Expression of CD4 on peripheral blood granulocytes

Cancer Genetics and Cytogenetics 136 (2002) 38–42

Expression of CD4 on peripheral blood granulocytes: a novel finding in a case of myelodysplastic syndrome in association with t(5;12) M. Tarek Elghetanya, Claudia P. Molinaa, Jyoti Patela, Joe Martineza, Hanna Schwaba, Gopalrao V.N. Velagaletia,b,* a

Department of Pathology, The University of Texas Medical Branch, 301 University Boulevard, Galveston, TX, USA Department of Pediatrics, The University of Texas Medical Branch, 301 University Boulevard, Galveston, TX, USA Received 15 October 2001; received in revised form 3 January 2002; accepted 4 January 2002.

b

Abstract

Myelodysplastic syndromes (MDS) are associated with cell maturation defects that can manifest as abnormal surface antigen expression. We describe a patient with refractory anemia with excess blasts, who presented with infection and extensive dysplastic features in peripheral blood granulocytes. The granulocytes expressed CD11b, CD13, CD15, CD33, and CD43. The granulocytes also expressed CD4 antigen. Cytogenetic analysis showed a clonal t(5;12)(q33;p13). The patient improved on antibiotics with partial improvement in the dysplastic features. However, shortly after, the patient experienced paravertebral extramedullary blast transformation followed by a leukemia phase of acute monoblastic leukemia. The patient died a few days later. This is the first report describing anomalous expression of CD4 on granulocytes in MDS. Since the breakpoint on chromosome 12 is near the CD4 gene, which is mapped to 12p12, we hypothesize that dysregulation of the CD4 gene may have occurred resulting in its persistent expression on mature and maturing granulocytes. © 2002 Elsevier Science Inc. All rights reserved.

1. Introduction The myelodysplastic syndromes (MDS) are a group of clonal stem cell disorders, which are characterized by defective cell maturation, increased apoptosis in bone marrow cells, and increased risk for developing acute leukemia [1]. The defective cell maturation can manifest itself in several aspects including morphologic, cytochemical, surface antigens, and immunohistochemical anomalies [2–5]. Studying surface marker abnormalities in MDS continues to provide insight regarding defective programming of cell maturation [4]. One of the most rare granulocytic abnormalities in MDS is the expression of lymphoid associated antigens on mature and maturing myelomonocytic cells. Expression of CD3 on peripheral blood monocytes has been described in chronic myelomonocytic leukemia (CMML) [6]. Another study reported the expression of CD20 on bone marrow CD14-positive mononuclear cells in a patient with CMML [7]. CD4 expression on normal granulocytes has not been reported in humans with the exception of eosinophils. The only mammalian expression of CD4 on neutrophilic granu-

* Corresponding author. Tel.: 409-772-3466; fax: 409-772-9595. E-mail address: [email protected] (G.V.N. Velagaleti).

locytes has been reported in dogs [8]. In this study, we report the novel finding of surface expression of CD4 on peripheral blood granulocytes in a case of MDS. Our patient also showed a clonal abnormality of t(5;12)(q33;p13) in unstimulated peripheral blood lymphocytes. Since the CD4 gene has been mapped to 12p12→pter, it is possible that this translocation may have a role in this unusual finding of CD4 expression on peripheral blood granulocytes. 2. Case history A 52-year-old male presented with a 1-month history of fever, chills, and shortness of breath. On admission the patient had fever of 38.4C, an oral abscess, and oral white plaques of candidiasis. Cultures of the abscess grew Stenotrophomonas maltophilia. Bilateral pleural effusion was also detected but no organism was isolated by culture. The initial complete blood count (CBC) was as follows: white blood cells (WBC) 21.9103/l; red blood cells (RBC) 2.59106/l; hemoglobin (Hb) 7.4 g/dl; hematocrit (Hct) 22.8%; mean cell volume (MCV) 88 fl; mean cell hemoglobin (MCH) 28.6 pg; mean cell hemoglobin concentration (MCHC) 32.5%; red cell distribution width (RDW) 15.7; platelets (Plt) 126103/l. Differential count showed: 18% segmented neutrophils, 10% band neu-

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M.T. Elghetany et al. / Cancer Genetics and Cytogenetics 136 (2002) 38–42

trophils, 16% lymphocytes, 9% monocytes, 2% eosinophils, and 45% of cells that were difficult to classify but were thought to represent dysplastic hypogranular maturing granulocytes to the extent of raising the possibility of hypogranular form of acute promyelocytic leukemia. The patient was transfused with 2 units of packed red cells. A bone marrow examination produced a limited number of cells with morphology similar to peripheral blood dysplastic cells; however, blasts were 8% and the diagnosis of refractory anemia with excess blasts (FAB classification) was made. The patient received systemic broad-spectrum antibiotics and local antifungal treatment. Ten days after therapy the patient’s general condition improved and the fever subsided. The CBC was as follows: WBC 3.6103/l, RBC 3.34106/l, Hb 9.2 g/dl, Hct 28.6%, MCV 85.9 fl, RDW 14.7, and Plt 121103/l. The differential count was 29% segmented neutrophils, 13% band neutrophils, 4% neutrophilic metamyelocytes, 6% neutrophilic myelocytes, 1% blasts, 24% lymphocytes, 21% monocytes, 1% eosinophils, and 1% basophils. The dysplastic features of granulocytes partially improved. Two weeks later, the patient experienced back pain. An MRI study revealed a paravertebral soft tissue mass at T10 through T12. A biopsy of the mass showed sheets of blasts, which were positive for lysozyme and CD68 with areas of focal positivity for myeloperoxidase suggestive of extramedullary transformation to acute monoblastic leukemia. The white cell count gradually increased over several days with increased blasts reaching as high as 53%. The patient had fever and weakness of lower extremities. Treatment with broad-spectrum antibiotics and radiation therapy to the vertebral mass was given. The patient’s condition deteriorated rapidly and he died a few days later.

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3. Materials and methods The peripheral blood granulocytes were further characterized at diagnosis using flow cytometry and cytochemistry. The bone marrow aspirate produced a small number of cells and similar studies could not be performed. Flow cytometry was performed using lysed whole blood technique on EDTAanticoagulated peripheral blood within 3 hours of collection. The following panel of antibodies were used: CD3, CD4, CD8, CD11b, CD13, CD14, CD24, CD33, CD34, CD35, CD43, CD45, CD49d, CD64 (Becton Dickinson Biosciences, San Jose, CA, USA), CD15 (Beckman Coulter Inc., Miami, FL, USA), CD16 (Ortho Diagnostic-Systems Inc., Raritan, NJ, USA), and HLA-DR (Caltag Laboratories, Burlingame, CA, USA). A three-color analysis was performed using the FACScan (Becton Dickinson Biosciences). Cytochemical lineage identification was attempted using the dual specific and nonspecific esterase staining (combined naphtol ASD chloroacetate esterase and -naphthyl acetate esterase) as previously described [3]. Cytochemical stains were performed on air-dried peripheral blood smears. Twenty-four–hour unstimulated bone marrow and peripheral blood lymphocyte cultures were set up and harvested using standard procedures. The slides were G-banded using pancreatin-Giemsa method. For chromosome analysis, a total of 20 metaphases were analyzed. Fluorescence in situ hybridization (FISH) studies were performed with -satellite probes targeting the centromeres of chromosomes 7 (D7Z1), 8 (D8Z1), and locus specific probes for ABL (9q34), BCR (22q11.2), PML (15q22), and RARA (17q21). All the probes were obtained from Vysis (Vysis Inc., Downers Grove, IL, USA) and FISH

Fig. 1. Peripheral blood smear with hypogranular granulocytes (arrow) and monocytoid cells (arrowheads). Wright-Giemsa stain, 1000.

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4. Results

Fig. 2. Partial karyotype showing the clonal t(5;12). The translocated chromosomes are placed on the right. The breakpoints are shown on the ideograms.

studies were carried out per the manufacturer’s recommendations. In brief, slides were denatured in 70% formamide/ 2SSC at 70C for 2 minutes followed by dehydration in grades of alcohol. The probes were denatured at 72C in a waterbath for 5 minutes and hybridized overnight to target areas on the denaturated slide. Posthybridization washes included a 0.4SSC/Tween 20 for 1 minute at 72C, followed by 2SSC wash at room temperature for 1 minute. The slides were counterstained with DAPI and visualized under a Nikon photomicroscope equipped with epifluorescence. A minimum of 200 interphase nuclei were analyzed for each probe.

Most granulocytes had hypogranular cytoplasm and/or pelgeroid nuclei (Fig. 1). Some granulocytes had prominent primary granules with weakly staining secondary granules and abnormal nuclear maturation with occasional nuclear indentation. Bone marrow cultures did not yield any analyzable metaphases. However, chromosome analysis from the peripheral blood showed 9 cells with normal 46,XY karyotype. The remaining 11 cells showed an apparently balanced reciprocal translocation involving chromosomes 5 and 12 with breakpoints at 5q33 and 12p13. The karyotype was interpreted as 46,XY,t(5;12)(q33;p13)[11]/46,XY[9] (Fig. 2) [9]. FISH analysis on interphase nuclei from the bone marrow cultures showed only normal hybridization with all the probes tested, indicating absence of trisomy 8, monosomy 7, BCR/ABL, and PML/RARA fusion. By flow cytometry, the granulocytes were identified using their scatter properties in addition to CD45-side scatter gating (Fig. 3A). The granulocyte gate contained no more than 4% monocytes as identified by the percentage of cells that are CD24, CD14 (Fig. 3B). The gate contained less than 2% eosinophils, which typically have a CD49d, CD64 phenotype. More than 80% of granulocytes were CD24 with more than 50% coexpressing CD24 and CD4

Fig. 3. Flow cytometry study of peripheral blood granulocytes. (A) Granulocyte gate using CD45-side scatter. (B) Gated cells showing positive CD24 and negative CD14 immunophenotype. (C) Majority of gated cells are positive for both CD4 and CD24. (D) CD4-positive cells are negative for HLA-DR.

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Fig. 4. Flow cytometry of normal granulocytes: positive for CD24 and negative for CD4.

(Fig. 2C). The CD4 cells were HLA-DR (Fig. 2D). Virtually all cells were positive for CD43, CD33, CD13, CD15, and CD11b, while negative for CD34, CD3, and CD8. Normal granulocytes are negative for CD4 (Fig. 4). Approximately 30% of granulocytes expressed CD16. Cytochemical stains showed 85% of the cells to be positive for specific esterase, 2% positive for nonspecific esterase, and 3% with dual esterase positivity (Fig. 5). 5. Discussion CD4 is a single polypeptide initially described as a marker for MHC class II-restricted helper T cells [10]. CD4 expres-

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sion has been later reported on a number of cells that do not express T-cell receptor, including megakaryocytes, eosinophils, monocytes, and Langerhans cells [11]. In addition, other studies indicated the expression of CD4 on early multipotent cells in the thymus, bone marrow, and fetal liver, and on bone marrow erythroid precursors [11,12]. Yet, with the exception of canine neutrophilic granulocytes, CD4 expression has not been reported on other mammalian neutrophilic granulocytes, including humans [8]. This case represents a novel finding of CD4 expression on neutrophilic granulocytes in MDS. Neutrophilic granulocytes have been positively identified using forward and side scatter properties, CD45 and side scatter properties, and CD24, which is characteristic of granulocytes and is not expressed by monocytes [13]. In addition, cells expressing CD4 were negative for CD14 and HLA-DR excluding monocytic differentiation. Studying CD4 expression in myeloid malignancies has been limited to blasts. CD4 blasts have been reported in as high as 68% of cases of acute myeloid leukemia [14–16]. In fact, CD4 expression, in absence of CD2, has been suggested to be a marker for myeloid differentiation of acute leukemia [14]. The clonal t(5;12)(q33;p13) seen in our patient has been reported in patients with myeloid malignancies showing the features of both myeloproliferative disorders (MPD) and MDS [17]. Since most of these patients present with eosinophilia and other clinical features that are suggestive of both MPD and CMML, it is difficult to classify these patients using the French–American–British (FAB) classification [18,19]. The translocation has been shown to result in fusion of ETV6, a putative transcription factor gene belonging to ETS family of genes at 12p13 and platelet derived growth

Fig. 5. Bone marrow aspirate showing cells (arrows) with positive reaction for dual esterase (naphthol ASD chloroacetate esterase, blue; -naphthyl acetate esterase, brown). Dual esterase stain, 1000.

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factor receptor  (PDGFRB) gene at 5q33 [20]. The same translocation was also seen in a few leukemic cell lines such as NALM-6, PBE1 and SUP-B26/B-28 with features of pre B-acute lymphoblastic leukemia (ALL), leading some investigators to propose possible involvement of ETV6 and PDGFRB in pre B-ALL [21]. However, recent studies have shown that the t(5;12) seen in these pre B-ALL cell lines is molecularly different from the t(5;12) seen in CMML and other unusual MPD/MDS patients [22]. The t(5;12) in these pre-ALL leukemic cell lines is associated with concurrent cryptic deletions involving both chromosomes at 5q31q33 and 12p12. These studies have shown that neither of the genes, ETV6 and PDGFRB that are implicated in the unusual cases of MPD/MDS, is involved in these pre B-ALL cell lines. They further suggested that the breakpoint on chromosome 12 was located more proximally at p12; however, this study [22] did not identify the genes involved, if any, in this translocation associated with the pre-B ALL cell lines. Interestingly, the CD4 gene is mapped to chromosome 12p12→pter [23]. In light of the studies showing that the breakpoint on chromosome 12 is more proximal at p12, it is possible that the clonal translocation (5;12) seen in our patient may have altered the CD4 gene and its expression leading to this novel finding. It is also significant that the deletion 5q31q33 seen in pre B-ALL cell lines reportedly overlaps the 5q deletion seen in 5q-refractory anemia patients [22], since the initial diagnosis of our patient was refractory anemia with excess blasts. In conclusion, CD4 expression on mature and maturing granulocytes in MDS could be viewed as anomalous expression of an immature marker beyond its specific stage of maturation similar to persistent expression of HLA-DR in MDS [4]. This rare finding may be explained on the basis of the clonal t(5;12).

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