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\begin{document}

\title{Inotuzumab ozogamicin is an effective treatment for CD22-positive acute
undifferentiated leukemia: A case report}



\author[1]{Ryo Akazawa}%
\author[1]{Itaru Kato}%
\author[1]{Hirohito Kubota}%
\author[1]{Kiyotaka Isobe}%
\author[2]{Hiroaki Masuno}%
\author[2]{Masamitsu Mikami}%
\author[2]{Mitsutaka Shiota}%
\author[1]{Kagehiro Kozuki}%
\author[1]{Naoko Kawabata}%
\author[1]{Kuniaki Tanaka}%
\author[3]{Satoshi Saida}%
\author[1]{Katsutsugu Umeda}%
\author[1]{Hidefumi Hiramatsu}%
\author[1]{Souichi Adachi}%
\author[1]{Junko Takita}%
\affil[1]{Graduate School of Medicine, Kyoto University}%
\affil[2]{Kitano Hospital, The Tazuke Kofukai, Medical Research Institute}%
\affil[3]{Graduate School of Medicine Kyoto University}%


\vspace{-1em}



  
  \date{May 25, 2024}


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\selectlanguage{english}
\begin{abstract}
Acute undifferentiated leukemia (AUL) is a rare subtype of leukemia that
expresses no lineage-specific markers; no optimal treatment for AUL has
been established. Here, we report a 16-year-old female with
CD22-positive refractory AUL who responded well to inotuzumab ozogamicin
(InO). Minimal residual disease negativity was achieved using InO,
followed by HLA-mismatched unrelated bone marrow transplantation (BMT).
Although grade II veno-occlusive disease/sinusoidal obstruction syndrome
occurred, it improved immediately. She remained disease-free at 10
months post-BMT, without severe complications (grade III--IV). This case
demonstrates the feasibility of a treatment strategy using InO against
CD22-positive AUL.%
\end{abstract}%



\sloppy


\textbf{BRIEF REPORT}

\textbf{Inotuzumab ozogamicin is an effective treatment for
CD22-positive acute undifferentiated leukemia: A case report}

\textbf{Running title:} Inotuzumab ozogamicin for CD22-positive AUL

Ryo Akazawa,\textsuperscript{1} Itaru Kato,\textsuperscript{1} Hirohito
Kubota,\textsuperscript{1} Kiyotaka Isobe,\textsuperscript{1} Hiroaki
Masuno,\textsuperscript{2} Masamitsu Mikami,\textsuperscript{2}Mitsutaka
Shiota,\textsuperscript{2} Kagehiro Kohzuki,\textsuperscript{1} Naoko
Kawabata,\textsuperscript{1} Kuniaki Tanaka,\textsuperscript{1} Satoshi
Saida,\textsuperscript{1} Katsutsugu Umeda,\textsuperscript{1} Hidefumi
Hiramatsu,\textsuperscript{1} Souichi Adachi,\textsuperscript{3} and
Junko Takita\textsuperscript{1}

\textsuperscript{1}Department of Pediatrics Graduate School of Medicine,
Kyoto University

\textsuperscript{2}Department of Pediatrics, Kitano Hospital, The Tazuke
Kofukai, Medical Research Institute

\textsuperscript{3}Department of Human Health Sciences, Graduate School
of Medicine, Kyoto University

Corresponding author: Itaru Kato

Department of Pediatrics, Graduate School of Medicine, Kyoto University,
54 Kawahara-cho, Shogoin, Sakyo-ku, Kyoto, 606-8507, Japan

Phone: +81-75-751-3290; Fax: +81-75-752-2361

E-mail address: itarkt@kuhp.kyoto-u.ac.jp

Text word count: abstract, 95 words; main text, 1,104 words

Number of tables and figures: 0 tables; 2 figures

\textbf{Abbreviations}\selectlanguage{english}
\begin{longtable}[]{@{}ll@{}}
\toprule
ALL & Acute lymphoblastic leukemia\tabularnewline
\midrule
\endhead
AML & Acute myeloid leukemia\tabularnewline
AUL & Acute undifferentiated leukemia\tabularnewline
BM & Bone marrow\tabularnewline
BMA & Bone marrow aspiration\tabularnewline
BMT & Bone marrow transplantation\tabularnewline
CR & Complete remission\tabularnewline
EBMT & European Society for Blood and Marrow
Transplantation\tabularnewline
GVHD & Graft-versus-host disease\tabularnewline
InO & Inotuzumab ozogamicin\tabularnewline
MPO & Myeloperoxidase\tabularnewline
MRD & Minimal residual disease\tabularnewline
TAC & Tacrolimus\tabularnewline
VOD/SOS & Veno-occlusive disease/sinusoidal obstruction
syndrome\tabularnewline
WHO & World Health Organization\tabularnewline
\bottomrule
\end{longtable}

\textbf{Keywords:} acute undifferentiated leukemia, inotuzumab
ozogamicin, stem cell transplantation, veno-occlusive disease/sinusoidal
obstruction syndrome



\textbf{ABSTRACT}

Acute undifferentiated leukemia (AUL) is a rare subtype of leukemia that
expresses no lineage-specific markers; no optimal treatment for AUL has
been established. Here, we report a 16-year-old female with
CD22-positive refractory AUL who responded well to inotuzumab ozogamicin
(InO). Minimal residual disease negativity was achieved using InO,
followed by HLA-mismatched unrelated bone marrow transplantation (BMT).
Although grade II veno-occlusive disease/sinusoidal obstruction syndrome
occurred, it improved immediately. She remained disease-free at 10
months post-BMT, without severe complications (grade III--IV). This case
demonstrates the feasibility of a treatment strategy using InO against
CD22-positive AUL.



\textbf{INTRODUCTION}

Acute undifferentiated leukemia (AUL) is a rare subtype of acute
leukemia that expresses neither lymphoid nor myeloid lineage-specific
markers. AUL is reported to be 0.2--1\% of all acute
leukemias.\textsuperscript{1} Due to the rarity of AUL, an optimal
treatment regimen has not been established. According to the treatment
protocol for mixed phenotype leukemia, first-line chemotherapy is
usually acute lymphoblastic leukemia (ALL)-type therapy, with patients
who cannot achieve complete remission (CR) considered for acute myeloid
leukemia (AML)- or combined-type therapy;\textsuperscript{2,3}however,
when first- and second-line treatment are ineffective, the choice of
alternative therapy is very limited.

In the 2016 revised World Health Organization (WHO) classification,
multiple antigens are required for B-lineage identification, including a
combination of CD19 with CD79a, cytoplasmic (cy) CD22, and/or
CD10.\textsuperscript{4} Expression of limited B-lineage-associated
antigens, such as CD22, has been reported in some cases of
AUL.\textsuperscript{5}

CD22 is a glycoprotein widely expressed on normal B cells, as well as
B-ALL cells.\textsuperscript{6} Inotuzumab ozogamicin (InO) is a
CD22-targeted antibody-drug conjugate of calicheamicin, which is
approved for treatment of relapsed/refractory CD22-positive B-cell ALL.
Bhojwani et al. reported that use of InO for 51 children with
relapsed/refractory ALL, who were heavily pretreated, resulted in 67\%
of patients achieving CR, and InO was
well-tolerated;\textsuperscript{7}however, no study has reported the
efficacy of InO for CD22-positive AUL. Here, we report a case of
CD22-positive AUL resistant to ALL- and AML-type therapy that achieved
minimal residual disease (MRD)-negative CR using InO and who was able to
undergo stem cell transplantation.

\textbf{RESULTS}

\textbf{Case report}

A 15-year-old female presented to our hospital for evaluation of dyspnea
on effort and pancytopenia. Bone marrow aspiration (BMA) showed 76.6\%
blasts, which were negative for myeloperoxidase (MPO) and 2--3 times the
size of small lymphocytes (Fig. 1A, 1B, and 1C). Flow cytometric
analysis revealed that the blasts expressed surface cyCD22, cyCD79a,
CD34, CD38, and HLA-DR, and partially expressed CD19, but lacked cyCD3,
MPO, and specific features of other lineages, such as monocytes,
megakaryocytes, or plasmacytoid dendritic cells. Karyotype analysis
revealed 46, XX, add(2)(q31), add(6)(p21), add(16)(q22),
del(20)(q11.2q13.3). No known chimeric transcripts (e.g., rearrangements
of \emph{KMT2A} , or \emph{BCR-ABL1} ) were detected. Immunophenotype
analysis met the criteria of B-cell precursor ALL,\textsuperscript{4}and
she received ALL-oriented induction therapy;\textsuperscript{8}however,
BMA after induction chemotherapy showed \textgreater{} 90\% blasts,
which expressed only CD34, CD7, and CD13, and partially expressed
cyCD79a and HLA-DR, but not CD19 or surface and cyCD22. Given the lack
of expression of lineage-specific markers and the fact that less than
two myeloid associated markers were detected,\textsuperscript{5} she was
diagnosed with AUL and treated with AML-oriented
chemotherapy.\textsuperscript{9} After re-induction therapy, partial
remission was achieved, with approximately 10\% blasts in the bone
marrow (BM); however, she could not obtain morphological CR after two
courses of consolidation therapy. She subsequently received a
clofarabine-containing regimen (clofarabine 40 mg/m\textsuperscript{2},
cyclophosphamide 400 mg/m\textsuperscript{2}, and etoposide 150
mg/m\textsuperscript{2}, daily for 5 consecutive days), which is used
for relapsed/refractory ALL and AML;\textsuperscript{10,11} however, she
achieved only partial remission (8.0\% blasts in the BM). CD22
expression was transiently lost after induction therapy, but the blasts
became positive for surface CD22 again and remained negative for CD19
after re-induction therapy (Figure. 1D, 1E, and 1F). Hence, based on the
persistent expression of CD22 after re-induction therapy, she received
InO (0.8 mg/m\textsuperscript{2} on day +1, and 0.5
mg/m\textsuperscript{2} on days +8 and +15) at 16 years of age, which
has approval for use in young adults by the Patient Safety Unit, Kyoto
University Hospital. She experienced an acute adverse effect of slight
transient elevation of aspartate aminotransferase and alanine
aminotransferase (grade I, National Cancer Institute Common Terminology
Criteria for Adverse Events, version 5). Hematological toxicity was only
thrombocytopenia, which recovered within approximately 3 weeks. After
one course of InO, she attained MRD-negative CR, as determined by flow
cytometry. Therefore, she proceeded to receive bone marrow
transplantation (BMT) from an HLA-7/8 allele-matched unrelated donor,
with a myeloablative conditioning regimen consisting of total body
irradiation (12 Gy in 6 fractions) and melphalan (180
mg/m\textsuperscript{2}). Graft-versus-host disease (GVHD) prophylaxis
consisted of tacrolimus (TAC) and short-course methotrexate (15
mg/m\textsuperscript{2} on day +1 and 10 mg/m\textsuperscript{2} on days
+3, +6, and +11). She received low molecular-weight heparin,
ursodeoxycholic acid, and antithrombin for veno-occlusive disease
(VOD)/sinusoidal obstruction syndrome (SOS) prophylaxis. She was infused
with a cell dose of 2.4 \selectlanguage{ngerman}× 10\textsuperscript{8}/kg body weight. From day
+6, she had fever with fluid retention and a rash on her face and
extremities, and received methylprednisolone for pre-engraftment immune
reaction. Neutrophil engraftment was confirmed on day +19. From day +25,
she became platelet transfusion-dependent, with elevated lactase
dehydrogenase and undetectable serum haptoglobin. Although she did not
have findings of increased schistocytes or renal dysfunction, we
considered probable thrombotic microangiopathy. Approximately 3 weeks
after reduction of TAC, she responded to platelet transfusion, with
recovery of serum haptoglobin level. From day +35, she experienced
abdominal swelling and her body weight increased by 5--6\%. Abdominal
ultrasonography revealed ascites and slight hepatomegaly, without
diminished portal venous flow. She never exhibited elevated serum
bilirubin or liver tenderness. She met the diagnostic criteria for
pediatric VOD/SOS (grade II), suggested by the European Society for
Blood and Marrow Transplantation (EBMT).\textsuperscript{12} She
received recombinant human soluble thrombomodulin and coadministration
of albumin and furosemide for 1 week, and the ascites and hepatomegaly
resolved in a few weeks.\textsuperscript{13} She presented with grade II
acute GVHD (stage 2 skin and stage 1 gastrointestinal) from around day
+36 and was treated with prednisolone and short-course methotrexate (7.5
mg/m\textsuperscript{2} on days +70, +77, and +84). She responded well
to prednisolone; however, there was repeated acute GVHD after weaning
off prednisolone. At last follow up, she was 10 months post-BMT and
received prednisolone and tacrolimus without GVHD. She was free from
disease, with no other severe complications.

\textbf{DISCUSSION}

Because AUL is an extremely rare disease, optimal treatment has not been
established and, particularly in relapsed/refractory cases, therapy
options are limited. A few B-lineage associated markers, such as CD22,
are expressed in some cases of AUL.\textsuperscript{4} Weinberg et al.
reported that 6 of 24 (25\%) AUL cases showed partial or full CD22
co-expression,\textsuperscript{5} and in our case, CD22-positive AUL
indicated a treatment strategy using InO against relapsed/refractory
disease.

Use of InO increases the risk of VOD/SOS after stem cell
transplantation.\textsuperscript{14} Our patient also met the proposed
EBMT pediatric criteria for VOD/SOS.\textsuperscript{12} Although the
severity grading was moderate (grade II) and VOD/SOS improved
immediately with symptomatic therapy in this case, careful judgment
should be made regarding administration of InO for AUL. Further
accumulation of cases is needed to allow evaluation of the efficacy and
safety of InO as a bridging therapy to stem cell transplantation in
patients with CD22-positive AUL.

\textbf{CONFLICT OF INTEREST}

The authors declare that there is no conflict of interest.

\textbf{ACKNOWLEDGMENTS}

We would like to thank Takao Deguchi for help with immunodiagnostic
analysis, and Makiko Yamazaki for help with morphological analysis.

\textbf{REFERENCES}

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2. Qasrawi A, Gomes V, Chacko CA, et al. Acute undifferentiated
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3. Hrusak O, de Haas V, Stancikova J, et al. International cooperative
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4. Arber DA, Orazi A, Hasserjian R, et al. The 2016 revision to the
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5. Weinberg OK, Hasserjian RP, Baraban E, et al. Clinical,
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leukemia and comparison to acute myeloid leukemia with minimal
differentiation: a study from the bone marrow pathology group. \emph{Mod
Pathol} . 2019;32(9):1373-1385.

6. Shah NN, Stevenson MS, Yuan CM, et al. Characterization of CD22
expression in acute lymphoblastic leukemia. \emph{Pediatr Blood Cancer}
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7. Bhojwani D, Sposto R, Shah NN, et al. Inotuzumab ozogamicin in
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8. Hasegawa D, Imamura T, Yumura-Yagi K, et al. Risk-adjusted therapy
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9. Suzuki N, Yumura-Yagi K, Yoshida M, et al. Outcome of childhood acute
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10. Locatelli F, Testi AM, Bernardo ME, et al. Clofarabine,
cyclophosphamide and etoposide as single-course re-induction therapy for
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11. Miano M, Pistorio A, Putti MC, et al. Clofarabine, cyclophosphamide
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12. Corbacioglu S, Carreras E, Ansari M, et al. Diagnosis and severity
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pediatric patients: a new classification from the European society for
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13. Yakushijin K, Ikezoe T, Ohwada C, et al. Clinical effects of
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14. McDonald GB, Freston JW, Boyer JL, DeLeve LD. Liver Complications
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2019;69(2):831-844.

\textbf{FIGULRE LEGENDS}

\textbf{Figure 1.} (A--C) Microscopic images of bone marrow smears at
onset. Blasts were negative for MPO and 2--3 times the size of small
lymphocytes with a 60--90\% nucleus-to-cytoplasm ratio, delicate
chromatin, irregular nuclear contours, prominent nucleoli, and no
cytoplasmic granules. (A) May-Giemsa-stained sample; magnification,
×200. (B) May-Giemsa-stained sample; magnification, ×1000. (C)
MPO-stained sample; magnification, ×400. (D--F) Flow cytometric analysis
before administration of InO, showing a population of blasts expressing
surface CD22 and dim CD45, but lacking CD19.

\textbf{Figure 2.} Clinical course until day +100 post transplantation.
PIR, pre-engraftment immune reaction; GVHD, graft-versus-host disease;
VOD, veno-occlusive disease; SOS, sinusoidal obstruction syndrome; rhTM,
recombinant human soluble thrombomodulin; MTX, methotrexate; mPSL,
methylprednisolone; PSL, prednisolone; TAC, tacrolimus.\selectlanguage{english}
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