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Double Minutes and MYC Amplification A Combined May-Grünwald Giemsa and Fluorescence In Situ Hybridization Study K. F. Wong, MD, Lisa L. P. Siu, PhD, and W. S. Wong, MBBS From the Department of Pathology, Queen Elizabeth Hospital, Hong Kong, China. Key Words: Hematopathology; Genetics; Fluorescence in situ hybridization DOI: 10.1309/AJCPWUBGT7C0LHIN

ABSTRACT Objectives: To report the demonstration of double minutes with MYC amplification in a case of myeloproliferative neoplasm with monocytosis in transformation by a combination of standard karyotyping and interphase and metaphase fluorescence in situ hybridization (FISH). Methods: To determine the lineage involvement, we applied combined morphology and an interphase FISH study using dual-color break-apart probes for MYC on peripheral blood film. Results: MYC amplification was demonstrated in both myeloid and monocytic cells but not lymphocytes. The MYC amplification was not associated with loss of MYC signals at the homologous 8q24 regions where the genes were located. Furthermore, the extent of MYC amplification has been shown to diminish as the granulocytes mature. Conclusions: Combined morphology and FISH study has shown a pluripotent myeloid disorder and also an inverse relationship between cell maturity and MYC amplification.

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A case of probable chronic myelomonocytic leukemia (CMML) was found to have multiple clonally occurring double minutes on standard karyotyping. Double minutes are rarely found in hematologic malignancies, but when present, they are associated with an adverse clinical outcome.1 The MYC gene, situated at 8q24, has been reported to be the most frequently amplified region in hematologic malignancies with double minutes.2,3 MYC amplification has been demonstrated in the present case by interphase and metaphase fluorescence in situ hybridization (FISH) using the dual-color break-apart rearrangement probes for MYC. We have further explored the application of combined morphology and FISH study to determine the lineage involvement and the relationship between the extent of MYC amplification and cell maturity.

Case Report An 86-year-old woman presented with fever and palpitation. Clinical examination was unremarkable, and there was no lymphadenopathy or organomegaly. Peripheral blood counts were as follows: hemoglobin, 5 g/ dL; platelets, 37 ×109/L; and leukocytes, 222.4 ×109/L. Examination of the blood film showed a constellation of granulocytes and monocytes in their mature and immature forms. Some cells contained micronucleus-like structures ❚Image 1❚. A 200-cell differential count showed the following: neutrophils, 32%; lymphocytes, 4%; monocytes, 12%; eosinophils, 1%; metamyelocytes, 5%; myelocytes, 39%; promyelocytes, 3%; and blasts, 4%. Some giant platelets were found. A diagnosis of CMML in transformation © American Society for Clinical Pathology

AJCP / Case Report

was suspected. In view of the patient’s age, however, further investigations such as bone marrow aspiration were declined. Conventional cytogenetics performed by overnight fluorodeoxyuridine‑synchronized culture of the peripheral blood showed the presence of one to nine clonally occurring double minutes, 46,XX,1~9dmin[17], and more than 40 double minutes could be observed in isolated metaphases ❚Image 2❚. Reverse transcription–polymerase chain reaction for BCR-ABL1 was negative.

monocytoid cells) but was not found in small lymphocytes, which showed two MYC signals. Some segmented neutrophils also harbored more than two MYC signals, but the extent of MYC amplification (signified by the number of MYC signals) in mature granulocytes was much less compared with the morphologically less mature cells ❚Image 4❚. The micronuclei found in some of the immature myeloid cells were also shown to contain MYC signals ❚Image 5❚.

Materials and Methods FISH Interphase and metaphase FISH with dual-color breakapart rearrangement probes for MYC (with SpectrumOrange-labeled 5′ and SpectrumGreen-labeled 3′ regions; Vysis, Abbott Park, IL) was performed on the cytogenetic preparation. Inverted 4′,6-diamidino-2-phenylindole (DAPI) images on metaphases were used to confirm the location of the MYC signals. Combined Morphology and FISH A sequential morphology and FISH study was performed on unmanipulated peripheral blood cells according to standard protocols.4,5 Briefly, the peripheral blood smears were first stained with May-Grünwald Giemsa (MGG) and examined under a light microscope. Selected nucleated blood cells were located with the coordinates recorded and their images were captured by digital camera. The smears were then rinsed with methanol/acetic acid for destaining and followed by pretreatment with protease. They were then processed for interphase FISH with the dual-color break-apart rearrangement probes for MYC. The cells were relocated and their fluorescence signals were studied and captured.

❚Image 1❚ Peripheral blood showing a constellation of granulocytes and monocytes in their mature and immature forms. A promonocyte had two micronuclei in its cytoplasm (arrow) (May-Grünwald Giemsa, ×1,000).

Results Interphase FISH showed MYC amplification with more than two MYC signals in 185 (92.5%) of 200 cells counted. Most cells had over five MYC signals. Metaphase FISH with inverted DAPI banding showed that the MYC amplification was confined to the double minutes. Normal MYC signals (without break-apart signals) were detected in the two apparently normal homologs of chromosome 8 with ish 8q24(MYCx2),dmin(MYCx1~43)[20] ❚Image 3❚. The combined morphology and FISH study on the intact nucleated blood cells showed that the MYC amplification was confined to myeloid cells (including monocytes and immature © American Society for Clinical Pathology

❚Image 2❚ Metaphase spread showing more than 30 double minutes in an otherwise normal karyotype. G-band with trypsin Giemsa (×1,000).

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❚Image 3❚ A composite image showing (A) the presence of MYC signals in the double minutes and (B) the two normal homologs of chromosome 8 (arrows). Metaphase fluorescence in situ hybridization with dual-color break-apart MYC rearrangement probes (Vysis) and inverted 4′,6-diamidino-2-phenylindole (×1,000).

Discussion Double minutes are small paired acentric chromatin structures that reflect extra-chromosomal gene amplification. They are found in less than 1% of acute myeloid leukemia (AML), mostly in elderly female patients with poor survival.1,6 It has been shown that the MYC gene, situated at 8q24, was the most frequently amplified region in cases of AML and myelodysplastic syndrome with double minutes detected by standard karyotyping. Some cases have been shown to be associated with loss of the MYC signal in chromosome 8,1,7 leading to the postulation that the MYC gene was excised and amplified to form double minutes. MYC amplification has been associated with disease progression and poor prognosis. In this report, we described the occurrence of double minutes as the sole cytogenetic abnormality in an elderly woman with probable CMML in transformation. CMML is a clonal hemopoietic stem cell neoplasm, but unlike chronic myelogenous leukemia, it involves only the myeloid lineage and not the lymphoid cells.8 To our knowledge, double minutes have been reported only in two patients with CMML, which is also the only non-AML that has been described with double minutes as an isolated change.9,10 This has thus prompted our group to perform a combined MGG and FISH study. The combined MGG and interphase FISH study was first described two decades ago,4 but its clinical utility has not been fully realized. The method, which allows direct correlation between morphologically identified cells and MYC amplification without the need of culturing and 282 282

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immunophenotyping, is a sensitive and specific technique for (1) identification of cell lineage involvement by specific genetic abnormalities and (2) cytogenetic characterization of morphologically atypical cells or rare blast cells. The combined MGG and FISH study has confirmed the presence of MYC amplification in both myeloid and monocytic cells but not in the lymphocytes in the present case. Extrusion of the double minutes and the associated MYC amplification in the form of micronuclei was also observed. Since the neutrophils contained fewer double minutes, it is apparent that the neoplastic clones with more double minutes have failed to further mature and remain as blasts, whereas clones with fewer double minutes (and therefore less advanced in clonal evolution) still retain the capability to mature. This is contrary to a previous report that failed to demonstrate any relationship between supernumerary chromosome (trisomies 8 and 9) and cellular maturation.5 Our findings thus concur with a previous report that expulsion of the amplified MYC gene in the form of micronuclei was associated with reduced tumorigenicity and phenotypic reversion.11 In view of the inverse relationship between cell maturity and MYC amplification, it is tempting to speculate that targeted therapy against the MYC gene may be useful in cases of myeloid neoplasms with double minutes as the sole abnormality that is associated with MYC amplification. In fact, it has been shown in the HL-60 cell line that loss of MYC copies after drug treatment was associated with differentiation to granulocytes.12

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❚Image 4❚ A composite image showing (A, C) MYC amplification in myeloid cells but not in small lymphocytes and (B, D) reduced MYC signals with cell maturation. Combined May-Grünwald Giemsa and interphase fluorescence in situ hybridization with dual-color break-apart MYC rearrangement probes (Vysis) (×1,000). A

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❚Image 5❚ A composite image showing (A) a promonocyte with micronuclei (arrow) harboring (B) MYC signals. Combined May-Grünwald Giemsa and interphase fluorescence in situ hybridization with dual-color break-apart MYC rearrangement probes (Vysis) (×1,000). © American Society for Clinical Pathology

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Address reprint requests to Dr K. F. Wong: Dept of Pathology, Queen Elizabeth Hospital, 30 Gascoigne Rd, Kowloon, Hong Kong SAR, China; [email protected].

References 1. Thomas L, Stamberg J, Gojo I, et al. Double minute chromosomes in monoblastic (M5) and myeloblastic (M2) acute myeloid leukemia: two case reports and a review of literature. Am J Hematol. 2004;77:55-61. 2. Rayeroux KC, Campbell LJ. Gene amplification in myeloid leukemias elucidated by fluorescence in situ hybridization. Cancer Genet Cytogenet. 2009;193:44-53. 3. Fugazza G, Bruzzone R, Puppo L, et al. Amplified c-MYC sequences localized by fluorescence in-situ hybridization on double minute chromosomes in acute myeloid leukemias. Leuk Res. 1997;21:703-709. 4. van Lom K, Hagemeijer A, Smit EM, et al. In situ hybridization on May-Grünwald Giemsa–stained bone marrow and blood smears of patients with hematologic disorders allows detection of cell-lineage-specific cytogenetic abnormalities. Blood. 1993;82:884-888. 5. Fugazza G, Bruzzone R, Dejana AM, et al. Cytogenetic clonality in chronic myelomonocytic leukemia studied with fluorescence in situ hybridization. Leukemia. 1995;9:109-114. 6. Marinello MJ, Bloom ML, Doeblin TD, et al. Double minute chromosomes in human leukemia. N Engl J Med. 1980;303:704.

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7. Bruyère H, Sutherland H, Chipperfield K, et al. Concomitant and successive amplifications of MYC in APL-like leukemia. Cancer Genet Cytogenet. 2010;197:75-80. 8. van Kamp H, Fibbe WE, Jansen RP, et al. Clonal involvement of granulocytes and monocytes, but not of T and B lymphocytes and natural killer cells in patients with myelodysplasia: analysis by X-linked restriction fragment length polymorphisms and polymerase chain reaction of the phosphoglycerate kinase gene. Blood. 1992;80:1774-1780. 9. Sambani C, Trafalis DT, Vessalas G, et al. Trisomy 6 and double minute chromosomes in a case of chronic myelomonocytic leukemia. Cancer Genet Cytogenet. 1998;106:180-181. 10. Paulsson K, Lassen C, Kuric N, et al. MYC is not overexpressed in a case of chronic myelomonocytic leukemia with MYC-containing double minutes. Leukemia. 2003;17:813-815. 11. Villa O, Salido M, Pérez-Vila ME, et al. Blast cells with nuclear extrusions in the form of micronuclei are associated with MYC amplification in acute myeloid leukemia. Cancer Genet Cytogenet. 2008;185:32-36. 12. Shima H, Nakayasu M, Aonuma S, et al. Loss of the MYC gene amplified in human HL-60 cells after treatment with inhibitors of poly(ADP-ribose) polymerase or with dimethyl sulfoxide. Proc Natl Acad Sci U S A. 1989;86:7442-7445.

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Double minutes and MYC amplification: a combined May-Grunwald Giemsa and fluorescence in situ hybridization study.

To report the demonstration of double minutes with MYC amplification in a case of myeloproliferative neoplasm with monocytosis in transformation by a ...
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