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Demonstration of clonality in neutrophils using FISH in a case of chronic neutrophilic leukemia MK Froberg1, RD Brunning1, P Dorion2, CE Litz1 and E Torlakovic1

1Division of Special Hematology, Department of Laboratory Medicine and Pathology, University of Minnesota, Minneapolis, MN; and 2Department of Laboratory Medicine, Geisinger Medical Center, Danville, PA, USA

A patient previously diagnosed with chronic neutrophilic leuke- Materials and methods mia (CNL) was studied using ﬂuorescent in situ hybridization (FISH) to determine clonality of neutrophils. By cytogenetic Patient and control samples studies the patient’s blood and bone marrow had an 11q14 deletion and were negative for the Philadelphia (Ph) chromosome. FISH was performed on peripheral blood smears using Buffy-coat smears prepared from EDTA anticoagulated blood probes for the bcr/abl translocation and a probe for 11q23 were studied from the patient and controls. Positive control (MLL). The patient’s white blood cells were negative for the consisted of a patient previously diagnosed with CML with bcr/abl translocation; neutrophils and eosinophils, but not lym- cytogenetically documented t(9;22). A patient without CML phocytes, were monosomic for the 11q23 probe indicating a for the bcr/abl probe and two patients with reactive neutrophi- clonal population within the neutrophil population. lia for the MLL probe served as negative controls. Smears were Keywords: chronic neutrophilic leukemia; FISH; Ph chromosome; 11q stored in a desiccator at ambient temperature. The oldest slide used in the study was stored for 3 weeks. Smears were ﬁxed in acetone for 10 min and air-dried.

Introduction Methodology

Chronic neutrophilic leukemia (CNL) is a rare hematologic Fluorescent in situ hybridization (FISH): FISH was perfor- disorder of uncertain pathogenesis characterized by persistent med with MLL (11q23) and Mbcr/abl translocation DNA pro- neutrophilia without an identifiable cause. The leukocytosis bes (Oncor, Gaithersburg, MD, USA) according to the manu- consists principally of mature neutrophils without immature facturer’s instructions. The digoxigenin-labeled MLL probe neutrophils, myeloblasts, increased basophils or monocytes. was detected by incubation with 20 ␮l of rhodamine-labeled The Philadelphia (Ph) chromosome is absent. These features anti-digoxigenin (Oncor); the Mbcr/abl probes were detected distinguish CNL from chronic myelogenous leukemia (CML), by incubation with a mixture of equal parts (20 ␮l each/slide) atypical chronic myelogenous leukemia (aCML), and chronic of rhodamine-anti-digoxigenin and fluorescein-avidin myelomonocytic leukemia (CMML) as defined by the FAB (Oncor). The appropriate probes were applied to the slides 1 group. Additionally, patients usually have hepatospleno- which were then covered with plastic coverslips and incu- megaly, elevated leukocyte alkaline phosphatase (LAP), bated in a moist chamber for 30 min at 37°C. Subsequently, increased serum uric acid and bone marrow granulocyte coverslips were removed and the slides washed three times in 2 hyperplasia. We (MKF and PD) have previously described a PBS for 2 min per wash. Negative controls were treated the 3 patient with features of CNL. Briefly the patient presented same. with anemia refractory to iron therapy without an identifiable source of bleeding. Her bone marrow was Ph chromosome negative by cytogenetic studies on five occasions spanning an Evaluation of signals: Fluorescent probe signals were 8 year period. She gradually developed a leukocytosis with a evaluated with an Olympus B-Max microscope with 100 × maximum WBC of 84.8 × 109/l consisting of greater than 90% objective U Plan Apo-Chromat Oil Immersion ‘Universal’ mature neutrophils without immature neutrophilia, mono- objective and 10 × eyepieces as previously described.4 For cytosis or basophilia (Figure 1). Splenomegaly, increased leu- the MLL 11q23 probe, cells were counted as having one or kocyte alkaline phosphatase score and increased serum uric two signals. A single signal was considered positive for mono- acid were present; an 11q14 deletion was consistently present somy of the distal portion of the long arm of chromosome 11 in karyotypes of bone marrow. We evaluated clonality of neu- consistent with the patient’s known 11q14 deletion. For the trophils using FISH with 11q23, bcr and abl probes on periph- Mbcr/abl translocation, signals were evaluated for the percent- eral blood smears. age showing fused or juxtaposed signals; ie fused red and green labeled probes were yellow, or red and green signals which were very close but without a color change. Fusion or overlapping of these signals was interpreted as positive for the Ph chromosome.

Correspondence: MK Froberg, Department of Laboratory Medicine Results and Pathology, University of Minnesota–Duluth, School of Medicine, 10 University Dr., Duluth, MN 55812, USA; Fax: 726 7559 FISH performed with the bcr/abl probes demonstrated color Received 16 October 1997; accepted 20 November 1997 change or overlapping of signals in ﬁve of 300 (1.6%) neutro- Brief communication MK Froberg et al 624

Figure 1 Peripheral blood smear of patient with chronic neutrophilic leukemia showing leukocytosis consisting of mature neutrophils. Red blood cell anisocytosis and pokilocytosis are marked. Original magniﬁcation × 200.

phils from the patient with CNL and 296 of 300 (98.6%) neu- Discussion trophils from a patient with CML, while a negative control stained as expected. With the 11q23 probe, 299 of 300 neu- Chronic neutrophilic leukemia is a rare hematologic disorder trophils and all cells identified as eosinophils from the patient first described by Tuohy5 in 1920. Subsequently, occasional with CNL had a single fluorescent signal (Figure 2). A single cases of CNL have been reported, but a clear understanding neutrophil had two signals. Occasional small cells with scant of its pathogenesis and proper classification have remained in cytoplasm and a round nucleus interpreted as lymphocytes all doubt. The Ph chromosome, monocytosis and basophilia are had two signals (Figure 3). FISH performed with the 11q23 not present and lack of these features serve to distinguish CNL probe on blood smears from two patients with reactive leuko- from CML, aCML and CMML. Chronic neutrophilic leukemia cytosis had two signals present within a combined total of 99 must also be distinguished from a leukemoid reaction, neces- of 100 (99%) neutrophils. sitating a search for infectious or reactive causes of the persist-

Figure 2 FISH using an 11q23 probe on blood smear from patient with CNL. A single red signal is present in the nucleus of one eosinophil and four neutrophils. Original magniﬁcation × 1000. Brief communication MK Froberg et al 625

Figure 3 A single signal is present in a neutrophil, while two signals are seen in a lymphocyte from patient with CNL. Original magnification × 1000. ent neutrophilia. Cases of CNL have been associated with patient had hepatosplenomegaly, marrow granulocyte hyper- polycythemia vera, myelodysplasia, multiple myeloma and plasia and an increased LAP score. WBC reached 100 × 109/l paraproteinemias of undetermined significance.6–11 with 98.5% mature neutrophils. No rearrangement of the M- Cytogenetic studies have shown the absence of the Ph chro- bcr region was detected by Southern analysis with the M-bcr mosome, and most cases of CNL studied have shown a nor- probe. Standen et al11 using restriction fragment length poly- mal karyotype.8–10 Recent case studies have confirmed the morphism and X inactivation of the X-linked probe M27␤ absence of the bcr/abl rearrangement by molecular tech- found a polyclonal pattern in a patient with CNL and multiple niques.6,9,12 Tanzer et al13 described a 72-year-old male with myeloma suggesting that the neutrophilia in this patient was a WBC of 60 × 109/l with 96% mature neutrophils, spleno- reactive. This patient had a paraproteinemia with Bence–Jones megaly and myeloid hyperplasia of the bone marrow. In five protein in the urine and a leukocytosis with 91% neutrophils. of 15 marrow cells karyotyped, one chromosome 2 was Following treatment with melphalan and prednisone, the neu- abnormally large; but they were unable to define the exact trophil count returned to normal, supporting a reactive etio- defect. The Ph chromosome was not identified. Donato et al14 logy for the neutrophilia. described a patient with features of CNL and trisomy 9 with A single fluorescent signal was present in over 99% of the partial deletion of the long arm of 20. A patient with features present patient’s neutrophils and all eosinophils studied from of CNL and a monoclonal gammopathy had a balanced trans- a peripheral blood smear using FISH with an 11q23 probe. location between chromosomes 1 and 20.15 Masini et al16 Monosomy for the distal long arm of chromosome 11 is con- described a patient with a plasma cell dyscrasia, hepato- sistent with this patient’s established 11q14 deletion repeat- splenomegaly, marrow myeloid hyperplasia, elevated serum edly demonstrated by cytogenetic studies of her bone marrow. vitamin B12, elevated LAP score and leukocytosis with a WBC Lymphocytes had two signals indicating they were not of 21 × 109/l with 85% neutrophils. Cytogenetic studies of the involved by the clonal process, although only small numbers marrow aspirate showed a loss of the Y chromosome. Orazi were present for evaluation. Neutrophils also lacked the et al17 reported a patient with splenomegaly, marrow myeloid bcr/abl rearrangement. To our knowledge this represents the hyperplasia, and an increased LAP score. The WBC was first demonstration by FISH of monoclonality of neutrophils in 51.6 × 109/l with 92% mature neutrophils. Three of eight mar- a patient with the characteristic features of chronic neutro- row cells karyotyped showed trisomy 8 without the Ph chro- philic leukemia; as well as the lack of the Ph chromosome by mosome. Hasle et al18 described a 15-year-old female and 25- FISH. This study reaffirms that at least in some patients, CNL year-old male with some signs of CNL and karyotypic abnor- represents a clonal disorder. malities. One patient had a neutrophilia composed of mature granulocytes and trisomy 21 which evolved to tetrasomy 21. The other patient had a modest neutrophilia, low LAP score References and normal karyotype at presentation. Three months later this evolved to acute myelomonocytic leukemia (AML-M4) with 1 Bennet JM, Catovsky, D, Daniel MT, Flandrin G, Galton DAG, t(2;2) (q32;p24). Gralnick H, Sultan C, Cox C. The chronic myeloid leukaemias: Recently, Kwong and Cheng19 demonstrated monoclonal guidelines for distinguishing chronic granulocytic, atypical chronic myeloid, and chronic myelomonocytic leukaemia. Pro- hematopoiesis in a patient with features of CNL using methyl- posals by the French–American–British Cooperative Group. Br J ation status of X-linked hypoxanthine phosphoribosyl transfer- Haematol 1994; 87: 746–754. ase gene following restriction endonuclease digestion. This 2 You W, Weisbrot IM. Chronic neutrophilic leukemia. Report of Brief communication MK Froberg et al 626 two cases and review of the literature. Am J Clin Pathol 1979; 72: leukaemia associated with myeloma: analysis using the X-linked 233–242. probe M27␤. J Clin Pathol 1993; 46: 297–298. 3 Pascucci M, Dorion P, Makary A, Froberg MK. Chronic neutro- 12 Storek J. Chronic neutrophilic leukemia: case report documenting philic leukemia evolving from a myelodysplastic syndrome. Acta the absence of bcr-abl rearrangement. Am J Hematol 1992; 41: Haematol 1997; 98: 163–166. 304. 4 Torlakovic E, Litz CE, McClure JS, Brunning RD. Direct detection 13 Tanzer J, Harel P, Boiron M, Bernard J. Cytochemical and cyto- of the Philadelphia chromosome in CD 20-positive lymphocytes in genetic findings in a case of chronic neutrophilic leukaemia of chronic myeloid leukemia by tri-color immunophenotyping/FISH. mature cell type. Lancet 1964; 1: 387–388. Leukemia 1994; 8: 1940–1943. 14 Donato CD, Gianfranco C, Lazzari S, Scarduelli L, Vignoli R, Buia 5 Tuohy EL. A case of splenomegaly with polymorphonuclear neu- M, Tramaloni C, Maccari S, Plancher AC. Chronic neutrophilic trophil hyperleukocytosis. Am J Med Sci 1920; 160: 18–25. leukemia: description of a new case with karyotypic abnormali- 6 Foa P, Iurlo A, Saglio G, Guerrasio A, Capsoni F, Maiolo AT. ties. Am J Clin Pathol 1986; 85: 369–371. Chronic neutrophilic leukemia associated with polycythemia vera: 15 Mehrotra PK, Winfield DA, Fergusson LH. Cellular abnormalities pathogenetic implications and therapeutic approach. Br J Haema- and reduced colony-forming cells in chronic neutrophilic leuke- tol 1991; 78: 286–288. mia. Acta Haematol 1985; 73: 47–50. 7 Lugassy G, Farhi R. Chronic neutrophilic leukemia associated with 16 Masini L, Salvarani C, Macchioni P, Baldi G, Rossi F, Croci G, polycythemia vera. Am J Hematol 1989; 31: 300–301. Prandi S, Davoli C, Portioli I, Gobbi F. Chronic neutrophilic leuke- 8 Cervantes F, Marti JM, Rozman C, Monforte R, Feliu E, Vives-Cor- mia (CNL) with karyotypic abnormalities associated with plasma rons JL. Chronic neutrophilic leukemia with marked myelodys- cell dyscrasia: a case report. Haematologica 1992; 77: 277–279. plasia terminating in blast crisis. Blut 1988; 56: 75–78. 17 Orazi A, Cattoretti G, Sozzi G. A case of chronic neutrophilic 9 Zittoun R, Rea D, Hoang Ngoc L, Ramond S. Chronic neutrophilic leukemia with trisomy 8. Acta Haematol 1989; 81: 148–151. leukemia. A study of four cases. Ann Hematol 1994; 68: 55–60. 18 Hasle H, Olesen G, Kerndrup G, Phillip P, Jacobsen N. Chronic 10 Standen GR, Jasani B, Wagstaff M, Wardrop CAJ. Chronic neutro- neutrophilic leukaemia in adolescence and young adulthood. Br philic leukemia and multiple myeloma. An association with ␭ light J Haematol 1996; 94: 628–630. chain expression. Cancer 1990; 66: 162–166. 19 Kwong YL, Cheng G. Clonal nature of chronic neutrophilic leuke- 11 Standen GR, Steers FJ, Jones L. Clonality in chronic neutrophilic mia. Blood 1993; 82: 1035–1036.