NUP98-NSD1 Driven MDS/MPN in Childhood Masquerading As JMML

1 | able fi BCR/ 95%) ltrate, fi colony- > – mutation she received RAS Most patients ow cytometry 8 6 fl p.Gly12Val at an 0.5% with a subset † ∼ After 1 cycle of che- were tested using next 7 NRAS udarabine and cytarabine to fl NPM1 † ,and RESULTS METHODS mutation was no longer detectable and CASE REPORT CEBPA Here, we report a patient who was suspected myeloblast population of s guardians provided informed consent to ’ 2 + Inge Behroozfard, BS,* NRAS fusion detected on RNA sequencing (RNASeq) † -ITD, BSERVATIONS but the majority of patients with JMML ( O Elizabeth P. Young, MD,* L with 20% monocytes) and elevated age-adjusted hemo- FLT3 4,5 µ † The patient was treated with A 2-year-old girl presented after 6 months of progressive The patient . Biopsy of a scalp lesion revealed a myelomonocytic in reduce her disease burden before HCT. generation sequencing and weresequencing all panel normal. detected An institutional a DNA mutation in was detected. Arequiring presumptive diagnosis ofstimulating HCT MDS/MPN-unclassi factor hypersensitivity testing was was not performed. made. Granulocyte-macrophage allele frequencyDiagnosis of of JMML 8%, wassplenomegaly suspected a at although time the of buccal patient diagnosis did and germline only not a have subclonal sample was negative. have somatic or germline mutationsproteins detected signaling in in genes the encoding RAS/MAPK pathway. this study inDNA and accordance RNA with werebuccal extracted the using and standard Declaration methods bone from whole of marrow genome Helsinki. mononuclear sequencingdetails, cells. were RNASeq see performed. and Content Supplemental For 1 Methods, (http://links.lww.com/JPHO/A394). additional Supplemental Digital of havingNUP98-NSD1 JMMLthat but has not been was previously reported in found MDS/MPN. to have a cryptic globin F (37.1%). Boneincreased marrow aspirate promonocytes, revealed dysplastic 5%ABL myeloid features, blasts, andconsistent absence with of leukemiafor cutis. Abdominal hepatosplenomegaly. ultrasoundabnormal was Cytogenetic cell negative yotype. with analysis a showed 44,X,-X,del(9)(q13q22),-16[1],46,XX[20] kar- a single symptoms including fatigue, pallor, andfound gingival hyperplasia, and to was (85.2 K/ have severe anemia, thrombocytopenia, leukocytosis with JMMLplantation (HCT), receive whichtreatment allogeneic currently option. remains hematopoietic the only cell curative trans- blasts to rule outJMML AML. Translocations have been reported in showing immunophenotypicCD7 abnormalities and with CD15expression. After dim expression conditioning with busulfan, andmelphalan, partial cyclophosphamide, moderate and to rabbit bright antithymocyte CD123 globulin, cytogenetic analysisrevealed was a CD34 normal. However, motherapy, the 2 2020 www.jpho-online.com able, Julia Chu, MD,* and Elliot Stieglitz, MD* Bone ABORATORY fi E. Alejandro Sweet-Cordero, MD,* 3 ’’ † L ‡ s Hospital; ’ Stanley G. Leung, BA,* Department of Alex G. Lee, PhD,* † ‡ † ,MD, Driven MDS/MPN in Childhood fi s website, www.jpho- ’ 000) – Masquerading as JMML l disease in early childhood. MDS/MPN is a rare diagnosis ncreased immature cells in the LINICAL AND ave an increased risk of pro- 1 C 2020;00:000 Volume 00, Number 00, Maria Maruf 1 to 4 cases per million, best exem- ∼ ict of interest. fl Astrid Behnert, MD,* Christopher C. Dvorak, MD,* Marcus R. Breese, PhD,* NUP98-NSD1 Most children also present with splenomegaly, 2020 Wolters Kluwer Health, Inc. Unauthorized reproduction of this article is prohibited. fusion, a translocation thought to exclusively occur in 2 MDS/MPN, JMML, NUP98-NSD1 r Overlapping myelodysplastic/myeloproliferative neoplasms

yelodysplastic/myeloproliferative neoplasmsMPN) are (MDS/ clonal hematopoietic disorders characterized ed in rare cases. Here, we report a 2-year-old patient who ﬁ Helen Diller Comprehensive Cancer Center, University of

ed by JMML, a frequently fata California San Francisco, San Francisco; and San Francisco, CA 94158 (e-mail: [email protected]). citations appear in theand PDF printed versions text of and this are article provided on the in journal the HTML † Pediatric Subspecialty, Kaiser Permanente, Oakland, CA. Cancer Aid (A.B.). online.com. ﬁ J Pediatr Hematol Oncol which is reserved for myeloidcriteria neoplasms for not meeting any diagnostic of the other 3. J Pediatr Hematol Oncol Reprints: Elliot Stieglitz, MD, UCSF, Stieglitz Lab 1450 3rd St, Box 3112, Supplemental Digital Content is available for this article. Direct URL Received for publication MayFrom 11, 2020; the accepted *Department June 28, of 2020. Pediatrics, Benioff Children Supported by the Frank A. Campini Foundation (E.S.) andThe the authors German declare no con Children with JMMLthrombocytopenia, typically presentmonocytosis. and with fever, anemia, leukocytosis with peripheral

Copyright © 2020 Wolters Kluwer Health, Inc. All rights reserved. M ( Summary: (MDS/MPN) aremyelodysplasia clonal and hematopoietic myeloproliferation.MDS/MPN disorders The in with only well-characterized aggressive features children disorder of of is infants andof juvenile this toddlers. disease The myelomonocytic is biochemical hyperactivation hallmark caused leukemia, of the by Ras/MAPK an signaling mutations pathway patients. Translocations in involving Ras receptor tyrosineidenti pathway kinases have genes been in more than 90% of presented withNUP98-NSD1 MDS/MPN drivenpatients with by acute myeloid leukemia. aKey cytogenetically Words: cryptic in children, occurring in pli while others develop it within a few weeks of diagnosis. by aberrant proliferation with i peripheral blood ascytopenias. well These as varying disordersgression degrees to h of acutedivided dysplasia myeloid into and atypical leukemialomonocytic chronic (AML) myeloid and leukemia, leukemia,(JMML), chronic are mye- juvenile and further a myelomonocytic fourth leukemia category, MDS/MPN-unclassi marrow aspirate typically revealsmyelo- varying or degrees megakaryopoiesis of but abnormal must display fewer than 20% Copyright Downloaded from https://journals.lww.com/jpho-online by BhDMf5ePHKav1zEoum1tQfN4a+kJLhEZgbsIHo4XMi0hCywCX1AWnYQp/IlQrHD3yRlXg5VZA8uSeVZO4Jyms7nBsZRWzUYTPpeZo3zORAmRpmoMPh4w5Q== on 09/01/2020 Downloaded from https://journals.lww.com/jpho-online by BhDMf5ePHKav1zEoum1tQfN4a+kJLhEZgbsIHo4XMi0hCywCX1AWnYQp/IlQrHD3yRlXg5VZA8uSeVZO4Jyms7nBsZRWzUYTPpeZo3zORAmRpmoMPh4w5Q== on 09/01/2020 Behnert et al J Pediatr Hematol Oncol Volume 00, Number 00, ’’ 2020

AB MWM1 2

574 bp

FIGURE 1. A, Reverse transcription polymerase chain reaction (RT-PCR) product of ∼574 base pair on 1.5% agarose gel (MWM, molecular weight marker; lane 1: 15 µL of RT-PCR product loaded; lane 2: 5 µL of RT-PCR product loaded). B, Schematic illustration of NUP98-NSD1 fusion.

allogeneic HCT from a 12/12 human leukocyte antigen-matched Whole genome sequencing from diagnosis also confirmed the unrelated donor. Shortly after HCT, research-grade RNA presence of the NRAS mutation and absence of a FLT3-ITD but sequencing using diagnostic material returned, demonstrating a did not reveal any additional mutations. Clinical-grade RT-PCR NUP98-NSD1 gene fusion. Direct sequence analysis revealed an wasthenusedtoconfirm the NUP98-NSD1 fusion using diag- in-frame gene fusion between the NUP98 gene on exon 12 nostic material which allowed for posttransplant monitoring of (chr11:3744509) and NSD1 on exon 6 (chr5:177235821) transcript levels. (Fig. 1B). Reverse transcription polymerase chain reaction The patient’s treatment course was complicated by (RT-PCR)wasusedtovalidatethisfinding using primers veno-occlusive disease and transplant-associated thrombotic flanking NUP98 and NSD1 genes, resulting in a band of ∼574 microangiopathy, which were successfully treated with base pairs (Fig. 1A). The expected sequence was confirmed by defibrotide and eculizumab. The patient is currently alive Sanger sequencing. Principal component analysis of RNASeq and well with absence of NUP98-NSD1 fusion expression by data from patients with JMML, AML, and acute lymphoblastic RT-PCR 5 months after HCT (Fig. 3). However, the patient leukemia showed that this patient clustered closer to other continues to evidence mixed chimerism in her CD3+ cells patients with AML comparedtothosewithJMML(Fig.2). despite rapid taper of tacrolimus. In an effort to achieve full

UPN3111 10

Disease acute lymphoblastic leukemia acute myeloid leukemia juvenile myelomonocytic leukemia Dim2 (7.2%) 0

−10

−20

−20 −10 0 10 20 Dim1 (11.5%)

FIGURE 2. Principal component analysis of RNASeq data from patients with acute lymphoblastic leukemia (n = 878, highlighted in red), patients with acute myeloid leukemia (n = 538, highlighted in yellow), and patients with juvenile myelomonocytic leukemia (n = 20, highlighted in blue). Our patient with NUP98-NSD1 MDS/MPN is indicated by a black arrow.

2 | www.jpho-online.com Copyright © 2020 Wolters Kluwer Health, Inc. All rights reserved. Copyright r 2020 Wolters Kluwer Health, Inc. Unauthorized reproduction of this article is prohibited. J Pediatr Hematol Oncol Volume 00, Number 00, ’’ 2020 NUP98-NSD1 Fusion in Pediatric MDS/MPN

AZA AZA + + DLI DLI DLI DLI

Flu/Ara-C

Days relative to HCT Diagnosis -20HCT +30 +90 +150 +180

Blasts by flow cytometry 5.0% 1.8% 0.0% 0.0% 0.0% 0.0%

NRAS MAF 8% 0% 0% n/a n/a n/a

Relative NUP98-NSD1 expression 100% n/a 0%* 0%* 0%* 0%*

Donor chimerism CD33+ n/a n/a 99% 97% 99% 99%

Donor chimerism CD3+ n/a n/a 56% 76% 82% 98%

FIGURE 3. Schematic timeline of patient’s clinical course. Timepoints shown are relative to HCT. *NUP98-NSD1 expression showed a log reduction of > 4 from baseline. Ara-C indicates cytarabine; AZA, azacitidine; DLI, donor lymphocyte infusion; Flu, fludarabine; HCT, hematopoietic cell transplantation; MAF, mutant allele frequency.

donor chimerism, the patient has received 2 cycles of aza- clinical management of this patient. With knowledge of the citidine followed by donor lymphocyte infusions but NUP98-NSD1 fusion, chemotherapy would have been remains mixed to date (Fig. 3).9 continued until RT-PCR was negative, as achieving a molecular remission before transplant has been associated DISCUSSION with improved outcomes in both JMML and AML.19,20 In NUP98-NSD1 istheresultofachromosomaltrans- addition, it would have prompted use of a less intensive location (t5;11)(q35;p15.5) that generates a fusion between the myeloablative conditioning regimen, which may have spared some of the patient’s post-HCT toxicities. Lastly, this N-terminus of nucleoporin 98 (NUP98) and the C-terminus of fi fi the nuclear receptor binding SET domain protein 1 (NSD1). nding was of bene t by offering the ability to track the patient’s disease status more accurately post-HCT using Because both NUP98 and NSD1 are located in the telomeric fi region of chromosomes 11 and 5, respectively, fusions RT-PCR. In summary, this represents the rst case of a NUP98-NSD1 involving these 2 genes are cryptic by conventional kar- fusion in a pediatric MDS/MPN patient and yotyping. NUP98-NSD1 is the most frequent gene fusion in expands the spectrum of fusions in this category. cytogenetically normal AML,10 but has not yet been reported in MDS, MPN, or overlapping MDS/MPN including REFERENCES JMML. Patients with NUP98-NSD1 AML have significantly 1. Clara JA, Sallman DA, Padron E. Clinical management of higher white blood cell count at diagnosis and frequent FAB- myelodysplastic syndrome/myeloproliferative neoplasm overlap M4/M5 morphology compared to patients without the syndromes. Cancer Biol Med. 2016;13:360–372. fusion.11 The presence of NUP98-NSD1 is also reported to be 2. Chang TY, Dvorak CC, Loh ML. Bedside to bench in juvenile a poor prognostic marker in pediatric and adult AML.11,12 myelomonocytic leukemia: insights into leukemogenesis from a – However, it was later shown that pediatric patients with rare pediatric leukemia. Blood. 2014;124:2487 2497. 3. Niemeyer CM, Arico M, Basso G, et al. Chronic myelomono- NUP98-NSD1 fusions frequently have cooccurring FLT3- cytic leukemia in childhood: a retrospective analysis of 110 cases. ITD alterations and while the cooccurrence in patients is European Working Group on Myelodysplastic Syndromes in associated with lower overall and event-free survival, patients Childhood (EWOG-MDS). Blood. 1997;89:3534–3543. with NUP98-NSD1 alone have outcomes similar to patients 4. Byrgazov K, Kastner R, Dworzak M, et al. A novel fusion gene without the fusion.13 NDEL1-Pdgfrb in a patient with JMML with a new variant of To date, 31 different fusion partners of NUP98 have been TKI-resistant mutation in the kinase domain of PDGFRβ. identified and the resulting fusion products are thought to lead to Blood. 2014;124:613. epigenetic dysregulation of different leukemia-associated genes 5. Chao AK, Meyer JA, Lee AG, et al. Fusion driven JMML: a including HOXA7 and HOXA9.11,14 The NUP98-NSD1 fusion novel CCDC88C-FLT3 fusion responsive to sorafenib identified by RNA sequencing. Leukemia. 2020;34:662–666. has been shown to be sufficient to cause malignant trans- 14 6. Murakami N, Okuno Y, Yoshida K, et al. Integrated molecular formation of normal hematopoietic cells. Additional somatic profiling of juvenile myelomonocytic leukemia. Blood. 2018;131: mutations in other genes including NRAS and more commonly 1576–1586. FLT3-ITD have been identified and are described as secondary 7. Stieglitz E, Ward AF, Gerbing RB, et al. Phase II/III trial of a events promoting malignant transformation.15,16 Avarietyof pre-transplant farnesyl transferase inhibitor in juvenile myelo- NUP98 fusions have been reported in MDS.17 One previous case monocytic leukemia: a report from the Children’s Oncology report describes a NUP98-HOXA11 fusion in a 3-year-old girl Group. Pediatr Blood Cancer. 2015;62:629–636. with JMML who also had a heterozygous mutation in NRAS p. 8. Dvorak CC, Satwani P, Stieglitz E, et al. Disease burden Gly13Asp.18 However, the NUP98-NSD1 gene fusion detected and conditioning regimens in ASCT1221, a randomized phase II trial in children with juvenile myelomonocytic leukemia: a in our patient has only been reported in AML. ’ fi Children s Oncology Group study. Pediatr Blood Cancer. 2018; This case highlights the bene t of RNASeq in pediatric 65:e27034. patients who present with diagnostic uncertainty and the 9. Horn B, Petrovic A, Wahlstrom J, et al. Chimerism-based pre- need for rapid turn-around-times in clinical laboratories. emptive immunotherapy with fast withdrawal of immunosuppression Earlier identification of this fusion would have altered the and donor lymphocyte infusions after allogeneic stem cell

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