PB1686 USE OF FLOW CYTOMETRY AND IMMUNOHISTOCHEMISTRY TO RULE OUT CNS RELAPSE OF ACUTE LYMPHOBLASTIC LEUKEMIA
Authors: M. Moschovi, C. Kelaidi, A. Zampogiannis, N. Tourkantoni, M. Tzanoudaki, M. Kanariou, K. Stefanaki, G. Paterakis
Journal: HemaSphere (2019)

Background: Increased cerebrospinal fluid (CSF) cellularity in an otherwise asymptomatic patient with acute lymphoblastic leukemia (ALL) may indicate CNS disease. Aims: To describe how flow cytometry and immunohistochemistry helped in ruling out suspicion of CNS relapse because of unexpected CSF findings in a child with acute lymphoblastic leukemia in CR during maintenance phase of chemotherapy. Methods: Examination of cytospin CSF samples with flow cytometry, fluorescent in vitro hybridization (FISH) for centromeres of chromosomes 7 and 8 on 200 cells and immunohistochemistry for CD10 and terminal deoxynucleotidyl transferase (TdT). Results: A 15-year old girl was diagnosed in 2005 with B common ALL with hyperdiploidy including trisomy 8, and no CNS involvement. She was treated according to a modified New York II protocol with good prednisone response and negative flow cytomerty minimal residual disease (MRD) on day 33, and prophylactic cranial radiation of 1200 cGy. She went on with maintenance treatment consisting of 6-mercaptopurine 300 mg/m2/d d0-3, cyclophosphamide 1200 mg/m2 d4, vincristine 1.5 mg/m2 d11, 18, 25, prednisonse 180 mg/m2/d d18-25, methotrexate 200 mg/m2 over 4 h d25, daunorubicin 20 mg/m2 d40, 41, cytosine arabinoside 100 mg/m2, thioguanine 40 mg/m2/dose 6 doses d42-44, methotrexate IT d60/0. Days 0 and 60 CSF findings were normal. However, on day 0 of the subsequent cycle, increased ceullarity was found incidentally. CSF cell number was 45/μL and increased up to 130/μL despite additional methotrexate IT injections. Examination of cytospin preparations showed 75% lymphocytes and 25% monocytes/macrophages. Morphological discrimination between lymphoblasts and mature lymphocytes was difficult. Protein level in CSF was mildly increased up to 71 mg/dL and glucose level in CSF was normal. The patient was free of symptoms and the neuroimaging was normal. Immunohistochemistry on cytospin preparation showed no TdT expression on lymphocytes. Flow cytometry of CSF using the initial immunophenotype showed normal lymphocytes and monocytes and no leukemic blasts. FISH on a cytospin preparation to detect trisomy 8, which was present at diagnosis, and using as an internal negative control chromosome 7 ploidy, was normal with two signals for chromosomes 8 and 7, respectively. Those results in addition to increasing cellularity despite intrathecal chemotherapy and progressive enrichment of CSF in monocytes and uneventful clinical course and undetactable MRD in bone marrow, led to the conclusion that high CSF high cellularity was of reactive nature. CSF cellularity returned to normal within 2 weeks. The patient completed maintenance treatment and remains in persistent complete first remission for 10 years. Summary/Conclusion: Incidental high CSF cellularity arousing suspicion of CNS relapse may be misleading. Sensitive methods of immunophenotyping and genotyping at the cellular level provide confidence in diagnosing reactive CSF pleocytosis secondary to previous intrathecal chemotherapy and avoid unnecessary treatment plan modifications.