\documentclass[10pt]{article}

\usepackage{fullpage}
\usepackage{setspace}
\usepackage{parskip}
\usepackage{titlesec}
\usepackage[section]{placeins}
\usepackage{xcolor}
\usepackage{breakcites}
\usepackage{lineno}
\usepackage{hyphenat}





\PassOptionsToPackage{hyphens}{url}
\usepackage[colorlinks = true,
            linkcolor = blue,
            urlcolor  = blue,
            citecolor = blue,
            anchorcolor = blue]{hyperref}
\usepackage{etoolbox}
\makeatletter
\patchcmd\@combinedblfloats{\box\@outputbox}{\unvbox\@outputbox}{}{%
  \errmessage{\noexpand\@combinedblfloats could not be patched}%
}%
\makeatother


\usepackage{natbib}




\renewenvironment{abstract}
  {{\bfseries\noindent{\abstractname}\par\nobreak}\footnotesize}
  {\bigskip}

\titlespacing{\section}{0pt}{*3}{*1}
\titlespacing{\subsection}{0pt}{*2}{*0.5}
\titlespacing{\subsubsection}{0pt}{*1.5}{0pt}


\usepackage{authblk}


\usepackage{graphicx}
\usepackage[space]{grffile}
\usepackage{latexsym}
\usepackage{textcomp}
\usepackage{longtable}
\usepackage{tabulary}
\usepackage{booktabs,array,multirow}
\usepackage{amsfonts,amsmath,amssymb}
\providecommand\citet{\cite}
\providecommand\citep{\cite}
\providecommand\citealt{\cite}
% You can conditionalize code for latexml or normal latex using this.
\newif\iflatexml\latexmlfalse
\providecommand{\tightlist}{\setlength{\itemsep}{0pt}\setlength{\parskip}{0pt}}%

\AtBeginDocument{\DeclareGraphicsExtensions{.pdf,.PDF,.eps,.EPS,.png,.PNG,.tif,.TIF,.jpg,.JPG,.jpeg,.JPEG}}

\usepackage[utf8]{inputenc}
\usepackage[ngerman,greek,english]{babel}



\usepackage{float}








\begin{document}

\title{Refractory Pediatric Acute Myeloid Leukemia expressing NUP98-NSD1 Fusion
Gene Conquered by Venetoclax and DCAG}



\author[1]{Hongbo Chen}%
\author[1]{Huan Xu}%
\author[1]{Hui Yu}%
\author[1]{jiawei xu}%
\author[1]{Xiaoyan Wu}%
\author[1]{Fen Zhou}%
\author[1]{Shuo Tang}%
\author[1]{Xingxing Feng}%
\author[1]{Quan Luo}%
\author[1]{Bingyu Zhang}%
\author[1]{Runming Jin}%
\affil[1]{Wuhan Union Hospital}%


\vspace{-1em}



  
  \date{May 20, 2024}


\begingroup
\let\center\flushleft
\let\endcenter\endflushleft
\maketitle
\endgroup





\selectlanguage{english}
\begin{abstract}
Pediatric AML with NUP98-NSD1 fusion defines a high-risk subset with a
low remission and high relapse rates. There are a few studies on
successful treatment of high-risk AML expressing NUP98-NSD1. We report a
refractory pediatric case who received successful treatment with Bcl-2
inhibitor, venetoclax plus DCAG chemotherapy followed by HSCT, after two
failure courses of conventional chemotherapies. Our case suggests that
the combination of venetoclax and DCAG chemotherapy may be an effective
salvage strategy to refractory AML with NUP98-NSD1. More studies and
clinical trials should focus on the synergistic effect of Bcl-2
inhibitors and hypomethylation agents, especially in pediatric AML.%
\end{abstract}%



\sloppy


\textbf{Introduction}

Acute myeloid leukemia (AML) is a clonal hematopoietic stem cell
malignancy, which is characterized by an accumulation of immature
progenitor cells with arrested differentiation causing suppression of
hematopoiesis {[}1{]}. Current treatment strategies for patients with
AML include combination chemotherapy, hypomethylation agents (HMAs),
and/or hematopoietic stem cell transplantation (HSCT), which are
generally selected based on clinical, hematological and genetic
prognostic indicators {[}2{]}.

Nucleoporin 98 gene (NUP98) is fused to a variety of partner genes in a
spectrum of hematologic malignancies, especially pediatric leukemias
with poor prognosis {[}3{]}. Nuclear receptor-binding SET domain protein
1 (NSD1) is a member of histone methyltransferases family, which are
essential in development and are mutated in AML, multiple myeloma, and
lung cancers {[}4{]}. About 5\% of human AMLs harbor the t (5;11) (q35;
p15.5) translocation, which generates NUP98-NSD1 fusion gene {[}5{]}.
Gang et al. identified that NUP98-NSD1 induced AML and sustained
self-renewal of myeloid stem cells in vitro {[}4{]}. Later several
largescale cohort studies of patients with AML have confirmed that the
presence of NUP98-NSD fusion defines a high-risk leukemia subset with a
low remission and high relapse rates {[}6, 7{]}.

Despite our current knowledge of the molecular mechanisms and
co-occurring genetic events of AML with NUP98-NSD1 fusion, the ability
to effectively treat patients with these translocations is very limited.
Current therapy strategies for AML patients with NUP98-NSD1 fusion often
employ chemotherapy followed by hematopoietic stem cell transplantation
(HSCT) during the first complete molecular remission. However, many
patients could not achieve complete remission (CR) after conventional
chemotherapy. For cases with poor remission status, salvage
transplantation tends to have low success rates and high recurrence
rates {[}8{]}.

Here we present a case of successful treatment with decitabine,
aclacinomycin, cytosine arabinoside (Ara-C), granulocyte-colony
stimulating factor (DCAG) plus Bcl-2 inhibitor, venetoclax in a patient
with AML (M2) expressing NUP98-NSD1 fusion, followed by allogeneic HSCT,
who had never achieved CR after two courses of conventional chemotherapy
regimens.

\textbf{Case Description}

A 3-year-old boy presented with fever for 4 days was admitted to our
hospital. The blood count showed elevated white blood cells (WBC,
151G/L), decreased hemoglobin (Hb, 55g/L), neutrophil and lymphocyte
percentage was 7.5\% and 91.3\%, respectively. The peripheral blood and
bone marrow smear showed blasts were 79\% and 73.5\%, respectively. The
flow cytometry of bone marrow identified 79.5\% abnormal cell population
of nuclear cells, which was positive for HLA-DR (91.94\%), CD13
(76.22\%), CD33 (94.25\%), CD34 (30.43\%), CD38 (79.99\%), CD117
(96.76\%), CD123 (98.93\%), MPO (47.52\%). The boy was diagnosed with
AML (M2) and hyperleukemia, tentatively classified as intermediate risk.

Then the boy accepted the routine chemotherapy regimens according to
China Childhood Leukemia Collaborative Group (CCLG)-AML 2019 guideline,
which included Daunorubicin, Ara-C, Homoharringtonine (HHT) (DAH),
followed by the second course of chemotherapy, IAH (Idarubicin, Ara-C,
HHT). However, the boy could not achieve CR after two courses of
chemotherapies. The results of the deep sequencing and RNA sequencing of
hematological malignancies of this boy came out and found GATA2 mutation
(NM\_032638:exon6:c.1154C>T:p.P385L) and NUP98-NSD1 fusion gene.
Consequently, the patient was classified as high-risk AML with
NUP98-NSD1 fusion gene. The clinicians thought the routine chemotherapy
could not help the patient, and more effective chemotherapy strategies
must be employed. Thus, the clinicians collected the primary tumor cells
of the patients to carry out a high-throughput drug sensitivity analysis
(HDS) as a result of high-risk NUP98-NSD1 fusion.

Subsequently, the eight highly sensitive chemotherapy regimens were
screened out according to the high-throughput drug sensitivity analysis
of the patient's primary tumor cells (\textbf{Table 1} ). However, the
first four chemotherapy regimens contain the drugs that have been
already employed in the two courses of conventional chemotherapy and
have been proved ineffective. Besides, the NUP98-NSD1 fusion gene causes
H3K36 methylation, resulting in leukemogenesis. Therefore, the
clinicians finally chose the combination chemotherapy regimens of Bcl-2
inhibitor and DCAG, which includes hypomethylation agent decitabine
(20mg/m\textsuperscript{2}), aclacinomycin (10mg/m\textsuperscript{2}),
Ara-C (10mg/m\textsuperscript{2}), granulocyte-colony stimulating factor
(125ug) and venetoclax tablets (DCAG plus venetoclax). Then the MRD
gradually decreased to less than 10\textsuperscript{-4} on days 28 after
DCAG plus venetoclax treatment. On the 35th day of this treatment, both
of the MRD and bone marrow blasts became negative, and the NUP98-NSD1
fusion ratio decreased to 2.34\%. The patient finally achieved CR after
DCAG plus venetoclax therapy, then he underwent allo-HSCT with his
HLA-haploidentical father as the donor. One month after transplantation,
the blood count of the patient returned to normal, both of the
peripheral blood and bone marrow blasts were negative, and flow
cytometry showed MRD was less than 10\textsuperscript{-4}. Besides, IDH1
mutation, GATA2 mutation and NUP98-NSD1 fusion gene were both negative.
The clinical course of this patient can be concluded in \textbf{Fig. 1}
. This patient will have regular bone marrow examinations to be alert
for recurrence.

\textbf{Discussion}

Targeting signaling pathways that regulate cell survival and death has
emerged as an effective therapeutic strategy in patients with AML.
Recently, numerous clinical trials have shown that the specific Bcl-2
inhibitor venetoclax combined with HMAs is effective in the treatment of
relapsed and primary refractory AML. DiNardo et al. demonstrated that
overall survival (OS) was longer and the incidence of remission was
higher among patients who received azacitidine plus venetoclax than
among those who received azacitidine alone in a randomized clinical
trial (NCT02993523) {[}9{]}. In a phase 3 clinical trial, Wei et al.
identified that venetoclax plus low-dose cytarabine (LDAC) improved
remission rate and OS compared to LDAC alone (NCT03069352) {[}10{]}.
Then Karol et al. demonstrated the safety and activity of venetoclax
plus chemotherapy in pediatric patients and indicated that this
combination could be employed in newly diagnosed pediatric patients with
high-risk AML {[}11{]}. Overall, venetoclax in combination with HMAs
improves the prognosis of older patients with AML, but there are a few
clinical trials about pediatric AML.

Our patient was diagnosed with NUP98-NSD1 fusion gene, IDH1 mutation,
and GATA2 mutation by RNA sequencing, which make him to be classified to
high-risk group. Isocitrate dehydrogenase 1 and 2 (IDH1 and IDH2) gene
mutations were reported in 19\% of newly diagnosed AML patients
{[}12{]}. Though the drugs targeting IDH1 mutation ivosidenib have been
approved, it is too expensive and unacquirable for the patient. Besides,
the IDH1 mutation frequency became very low after two courses of
conventional chemotherapy. However, the MRD was still very high (50\%),
which was mainly caused by the high expression level of NUP98-NSD1
fusion gene (75.94\%). Kivioja et al. screened patient cells and
engineered cell models with over 300 compounds, and found that the cells
expressing NUP98-NSD1 had significantly increased sensitivity to Bcl-2
inhibitors {[}13{]}. However, there are no reports on venetoclax plus
decitabine use in pediatric patients with AML expressing NUP98-NSD1
fusion.

Here we firstly employed DCAG plus venetoclax to treat the patient with
NUP98-NSD1 fusion who could not achieve CR after two courses of
chemotherapy. The combination therapy of Bcl-2 inhibitor venetoclax and
HMAs (decitabine) had a promising outcome, and the patient achieved CR
which made him to be able to accept HSCT. Overall, venetoclax could be
effective and safe for treating pediatric AML expressing NUP98-NSD1
fusion. Therefore, establishing the appropriate dosage, efficacy, and
safety of venetoclax in pediatric patients through clinical trial is
required. Further genetic and pharmacological studies of AML with
NUP98-NSD1 fusion are urgently needed to improve patient outcomes.

\textbf{Ethics Statement}

The patients provided their written informed consent to participate in
this study. Written informed consent was obtained from the individual(s)
for the publication of any potentially identifiable images or data
included in this article.

\textbf{Conflict of Interest}

The authors declare that the research was conducted in the absence of
any commercial or financial relationships that could be construed as a
potential conflict of interest.

\textbf{Author Contributions}

HX and HY collected the data and drafted the manuscript, and are the
co-first authors. JX, XW, FZ, ST, XF, QL, BZ helped collect the data and
discussed the case. RJ and HC reviewed and finalized the manuscript. All
authors had the approval of the version to be published.

\textbf{Funding}

This work is supported by the National Natural Science Foundation of
China (No. 31701207) and Chen Xiao-ping Foundation for the Development
of Science and Technology of Hubei Province, China.

\textbf{References}

1. Dohner H, Wei AH, Lowenberg B. Towards precision medicine for AML.
Nature reviews Clinical oncology. 2021;18(9):577-90.

2. Short NJ, Konopleva M, Kadia TM, Borthakur G, Ravandi F, DiNardo CD,
et al. Advances in the Treatment of Acute Myeloid Leukemia: New Drugs
and New Challenges. Cancer Discov. 2020;10(4):506-25.

3. Michmerhuizen NL, Klco JM, Mullighan CG. Mechanistic insights and
potential therapeutic approaches for NUP98-rearranged hematologic
malignancies. Blood. 2020;136(20):2275-89.

4. Wang GG, Cai L, Pasillas MP, Kamps MP. NUP98-NSD1 links H3K36
methylation to Hox-A gene activation and leukaemogenesis. Nat Cell Biol.
2007;9(7):804-12.

5. Cerveira N, Correia C, D\selectlanguage{ngerman}ória S, Bizarro S, Rocha P, Gomes P, et al.
Frequency of NUP98-NSD1 fusion transcript in childhood acute myeloid
leukaemia. Leukemia. 2003;17(11):2244-7.

6. Bisio V, Zampini M, Tregnago C, Manara E, Salsi V, Di Meglio A, et
al. NUP98-fusion transcripts characterize different biological entities
within acute myeloid leukemia: a report from the AIEOP-AML group.
Leukemia. 2017;31(4):974-7.

7. Niktoreh N, Walter C, Zimmermann M, von Neuhoff C, von Neuhoff N,
Rasche M, et al. Mutated WT1, FLT3-ITD, and NUP98-NSD1 Fusion in Various
Combinations Define a Poor Prognostic Group in Pediatric Acute Myeloid
Leukemia. Journal of oncology. 2019;2019:1609128.

8. Tong Wang, Jingbo Ni, Xinyu Wang, Yan Dai, Xiaoli Ma, Yunchao Su, et
al. Observation on genetic characteristics and clinical efficacy of
NUP98-NSD1 positive children with acute myeloid leukemia. Chinese
Journal of Medicine. 2019;(36):2820-2825.

9. DiNardo CD, Jonas BA, Pullarkat V, Thirman MJ, Garcia JS, Wei AH, et
al. Azacitidine and Venetoclax in Previously Untreated Acute Myeloid
Leukemia. The New England journal of medicine. 2020;383(7):617-29.

10. Wei AH, Montesinos P, Ivanov V, DiNardo CD, Novak J, Laribi K, et
al. Venetoclax plus LDAC for newly diagnosed AML ineligible for
intensive chemotherapy: a phase 3 randomized placebo-controlled trial.
Blood. 2020;135(24):2137-45.

11. Karol SE, Alexander TB, Budhraja A, Pounds SB, Canavera K, Wang L,
et al. Venetoclax in combination with cytarabine with or without
idarubicin in children with relapsed or refractory acute myeloid
leukaemia: a phase 1, dose-escalation study. The Lancet Oncology.
2020;21(4):551-60.

12. Chotirat S, Thongnoppakhun W, Promsuwicha O, Boonthimat C,
Auewarakul CU. Molecular alterations of isocitrate dehydrogenase 1 and 2
(IDH1 and IDH2) metabolic genes and additional genetic mutations in
newly diagnosed acute myeloid leukemia patients. Journal of hematology
\& oncology. 2012;5:5.

13. Kivioja JL, Thanasopoulou A, Kumar A, Kontro M, Yadav B, Majumder
MM, et al. Dasatinib and navitoclax act synergistically to target
NUP98-NSD1(+)/FLT3-ITD(+) acute myeloid leukemia. Leukemia.
2019;33(6):1360-72.

\textbf{Figure Legends}

\textbf{Figure 1.} Clinical course of the patient. DAH, daunorubicin (40
mg/m\textsuperscript{2}), cytosine arabinoside
(100mg/m\textsuperscript{2}), homoharringtonine (3
mg/m\textsuperscript{2}); IAH, idarubicin (10 mg/m\textsuperscript{2}),
cytosine arabinoside (100mg/m\textsuperscript{2}), homoharringtonine (3
mg/m\textsuperscript{2}); DCAG, decitabine (20 mg/m\textsuperscript{2}),
aclacinomycin (10 mg/m\textsuperscript{2}), cytosine arabinoside (10
mg/m\textsuperscript{2}), granulocyte-colony stimulating factor (125\selectlanguage{greek}μ\selectlanguage{english}g).\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/Figure-1/Figure-1}

\end{center}
\end{figure}

\textbf{Hosted file}

\verb`TABLE 1.docx`  available at \url{https://authorea.com/users/323903/articles/571884-refractory-pediatric-acute-myeloid-leukemia-expressing-nup98-nsd1-fusion-gene-conquered-by-venetoclax-and-dcag}

\selectlanguage{english}
\FloatBarrier
\end{document}

