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case study [hematology | cytology] Leukocytosis, Thrombocytopenia, and Hepatosplenomegaly In A Neonate With Down Syndrome Lugen Chen, MD, PhD,1 Qinglong Hu, MD,1 Harshad O Shah, PhD,1 Jen H Lin, MD1,2 1Departments of Pathology and Laboratory Medicine, Nassau University Medical Center, 2The Health Science Center of the State University of New York at Stony Brook, East Meadow, NY DOI: 10.1309/UN80NFB0GQTM3XJH Patient Full-term male newborn. chlamydia, and a purified protein derivative (PPD) skin test was negative. At the time of delivery, the baby was floppy with no spontaneous respirations. His Apgar score was 1 at 2 minutes and 10 at 5 minutes after birth. He had 1 healthy sibling, and there was no history of familial or genetic disorders. Physical Examination He was intubated, afebrile, and responsive to minimal stimuli. He had scattered rashes over his entire body, lowset ears, epicanthic folds, hypoplastic maxilla, muscular hypotonia, abundant neck skin, and simian creases. There was hepatosplenomegaly with a palpable liver and spleen, 2 cm (liver) and 5 cm (spleen) below the right and left costal margins, respectively. Chief Complaint Respiratory distress, meconium aspiration, and blasts in a peripheral blood smear. Medical History A 2,300-gram baby boy with breach presentation and protrusion of an arm, leg, and umbilical cord in the vaginal canal was delivered by Caesarean section at 39 weeks of gestation. His 20-year-old G2P1 mother received routine prenatal care, and the progression of her pregnancy occurred normally. Her blood type was group B/Rh negative, and she received Rh immunoglobulin at 28 weeks of gestation. The mother’s laboratory tests were negative for human immunodeficiency virus (HIV) antibodies, hepatitis B surface antigen (HBsAg), syphilis, gonorrhea, and Questions: 1. What are this patient’s most striking clinical and laboratory findings? 2. How do you explain this patient’s most striking clinical and laboratory findings? 3. Based on the cytochemical and immunocytochemical findings, what type of blasts are present in this patient’s peripheral blood and bone marrow? 4. What is this patient’s diagnosis? 5. Does this patient have a true leukemia? 6. Is it important to know whether or not this patient has a true leukemia? 7. How should this patient be treated and what is the most likely clinical course of this patient’s disease? T1 Principal Laboratory Findings Test Patient’s Result “Normal” Reference Range Neonatal Age @Birth 3 wk 15 wk @Birth 3 wk 15 wk Hematology WBC count Blast count Platelet count Hemoglobin Peripheral blood smear findings: 542 47.7 34.1 13.5 9.0–30.0 5.0-20.0 6.0-17.5 x 103/µL 51 26 0 ———————————0%————————————— 50.0 319 392 ———————150-450 x 103/µL—————————— 17.2 14.8 12.6 17.1-21.5 12.1-18.0 10.0-13.0 g/dL 44 NRBCs/100 WBCs and blasts resembling lymphoblasts [I1A], occasionally demonstrating ectocytoplasmic projections [I1B] Cytochemical characteristics of blastoid cells after staining using: α-Naphthyl acetate esterase Positive ASD-chloroacetate esterase Negative Acid phosphatase Paranuclear Golgi zone staining [I1C] Immunocytochemical findings: Positive for CD61 [I1D] Negative for CD3, CD10, and CD20 Bone marrow aspirate findings: 27% blasts, morphologically similar to those seen in the peripheral blood smear Cytogenetics Karyotype (all cells)* 47XY+21 *Performed on nucleated cells from culture of a peripheral blood sample from the patient with phytohemagglutinin followed by chromosome isolation and banding using the trypsin-Giemsa method. WBC, white blood cell; NRBCs, nucleated red blood cells; CD, cluster of differentiation. laboratorymedicine> september 2004> number 9> volume 35 © Downloaded from http://labmed.oxfordjournals.org/ by guest on October 25, 2016 Principal Laboratory Findings [T1] and [I1]. Possible Answers: 1. Scattered rashes over his entire body, low-set ears, epicanthic folds, hypoplastic maxilla, muscular hypotonia, abundant neck skin, simian creases, and hepatosplenomegaly; increased WBC count; decreased platelet count; increased number of nucleated red blood cells (NRBCs) and a transiently increased number of lymphoblastoid cells in peripheral blood with similarly appearing blast cells in a bone marrow aspirate; and abnormal karyotype (47XY+21) [T1]. 3. The lineage of blasts is megakaryoblastic. Morphologically, megakaryoblasts are usually medium to large in size (12 to 18 A B C D Downloaded from http://labmed.oxfordjournals.org/ by guest on October 25, 2016 2. This patient’s phenotypic clinical features (ie, low set ears, epicanthic folds, hypoplastic maxilla, muscular hypotonia, abundant neck skin, and simian creases) are consistent with Down syndrome as confirmed by the trisomy 21 karyotype, while the transiently increased number of peripheral blood blast cells is consistent with transient myeloproliferative disorder (TMD). µm in diameter) with a round or slightly indented nucleus that displays fine chromatin and 1 to 3 conspicuous nucleoli. The cytoplasm of these cells is basophilic, agranular, and may show distinct blebs or pseudopods. In some cases, however, megakaryoblasts are predominantly small with a high nuclearcytoplasmic (N/C) ratio, resembling lymphoblasts.1 Cytochemical diagnostic features of megakaryocytes are positive staining for α-naphthyl acetate esterase and negative staining using a granule-specific esterase. Characteristically, acid phosphatase staining of these cells is strongly positive and predominantly localized in the Golgi zone.2 Immunophenotyping of megakaryoblasts usually indicates expression of CD34, a marker for primitive blasts, CD33 and/or CD13 (ie, myeloid differentiated antigens), and at least 1 platelet-associated antigen (CD36, CD41a, CD41b, or CD61) with occasional aberrant expression of CD7 (a T-cell associated antigen) and CD56.3-6 In addition, a proportion of these type of blast cells also express glycophorin A (an erythroid marker)7 and erythroid-specific mRNAs such as gamma-globin and erythroid δ-aminolevulinate synthetase.8 The immunophenotypic profile tends to favor that blasts in TMD are derived 543 [I1] Patient’s hematological, cytochemical, and immunocytochemical peripheral blood findings illustrating (A) a typical blast cell with fine nuclear chromatin, a high nuclear-cytoplasmic (N/C) ratio, and morphology resembling a lymphoblast; (B) a blast cell with distinct blebs or pseudopods; (C) paranuclear Golgi zone staining of a blast cell with acid phosphatase; (D) positive staining of blast cells for CD61. All images, 1,000x magnification. © laboratorymedicine> september 2004> number 9> volume 35 from an immature megakaryocytic or a common erythroid megakaryocytic lineage. Ultrastructurally, megakaryoblasts typically demonstrate the presence of platelet peroxidase (PPO) within the nuclear envelope and in the endoplasmic reticulum but not in the granules of the cytoplasm.9 In this case, the combination of cytochemical (ie, positive staining for α-naphthyl acetate esterase, negative staining for ASDchloroacetate esterase, and paranuclear Golgi zone staining with acid phosphatase) and immunocytochemical findings (ie, positive for CD61 and negative for CD3, CD10, and CD20 [T1]) confirms the lineage of these blast cells as megakaryocytic. 546 5. Yes, TMD is a true leukemia-showing clonal proliferation. The unique feature of TMD in a Down syndromeaffected individual is its spontaneous resolution. The hypothesis to explain this phenomenon is that the TMD clone is derived from fetal hematopoietic cells in the liver rather than in the bone marrow; thus, spontaneous resolution occurs due to the turning off of liver hematopoiesis shortly after birth.12 This hypothesis is consistent with the lower blast count in the bone marrow than in the peripheral blood that is observed often in cases of TMD associated with Down syndrome. Moreover, it is well known that neonates and infants with Down syndrome have a significant risk of developing leukemia. There is at least a 20-fold increased incidence of leukemia in Down syndrome-affected individuals compared with cytogenetically normal individuals. In addition, TMD occurs in approximately 10% of patients with Down syndrome. Although the majority of such TMD cases resolve spontaneously, approximately 25% of patients who recover from TMD relapse to acute megakaryocytic leukemia (AMKL) in the first 4 years of life.10,13 One of the most popular models to explain the increased incidence of TMD in Down syndrome-affected individuals is increased gene dosage of a leukemia predisposition gene or a hematopoiesis regulatory gene on chromosome 21 that stimulates abnormal proliferation of hematopoietic stem 6. Yes, differentiation of TMD from AMLK and true congenital leukemia (TCL) has clinical significance due to the different prognoses and treatments for these diseases. This differentiation is complicated by the fact TMD blasts are morphologically indistinguishable from AMKL blasts.1 However, the main difference in the clinical presentation of these disorders is the age of onset, with TMD occurring at birth or in the first few days of life and AMLK often developing after 1 year of age.11 Blast cells associated with these 2 disorders share some immunophenotypic features, including positivity for CD38, CD33, CD36, CD45, CD34, CD41, CD61, and CD7 and negativity for CD14, CD15, and CD16. The major immunophenotypic difference between them is the expression of CD13 and CD11b in AMLK. Other biologic markers to distinguish TMD and AMLK include telomerase activity which is higher in myeloid cells from patients with malignant AMLK and lower in cells from patients with TMD, a relatively benign disorder.19 In contrast to AMLK and TMD, TCL represents a distinctive category of neonatal myeloproliferative disorders. It is usually found in neonates without Down syndrome and shows a more aggressive clinical course. Patients with TCL usually have marked hepatosplenomegaly and typical skin leukemic infiltrates—generalized multiple red-blue nodules of “blueberry muffin lesions.”20 The majority of TCL cases express monocytic or myelomonocytic CD markers, and occasionally, biphenotypic (ie, myelocytic and lymphocytic) markers.21 Chemotherapy is the treatment of choice for TCL. 7. Close observation and supportive care are the treatments of choice in this patient, since infants with this disorder are typically healthy except for the rare presence of erythematous skin plaques and rashes and a mild to moderate degree of hepatosplenomegaly.22 Most cases of TMD in patients with Down syndrome follow a self-limited benign course with spontaneous resolution of the signs and symptoms. Even after the disappearance of laboratorymedicine> september 2004> number 9> volume 35 © Downloaded from http://labmed.oxfordjournals.org/ by guest on October 25, 2016 4. Diagnosis: Down syndrome with TMD. The presence of a large number of megakaryocytic blasts (51%) in our patient’s peripheral blood and a lesser number (27%) of similar blasts in his bone marrow, which spontaneously disappeared from the peripheral blood 15 weeks later without chemotherapy, is a typical course for TMD in a neonate with Down syndrome. Moreover, TMD is a disorder found only in neonates with Down syndrome. Transient myeloproliferative disorder (TMD) is characterized by leukocytosis with blasts expressing megakaryocytic antigens in both the peripheral blood and bone marrow with various degrees of thrombocytopenia, anemia, and hepatosplenomegaly.10 The fact that a higher blast count is seen in the peripheral blood than in the bone marrow is a classic feature of TMD.11 In most TMD cases, spontaneous regression of the signs and symptoms occurs within 4 months of the initiation of supportive therapy.10 cells in infancy, frequently resulting in TMD.14 Recent molecular studies have demonstrated that blast cells from both TMD- and AMKL-affected individuals contain mutations in the GATA1 gene,15-17 the gene that encodes the essential hematopoietic transcript factor, GATA1 (GATA binding protein 1 or globin transcription factor 1). The GATA binding protein plays a key role in the maturation of erythroid cells and megakaryocytes. More recently, a “multiple hit” model has been proposed to explain the leukemogenesis of TMD and AMLK.12,18 In this model, trisomy 21 is the initial event, which may provide a survival advantage to progenitors by increased dosage of genes on chromosome 21, whereas GATA1 mutations contribute to leukemogenesis by blocking megakaryocytic differentiation. Enhanced proliferation and disrupted differentiation of megakaryocytes are considered adequate events for the induction of TMD. Additional acquired lesions (such as a p53 mutation, altered telomerase activity, or additional genetic abnormalities) lead to overt AMLK. blasts from the peripheral blood, a close follow-up of these patients is recommended at least for the first 3 years of life because of the potential for developing acute leukemia, particularly AMKL.13 Moreover, some cases of TMD are complicated by severe, possible lethal sequelae, including progressive hepatic fibrosis, hydrops, and/or cardiopulmonary failure. Low-dose Ara-C therapy has been found to be effective in the treatment of these sequelae.23 Currently, there are 4 primary indications for using low-dose Ara-C in patients with TMD: 1) hyperleukocytosis (ie, a leukemic blast count exceeding 100,000/µL), 2) organ involvement with signs of impairment, 3) hydrops faetalis at birth, and 4) the presence of a disseminated intravascular coagulopathy.12 Downloaded from http://labmed.oxfordjournals.org/ by guest on October 25, 2016 Patient’s Treatment and Course On the 10th day of life, the infant’s skin rashes became coalescent with crusty, dry, and yellowish exudates, for which antibiotics and supportive therapy were initiated. Three (3) weeks later, the skin rashes disappeared. The patient had been doing well on the last clinic visit at age 27 months with unremarkable laboratory results. Thereafter, the infant was lost to follow-up. 13. Zipursky A. Transient leukaemia - A benign form of leukaemia in newborn infants with trisomy 21. Br J Haematol. 2003;120:930-938. 14. Abe K, Kajii T, Niikawa N. Disomic homozygosity in 21-trisomic cells: A mechanism responsible for transient myeloproliferative syndrome. Hum Genet. 1989;82:313-316. 15. Mundschau G, Gurbuxani S, Gamis AS, et al. Mutagenesis of GATA1 is an initiating event in Down syndrome leukemogenesis. Blood. 2003;101:42984300. 16. Hitzler JK, Cheung J, Li Y, et al. GATA1 mutations in transient leukemia and acute megakaryoblastic leukemia of Down syndrome. Blood. 2003;101:4301-4304. 17. Xu G, Nagano M, Kanezaki R, et al. Frequent mutations in the GATA-1 gene in the transient myeloproliferative disorder of Down syndrome. Blood. 2003; 102:2960-2968. 18. Gurbuxani S, Vyas P, and Crispino JD. Recent insights into mechanisms of myeloid leukemogenesis in Down syndrome. Blood. 2004;103:399-406. 19. Holt SE, Brown EJ, Zipursky A. Telomerase and the benign and malignant megakaryoblastic leukemias of Down syndrome. J Pediatr Hematol Oncol. 2002;24:14-17. 20. Resnick KS, Brod BB. Leukemia cutis in congenital leukemia: Analysis and review of the world literature with report of an additional case. Arch Dermatol. 1993;129:1301-1306. 21. Brissette M. Simurdak J, Larsen R. Immunophenotyping of congenital leukemia. Cytometry. 1995; 22:180-181. 22. de Tar MW, Bittman W, Gilbert J. Transient myeloproliferative disease of the newborn: Case report with placental, cytogenetic, and flow cytometric findings. Hum Pathol. 2000;31:396-398. 23. Al-Kasim F, Doyle JJ, Massey GV, et al. Incidence and treatment of potentially lethal diseases in transient leukemia of Down syndrome: Pediatric oncology group study. J Pediatr Hematol Oncol. 2002;24:9-13. Keywords: transient myeloproliferative disorder, Down syndrome, acute megakaryocytic leukemia, true congenital leukemia, cluster of differentiation 1. Litz CE, Davies S, Brunning RD, et al. Acute leukemia and the transient myeloproliferative disorder associated with Down syndrome: Morphologic, immunophenotypic and cytogenetic manifestations. Leukemia. 1995;9:1432-1439. 2. Pombo De Oliveira MS, Gregory C, Matutes E, et al. Cytochemical profile of megakaryoblastic leukemia: A study with cytochemical methods, monoclonal antibodies and ultrastructural cytochemistry. J Clin Pathol. 1987;40:663-669. 3. Karandikar NJ, Aquino AB, McKenna RW, et al. Transient myeloproliferative disorder and acute myeloid leukemia syndrome: An immunophenotyic analysis. Am J Clin Pathol. 2001;116:204-210. 4. Slørdhl SH, Smeland EB, Holte H, et al. Leukemic blasts with markers of four cell lineages in Down’s syndrome (“megakaryoblastic leukemia”). Med Pediatr Oncol. 1993;21:254-258. 5. Domenico DR, Dizikes GJ, Melnyk AR, et al. Pseudoleukemia in Down’s syndrome: Analysis of immunophenotype and gene rearrangement. Am J Clin Pathol. 1989;91:709-714. 6. Yumura-Yagi K, Hara J, Kurahashi H, et al. Mixed phenotype of blasts in acute megakaryocytic leukemia and transient abnormal myelopoiesis in Down’s syndrome. Br J Hematol. 1992;81:520-525. 7. Bozner P. Transient myeloproliferative disorder with erythroid differentiation in Down syndrome. Arch Pathol Lab Med. 2002;126:474-477. 8. Ito E, Kasai M, Hayashi Y, et al. Expression of erythroid-specific genes in acute megakaryoblastic leukemia and transient myeloproliferative disorder in Down’s syndrome. Br J Haemtol. 1995;90:607-614. 9. Choate JJ, Domenico DR, McGraw TP, et al. Diagnosis of acute megakaryoblastic leukemia by flow cytometry and immunoalkaline phosphatase technique: Utilization of new monoclonal antibodies. Am J Clin Pathol. 1988;89:247-253. 10. Zipursky A, Brown E, Christensen H, et al. Leukemia and/or myeloproliferative syndrome in neonates with Down syndrome. Sem Perinat. 1997;21:97-101. 11. Hayashi Y, Eguchi M, Sugita K, et al. Cytogenetic findings and clinical features in acute leukemia and transient myeloproliferative disorder in Down’s syndrome. Blood. 1988;72:15-23. 12. Gamis AS, Hilden JM. Transient myeloproliferative disorder, a disorder with too few data and many unanswered questions: Does it contain an important piece of the puzzle to understanding hematopoiesis and acute myelogenous leukemia? J Pediatr Hematol Oncol. 2002;24:2-5. © laboratorymedicine> september 2004> number 9> volume 35 547