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\begin{document}

\title{\emph{KMT2A}-rearranged diffuse large B-cell lymphoma in a child: a case
report and molecular characterization}



\author[1]{Shota Kato}%
\author[1]{Yasuo Kubota}%
\author[1]{Masahiro Sekiguchi}%
\author[1]{Kentaro Watanabe}%
\author[1]{Aya Shinozaki-Ushiku}%
\author[1]{Junko Takita}%
\author[1]{Mitsuteru Hiwatari}%
\affil[1]{The University of Tokyo}%


\vspace{-1em}



  
  \date{May 08, 2024}


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\selectlanguage{english}
\begin{abstract}
A 19-month-old female with a right temporal bone tumor was diagnosed
with diffuse large B-cell lymphoma by tumor biopsy. Leukemic conversion
occurred after chemotherapy for mature B-cell lymphoma. Additional
chemotherapy was unsuccessful and resulted in death of the patient owing
to disease progression. We detected the \emph{KMT2A}-\emph{MLLT3} fused
transcript in the bone marrow of the patient with primary and recurrent
cancer. RNA sequencing of the bone marrow with recurrent cancer showed
similarities with \emph{KMT2A}-rearranged leukemia, such as
overexpression of \emph{MEIS1} and \emph{MEF2C}, which was attributable
to the aggressive course.%
\end{abstract}%



\sloppy


\textbf{INTRODUCTION}

Diffuse large B-cell lymphoma (DLBCL) is the most common type of
non-Hodgkin lymphoma in adults, while it is rare in children. DLBCL is a
heterogeneous subtype of mature B-cell lymphoma characterized by the
diffuse proliferation of B-cell antigen-expressing lymphoma cells larger
than histiocyte nuclei. DLBCL is associated with frequent gene
translocations, such as \emph{IGH} -\emph{BCL6} , \emph{IGH}
-\emph{BCL2} , and \emph{IGH} -\emph{MYC} .\textsuperscript{1}

\emph{KMT2A} rearrangement is associated with leukemogenesis in acute
lymphoblastic leukemia (ALL) and acute myeloid leukemia.
\emph{KMT2A}rearrangement in acute leukemia accounts for 5--10\% of the
total cases and correlates with poor prognosis.\textsuperscript{2}
However,\emph{KMT2A} -rearranged lymphoma has not been studied in
detail.\emph{KMT2A} -rearranged lymphomas, especially \emph{KMT2A-}
rearranged DLBCL, are less frequent than \emph{KMT2A} -rearranged
leukemia and the majority of those is constituted by lymphoblastic
lymphoma.\textsuperscript{3-8} \emph{KMT2A} -rearranged DLBCL can be
detected in a few adult and pediatric patients.\textsuperscript{9,10}

Herein, we describe a pediatric case of \emph{KMT2A} -rearranged DLBCL.
We performed RNA sequencing (RNA-seq) to understand the molecular
biology of \emph{KMT2A} -rearranged DLBCL.

\textbf{METHODS}

This study was approved by the Human Genome, Gene Analysis Ethics
Committee of the University of Tokyo (approval number G0948). Bone
marrow (BM) samples collected were analyzed after obtaining written
informed consent from legal guardians of the patient.

Total RNA was extracted from recurrent BM and checked for integrity and
concentration using the Agilent Tapestation (Agilent, Santa Clara, CA,
USA). RNA-seq libraries were constructed using the NEBNext Ultra RNA
library prep kit compatible with the Illumina platform (New England
BioLabs, Ipswich, MA, USA). Next-generation sequencing was performed
using the Illumina NovaSeq 6000 platform with a standard 101 bp
paired-end read protocol according to the manufacturer's instructions.
Reference genome (hg19) alignment and fusion gene detection were
performed using Genomon v2.6.2. For expression analysis, fragments per
kilobase of exon per million reads mapped (FPKM) were used as normalized
count data. T-distributed stochastic neighbor embedding (t-SNE) was
performed using R (v3.6.1) package Rtsne to reduce dimensions. For
t-SNE, RNA-seq data of DLBCL and \emph{KMT2A} -rearranged ALL were
obtained from published studies (accession number GSE147986 from Gene
Expression Omnibus {[}ncbi.nlm.nih.gov/geo{]} for DLBCL and a personal
communication for \emph{KMT2A} -rearranged ALL).\textsuperscript{11,12}

Primary and recurrent BM samples were subjected to reverse transcription
polymerase chain reaction (RT-PCR) and Sanger sequencing of\emph{KMT2A}
-\emph{MLLT3} and internal tandem duplication (ITD) and variants in N676
and D835 of \emph{FLT3} . ITD and the variants of\emph{FLT3} are
additional gene alterations in \emph{KMT2A} -rearranged
leukemia.\textsuperscript{13,14} Supplemental Table S1 lists the primer
sequences.

\textbf{RESULTS}

A 19-month-old female with right facial nerve palsy was referred to the
University of Tokyo Hospital. She had no personal or family history of
cancer. Magnetic resonance imaging revealed a tumor lesion that filled
the right temporal bone (Fig. 1A). Enhanced computed tomography showed
bilateral renal involvement (Fig. 1B). Lymph nodes and the thymus were
intact. Biopsy of the tumor tissue revealed DLBCL consisting of diffuse
proliferation of centroblasts (Figs. 1C--F). The tumor infiltrated the
central nervous system and was diagnosed as Murphy stage IV DLBCL. BM
samples aspirated from the bilateral ilia showed normal karyotype and no
morphological evidence of tumor invasion.

She was administered short-pulse intensive chemotherapy to treat mature
B-cell lymphoma.\textsuperscript{15} Cytology of the cerebrospinal fluid
was normal one month after initiating chemotherapy. The tumor nearly
dissolved after chemotherapy, except for a small residual lesion in the
right mastoid observed by gadolinium enhancement magnetic resonance
imaging. Although discharged seven months after diagnosis, leukocytosis
with blast cells in peripheral blood developed 2 months after completion
of the chemotherapy. Flow cytometry showed abnormal lymphocytes with
characteristics of the mature B-lineage (CD10\textsuperscript{+},
CD19\textsuperscript{+}, and CD20\textsuperscript{+}, partially positive
for immunoglobulin light chain lambda, and CD34\textsuperscript{-}) in
the BM. G-banding analysis and fluorescence \emph{in situ} hybridization
showed addition of 11q23 and \emph{KMT2A} rearrangement, respectively.
RT-PCR confirmed the presence of the \emph{KMT2A} -\emph{MLLT3} fusion
not only in sample at the recurrent timepoint, but also in that upon
initial presentation, which demonstrated normal morphology and
karyotype. Since leukocytosis worsened after reinitiating ALL-specific
chemotherapy,\textsuperscript{16} she underwent hematopoietic cell
transplantation during non-remission that resulted in the expansion of
blast cells after neutrophil engraftment. She continued palliative
chemotherapy with bortezomib. Unfortunately, the disease progressed and
she died 18 months after diagnosis.

RNA-seq on BM during recurrent cancer identified the\emph{KMT2A}
-\emph{MLLT3} fusion including exons 1--9 and 6--11 of\emph{KMT2A} and
\emph{MLLT3} , respectively, which was validated by Sanger sequencing
(Figs. 2A--B). Other fusions, such as\emph{IGH} -\emph{BCL6} ,
\emph{IGH} -\emph{BCL2} , and\emph{IGH} -\emph{MYC} , were not detected
and no mutations were found in\emph{KRAS} , \emph{NRAS} , \emph{PTPN11}
, and \emph{FLT3} . ITD and the N676 and D835 variants of \emph{FLT3}
were not identified by RT-PCR and Sanger sequencing of the primary or
recurrent BM.

\emph{MEIS1} and \emph{MEF2C} , which are highly expressed
in\emph{KMT2A} -rearranged leukemia,\textsuperscript{17} were
overexpressed in the BM during cancer recurrence (60 and 83 FPKM,
respectively); however, expression of posterior \emph{HOXA} genes
(\emph{HOXA6} ,\emph{7} , \emph{9} , and \emph{10} ), also upregulated
in\emph{KMT2A} -rearranged leukemia, were not detected.
\emph{IGLL5}levels, involved in the expression of \emph{IGLJ1} and
\emph{IGLC1} , were elevated (400 FPKM), whereas the expression of
\emph{CD34} and\emph{DNTT} (encoding terminal deoxynucleotidyl
transferase) was not elevated (0.11 and 0.80 FPKM, respectively). t-SNE
plots showed that our patient formed a part of the \emph{KMT2A}
-rearranged ALL, but not DLBCL, cluster (Fig. 2C).

\textbf{DISCUSSION}

We have described an intractable case of pediatric DLBCL
with\emph{KMT2A} rearrangement. The patient showed histological
similarities with DLBCL; however, she exhibited disease relapse with
leukemic conversion soon after chemotherapy against mature B-cell
lymphoma. She was resistant to the salvage chemotherapy.

The \emph{KMT2A} -\emph{MLLT3} fusion in BM motivated us to further
investigate the biological features of the cancer. RNA-seq did not
identify other fusions associated with DLBCL, but revealed the
overexpression of genes associated with \emph{KMT2A} -rearranged
leukemia, such as \emph{MEIS1} .\textsuperscript{17} Moreover, the
patient formed a part of the \emph{KMT2A} -rearranged ALL cluster in the
t-SNE plot. These results suggested that \emph{KMT2A} -rearranged DLBCL
shares biological features with \emph{KMT2A} -rearranged leukemia,
thereby resulting in resistance to treatment.

In contrast, the posterior \emph{HOXA} cluster, upregulated
in\emph{KMT2A} -rearranged leukemia, was not
overexpressed.\textsuperscript{17} Posterior \emph{HOXA} is maximally
expressed at the progenitor stage;\textsuperscript{18} therefore, the
low expression of posterior \emph{HOXA} in our patient could be
attributed to the maturity of lymphoma cells (confirmed by flow
cytometry and RNA-seq). Although \emph{KMT2A} rearrangement is assumed
to occur in early B-cell precursors, additional genetic aberrations,
such as mutations in RAS pathway genes or tyrosine kinase domain
mutations or ITD in \emph{FLT3} , are pivotal for
leukemogenesis.\textsuperscript{13,14,19,20} \emph{KMT2A} -rearranged
B-lineage ALL mainly includes pro-B-ALL, the most immature phenotype of
precursor B-cell ALL;\textsuperscript{19} whereas some\emph{KMT2A}
-rearranged lymphoblastic lymphoma has a mature B-cell
phenotype.\textsuperscript{4,6} Thus, the stage of differentiation in
which aberrations other than \emph{KMT2A} rearrangement occur might
determine disease phenotype, i.e., development of ALL or lymphoma.

\emph{KMT2A} -rearranged DLBCL has been reported in two adults and one
child.\textsuperscript{9,10} The adults maintained remission after
R-CHOP chemotherapy (cyclophosphamide, doxorubicin, vincristine,
prednisolone, and rituximab).\textsuperscript{9} The pediatric case
demonstrated poor response to mature B-cell lymphoma-specific
chemotherapy and was in remission after high-dose cytarabine and oral
maintenance chemotherapy with 6-mercaptopurine and
methotrexate.\textsuperscript{10} This difference in therapeutic
response between the adult and pediatric cases, including our patient,
may be attributed to differences in their genetic background. However,
previous reports have not identified genetic alterations other
than\emph{KMT2A} rearrangement.

In conclusion, although further investigation is warranted, there might
be similarities between the characteristics of \emph{KMT2A} -rearranged
DLBCL and leukemia at the molecular level.

\textbf{Acknowledgements:}

We acknowledge M. Matsumura and F. Saito for their technical assistance
and Editage (www.editage.jp) for the English language review.

\textbf{Conflict of interest:}

The authors declare no conflict of interest.

\textbf{References}

1. Li S, Young KH, Medeiros LJ. Diffuse large B-cell
lymphoma.\emph{Pathology.} 2018;50(1):74-87.

2. Yokoyama A. Molecular mechanisms of MLL-associated leukemia.\emph{Int
J Hematol.} 2015;101(4):352-361.

3. Shafer D, Wu H, Al-Saleem T, et al. Cutaneous precursor B-cell
lymphoblastic lymphoma in 2 adult patients: clinicopathologic and
molecular cytogenetic studies with a review of the literature.\emph{Arch
Dermatol.} 2008;144(9):1155-1162.

4. Takachi T, Iwabuchi H, Imamura M, Imai C. Lymphoblastic lymphoma with
mature B-cell immunophenotype and MLL-AF9 in a child. \emph{Pediatr
Blood Cancer.} 2011;57(7):1251-1252.

5. Quigg TC, Haddad NG, Buchsbaum JC, Shih CS. Hypothalamic obesity
syndrome: rare presentation of CNS+ B-cell lymphoblastic
lymphoma.\emph{Pediatr Blood Cancer.} 2012;59(5):930-933.

6. Ahlmann M, Meyer C, Marschalek R, et al. Complex MLL rearrangement in
non-infiltrated bone marrow in an infant with stage II precursor
B-lymphoblastic lymphoma. \emph{Eur J Haematol.} 2014;93(4):349-353.

7. Larish AM, Dolan M, Casey T, Perkins JL. Bilateral ovarian B-lineage
lymphoblastic lymphoma with MLL gene rearrangement: a novel case in
infancy. \emph{J Pediatr Hematol Oncol.} 2015;37(4):e215-217.

8. Thandla S, Alashari M, Green DM, Aplan PD. Therapy-related T cell
lymphoblastic lymphoma with t(11;19)(q23;p13) and MLL gene
rearrangement. \emph{Leukemia.} 1999;13(12):2116-2118.

9. Gindin T, Murty V, Alobeid B, Bhagat G. MLL/KMT2A translocations in
diffuse large B-cell lymphomas. \emph{Hematol Oncol.}2015;33(4):239-246.

10. Hiroki H, Yoshimi A, Kato K, Yanai T, Koike K. Primary Cutaneous
Diffuse Large B-Cell Lymphoma With KMT2A-MLLT3: An Infantile Case
Study.\emph{J Pediatr Hematol Oncol.} 2019;41(8):657-658.

11. Yan W, Jiang X, Wang W, et al. Cell-of-Origin Subtyping of Diffuse
Large B-Cell Lymphoma by Using a qPCR-based Gene Expression Assay on
Formalin-Fixed Paraffin-Embedded Tissues. \emph{Front
Oncol.}2020;10:803.

12. Hirabayashi S, Ohki K, Nakabayashi K, et al. ZNF384-related fusion
genes define a subgroup of childhood B-cell precursor acute
lymphoblastic leukemia with a characteristic
immunotype.\emph{Haematologica.} 2017;102(1):118-129.

13. Hyrenius-Wittsten A, Pilheden M, Sturesson H, et al. De novo
activating mutations drive clonal evolution and enhance clonal fitness
in KMT2A-rearranged leukemia. \emph{Nat Commun.} 2018;9(1):1770.

14. Tsutsumi S, Taketani T, Nishimura K, et al. Two distinct gene
expression signatures in pediatric acute lymphoblastic leukemia with MLL
rearrangements. \emph{Cancer Res.} 2003;63(16):4882-4887.

15. Tsurusawa M, Mori T, Kikuchi A, et al. Improved treatment results of
children with B-cell non-Hodgkin lymphoma: a report from the Japanese
Pediatric Leukemia/Lymphoma Study Group B-NHL03 study. \emph{Pediatr
Blood Cancer.} 2014;61(7):1215-1221.

16. Takahashi H, Kajiwara R, Kato M, et al. Treatment outcome of
children with acute lymphoblastic leukemia: the Tokyo Children's Cancer
Study Group (TCCSG) Study L04-16. \emph{Int J
Hematol.}2018;108(1):98-108.

17. Krivtsov AV, Twomey D, Feng Z, et al. Transformation from committed
progenitor to leukaemia stem cell initiated by MLL-AF9.
\emph{Nature.}2006;442(7104):818-822.

18. Forsberg EC, Prohaska SS, Katzman S, Heffner GC, Stuart JM, Weissman
IL. Differential expression of novel potential regulators in
hematopoietic stem cells. \emph{PLoS Genet.} 2005;1(3):e28.

19. Jansen MW, Corral L, van der Velden VH, et al. Immunobiological
diversity in infant acute lymphoblastic leukemia is related to the
occurrence and type of MLL gene rearrangement.
\emph{Leukemia.}2007;21(4):633-641.

20. Andersson AK, Ma J, Wang J, et al. The landscape of somatic
mutations in infant MLL-rearranged acute lymphoblastic
leukemias.\emph{Nat Genet.} 2015;47(4):330-337.

\textbf{Legends}

\textbf{FIGURE 1} A, Magnetic resonance T2-weighted imaging of the head.
The tumor occupied the right external acoustic meatus, tympanic cavity,
mastoid cells, and internal acoustic meatus with invasion into the right
temporal muscle (arrow). B, Contrast-enhanced abdominal computed
tomography. The kidneys were bilaterally enlarged with poorly enhanced
lesions. C--F, Histopathologic findings of the tumor tissue. C,
Hematoxylin and eosin staining. Infiltration of medium- to large-sized
centroblasts. D, Immunohistochemical staining for MIB-1. The MIB-1
proliferation index was 80--90\%. E and F, Immunohistochemical staining
for CD20 (E) and BCL2 (F). The centroblasts were positive for CD10,
CD20, CD79a, and BCL2 and negative for CD3, CD5, terminal
deoxynucleotidyl transferase, and Epstein-Barr virus-encoded small
RNA\emph{in situ} hybridization.

\textbf{FIGURE 2} A, DNA, mRNA, and protein conformation of
the\emph{KMT2A} -\emph{MLLT3} fusion. MBM, MENIN-binding motif; IBM,
integrase domain-binding motif; zf, zinc finger; AHD, ANC1 homology
domain. B, Sanger sequencing showing the fusion between exon 9
of\emph{KMT2A} and exon 6 of \emph{MLLT3} . C, t-SNE plot generated
using the RNA-seq data from the patient and patients with DLBCL
and\emph{KMT2A} -rearranged ALL obtained from published
studies.\textsuperscript{11,12}

\textbf{SUPPLEMENTAL TABLE S1} Primer sequences for reverse
transcription-polymerase chain reaction.\selectlanguage{english}
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