Cytometry Part B (Clinical Cytometry) 80B:122–124 (2011)

Flow Cytometric Detection of DegranulatedBasophils in Chronic Myeloid Leukemia in

Accelerated Phase

Alessandra Stacchini,1* Anna Demurtas,1 and Laura Godio2

1Flow Cytometry Unit, Molinette Hospital, Turin, Italy2Laboratory of Pathology, Department of Pathology, Molinette Hospital, Turin, Italy

We report a rare case of chronic myeloid leukemia in accelerated phase with basophilic transforma-tion, in which basophils exceeding 70%, were detectable only by flow cytometry because of their mor-phologic atypicality and degranulation. VC 2010 International Clinical Cytometry Society

Key terms: chronic myeloid leukemia; accelerated phase; basophils; flow cytometry

How to cite this article: Stacchini A, Demurtas A, Godio L. Flow cytometric detection of degranulated basophilsin chronic myeloid leukemia in accelerated phase. Cytometry Part B 2011; 80B: 122–124.

Chronic myeloid leukemia (CML) is a clonal disorderof the pluripotent stem cells (1). The initial indolentphase of CML is unstable and the disease progresses toaccelerated and blast phases that may affect any myeloidlineage. The accelerated phase is characterized by moreaggressive disease, less responsive to therapy; basophilictransformation is a rare but dramatic finding in theaccelerated phase of CML (2). We report a rare case ofCML in accelerated phase, in which basophils in the pe-ripheral blood exceed 70%. The basophils were notidentifiable in blood smears because of morphologic aty-picality and degranulation, but were evident by flowcytometry (FC) having peculiar side scatter propertiesalong with a CD45 and CD123 characteristic expression.

CASE REPORT

A 78-year-old man was initially admitted at MolinetteHospital in November 2006 because of leukocytosis. Onphysical examination, no hepatosplenomegaly was evi-dent. Complete blood count revealed a hemoglobin of7.4 g/dL, a white cell count (WBC) of 27.7 � 109/L, anda platelet count of 174 � 109/L. The differential revealed68% neutrophils, 6% lymphocytes, 6% monocytes, 2%eosinophils, 0% basophils, and 18% immature cells (12%metamyelocytes and 6% myelocytes). The bone marrow(BM) aspirate showed hypercellularity with a markedlyincreased myeloid elements. On biopsy, the BM wasstrikingly hypercellular with pronounced granulocytichyperplasia, thick paratrabecular cuff of immature mye-loid cells, and numerous small hypolobulated

megakaryocytes (dwarf megakaryocytes). The networkof reticulin fibers was minimally increased. The karyo-type of the BM was 46xy, t(9;22)(q34;q11). A diagnosisof CML was posed and treatment with Imatinib wasstarted. He remained in stable condition until April2010, when he suffered of heavy anaemization. Com-plete blood count revealed a hemoglobin of 8.8 g/dL,WBC of 31.55 � 109/L, and a platelet count of 66 �109/L. The differential revealed 80% neutrophils, 1% lym-phocytes, 7% monocytes, 0% eosinophils, 0% basophils,and 12% immature cells. Peripheral blood morphologicinspection evidenced granulocytes with atypical featuresand circulating immature myeloid forms such as meta-(6%), myelo- (2%), pro-myelocytes (2%), and blasts (2%)(Fig. 1). FC evidence a small amount (2%) of circulat-ing myeloblasts, 22% of granulocytes with immatureelements (evaluated by combined expression ofCD66b and CD11b, not shown) and a population withmoderate CD45 expression (dimmer than lymphocytesand brighter than myeloblast) and low-side scatter,exceeding 70% (Fig. 2). Immunophenotypic analysisrevealed high expression of CD123, positivity for

*Correspondence to: Alessandra Stacchini, Department of Pathology,Molinette Hospital, via Santena 7, 10126 Turin, Italy.E-mail: astacchini@molinette.piemonte.itReceived 28 May 2010; Revision 28 July 2010; Accepted 4 August

2010Published online 15 September 2010 in Wiley Online Library

(wileyonlinelibrary.com).DOI: 10.1002/cyto.b.20566

Brief Communication

VC 2010 International Clinical Cytometry Society

CD11b, CD13, CD22 (dim expression), and CD33,

negativity for CD19, CD34, CD117, and HLA-DR, cor-

responding to the basophil’s phenotypic profile (3).

The BM biopsy demonstrated extensive fibrosis, a

blast count increased at 16% and basophil population

not detectable (Fig. 3).

DISCUSSION

According to the guidelines published by the WorldHealth Organization in 2008 (2), one or more of the fol-lowing criteria should be present in the acceleratedphase of CML (1): persistent or increasing WBC andspleen size unresponsive to therapy (2), persistent

FIG. 2. Multicolor flow cytometry of peripheral blood: basophils (identified by CD123, P1 region in red), granulocytes (identified by CD66b, P2region in green), myeloblasts (identified by CD34, P3 region in blue) are back-gated in the SSC/CD45 dot plot. SSC, side scatter; FITC, fluorescein isothio-cyanate; Pe-Cy7, tandem conjugate phycoerithrin-cyanin 7; APC, allophycocyanin; APC-Cy7, tandem conjugate allophycocyanin-phycoerithrin-cyanin 7.[Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

FIG. 1. Peripheral blood smear showing variations in basophils morphology and excess degranulation (arrows); in C, a circulating myeloblast is alsoevident (May-Grunwald-Giemsa stain; A, low magnification; B and C, high magnification). [Color figure can be viewed in the online issue, which isavailable at wileyonlinelibrary.com.]

FC DETECTION OF DEGRANULATED BASOPHILS IN CML 123

Cytometry Part B: Clinical Cytometry

trombocytosis >1000 � 109/L unrelated to therapy (3),persistent trombocytopenia <100 � 109/L unrelated totherapy (4), clonal cytogenetic evolution (5), blood baso-phils >20% (6), and myeloblasts ranging from 10% to19% in the blood or BM. Our patient exhibited bloodbasophils >70% detected by FC and BM blasts count of16%, thereby supporting the diagnosis of acceleratephase. These data in particular support the hypothesisof a transition between accelerated and blastic phase(2). FC was able to easily discriminate basophils frommyeloblast and lymphocytes in peripheral blood, despitemorphology was not as suggestive of the basophils dra-matic increase. The evaluation of immunophenothypicprofile of leukocyte population can aid in the identifica-tion of blasts or basophils, frequently affected by mor-phologic aberrancies in chronic myeloproliferativedisease (4), contributing in the identification of acceler-ated or blastic phase.

In this patient, also extensive collagen fibrosis wasobserved in the accelerated phase. BM fibrosis is a typi-cal complication of CML and a poor prognostic factor(5). What causes marrow fibrosis in CML is unknown,but may be possibly mediated by various cytokines suchas platelet-derived growth factor produced by megakar-yocytes and stimulation of fibroblasts. Elevated levels ofthrombopoietin and its receptor (C-MPL protein) werefound in CML and correlates with thrombocytosis (6). Inour patient, numerous dwarf megakaryocytes were pres-ent at diagnosis and incremented to small clusters in the

accelerated phase. We retain the increased number ofmegakaryocytes responsible of the extensive fibrosisobserved in the accelerated phase; therapy with Imati-nib in this case did not guarantee against evolution ofmarrow fibrosis, as reported by others (7).

In conclusion, we present an unusual CML case inwhich BM biopsy evaluation and FC analysis of bloodleukocytes, both concurred in the identification of theaccelerated phase.

LITERATURE CITED

1. Mughal TI, Goldman JM. Chronic myeloid leukemia: Why does itevolve from chronic phase to blast transformation? Front Biosci2006;11:198–208.

2. Swerdlow H, Campos E, Harris NL, Jaffe ES, Pileri SA, Stein H, ThieleJ, Wardiman JW, editors. World Health Organization Classification ofTumours of Haemopoietic and Lymphoid tissues, 4th ed. Lyon,France: IARC Press; 2008. pp 32–37.

3. Han X, Jorgensen J, Brahmandn A, Schlette E, Huh YO, Shi Y, AgawuS, Chen W. Immunophenotypic study of basophils by multiparameterflow cytometry. Arch Pathol Lab Med 2008;132:813–819.

4. Foucar K. Basophils disorders. In: Bone Marrow Pathology, 2nd ed.Chicago, USA: ASCP Press; 2005. pp 165–166.

5. Buesche G, Hehlmann R, Hecker H, Heimpel H, Heinze B, SchmeilA, Pfirrmann M, Gomez G, Tobler A, Herrmann H, Kappler M, Has-ford J, Buhr T, Kreipe HH, Georgii A. Marrow fibrosis, indicator of ther-apy failure in chronic myeloid leukemia—prospective long-term resultsfrom a randomized-controlled trial. Leukemia 2003;17:2444–2453.

6. Kaban K, Kantarijan H, Talpaz M, O’Brien S, Cortes J, Giles FJ, PierceS, Albitar M. Expression of thrombopoietin and its receptor (c-mpl)in chronic myelogenous leukemia: Correlation with disease progres-sion and response to therapy. Cancer 2000;88:570–576.

7. Buesche G, Ganser A, Schlegelberger B, von Neuhoff N, Gadzicki D,Hecker H, Bock O, Frye B, Kreipe H. Marrow fibrosis and itsrelevance during imatinib treatment of chronic myeloid leukemia.Leukemia 2007;21:2420–2427.

FIG. 3. The bone marrow biopsy shows a diffuse fibrosis (A: H&E, �20) and excess of myeloblast (B: CD34 immunostaining, �20). No basophilswere evident. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

124 STACCHINI ET AL.

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