34;p13)t(9;22)(p13; q11)t(9;22)(q34;q11), der(11)t(9;11)(p13;p13)t(9;11)(q34;p13), der(22)t(9;22)(p13;q11)t(9;22)(q34;q11).
The FISH technique was used as an adjunct to conventional cytogenetic studies for confirmation of the results. The wcp probes were used to determine the exact nature of the complex rearrangements (Figure).
The FISH analysis revealed complex rearrangements involving the breakage and reunion of the 3 chromosomes in complex translocations as follows: ish der(9)t(9;22)(p13; q11)(wcp22+)t(9;11)(q34;p13)(wcp11+), der(11)t(9;11)(p13; p13)(wcp9+), der(22)t(9;22)(q34;q11)(wcp9+).
Since diagnosis, the patient's leukocytosis has responded to chemotherapy and he is waiting to receive a bone marrow transplant from an HLA-matched donor.
COMMENT
Involvement of chromosome 9 in Ph-positive CML patients is well known. The translocations noted here are, to the best of our knowledge, unique because they involved both arms of chromosome 9 in 4 different translocations. If this patient had been diagnosed with Ph-positive CML and subsequently developed a change in karyotype, his prognosis would be poor. However, these complex translocations occurred in the context of chronic-phase CML, and their clinical significance is unclear. It is important to note clonal evolution (ie, additional numerical or structural chromosomal abnormalities) in patients with CML because 50% of patients enter an accelerated phase after about 3 years. This phase is characterized by increasing anemia, thrombocytopenia, additional cytogenetic abnormalities, and eventual transformation to acute leukemia.8
This patient's leukocytosis has responded to initial therapy. The prognostic significance associated with these translocations is not yet apparent. Significant associations have been reported between cytogenetic responses of Ph-- positive CML and remission duration or survival.9 It is not possible to determine if these complex genetic translocations represent clonal evolution or a unique, initial presentation variant of the Ph chromosome. This study demonstrates how multiple cytogenetic techniques, such as conventional cytogenetics and FISH, are useful for specifically identifying chromosomal abnormalities in complex karyotypes.
This work was supported in part by the Olla Stribling Chair Fund for cancer research. The authors wish to recognize and thank the professional efforts of Kimberly Hayes, CLSp(CG), Lian Zhao, CLSp(CG), cytogenetics technologists, secretarial assistance of Pinkie Davis, Kris Smith, and clinical colleagues of the University of Texas MD Anderson Cancer Center.
References
1. Nowell PC, Hungerford DA. A minute chromosome in human granulocytic leukemia. Science. 1960;132:1497.
2. Rowley JD. A new consistent chromosomal abnormality in chronic myelogenous leukemia identified by quinacrine fluorescence and Giemsa staining. Nature. 1973;243:290-293.
3. de Klein A, Geurts Van Kessel A, et al. A cellular oncogene is translocated to the Philadelphia chromosome in chronic myelocytic leukemia. Nature. 1982; 300:765-767.
4. Groffen J, Stephenson JR, Heisterkamp N, de Klein A, Bartram CR, Grosveld G. Philadelphia chromosomal breakpoints are clustered within a limited region, bcr, on chromosome 22. Cell. 1984;36:93-99.
5. Shtivlman E, Gale RP, Dreazen O, et al. bcr-abl RNA in patients with chronic myelogenous leukemia. Blood. 1987;69:971-973.
6. Hild F, Fonatsch C. Cytogenetic peculiarities in chronic myelogenous leukemia. Cancer Genet Cytogenet. 1990;47:197-217.
7. Barch MJ. The Association of Cytogenetic Technologists Laboratory Manual. New York, NY: ACT, Inc, Raven Press; 1991:213-223.
8. Baccarani M for the Italian Cooperative study group on chronic myelogenous leukemia. Prognostic study of alpha-interferon (INF)-treated chronic myelogenous leukemia (CML) patients: II. Predicting survival. In: Proceedings of the 2nd International Conference on Chronic Myelogenous Leukemia, Bologna, Italy, 1992:163.
9. Hernandez J. White cells, lymph node and spleen. In: Cotran RS, Kumar V, Robbins SL, eds. Robbins Pathologic Basis of Disease. 5th ed. Philadelphia, Pa: WB Saunders Company; 1994:272, 654-655.
Accepted for publication August 8, 2000.
From the Department of Clinical Cytogenetics, Division of Pathology and Laboratory Medicine, Department of Hematopathology, The University of Texas, M. D. Anderson Cancer Center, Houston, Tex.
Reprints: Armand B. Glassman, MD, Olla S. Stribling, Distinguished Chair for Cancer Research, Division of Pathology and Laboratory Medicine, Department of Hematopathology Clinical Cytogenetics, Box 350, The University of Texas, M. D. Anderson Cancer Center, Houston, TX 77030 (e-mail: aglassma@mdanderson.org).
Copyright College of American Pathologists Mar 2001
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