Bumps on the Neck and Groin of a 2-Year-Old Male. Laboratory Findings: Table 1, Table 2; Figure 1; Image 1, Image 2, Image 3

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1 Bumps on the Neck and Groin of a 2-Year-Old Male 1 Erikakelly Strand, BS* Clinical History Patient: 2-year-old white male. Chief Complaint: Bumps on neck and groin. History of Present Illness: A 2-year-old boy arrived at the emergency department with a 1- to 2-week history of bumps on his neck and groin. He had been referred to the emergency department by his primary care physician (PCP) when the results of an in-office fingerstick blood test revealed a high white blood cell (WBC) count and a low platelet count. The parents of the patient say that he had had bilateral knee pain a few days previously, which had resolved spontaneously, and had had a cough for the past week. The parents mentioned that when their son had started coughing one week prior, they had contacted the PCP and had mentioned the bumps on his neck and his cough. The physician diagnosed the boy as having lymphadenopathy attributed to a viral illness. Medical and Family History: As of this writing, the patient s immunizations are up to date. He experiences seasonal allergies, with symptoms that include itchy eyes and runny nose. His recurrent ear infections have been resolved by pressure-equalization tubes and adenoidectomy. The patient s maternal grandmother and maternal uncle have long QT syndrome. His paternal great-grandfather has a low platelet count (specificity not given). The patient lives with his parents; as of this writing, his mother is currently at 31 weeks gestation, originally with triplets but currently with twins (one fetus recently died in utero). Physical Examination: During examination to determine the extent of symptoms, abnormalities were found in the respiratory, lymphatic, and musculoskeletal systems. The patient was observed to cough and have leg and knee pain; he also had swollen lymph glands. Laboratory Findings: Table 1, Table 2; Figure 1; Image 1, Image 2, Image 3 Additional Diagnostic Tests: Results of flow cytometric testing indicate an abnormal population in the R2 blast gate. These blasts, which accounted for 40% of the nucleated cells studied, tested positive for B-cell markers CD19 and CD22 and biphenotypic for CD20. The blast cells also tested positive for CD10 and terminal deoxynucleotidyl transferase (TdT). Keywords: Pre-B Acute Lymphocytic Leukemia, lymphadenopathy, pediatric DOI: /LMI17NQD1SBTNHYT Abbreviations PCP, primary care physician; WBC, white blood cell; TdT, terminal deoxynucleotidyl transferase; CBC, complete blood count; LDH, lactate dehydrogenase; BUN, blood urea nitrogen; PBS, peripheral blood smear; BM, bone marrow; FAB, French-American-British; WHO, World Health Organization; HLA-DR, human leukocyte antigen serotype DR; pre-b ALL, pre B-cell acute lymphoblastic leukemia; FISH, fluorescence in situ hybridization; B-ALL, B-cell ALL; CALLA, common ALL antigen; MLL, mixed-lineage leukemia; CNS, central nervous system; COG, Children s Oncology Group; mos, milliosmoles; NA, not applicable; +, present; c, cytoplasmic; +/, can be present;,absent; s, surface; PerCP, peridinin chlorophyll; FSC, forward scatter; FITC, fluorescein isothiocianate 1 Department of Biology, University of North Florida *To whom correspondence should be addressed: erikakelly@comcast.net Questions 1. What were the patient s most striking clinical and laboratory test findings? 2. What did the patient s peripheral blood smear and bone marrow test results indicate? 3. What would be the patient s diagnosis? 4. What was the most likely cause of the patient s condition? 5. What are some treatment regimens available for this patient? Answers 1. The patient s most striking clinical findings on initial complete blood count (CBC) testing were marked leukocytosis and marked thrombocytopenia. The patient s chemistry findings include increased glucose and lactate dehydrogenase (LDH) levels, as well as decreased blood urea nitrogen (BUN), creatinine, and osmolality (Table 1). Summer 2013 Volume 44, Number 3 Lab Medicine e57

2 A B Image 1 Patient s laboratory results via Wright-Giemsa stained peripheral blood smear (original magnification, 40). A, image illustrating leukocytosis, thrombocytopenia, and lymphoblasts. B, Bone marrow image illustrating hypercellularity and increased proliferation of lyphoblasts. Table 1. Initial Laboratory Findings Test Result Reference Range Chemistry Glucose mg/dl BUN mg/dl Creatinine mg/dl Osmolality mos/kg LDH IU/L Hematology WBC count K/µL Hemoglobin g/dl Hematocrit %-40.0% Platelet count K/µL Abbreviations: BUN, blood urea nitrogen; mos, milliosmols; LDH, lactate dehydrogenase; WBC, white blood cell. The abnormal elevated WBC count caused an automatic manual review of the patient s blood smear. Elevated LDH levels are an indicator for acute or chronic tissue damage and can be used to monitor the progression of certain cancers. 1 The decreased BUN and creatinine levels indicated that further testing was required. 2. The results of the patient s peripheral blood smear (PBS) revealed marked thrombocytopenia combined with leukocytosis. The differential showed a marked decrease in neutrophils, lymphocytes, and monocytes. Along with Table 2. Results of Peripheral Blood Smear and Bone Marrow Tests Cell Type Result (%) Reference Range, % Peripheral Blood Neutrophil Lymphocyte 1-71 Monocyte Band Metamyelocyte 2 0 Blast 74 0 Platelet sufficiency Decreased Adequate Polychromasia Few None-few Schistocytes Few None Bone marrow Blasts Lymphocytes Erythrocytes Granulocytic precursor Segmented neutrophils and bands the decreased relative WBC count, polychromasia and schistocytes were present. The most striking characteristic of the patient s PBS is a predominance (ie, 74%) of blasts (Image 1, part A and Table 2). The high blast percentage falsely elevated the WBC count in the initial CBC. The most striking characteristic of the bone marrow (BM) differential was marked hypercellularity, with a blast result of 94% (Image 1, part B and Table 2). Downloaded e58 Lab Medicine Summer 2013 Volume 44, Number 3

3 Image 2 Results of flow cytometry testing indicating an abnormal population of leukocytes in the R2 blast gate. PerCP, peridinin chlorophyll; FSC, forward scatter. A 1,000 SSC Height R5 400 R R3 R2 R1 CD45 PerCP B 1,000 SSC Height ,000 FSC Height Image 3 Flow cytometry results indicating the blast population was positive for markers CD19, CD22, CD10,TdT, and HLA-DR. CD19 CD19 PE CD5 CD10 FITC CD22 PE CD20 FITC CD38 CD34 CD33 CD16 FITC TdT FITC HLA-DR FITC 3. The patient s diagnosis at this point without the results of further testing would be acute leukemia. Initial diagnosis of leukemia according to the French-American-British (FAB) and World Health Organization (WHO) classifications require that blast percentage in the BM be at least 30% and 20%, respectively. 2,3 Flow cytometry testing indicated that the side scatter plot versus marker CD45, a common leukocyte antigen, showed an abnormal population of immature cells in the R2 blast gate, which accounted for 40% of the nucleated cells studied (Image 2). These blast cells were then further differentiated; it was noted that the B cells were positive for markers CD19, CD22, CD10, and TdT. These cells also tested positive for human leukocyte antigen serotype DR (HLA-DR) (Image 3). The test results indicate that the patient s acute leukemia was subtyped to pre B-cell acute lymphoblastic leukemia (Pre-B ALL). A fluorescence in situ hybridization (FISH) panel was performed; the results indicated no TEL/AML1, BCR/ ABL, or MLL gene arrangement. It was also noted that no trisomies were observed on chromosomes 4, 10, or 17. ALL is a malignant neoplasm of precursor B- or T-cells that affects mostly children, with a peak incidence occurring in individuals of approximately 2 to 4 years of age. B-cell ALL (B-ALL) accounts for approximately 80% to 85% of all newly diagnosed ALL cases every year. 3,4 The worldwide incidence is estimated at 1 to 4.75 per 100,000 people per year. 5 It has been reported that males are predominantly affected by B-ALL. 4,5 Patients often experience fever, bleeding, fatigue, and severe bone pain. Bone pain can be Summer 2013 Volume 44, Number 3 Lab Medicine e59

4 Table 3. Laboratory Findings Characteristic of ALL Peripheral Blood Bone Marrow Increased WBC Count Neutropenia Lymphoblasts Normocytic, normochromic anemia Thrombocytopenia Hypercellular Minimum 20% lymphoblasts (per WHO) Source: McKenzie, WBC, white blood cell; WHO, World Health Organization. a possible result of tumor infiltration or marrow necrosis. 4 Patients may have nodal and extranodal involvement. 3,5 Laboratory findings include a form of cytopenia, such as anemia or thrombocytopenia. The overall leukocyte count can be increased, decreased, or normal, with a median count of approximately 10 to /L (Table 3). 3-5 However, the presence of a high percentage of circulating blasts can often falsely elevate the total leukocyte count. 3 Blast cells are usually medium sized with scant cytoplasm, moderately condensed to dispersed chromatin, and nucleoli that may not be readily discernible. 5 Flow cytometry testing can help differentiate among the various forms of B-ALL by identifying the CD markers that exist on the leukocytes. For a patient to be diagnosed with B-ALL, most of the leukocytes must express specific markers, including CD19 and/or CD20, which are primarily found on cells committed to the B-cell maturation process. 4,5 Precursor-B lymphoblasts often present are TdT + and HLA-DR +. Moreover, the expression of CD10, the common ALL antigen (CALLA), differentiates intermediate pre-b from pro-b-all (Table 4) It is currently believed that ALL arises from mutations at critical steps in the cell-maturation process. 4 It is also believed that environmental factors contribute to ALL; however, this has not been fully elucidated. 6 These mutations have been detected in neonatal samples long before the onset of leukemia in approximately 90% of childhood ALL. 5,7 WHO currently classifies B-ALL into 5 categories according to which genetic abnormality is found (Table 5).However, these genotypes are comprised of only 60% of pre-b ALL; the remaining 40% are unknown or rare. 8 The Philadelphia chromosome occurs in only about 2-5% of ALL cases and is one of the rarest causes of leukemia. 4,9 Clinically, patients with this chromosome tend to be older and have a higher leukocyte count at the time Table 4. Cellular Markers Useful in Diagnosis and Classification of B ALL 3 Subgroup HLA DR TdT CD34 CD19 CD22 CD10 CD20 Pro-B (early precursor) (c) Intermediate pre-b (common ALL) + + +/ + +(c) + +/ Pre-B ALL (s) +/ + Abbreviations: B ALL, B cell acute lymphoblastic leukemia; HLA DR, human leukocyte antigen serotype DR; TdT, terminal deoxynucleotidyl transferase; +, present; c, cytoplasmic; ALL, acute lymphoblastic leukemia; +/, can be present;,absent; s, surface. Table 5. World Health Organization Classification of B-cell Acute Lymphoblastic Leukemia 3,10 Variable Cytogenetic Abnormality Genetic Alteration Risk Group Recommended Therapy Precursor t(9;22)(q34;q11) BCR/ABL (Philadelphia chromosome) High Allogeneic BM transplant B-cell ALL t(11;v)(11q23;var) MLL rearranged High Allogeneic BM transplant t(1;19)(q23;p13) E2A/PBX1 Intermediate Intensive therapy T(17;19) E2A/HLF Intermediate Intensive therapy t(12;21)(p12;q22) TEL/AML1 Low Antimetabolite therapy Abbreviations: BM, bone marrow; ALL, acute lymphoblastic leukemia; MLL, mixed-lineage leukemia. Downloaded e60 Lab Medicine Summer 2013 Volume 44, Number 3

5 of diagnosis. 9 It is important to diagnose this condition via FISH studies due to its poor prognosis. 4 Another rare genetic abnormality linked to pre-b ALL is mixed-lineage leukemia gene (MLL) rearranged, which accounts for approximately 6% of all cases. 3,4 Currently, more than 60 genes are in this category. However, a study 4 has shown that certain gene rearrangements have been observed to occur in approximately 60% of MLL-related leukemias. These leukemias tend to involve high WBC counts and often show central nervous system (CNS) involvement. 4 Both leukemias are also associated with poor prognosis. The most common genetic abnormality observed is t(12;21) (p12;q22), which produces the TEL-AML1 fusion gene. The expression of this gene has been associated with favorable prognosis, with an event-free survival rate of 90%. 3-5 The results of FISH studies indicated that the patient did not express any of the genetic abnormalities commonly used to categorize acute leukemia. However, a current study 8 has shown that more abnormalities have been observed to be recurrent and may be added to the current FISH panel for diagnostic testing in the future. 8 Further molecular studies are needed to properly assess whether the patient harbors any abnormalities. 5. Children diagnosed with ALL have approximately a 95% chance of remission if treated with combination chemotherapy. Of these treated patients, approximately 80% are disease-free 5 years after diagnosis. 4,5 Treatments today include systemic chemotherapy and CNS therapy. 4 A number of new treatments are available to newly diagnosed patients through Children s Oncology Group (COG) studies, which follow strict protocols and are offered to newly diagnosed patients who have not responded to treatment, have relapsed, or are in the maintenance phase. 1 The specific therapy recommended for patients is dependent on the risk group to which the patient belongs, according to the patient s genetic features (Table 5). Other factors, such as age and leukocyte count, are also taken into consideration. 10 Chemotherapy for ALL is divided into 3 stages. The first stage, induction therapy, is administered to eradicate the leukemic blast population. 3 The drugs administered during this period are usually a glucocorticoid, vincristine, and asparaginase. 10 The second stage, the CNS prophylactic stage, is administered because the most common site of relapse is the CNS 3 ; the drugs in this stage are administered at higher doses. Periodically, high-dose methotrexate (an antimetabolite) and 6-mercaptopurine (an immunosuppressant) can be used. 10 The final stage of treatment, maintenance chemotherapy, is designed to eradicate any remaining leukemic cells and to prolong remission. 3 To be cured, patients with pre-b ALL require a 2- to 2½-year course of therapeutic agents. It has been observed that males need to be treated longer than females because relapse can occur in the testes. 10 Acknowledgments The author would like to thank the Hematology Team at Baptist Hospital-Downtown in Jacksonville, Florida, for providing clinical information and patient test results. LM References 1. American Association for Clinical Chemistry. Retrieved April 4, 2013, from Lab Tests Online: analytes/ldh/tab/glance. 2. Leukemia & Lymphoma Society. Understanding Leukemia. Retrieved April 4, 2013, from the Leukemia & Lymphoma Society Web site. understandingleukemia. 3. McKenzie S, Williams J. Clinical Laboratory Hematology. 2nd edn. Upper Saddle River: Pearson Education; Foucar K, Reichard K, Czuchlewski D. Bone Marrow Pathology. 3rd edn. Chicago: ASCP Press; Swerdlow S, Campo E, Harris N, et al, eds. WHO Classification of Tumours of Haematopoietic and Lymphoid Tissues. Lyon, France: International Agency for Research on Cancer (IARC); Dorak MT, Lawson T, Machulla HKG, Darke C, Mills KI, Burnett AK. Unravelling an HLA-DR association in childhood acute lymphoblastic leukemia. Blood. 1999;94: Pui CH, Behm FG, Crist WM. Clinical and biologic relevance of immunologic marker studies in childhood acute lymphoblastic leukemia. Blood 1993;82: Meijerink JPP, den Boer ML, Pieters R. New genetic abnormalities and treatment response in acute lymphoblastic leukemia. Semin Hematol. 2009;46: Raimondi SC. Current status of cytogenetic research in childhood acute lymphoblastic leukemia. Blood. 1993;81: Rubnitz JE, Pui C-H. Childhood acute lymphoblastic leukemia. Oncologist. 1997:2: Summer 2013 Volume 44, Number 3 Lab Medicine e61