Unique morphologic and genetic characteristics of acute myeloid leukemia with chromothripsis: a clinicopathologic study from a single institution

Journal Pre-proof Unique morphologic and genetic characteristics of acute myeloid leukemia with chromothripsis: a clinicopathologic study from a single institution Juehua Gao, MD, PhD, Yi-Hua Chen, MD, Alain Mina, MD, Jessica K. Altman, MD, Kwang-Youn Kim, PhD, Yanming Zhang, MD, Xinyan Lu, MD, Lawrence Jennings, MD, PhD, Madina Sukhanova, PhD PII:

S0046-8177(20)30021-6

DOI:

https://doi.org/10.1016/j.humpath.2020.02.003

Reference:

YHUPA 4968

To appear in:

Human Pathology

Received Date: 17 September 2019 Revised Date:

2 February 2020

Accepted Date: 16 February 2020

Please cite this article as: Gao J, Chen Y-H, Mina A, Altman JK, Kim K-Y, Zhang Y, Lu X, Jennings L, Sukhanova M, Unique morphologic and genetic characteristics of acute myeloid leukemia with chromothripsis: a clinicopathologic study from a single institution, Human Pathology, https:// doi.org/10.1016/j.humpath.2020.02.003. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 Elsevier Inc. All rights reserved.

Unique morphologic and genetic characteristics of acute myeloid leukemia with chromothripsis: a clinicopathologic study from a single institution Juehua Gao, MD, PhD1, Yi-Hua Chen, MD1, Alain Mina, MD2, Jessica K. Altman, MD2, Kwang-Youn Kim, PhD3, Yanming Zhang, MD4, Xinyan Lu, MD1, Lawrence Jennings, MD, PhD1, Madina Sukhanova, PhD1 1

Department of Pathology, Northwestern University Feinberg School of Medicine, Chicago, IL Department of Medicine, Division of Hematology and Oncology, Northwestern University Feinberg School of Medicine 3 Department of Preventive Medicine – Biostatistics, Northwestern University Feinberg School of Medicine 4 Department of Pathology, Memorial Sloan Kettering Cancer Center, New York, NY 2

Corresponding Author: Madina Sukhanova, PhD Department of Pathology Northwestern University Feinberg School of Medicine 3030 East Chicago Ave, Ward 3-140 Chicago, IL 60611 Email: [email protected] Office: (312)926-3353 Running title: Morphologic and genetic characteristics of AML with chromothripsis

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Summary Chromothripsis is a unique type of genomic instability and is recognized in various cancers. In myeloid neoplasms (MNs) chromothripsis was linked to poor prognosis and specific genetic alterations [complex karyotype, 5q deletions, and loss of TP53]. However, the clinicopathologic features of MNs with chromothripsis have not been thoroughly characterized. We identified chromothripsis in 11 cases of MNs (9 AML and 2 MDS) and noted that all chromothripsis-positive AML cases were AML with myelodysplasia related changes (AML-MRC). We performed a comparative clinicopathologic and genetic characterization of AML-MRC cases with and without chromothripsis. AML-MRC with chromothripsis is associated with lower WBC and platelet counts and higher degree of karyotypic complexity. Chromothripsis in AML-MRC most frequently involves chromosomes 8 and 11 with consequent amplification of either MYC or KMT2A. Comparative morphologic assessment of blast morphology revealed unique features characteristic to AML-MRC with chromothripsis: a variable degree of cytoplasmic vacuolization, granulation, and blebbing. These morphologic markers in the context of AMLMRC may prompt additional studies to identify cases with chromothripsis.

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1. Introduction The theory of oncogenesis as a progressive multi-step accumulation of somatic genetic alterations has changed with the discovery of chromothripsis, a single catastrophic event of chromosome shattering that is characterized by tens to hundreds of locally clustered rearrangements affecting one or few chromosomes or chromosomal segments (1). Chromothripsis has been reported in a wide range of cancers, including lung, esophageal, bladder, prostate and breast adenocarcinomas, as well as glioblastomas (2-6), in association with an adverse prognosis, gaining a reputation of a high-risk marker. In hematopoietic neoplasms, chromothripsis was frequently noted in Acute Lymphoblastic Leukemia with iAMP21 (7), MDS with complex chromosomal aberrations, and AML with deletions at 5q and frequent loss of TP53 due to either mutations or deletions at 17p (8). The incidence of chromothripsis has been reported at approximately 6.6% in de novo AML (9) and chromothripsis was proposed as a prognostic marker of a worse overall survival in AML even in comparison to other high-risk entities. However, the clinicopathologic features of AML cases with chromothripsis are very limited. In this retrospective single institution study, we identified and thoroughly characterized clinical, morphologic, and genetic features of AML cases with chromothripsis.

2. Materials and Methods 2.1 Patients Chromosomal Microarray Analysis (CMA) was performed on a series of consecutive myeloid neoplasms newly diagnosed in our institution. Only the cases with available DNA material were included. In this series, 11 cases with chromothripsis were identified including 9 AML-MRC and 2 MDS. Whereas chromothripsis most often occurs in AML-MRC, we include 17 cases of AML-MRC without chromothripsis diagnosed within the same period of time as a comparison in this retrospective analysis. 3

The pathologic diagnoses were confirmed by examining the peripheral blood (PB) smear, bone marrow (BM) aspirate, and core biopsies, as well as by reviewing all ancillary studies including immunohistochemistry, flow cytometry, fluorescence in situ hybridization (FISH), and conventional cytogenetic analyses. The clinical risk stratification was determined according to the World Health Organization (WHO) 2016 classification system that relies on clinical features, morphology, immunophenotype and genetics.(10) Overall survival (OS) was assessed as the time in days from the time of diagnosis to the time of death or last follow-up. This study was approved by the Northwestern University Institutional Review Board. Student t test was performed to determine if two sets of data are significantly different. All tests were two-sided and a p-value <0.05 was considered significant.

2.2 Morphology Wright-Giemsa stained PB and BM aspirate smears, and hematoxylin-eosin stained core biopsy slides were reviewed by two hematopathology attendings (JG, YC) independently and blinded to the genetic results. The diagnosis and classification of each case was rendered based on standard criteria (11). Specific dysplastic features in each lineage were evaluated and scored as mild (<10%), moderate (1050%) and marked (>50%). The specific dysplastic features in the erythroid lineage included: 1) megaloblastoid changes; 2) multinucleation; 3) nuclear irregularities; and 4) pyknosis. Dysgranulopoiesis features included: 1) hypolobation; 2) hypogranulation; and 3) abnormal chromatin pattern. Dysmegakaryopoiesis included: 1) micromegakaryocytes; 2) hypolobation; and 3) separate lobes. Dysplasia in erythroid, granulocytic and megakaryocytic lineages were scored as 0 (negative), 1 (<10%), 2 (10-50%) or 3 (>50%). For each case, the dysplasia score was calculated by adding the scores of each lineage. Other features of the blasts were also evaluated including: 1) Cytoplasmic vacuolization; 2) Cytoplasmic granularity; 3) Auer rods; and 4) cytoplasmic membrane projections. These features were also scored as 0 (negative), 1 (<10%), 2 (10-50%) or 3 (>50%). For each case, the blast morphology score

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was calculated by adding scores of these specific features. The BM differential counts, myeloid to erythroid ratio, BM or core biopsy cellularity, presence or absence of ring sideroblasts, presence or absence of BM fibrosis were also recorded.

2.3 Flow cytometry Flow cytometric analyses were performed on fresh BM samples. Samples were processed using the whole blood lysis. The white blood cell suspension was mixed with the following fluorochromeconjugated anti-human monoclonal antibodies from Becton Dickinson Biosciences: CD34-BV421, CD13PE, CD3-APC-H7, CD5-APC, CD11b-APC, HLADR-APC-H7. The following antibodies were from Beckman Coulter: CD45-krome orange, CD117-PC7, MPO-PE, CD33-PC5.5, CD64-FITC, CD2-PC7, CD7-PE, TDT-FITC. The cells were incubated at room temperature for 20 minutes, washed in PBS with 0.2% NaAzide, and resuspended in 400 μL of 4% Para formaldehyde. Data acquisition was performed on a standard Canto II eight-color flow cytometer (Becton Dickinson Biosciences) employing 405-nm, 488-nm, and 635-nm excitation lasers using a standard optical detection configuration. Data analysis was performed using Kaluza software (Beckman Coulter) and FACSDiva software (Becton Dickinson Biosciences).

2.4 Cytogenetics and FISH Conventional cytogenetic analyses using the G-banding method were done as part of the routine clinical diagnostic work-up. Conventional cytogenetic analysis was performed on fresh BM aspirate samples after 24-hr or 48-hr unstimulated cultures following standard protocol. At least 20 metaphase cells were examined in each case, and the chromosomal abnormalities were described according to the International System for Human Cytogenetic Nomenclature (ISCN). FISH analyses were performed on the same fixed cells from BM samples collected at the time of diagnosis. A total of 200 interphase cells

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were evaluated independently by two technologists. FISH testing was performed using commercially available probes (Vysis/Abbott Molecular, Abbott Park, IL) according to the manufacturer’s instructions.

2.5 DNA isolation and Chromosomal Microarray Analysis

Genomic DNA was extracted with a Qiagen DNeasy Blood & Tissue Kit (Qiagen, Valencia, CA, USA), following the manufacturer’s instructions. CMA was performed using the Affymetrix CytoScanTM HD arrays (ThermoFisher Scientific, Santa Clara, CA), following the manufacturer’s recommendations. The data were analyzed with the Chromosome Analysis Suite (ChAS) software from Affymetrix. Copy number variants (CNVs) including copy number gains or losses, and stretches of copy neutral loss of heterozygosity (CN-LOH) were manually reviewed. Chromothripsis was assessed manually according to criteria reported by Korbel et al (12). The following criteria were used for analysis: more than 10 breakpoints with a minimum segment size of 10kb.

3. Results 3.1 Clinical patient characteristics and pathological assessment Through retrospective review, we identified 11 chromothripsis-positive cases of MNs which included 9 AML-MRC cases, 1 t-MDS case, and 1 case of MDS-EB1. The clinicopathologic features and genetic characterization of the 9 AML-MRC cases with chromothripsis were compared with 17 cases of AMLMRC without chromothripsis. Chromothripsis-positive AML-MRC cohort consisted of 6 male and 3 female patients with median age of 65 years of age; chromothripsis-negative AML-MRC cohort included 7 male and 9 female patients with median age of 59 years of age. Consistent with previously reported data, chromothripsis in AML-MRC has an association with more advanced age (9). All AML-MRC cases in the chromothripsis-positive cohort were classified as high risk: the two MDS cases included 1 high-risk and 1 very high-risk MDS. Of the AML-MRC cases without chromothripsis, 10 were classified as high risk, 6

4 as intermediate risk, 1 as low risk, and the risk assessment for 1 patient was unknown. Although AMLMRC in general is associated with very poor prognosis, patients with chromothripsis showed a tendency towards more rapid decline as compared to chromothripsis-negative AML-MRC cohort; median overall survival (OS) among AML-MRC patients with chromothripsis was 73 days vs 431 days for AML-MRC patients without chromothripsis (Figure 1). Strikingly, 3 out of 9 (33%) AML-MRC patients with chromothripsis died within 10 days after diagnosis and another 3 patients (33%) died within 3 months after diagnosis; this finding suggests an association of chromothripsis in AML-MRC with a high risk of imminent death and therapy resistance to existing therapy treatment. Comparative analysis of AML-MRC cases in chromothripsis-positive and chromothripsis-negative groups revealed that patients with chromothripsis tend to present with lower white blood cell (WBC) counts (10.9 versus 26.4) and lower platelet counts (60.9 versus 94.8), whereas hemoglobin levels did not reveal significant differences between both groups (8.1 versus 8.3), consistent with previously reported data [8]. Table 1 and Supplementary Table 1 show clinical characteristics of the AML-MRC patients with or without chromothripsis in our study. We detected that all chromothripsis-positive cases of AML-MRC displayed notable dysplasia. In 5 out of 9 cases, a diagnosis of AML-MRC could be established based solely on morphologic criteria, i.e. at least 50% of dysplastic cells observed in two or more hematopoietic lineages: 2 cases revealed erythroid and granulocytic dysplasia, 1 case was notable for erythroid and megakaryocytic dysplasia, and 2 cases had prominent dysplasia in all three lineages. Three out of the remaining 4 cases had few granulocytes or megakaryocytes for morphology evaluation and therefore the diagnosis of AML-MRC could not be established solely based on morphologic criteria; however, dysplasia was seen in at least one hematopoietic lineage in all these cases. In AML-MRC cases without chromothripsis, 4 out of 17 cases demonstrated significant multilineage dysplasia meeting the morphologic criteria for AML-MRC, 8 cases had somewhat mild dysplasia in one or two hematopoietic lineages, 1 case had no morphologic 7

evidence of dysplasia, and the remaining 4 cases showed few non-blast cells for morphologic evaluation although no obvious dysplasia was evident. The presence or absence of dysplasia and the degree of dysplasia does not seem to differ significantly in AML-MRC cases with or without chromothripsis (Supplementary Table 2).

Interestingly, the blasts in AML-MRC with chromothripsis revealed some unusual unique morphologic features: they tend to be larger in size than typical myeloid blasts, and have abundant light blue cytoplasm and coarsely clumped chromatin. Cytoplasmic vacuoles, which is a morphologic feature for dysplasia, is particularly enriched in cases with chromothripsis. Cytoplasmic vacuoles were present in 7 out of 9 cases (78%). They were often present in a great number and were variable in sizes with visible crisp borders. In approximately 44% of the cases (4 out of 9), we also noted coarse cytoplasmic granules and no Auer rods. All cases except one demonstrated varying degree of cytoplasmic protrusion known as blebbing, as well as increased cytoplasmic shedding in the background (Figure 2 and 3). Although erythroid dysplasia was a common finding, ring sideroblasts were not identified in these cases. All three morphologic features (coarse cytoplasmic granules, cytoplasmic vacuoles and cytoplasmic shedding) were identified to a variable degree in 7 out of 9 (78%) AML-MRC cases with chromothripsis and only in 1 out of 16 (6%) cases of AML-MRC without chromothripsis. Although none of the morphologic features by itself was specific enough to identify cases with chromothripsis, a combination of all these features seems quite characteristic of AML-MRC cases with chromothripsis. Figure 4 shows a representative case of acute myeloid leukemia with chromothripsis for illustration. A scoring system was used to evaluate these three morphologic features in the blasts by a hematopathologist blinded to the genetic results. A score of 0 to 3 was determined in 5 categories, based on the percentage of myeloid/erythroid/megakaryocytic dysplasia, cytoplasmic granularity and projections (0 as no evidence, 1 as 0-10%, 2 as 10-50% and 3 as >50%). Cases of AML-MRC with or without chromothripsis demonstrated comparable dysplasia scores (Supplementary Table 2, Figure 5A). However, cases of AML8

MRC with chromothripsis had an average blast morphologic score of 4.33, significantly higher than the average score of 1.82 in AML-MRC without chromothripsis (p=0.002) (Supplementary Table 2, Figure 5B). The morphologic appearance of the blasts, in some cases, raises the possibility of erythroid and megakaryocytic differentiation. The blasts were predominantly CD117+ (9 out of 9, 100%), CD34+ (8 out of 9, 89%), MPO+ (5 out of 9, 56%), and expressed myeloid antigens CD13 (9 out of 9, 100%) and CD33 (9 out of 9, 100%). Aberrant expression of B- or T-lymphoid antigens was seen in 67% of AML-MRC cases with chromothripsis and only in 22% of AML-MRC cases without chromothripsis. The immunophenotypic features, including the aberrant expression of T- or B-cell antigens were not statistically different in AML-MRC with or without chromothripsis. The blasts were typically negative for myeloperoxidase (MPO) by cytochemistry. Chromothripsis was also identified in two MDS cases including one with excess blasts. Both cases demonstrated evidence of dysplasia in multiple lineages. One of the two cases also showed all three features of cytoplasmic granules, vacuole and shedding. 3.2 Cytogenetics and SNP microarray We compared karyotypic complexity between both cohorts, defining cases with complex karyotype as those with 5 or more chromosomal abnormalities including structural rearrangements. All AML-MRC cases with chromothripsis (100%) revealed complex karyotypes with multiple numerical and structural abnormalities, including deletions of 5q31.1 and 17p13.1 regions, along with marker and ring chromosomes, whereas only 5 out of 16 chromothripsis-negative AML-MRC cases (31%) revealed complex karyotypes. AML-MRC patients with chromothripsis are also characterized by a significantly higher number of copy number alterations (CNAs), detected by CMA, compared to AML-MRC patients without chromothripsis. Average number of copy number gains, losses, and stretches of CN-LOH were 12.8, 25.8, and 2.9, respectively, in chromothripsis-positive AML-MRC cases vs 1.1, 3.0, and 0.7 in

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chromothripsis-negative cases (Table 2, Supplementary Table 3). Deletions at 5q were present in all 9 AML-MRC cases with chromothripsis (100%), which was not surprising in lieu of our finding that chromothripsis might have a tight association with AML-MRC entity, where losses at 5q, 7q and 17p are recurrent abnormalities. However, we still noted that the frequency of del(5q) was higher in AML-MRC chromothripsis-positive groups (100%) compared to chromothripsis-negative AML-MRC (7 out of 16, 44%) (Table 2, Figure 6 C, D). Here we confirm the presence of a minimal commonly-deleted region at 5q31.1-q33.1, reported previously (8), in all 9 chromothripsis-positive AML-MRC cases (100%) in our cohort. Only 5 out of 16 chromothripsis-negative AML-MRC cases (31%) harbored focal deletions at 5q, consistent with previous reports (14%) (9). Deletions at 17p, resulting in loss of the TP53 gene, were present in 6 out of 9 AML-MRC cases with chromothripsis (68%), but were not detected in any of AMLMRC cases without chromothripsis (0%) (Figure 6 C, D, Supplementary Tables 3 and 4). Detailed analysis of the observed CNVs in chromothripsis-positive vs chromothripsis-negative AML-MRC cases revealed apparently non-random enrichment of common gains at chromosomes 8, 11q, and 22 and focal deletions at 3p, 15q, 16, 17, and 18 in AML-MRC with chromothripsis in addition to recurrent losses at 5q and 7q (Figure 6). In our cohort of chromothripsis-positive AML-MRC cases, chromothripsis affects one chromosome (7/9 cases, 78%), two chromosomes (1/9 case, 11%), or three chromosomes (1/9 case, 11%). Chromothripsis affected chromosome 11 in 2/9 cases and either of chromosomes 1, 2, 3, 6, 8, 12, 13, 15, 17, and 20, each in one case (Supplementary Table 4). In agreement with previously reported data by Fontana et al, we noted that when affected by chromothripsis, chromosome 8 or 11 often acquires amplification of either MYC or KMT2A, respectively (9). In addition, we detected amplifications of other genes, i.e. RUNX1, DYRK1A, ERG, ETS2, as well as clusters of genes at 12p and 15q, in our study.

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4. Discussion Chromothripsis is a recently identified phenomenon characterized by massive rearrangements of one or several chromosomes as one single catastrophic event. Chromothripsis has been reported in a wide range of cancers and is thought to cause cellular transformation due to loss of tumor suppressor or amplification of an oncogene. The underlying mechanisms resulting in chromothripsis are still not known. Initial theory implied aberrant replication timing leading to genomic instability and the unique genetic signature of chromothripsis (13). Alternative theory suggested chromothripsis as result of a series of random non-homologous end joining after DNA damage (14). Recent studies have identified the association between chromothripsis with other genetic features such as hyper-diploidy (15), telomere crisis (16, 17), mutations in critical genes involved in genome stability such as TP53 (18, 19) and ATM (20). Chromothripsis has been proposed as a genomic marker of poor outcome (9), thus, it came of no surprise to note a presumably non-random enrichment of chromothripsis in a high-risk entity AML-MRC in our cohort. Taking into consideration fast deterioration of patients diagnosed with chromothripsispositive AML-MRC, it would be beneficial to identify chromothripsis at the time of diagnosis for proper prognostication. However, chromothripsis is a phenomenon that could only be identified through comprehensive genomic studies such as chromosomal microarray analysis or whole genome sequencing, which are not widely accessible and not offered as part of the initial work up. In this study, we attempted to look for unique clinical, morphologic or genetic features may help identify AML cases with chromothripsis during routine initial assessment, which may serve as a triaging tool and prompt additional genetic studies. Our study showed that dysplasia in AM-MRC with or without chromothripsis were common, the presence or absence, and the degree of dysplasia, cannot be used for confident identification of AML -MRC cases with chromothripsis. Interesting, we observed a set of morphologic characteristics of the blasts, namely cytoplasmic vacuoles, coarse cytoplasmic granules and cytoplasmic 11

blebbing, that were associated with this limited cohort of chromothripsis cases. The mechanism for these morphologic findings and its association with the chromothripsis is still unclear. Myeloid blasts often contain granules, the coarse granulation seen in these cases is uncommon, and unlikely to be Azurophilic granules as a myeloperoxidase cytochemical stain is typically negative. Some blasts show increased cytoplasmic protrusion and increased cytoplasmic shedding in the background, which may be attributed to the fragile cytoplasm of the leukemic blasts. The presence of these features, particularly in cases classified as AML-MRC, may prompt additional genetic studies for further evaluation of chromothripsis. We recognize that this association of these morphologic features with chromothripsis is hampered in this small series and will need to be validated in a larger prospective study. Our results support previously reported observations of association of complex karyotype with chromothripsis in AML (9). Thus, chromothripsis-positive AML-MRC cases in our study have a significantly higher number of copy number alterations than those without chromothripsis (Figure 2, Supplementary Table 1 and 2). In our cohort, a minimal common deleted region at 5q31.1, was identified in all cases with chromothripsis. This finding is in agreement with data previously reported by Fontana et al, who also identified the minimal common-deleted region at 5q31.1–5q33.1 in 24 out of 26 patients with chromothripsis (9). 5q31.1 region is a commonly deleted region in MDS and AML, likely resulting in haploinsufficiency of candidate genes in this region (21). In addition to these complex genetic changes, amplification of the MYC (8q24.2) and KMT2A (11q23.3) genes have also been detected in our cohort. The MYC oncogene was reported as commonly amplified in brain tumors with chromothripsis and was thought to stimulate division of damaged cells and increase the risks of genome chaos(22). In fact, amplification of oncogenes, such as EGFR, MDM2, MDM4, or CDK4 have been associated with chromothripsis in glioblastoma (23). Interesting, abnormalities affecting the TP53 gene (deletions and/or mutations) have been reported in AML cases with complex karyotype and dismal outcome (24, 25). Loss of this tumor suppressor gene in 68% (6/9) of chromothripsis-positive AML-MRC 12

cases vs 0% of chromothripsis-negative AML-MRC cases in our cohort, strongly supports the hypothesis of a tight link of TP53 with destabilization of genome, possibly resulting in chromothripsis. The prevalence of TP53 abnormalities in AML-MRC cases with chromothripsis would be expected to be higher than observed, if we tested not only for copy number loss of the TP53 locus, but also for point mutations in the TP53 gene, but the latter were not tested in this cohort and was not a goal of our study. Besides, although there is a strong association between TP53 abnormalities with chromothripsis (18, 19), there are many cancer types without TP53 mutations or deletions showing chromothripsis, suggesting that other pathways besides checkpoint mechanisms controlled by TP53, can be involved in the occurrence of chromothripsis. It is difficult to assess whether these multiple genetic alterations including chromothripsis occur is a step wise process or simultaneously as a result of global genetic chaos. But certain genetic changes such as TP53 and ATM mutations may lead to the disruption of checkpoint mechanism and open the gate to global genome instability. Non-random enrichment of commonly deleted or duplicated regions in our chromothripsis-positive AML-MRC cases is suggestive of the unique genetic signature associated with chromothripsis in AML-MRC and prompts additional genetic study to identify gene(s) responsible for treatment resistance and dismal outcome in patients with chromothripsis-positive AML-MRC. To our knowledge, this is the first study to explore the relationship of morphologic findings with chromothripsis in AML-MRC. A combination of morphologic features, such as cytoplasmic vacuoles, coarse granulation, and blebbing, has strong association with chromothripsis in AML-MRC. Similar to reported findings, AML-MRC with chromothripsis is frequently associated with complex karyotype including marker, derivative and ring chromosomes, which may reflect the underlying genomic instability. These unique genetic and morphologic features of the blasts, when identified in AML-MRC, may provide diagnostic clue and prompt additional genetic studies to identify these cases.

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Disclosure/Conflict of Interest The authors declare that they have no conflicts of interest related to published content in this article.

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Figure legend

Figure 1. Overall survival association in AML/MDS patients with chromothripsis (n=11, orange dotted line) and without chromothripsis (n=17, green solid line) after diagnosis. Figure 2. Bone marrow aspirate smears from patients with AML-MRC with chromothripsis revealed a distinct blast morphology (Giemsa, x1000). Figure 3. Bone marrow aspirate smears from patients with AML-MRC without chromothripsis revealed a typical myeloid blast morphology (Giemsa, x1000). Figure 4. A representative case of acute myeloid leukemia (case CP8) with chromothripsis. A. Bone marrow aspirate revealed increased blasts with clumped chromatin, grey cytoplasm, occasional cytoplasmic vacuoles and coarse granules (Giemsa, x1000). B. Conventional cytogenetics revealed a hypo-diploid karyotype with multiple structural and numerical abnormalities, including del(5q) and losses of chromosomes 14, 15, 17, and 19. C. Summary of copy number alterations and CN-LOH events observed in this case by CMA. D. Representation of chromosome 5 with del(5q). E. Representation of chromosome 15 affected by chromothripsis. Figure 5. Scatter plots of dysplasia scores and morphology scores in AML-MRC cases with or without chromothripsis. A. Dysplasia scores evaluated by the presence and degree of dysplasia in erythroid, granulocytic and megakaryocytic lineages (bar indicates mean value). B. Morphology scores evaluated by the presence and degree of cytoplasmic vacuoles, granules and membrane projections (bar indicates mean value). Figure 6. Summary of copy number alterations and CN-LOH events observed by CMA in cases of AMLMRC and MDS with and without chromothripsis (abnormalities detected in each individual case are listed in Supplementary Tables 1 and 3). A. Copy number gains in AML-MRC and MDS cases with chromothripsis. B. Copy number gains in AML-MRC and MDS cases without chromothripsis. C. Copy number losses in AML-MRC and MDS with chromothripsis. D. Copy number losses in AML-MRC and MDS cases without chromothripsis. E. CN-LOH events in AML-MRC and MDS cases with chromothripsis. F. CNLOH in AML-MRC and MDS cases without chromothripsis.

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Table 1. A comparison of clinical features in AML-MRC with or without chromothripsis

Parameter

AML-MRC with chromothripsis (N=9)

AML-MRC without chromothripsis (N=17)

Age at diagnosis (median, years)

61 (range 55~85)

58 (range 35~77)

Sex

5 males and 4 females

8 males and 9 females

WBC (k/uL)

10.9

26.4

HbB (g/dL)

8.1

8.3

MCV (fl)

97.1

92.8

Plt (k/L)

60.9

94.8

Intermediate (1), High (8)

Low (1), Intermediate (3), High (10), Unknown (2)

66 (9-680)

170 (169-1651)

CBC

Risk Groups

Median Survival Days

Table 2. Clinical, morphologic and genetic features in AML-MRC with or without chromothripsis Features Morphology

AML-MRC with chromothripsis (n=9)

AML-MRC without chromothripsis (n=17)

Dysplasia meeting the 5/9 (56%) diagnostic criteria of AML-MRC

4/17 (24%)

Cytoplasmic vacuolization, granulation and blebbing

7/9 (78%)***

1/17 (6%) ***

Immunophenotype Aberrant expression of CD7/CD5/CD2

6/9 (67%)

9/15 (60%)

Aberrant expression of CD19/CD79

2/9 (22%)

1/15 (7%)

Complex karyotype

9/9 (100%)

8/17 (47%)

5q deletions

8/9 (89%)

7/17 (41%)

7q deletions

2/9 (22%)

3/17 (15%)

5q/7q deletions

8/9 (89%)

9/17 (53%)

Marker chromosomes

7/9 (78%)

3/17 (18%)

Derivative chromosomes

6/9 (67%)

3/17 (18%)

Ring chromosomes

2/9 (22%)

1/17 (6%)

Marker/derivative/ring chromosomes

9/9 (100%)

5/17 (29%)

Deletion 17p (loss of TP53)

6/9 (68%)

0/17 (0%)

Average number of copy number gains

12.8 (3~33)

1.1 (0~6)

Average number of copy number losses

25.8 (8~53)

3 (0~14)

Average number of stretches of CN-LOH

2.9 (0~9)

0.7 (0~3)

Number of chromosomes affected by chromothripsis

2 cases with 2 chromosomes;

0

Cytogenetics

Microarray

9 cases with 1 chromosome *** p<0.01, student t-test