AJCP / Original Article Cytogenetic Variation of B-Lymphoblastic Leukemia With Intrachromosomal Amplification of Chromosome 21 (iAMP21) A Multi-Institutional Series Review Ryan C. Johnson, MD,1 Olga K. Weinberg, MD,2 Michael J. Cascio, MD,3 Gary V. Dahl, MD,1 Bryan A. Mitton, MD,1 Lewis B. Silverman, MD,2 Athena M. Cherry, PhD,1 1 1 Daniel A. Arber, MD, and Robert S. Ohgami, MD, PhD Downloaded from https://academic.oup.com/ajcp/article/144/1/103/1761609 by guest on 30 September 2021 From the 1Stanford University Medical Center, Stanford, CA; 2Boston Children’s Hospital, Boston, MA; and 3Oregon Health and Science University, Portland. Key Words: Hematopathology; Flow cytometry; Pediatric acute lymphoblastic leukemia; iAMP21; Cytogenetics and molecular genetics; Clinical and molecular epidemiology Am J Clin Pathol July 2015;144:103-112 DOI: 10.1309/AJCPLUYF11HQBYRB ABSTRACT The editors of the 2008 WHO Classification of Tumours of Haematopoietic and Lymphoid Tissues sought to place Objectives: B-lymphoblastic leukemia (B-ALL) with a priority on the most recent cytogenetic and molecular intrachromosomal amplification of chromosome 21 findings to establish a more definitive characterization and (iAMP21) is a relatively uncommon manifestation of classification of both acute lymphoblastic and acute myeloid acute leukemia and limited predominantly to the pediatric leukemias, with some leukemias defined by their specific population. Case-specific information regarding flow translocation independent of the blast count observed. This cytometric, morphologic, and laboratory findings of this classification often provides a characteristic cytogenetic cor- subtype of leukemia is currently lacking. relation to the morphologic and flow cytometric expression Methods: We searched the databases of three large pattern observed. For example, although B-lymphoblastic institutions for lymphoblastic leukemia with iAMP21 from leukemia/lymphoma (B-ALL) with t(5;14)(q31;q32) IL3- 2005 through 2012 and analyzed the clinicopathologic IGH accounts for less than 1% of all B-ALL cases, the features. characteristic consistent finding of an associated nonclonal eosinophilia merits inclusion of this leukemia as one with a Results: We identified 17 cases with five or more RUNX1 recurrent cytogenetic abnormality.1 More important, how- signals on interphase nuclei, 14 of which were consistent ever, classification of leukemias with recurrent cytogenetic with the Children’s Oncology Group (COG) definition for abnormalities serves to stratify subtypes of leukemia based iAMP21—namely, the presence of three or more RUNX1 on their overall prognosis and facilitate appropriate chemo- signals on one marker chromosome. These cases showed therapeutic regimen selection.2 a statistically significant lower peripheral WBC count and Other less common recurrent cytogenetic abnormalities older age at diagnosis compared with all pediatric cases have also been recently reported but have not yet become of B-ALL. We also identified three cases with increased established entities. Such is the case with B-ALL with intra- RUNX1 signals scattered on multiple marker chromosomes chromosomal amplification of chromosome 21 (iAMP21). that did not meet the COG definition of iAMP21 but showed These cases are identified on routine karyotype by the similar 21q instability and older age at presentation. absence of a second normal copy of chromosome 21 and Conclusions: Our findings not only demonstrate that B-ALL concurrent fluorescence in situ hybridization (FISH) studies, with iAMP21 is truly a distinct clinicopathologic entity but which reveal an abnormal number and pattern of RUNX1 also suggest that a subset of cases of B-ALL with iAMP21 (AML1) signals when probes for t(12;21) ETV6-RUNX1 are can show variable cytogenetic features. used. The current International Standard for Human Cyto- genetic Nomenclature for this cytogenetic finding is nuc ish (RUNX1 × n), where n is the number of identified RUNX1 signals per interphase nucleus; for enumerable RUNX1 © American Society for Clinical Pathology Am J Clin Pathol 2015;144:103-112 103 DOI: 10.1309/AJCPLUYF11HQBYRB Johnson et al / B-Lymphoblastic Leukemia With iAMP21 signals, the nomenclature is nuc ish (RUNX1 amp).3 For sim- responsible for the breakpoint in a study by Sinclair et al21 plicity, however, this has been referred to in the literature as revealed that other genes local to the region around RUNX1, iAMP21. Although B-ALL with iAMP21 is not recognized such as PDE9A, COL6A, and DSCAM, may be involved. by the 2008 edition of the WHO Classification of Tumours Among cases of B-ALL with iAMP21, some consistent of Haematopoietic and Lymphoid Tissues, various groups clinicopathologic findings have been noted8-16: B-ALL with have defined iAMP21 in studies by the presence of three or iAMP21 tends to present in older children with pancytope- more RUNX1 signals on an abnormal chromosome 21 and/ nia or mildly elevated WBC counts (usually ≤10 × 109/L, or five or more total signals per interphase nucleus.4-7 The with most ≤50 × 109/L) at diagnosis.22 A low WBC count at Children’s Oncology Group (COG), however, currently diagnosis in B-ALL in general usually results in a reduced requires the presence of three or more RUNX1 signals on a risk stratification for treatment. However, the prognosis of single-marker chromosome to qualify as iAMP21.5 patients with B-ALL with iAMP21 is consistently poor, The first reports of B-ALL with increased RUNX1 with patients demonstrating worse event-free survival (EFS) Downloaded from https://academic.oup.com/ajcp/article/144/1/103/1761609 by guest on 30 September 2021 signals were made serendipitously when FISH probes for and overall survival (OS) when treated with standard-risk the t(12;21)(q22;q22) ETV6/RUNX1 translocation revealed chemotherapy regimens.7 Although these children typically increased signals of the RUNX1 probe.8-16 All of these cases demonstrate hematologic disease remission following induc- showed, in common, a loss of a normal chromosome 21 with tion chemotherapy, the relapse rate is very high and, subse- the presence of a derivative marker chromosome. The num- quently, relatively refractory to treatment.7 However, more ber of RUNX1 probe signals was consistent in each patient recent work has shown that treating B-ALL with iAMP21 but varied highly between patients, ranging from four to 15 with high-risk chemotherapeutic regimens ameliorates the copies. It was thus proposed in these initial studies that the poor EFS and OS findings.4,5 derivative chromosome was composed of variable fragments Given the relative heterogeneity in RUNX1 signals per of the long arm of chromosome 21, which contained the marker chromosome reported between leukemic clones RUNX1 gene. It has since been estimated that B-ALL with within and between patients, as well as the current two iAMP21 accounts for 1% to 2% of all pediatric B-ALLs.7 working definitions of B-ALL with iAMP21, we sought to The cytogenetic underpinnings of increased RUNX1 determine the heterogeneity of iAMP21 in a limited multi- signals are not completely understood; however, the institutional series. In addition, we wanted to compare our most widely cited mechanism involves multiple breakage- relative frequency of B-ALL with iAMP21, as well as mor- fusion-bridge (BFB) cycles and chromosome region 21q phologic and flow cytometric data with series and studies instability. By morphologic analysis and whole-chromo- reported in the literature to date. some painting probes for chromosome 21, Kuchinskaya et al17 showed that bone marrow lymphoblasts and cul- tured lymphoblasts from patients with iAMP21 showed Materials and Methods abnormal anaphase bridges of chromosome 21 material between mitotically active cells in a subset of relapsed cases. Multiple chromosome color banding using seven Patient Cohort and Cytogenetic Studies probes spanning the 21q region on metaphase cells showed All aspects of this study were approved by the institu- highly variable arrangements of probes, including cases tional review boards of all participating institutions where where probes proximal to RUNX1 were identified distal to applicable. We reviewed the cytogenetics reports of all repeated sequences of RUNX1; this arrangement suggested adult and pediatric cases of B-ALL within the database of a repeated cycle of chromosomal inversion with subse- the Stanford University Department of Pathology, Boston quent duplication. Independent of the exact banding pat- Children’s Hospital, and Oregon Health Sciences University tern, all color band plots showed a highly unstable region from 2005 through 2012 and identified cases where four or of chromosome 21q, with most cases also showing loss of more RUNX1 signals per nucleus were identified on FISH telomeric or subtelomeric sequences of 21q.18 analysis with the RUNX1/ETV6 probe set. Additional back- While one might presume that RUNX1 plays a central ground information regarding the general demographics of role in the leukemogenesis of B-ALL with iAMP21 cases, all acute B-ALLs, both pediatric and adult, were also con- RUNX1 itself has not been shown to be overexpressed com- currently obtained to determine the frequency of increased pared with other subtypes of B-ALL in expression profile RUNX1 signals and those that meet COG criteria for B-ALL analysis.19,20 Closer examination into the border between with iAMP21. the common region of amplification and normal comple- For cases with increased RUNX1 signals meeting cri- ment of chromosome 21 by