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\begin{document}

\title{Emergence of a prominent myeloid clone in a ZNF384-rearranged B-cell
precursor acute lymphoblastic leukaemia post-corticosteroid pre-phase
therapy.}



\author[1]{Alpeshkumar Kapadia}%
\author[1]{Sreejesh Sreedharanunni}%
\author[1]{Safal  Muhammed}%
\author[1]{Indrani Karmakar}%
\author[1]{Sonia Rana}%
\author[1]{Prashant Sharma}%
\author[1]{Man Updesh Sachdeva}%
\author[1]{Amita Trehan}%
\affil[1]{Post Graduate Institute of Medical Education and Research}%


\vspace{-1em}



  
  \date{May 08, 2024}


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\selectlanguage{english}
\begin{abstract}
A 2-year-old girl with TCF3-ZNF384 re-arranged standard-risk B-cell
precursor acute lymphoblastic leukaemia (BCP-ALL) showed an apparent
lineage-switch to predominantly myeloid blasts following 7-days of
corticosteroid therapy. ZNF384-re-arranged leukemias are increasingly
recognized to present either as mixed-phenotype acute leukemia or as
BCP-ALL with pro-B immunophenotype. Lineage switch is a rare phenomenon
described at relapse or following CAR-T-cell therapy previously mostly
in KMT2A re-arranged leukemias; and now, also in ZNF384-rearranged
patients. It is previously unreported in any patient after pre-phase
corticosteroid therapy. Hematologists should be aware of this distinct
emerging entity among leukemias with multi-lineage differentiation
potential.%
\end{abstract}%



\sloppy


\textbf{Introduction}

\emph{ZNF384} (zinc finger protein 384) re-arrangements are recently
recognized cytogenetic abnormalities that occur in as many as 48\% of
mixed-phenotype acute leukaemia (MPAL), B/myeloid and 4\% of B-cell
precursor acute lymphoblastic leukaemia (BCP-ALL)\textsuperscript{1}.
Several fusion partners of \emph{ZNF384} have been described, the most
common being \emph{TCF3,} but also \emph{EP300, ESWR1, TAF15, CREBBP,
ARID1B, SYNRG} and \emph{BMP2K} \textsuperscript{1--3} We report a
challenging case with \emph{TCF3-ZNF384} rearrangement that initially
presented as BCP-ALL but showed the emergence of a large myeloid blast
subset following one week of high-dose steroid therapy.

\textbf{Case History}

A two-and-a-half-year-old female child presented with fever and pallor
of 1-week duration. Examination revealed hepatomegaly (span 11 cm) and
splenomegaly (2 cm below the left subcostal margin). The hemoglobin was
46 g/L, platelet count 14.0 x10\textsuperscript{9} /L and total
leucocyte count 14.7 x 10\textsuperscript{9}/L. Peripheral blood film
showed 62\% circulating blasts (\textbf{Fig. 1A-B} ). Bone marrow
aspiration revealed 82\% blasts with scanty, agranular basophilic
cytoplasm without Auer rods; 2\% of the blasts showed cytochemical
myeloperoxidase positivity. On flow cytometry, the blasts expressed
bright CD19 and dim CD10 along with cytoplasmic CD79a and CD22. There
was cross-lineage antigen expression of CD33 (bright; 90\% cells) and
CD13 (dim; 14\% cells). The blasts were also positive for CD34, HLA-DR,
CD38, CD81 and CD86; and were negative for CD20, CD117, T-lineage
associated markers, monocytic \& megakaryocytic-lineage associated
markers. Cytoplasmic myeloperoxidase (anti-MPO) expression was also not
noted. A working diagnosis of BCP-ALL with aberrant CD33 expression was
rendered, following which the child was initiated on the Standard Risk
(SR) BCP-ALL protocol of the Indian Childhood Collaborative Leukemia
Group (ICiCLe), comprising of a pre-phase of prednisolone 60
mg/m\textsuperscript{2}/d in three divided doses. \textsuperscript{4}

Fluorescent \emph{in-situ} hybridization (FISH) testing with dual-colour
dual-fusion probes for \emph{BCR-ABL1} and \emph{ETV6-RUNX1,} dual
break-apart probes for \emph{KMT2A} and \emph{TCF3} (CytoTest Inc.
Rockville, United States) revealed presence of \emph{TCF3} translocation
in more than 80\% of cells (\textbf{Fig. 1E)} . Subsequently,
both\emph{TCF3-PBX1} and \emph{TCF3-HLF} translocations were excluded
using a tri-colour probe (Cytocell Ltd, Cambridge).

After one week of pre-phase steroid therapy, although the toddler
clinically appeared well, her leukocyte count increased to
63x10\textsuperscript{9}/L with 60\% circulating blasts. On peripheral
blood smear examination, in contrast to the morphology at diagnosis,
these blasts were larger with indented nuclei, prominent nucleoli and
moderate amount of granular basophilic cytoplasm without Auer rods. The
blasts showed unequivocal cytochemical myeloperoxidase positivity in
20\% of the cells (\textbf{Fig. 1C-D} ). The smaller lymphoblasts noted
at the time of diagnosis were absent. Repeat flow cytometry performed on
peripheral blood showed that 90\% of the blasts expressed myeloid
antigens (CD13, CD33, CD64, CD14, CD36, CD15 and cytoplasmic
myeloperoxidase. The remaining 10\% blasts showed the initial
immunophenotype of BCP-ALL. The initial cytochemical myeloperoxidase and
anti-MPO (flow cytometry) results were reviewed and the negative report
(\textless{}3\% positivity) was reverified.

Repeat FISH testing revealed the \emph{TCF3} rearrangement in blasts as
well as granulocytes \textbf{(Fig. 1F)} . In view of absence of
metaphase, further FISH testing was performed using a commercially
available dual-colour break-apart probe (ZytoVision GmbH, Germany) that
confirmed rearrangement of \emph{ZNF384} \textbf{(Fig. 1G)} \emph{.} The
diagnosis was revised to \emph{TCF3-ZNF384} {[}t(12;19)(p13;p13){]}
rearranged MPAL, B/myeloid. The patient's risk stratification was
upgraded to High-Risk (HR) and appropriate management
initiated\textsuperscript{4}.

Her remaining induction phase chemotherapy was interrupted by two
episodes of febrile neutropenia, both managed with antimicrobials.
End-of-induction bone marrow revealed 03\% blasts and no measurable
residual disease (MRD) on high-resolution multiparameter flow cytometry.
She had a stormy course prior to consolidation too, developing a fungal
brain abscess requiring surgical drainage, leading to a 2-week delay in
start of consolidation therapy. Currently, the patient is on an
augmented BFM (Berlin-Frankfurt-Munster) consolidation protocol and
continues her antifungal prophylaxis.

\textbf{Discussion}

\emph{TCF3-ZNF384} fusion gene resulting from a reciprocal translocation
between chromosomes 19p13 (\emph{TCF3} ) and 12p13 (\emph{ZNF384} )
appears to be a distinct category. Bearing similarities to\emph{KMT2A-}
rearranged acute leukemias, these too present either as BCP-ALL or MPAL,
B/myeloid type\textsuperscript{5} and are also associated with dim CD10
expression (pro-B immunophenotype) and upregulation of CD13 and/or
CD33\textsuperscript{1,2} as seen in our patient. About 2\% blasts
expressed myeloperoxidase, but the overall initial immunophenotype did
not meet the required criteria for diagnosis of MPAL. Such diagnostic
dilemmas with MPALs are frequent in routine practice and are also
well-documented in the literature\textsuperscript{2}. \emph{TCF3-ZNF384}
-positive BCP-ALLs are associated with higher total leukocyte counts,
poor response to steroids at day-8 and a relatively unfavourable
outcome\textsuperscript{1}. In our case too, the total leukocyte count
showed a fivefold increase by day 8, with a significant increase in
myeloblasts.

A lineage switch, i.e. conversion from lymphoid to myeloid
immunophenotype (and exceptionally, \emph{vice versa} ) in acute
leukemias is a rare phenomenon. It occurs more often in pediatric
leukemias, being reported mostly in patients with
\emph{KMT2A}re-arrangement\textsuperscript{6--8} but occasionally also
in patients with \emph{ZNF384} -rearranged acute
leukemias\textsuperscript{5,9}. The lineage switch mostly occurs at the
time of relapse. However, it is also reported in patients who have
received either CD19-targeting agents or chimeric antigen receptor
T-cell therapy\textsuperscript{7,8,10,11}.

Various mechanisms may explain the lineage switch from lymphoid to
myeloid or emergence of large myeloid clone following one week of
high-dose steroid therapy in our case. It could simply represent the
unmasking of a myeloid sub-clone that was initially outnumbered by the
preponderant lymphoid blasts. This is supported by the presence of 2\%
myeloperoxidase-positive blasts at diagnosis. An alternative explanation
might lie in the inherent plasticity of leukaemia stem cells for
divergent differentiation\textsuperscript{12}. The presence
of\emph{ZNF384} translocation initially noted in the lymphoid blasts and
subsequently shown to be present also in granulocytic cells may points
towards the origin of this leukemia from a primitive hematopoietic
precursor with divergent differentiation potential. In support of this
theory, gene expression profiles of \emph{ZNF384-} rearranged BCP-ALLs
as well as B/myeloid MPALs have revealed enrichment of stem cell
pathways.\textsuperscript{2} The stem cell origin probably leads to an
initial development of acute leukemia with single lineage
differentiation; but a therapeutic challenge may unravel its
multi-lineage differentiation potential.

In conclusion,\emph{ZNF384-} rearranged leukemias are a unique and
possibly under-recognized category of neoplasms with multi-lineage
differentiation potential. Both hematopathologists as well as
hemato-oncologists should be aware of the potential lineage switch that
may occur in this group of leukemias.

\textbf{Conflict of interest statement:} - All authors declare that they
do not have any conflict(s) of interest(s) to declare.

\textbf{Acknowledgement:} The authors are thankful to Dr Mayur Parihar,
Tata Medical Centre for performing FISH testing for
\emph{TCF3-HLF}translocation.

\textbf{References:}

1. 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;102:118-129.

2. Alexander TB, Gu Z, Iacobucci I, et al. The genetic basis and cell of
origin of mixed phenotype acute leukaemia. \emph{Nature} .
2018;562:373-379.

3. McClure BJ, Heatley SL, Kok CH, et al. Pre-B acute lymphoblastic
leukaemia recurrent fusion, EP300-ZNF384, is associated with a distinct
gene expression. \emph{Br J Cancer} . 2018;118:1000-1004.

4. Roy P, Narula G, Arora B, et al. Implementation of risk adapted
therapeutic strategy for childhood acute lymphoblastic leukaemia --
interim report of the pilot InPOG-ALL-15-01 study. \emph{Pediatr Hematol
Oncol J} . 2018;3:S19.

5. Oberley MJ, Gaynon PS, Bhojwani D, et al. Myeloid lineage switch
following chimeric antigen receptor T-cell therapy in a patient with
TCF3-ZNF384 fusion-positive B-lymphoblastic leukemia. \emph{Pediatr
Blood Cancer} . 2018;65:e27265.

6. Aldoss I, Song JY. Extramedullary relapse of KMT2A(MLL)-rearranged
acute lymphoblastic leukemia with lineage switch following
blinatumomab.\emph{Blood} . 2018;131:2507.

7. Balducci E, Nivaggioni V, Boudjarane J, et al. Lineage switch from B
acute lymphoblastic leukemia to acute monocytic leukemia with persistent
t(4;11)(q21;q23) and cytogenetic evolution under CD19-targeted
therapy.\emph{Ann Hematol} . 2017;96:1579-1581.

8. Rayes A, McMasters RL, O'Brien MM. Lineage Switch in MLL-Rearranged
Infant Leukemia Following CD19-Directed Therapy. \emph{Pediatr Blood
Cancer} . 2016;63:1113-1115.

9. Grammatico S, Vitale A, La Starza R, et al. Lineage switch from pro-B
acute lymphoid leukemia to acute myeloid leukemia in a case with
t(12;17)(p13;q11)/TAF15-ZNF384 rearrangement. \emph{Leuk Lymphoma} .
2013;54:1802-1805.

10. Zoghbi A, Zur Stadt U, Winkler B, M\selectlanguage{ngerman}üller I, Escherich G. Lineage
switch under blinatumomab treatment of relapsed common acute
lymphoblastic leukemia without MLL rearrangement. \emph{Pediatr Blood
Cancer} . 2017;64.

11. Jacoby E, Nguyen SM, Fountaine TJ, et al. CD19 CAR immune pressure
induces B-precursor acute lymphoblastic leukaemia lineage switch
exposing inherent leukaemic plasticity. \emph{Nat Commun} .
2016;7:12320.

12. Dorantes-Acosta E, Pelayo R. Lineage switching in acute leukemias: a
consequence of stem cell plasticity? \emph{Bone Marrow Res} .
2012;2012:406796.

\textbf{Figure legends:}

\textbf{Figure 1: (A-B)} Morphology of blasts (May-Grünwald Giemsa
stain; 1000x) and myeloperoxidase-staining (Hematoxylin counterstain) of
blasts at diagnosis and; \textbf{(C-D)} at day-8 post-corticosteroid
therapy. \textbf{(E)} Fluorescent \emph{in-situ} hybridization using
dual colour break apart probe showing deletion of telomeric end (3'end;
orange) of TCF3 gene on chromosome 19p13.3 indicating unbalanced
translocation in blasts and \textbf{(F)} granulocytes.
\textbf{(G)}Fluorescent \emph{in-situ} hybridization using dual colour
break apart probe showing an abnormal signal pattern indicative of
\emph{ZNF384}translocation (DAPI counterstain).

\textbf{Figure 2:} Immunophenotype of blasts - \textbf{(A-D)} at
diagnosis (CD19\textsuperscript{pos}
CD10\textsuperscript{dimpos}CD34\textsuperscript{pos}
CD33\textsuperscript{pos}CD13\textsuperscript{dim}
CD15\textsuperscript{neg}CyCD79a\textsuperscript{pos}
CyCD22\textsuperscript{pos}) \textbf{(E-H)}post-corticosteroid prephase
therapy (CD19\textsuperscript{neg}CD10\textsuperscript{neg}
CD34\textsuperscript{neg}CD33\textsuperscript{pos}
CD13\textsuperscript{pos}CD15\textsuperscript{pos}
CyCD79a\textsuperscript{neg}CyCD22\textsuperscript{neg})\selectlanguage{english}
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