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\begin{document}

\title{Mixed Phenotype Acute Leukemia with BCL11B Copy Gain: What is the Best
Strategy?}



\author[1]{Jinjun Cheng}%
\author[1]{Keri Toner}%
\author[1]{Reuven Schore}%
\affil[1]{Children's National Hospital Center for Cancer and Blood Disorders}%


\vspace{-1em}



  
  \date{May 26, 2024}


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\textbf{Mixed Phenotype Acute Leukemia with \emph{BCL11B} Copy Gain:
What is the Best Strategy?}

Keri Toner MD\textsuperscript{1, 2}, Reuven Schore
MD\textsuperscript{1,2} and Jinjun Cheng MD PhD\textsuperscript{1,3,4*}

\begin{enumerate}
\tightlist
\item
  Center for Cancer and Blood Disorders, Children's National Hospital,
  Washington, DC
\item
  Department of Pediatrics, The George Washington University School of
  Medicine and Health Sciences, Washington, District of Columbia, USA
\item
  Division of Pathology and Laboratory Medicine, Children's National
  Hospital, Washington, DC
\item
  Departments of Pathology and Pediatrics, The George Washington
  University School of Medicine and Health Sciences, Washington,
  District of Columbia, USA
\end{enumerate}

\textbf{*Correspondence to:}

Jinjun Cheng, MD, PhD

Pathology and Laboratory Medicine Division

Children's National Hospital

Room 1629, 111 Michigan Avenue NW

Washington, DC 20010-2970

Telephone: 202-476-4311

Fax: 202-476-4030

Email: jcheng2@childrensnational.org

\textbf{Word Count:} 849

\textbf{Tables:} 0

\textbf{Figures:} 1

\textbf{Running title:} \emph{BCL11B} copy gain in Mixed Phenotypic
Acute Leukemia

\textbf{Key words:} MPAL, BCL11B, FLT3-ITD, leukemia

\textbf{List of abbreviations:}\selectlanguage{english}
\begin{longtable}[]{@{}ll@{}}
\toprule
Abbreviation & Full term\tabularnewline
\midrule
\endhead
MPAL & Mixed Phenotype Acute Leukemia\tabularnewline
BCL11B & BCL11 Transcription Factor B\tabularnewline
FLT3-ITD & FLT3 internal tandem duplication\tabularnewline
COG & Children Oncology Group\tabularnewline
WHO & World Health Organization\tabularnewline
\bottomrule
\end{longtable}



\textbf{Mixed Phenotype Acute Leukemia with \emph{BCL11B} copy gain:
What is the best strategy?}

\emph{To the Editor:}

Mixed-phenotype acute leukemia (MPAL) is a rare, heterogeneous group of
leukemia that comprises admixture of myeloid and lymphoid blasts
(bilineal) or a single blast population co-expressing lymphoid and
myeloid markers (biphenotypic). The accurate diagnosis relies on the
immunophenotype of flow cytometric or immunostaining
findings.\textsuperscript{1-3} European Group of Immunological
Characterization of Leukemia (EGIL) was released in 1990s to establish
guidelines for the characterization of acute leukemia based on marker
expression,\textsuperscript{2} and the World Health Organization (WHO)
classification also acknowledges lineage-specific antibodies and genetic
markers.\textsuperscript{2} In brief, the assignment of myeloid lineage
depends on the expression of Myeloperoxidase (MPO); the assignment of
monocytic differentiation depends on at least 2 of the following:
Nonspecific Esterase, CD11c, CD14, CD64 or Lysosome; the assignment of
B-lineage depends on CD19 and more than 1 or 2 strong expression of
CD79a, cytoplasmic CD22 or CD10; the assignment of T-lineage depends on
strong cytoplasmic CD3 (strong is equal to brighter than the normal T
cells) or surface CD3 expression.\textsuperscript{1}

Recent genetic studies reveal recurrent aberrations in MPAL, which have
been integrated in the classification of MPAL with
\emph{BCR::ABL1}fusion, \emph{KMT2A} rearrangement, \emph{ZNF384}
rearrangement, and\emph{BCL11B} rearrangement.\textsuperscript{1} Within
these genetic aberrations, \emph{BCL11B} rearrangement is particularly
interesting. Inactivating mutations of \emph{BCL11B} have been
identified in up to 16\% of T-acute lymphoblastic leukemia (T-ALL),
indicating its likely role of tumor-suppressor gene for
T-ALL.\textsuperscript{4,5} However, novel \emph{BCL11B} rearrangements
have been identified in MPAL, early T-precursor-ALL, and in poorly
differentiated AML with aberrant T-cell antigen
expression.\textsuperscript{6-10}

The complex phenotype of MPAL creates not just a diagnostic challenge,
but challenges in determining and initiating optimal
therapy.\textsuperscript{11} Lack of prospective trials and varying
classification systems leads to inability to define standard therapy. A
recent retrospective study by an MPAL cohort from the Children's
Oncology Group (COG), suggests that ALL chemotherapy without HSCT may be
the preferred initial therapy for pediatric
MPAL.\textsuperscript{12}Ongoing efforts are underway within COG to
prospectively evaluate the use of high risk ALL therapy in MPAL. Here we
report a diagnostically challenging T/My MPAL patient with \emph{BCL11B}
copy gain.

\emph{Case Presentation:}

A 9-year-old male presented with lymphadenopathy, leukocytosis with
white blood cell count (WBC) 277 x10\textsuperscript{9}/L, Hemoglobin
6.1g/dL, and circulating blasts in peripheral blood. The flow cytometry
analysis of peripheral blood confirmed 85\% blasts expressing CD34,
CD13, CD117, CD15, HLA-DR partial, CD7 and CD2. Most blasts were
negative for MPO but equivocally express cytoplasmic CD3 (with an
intensity lower than background T cells). In addition, a small
population of discrete blast population (less than 5\%) were positive
for MPO. These flow cytometry findings raised differential diagnoses of
MPAL, early T precursor (ETP)-ALL or AML with aberrant T cell antigen
expression. A bone marrow biopsy was performed, and the flow cytometry
analysis of bone marrow revealed dim cytoplasmic CD3 on blasts with the
intensity equal to background T-cells. There was a discrete population
of blasts (5-10\%) with positive MPO expression. The findings supported
a T/My MPAL diagnosis based on the current WHO diagnostic criteria. The
immunostains of CD3 and MPO on bone marrow core biopsy also supported
the diagnosis. The genetic and cytogenetic karyotype studies on bone
marrow aspirate revealed a \emph{BCL11B} copy gain (rearrangement not
detected) and FLT3-ITD. The patient was treated on COGAALL1732 protocol
and while leukocytosis responded to initial treatment, on day three of
treatment, the patient was found on CT to have a large intraparenchymal
hemorrhage and died the following day.

We report a unique case demonstrating the diagnostic and treatment
challenge of MPAL with \emph{BCL11B} copy gain. MPAL comprises less than
5\% of pediatric acute leukemia.\textsuperscript{13} The current
diagnostic criteria of MPAL relies on flow cytometry,
immunohistochemistry or less frequently by cytochemical stain to
determine the blast lineage.\textsuperscript{7} However, it is
subjective regarding the percentage and intensity of antigen expression
when assigning the lineage. For instance, the peripheral blasts in our
patient revealed very dim expression of cytoplasmic CD3 and less than
5\% MPO+ blasts, which together created a diagnostic
challenge.\textsuperscript{14}

Because of the rarity of MPAL, there are frequently diagnostic
challenges that could lead to delay in treatment initiation as well as a
lack of prospective trial data to guide
therapy.\textsuperscript{11,12,15} \emph{BCL11B} rearrangement is known
to associate with a distinct subgroup of acute leukemias with a broadly
variable phenotype, including AML, ETP-ALL, and
MPAL.\textsuperscript{5,6,8,13,16} The functional consequence
of\emph{BCL11B} rearrangement is overexpression of\emph{BCL11B}
.\textsuperscript{8} Our patient had a \emph{BCL11B} copy gain without
known rearrangement, which might be an alternative mechanism of
\emph{BCL11B} overexpression. Moreover, the leukemias with\emph{BCL11B}
aberrations frequently contain FLT3-ITD and other high risk
mutations.\textsuperscript{8} An ex vivo drug-sensitivity profile study
revealed that FLT3 tyrosine kinase inhibitors are effective on these
leukemias with \emph{BCL11B} rearrangement and
FLT3-ITD,\textsuperscript{8} which may provide a novel therapy strategy
for MPAL with \emph{BCL11B} rearrangement in the future.

In summary, MPAL is a rare leukemia which presents diagnostic
challenges. We report a patient with an identified \emph{BCL11B} copy
gain without rearrangement present in T/myeloid MPAL. Identification of
this \emph{BCL11B} aberration may contribute to the current molecular
knowledge of MPAL, and ideally additional prospective studies of
patients with MPAL will more clearly define diagnostic criteria and
appropriate therapy.

\textbf{References:}

1. Khoury JD, Solary E, Abla O, et al. The 5th edition of the World
Health Organization Classification of Haematolymphoid Tumours: Myeloid
and Histiocytic/Dendritic Neoplasms.
\emph{Leukemia.}2022;36(7):1703-1719.

2. Swerdlow SH CE, Harris NL, Jaffe ES, Pileri SA, Stein H, et al.
.\emph{WHO Classification of Tumours of Haematopoietic and Lymphoid
Tissues, Revised Fourth Edition.} Vol 2. Lyon, France: IARC/WHO Press;
2017.

3. Weinberg OK, Arber DA. How I Diagnose Acute Leukemia of Ambiguous
Lineage. \emph{Am J Clin Pathol.} 2022;158(1):27-34.

4. Liu Y, Easton J, Shao Y, et al. The genomic landscape of pediatric
and young adult T-lineage acute lymphoblastic leukemia. \emph{Nat
Genet.} 2017;49(8):1211-1218.

5. Meijerink JPP. BCL11B, the Cerberus of human leukemia.
\emph{Blood.}2021;138(9):741-743.

6. Abbas S, Sanders MA, Zeilemaker A, et al. Integrated genome-wide
genotyping and gene expression profiling reveals BCL11B as a putative
oncogene in acute myeloid leukemia with 14q32
aberrations.\emph{Haematologica.} 2014;99(5):848-857.

7. Bendig S, Stengel A, Walter W, et al. Diagnostic challenge of
identifying cases with recurrent t(8;14)(q24.21;q32.2) Involving BCL11B
in acute leukemias of ambiguous lineage: an analysis of eight
patients.\emph{Leuk Lymphoma.} 2022;63(3):747-750.

8. Di Giacomo D, La Starza R, Gorello P, et al. 14q32 rearrangements
deregulating BCL11B mark a distinct subgroup of T-lymphoid and myeloid
immature acute leukemia. \emph{Blood.} 2021;138(9):773-784.

9. Montefiori LE, Bendig S, Gu Z, et al. Enhancer Hijacking Drives
Oncogenic BCL11B Expression in Lineage-Ambiguous Stem Cell
Leukemia.\emph{Cancer Discov.} 2021;11(11):2846-2867.

10. Wang W, Beird H, Kroll CJ, et al. T(6;14)(q25;q32) involves BCL11B
and is highly associated with mixed-phenotype acute leukemia,
T/myeloid.\emph{Leukemia.} 2020;34(9):2509-2512.

11. Maruffi M, Sposto R, Oberley MJ, Kysh L, Orgel E. Therapy for
children and adults with mixed phenotype acute leukemia: a systematic
review and meta-analysis. \emph{Leukemia.} 2018;32(7):1515-1528.

12. Orgel E, Alexander TB, Wood BL, et al. Mixed-phenotype acute
leukemia: A cohort and consensus research strategy from the Children's
Oncology Group Acute Leukemia of Ambiguous Lineage Task
Force.\emph{Cancer.} 2020;126(3):593-601.

13. Alexander TB, Gu Z, Iacobucci I, et al. The genetic basis and cell
of origin of mixed phenotype acute leukaemia.
\emph{Nature.}2018;562(7727):373-379.

14. Cheng J, Klairmont MM, Choi JK. Peripheral blood flow cytometry for
the diagnosis of pediatric acute leukemia: Highly reliable with rare
exceptions. \emph{Pediatr Blood Cancer.} 2019;66(1):e27453.

15. Rasekh EO, Osman R, Ibraheem D, et al. Acute lymphoblastic
leukemia-like treatment regimen provides better response in mixed
phenotype acute leukemia: a comparative study between adults and
pediatric MPAL patients. \emph{Ann Hematol.} 2021;100(3):699-707.

16. Duffield AS, Mullighan CG, Borowitz MJ. International Consensus
Classification of acute lymphoblastic leukemia/lymphoma. \emph{Virchows
Arch.} 2023;482(1):11-26.

\textbf{Figure Legend:}

\textbf{Figure 1.} Flow cytometry and FISH results of T/My mixed
phenotype acute leukemia. A: The peripheral blood flow cytometry
identified a major blast population expressing equivocal cytoplasmic CD3
(with intensity not reaching to the CD3 intensity of normal T cells),
and a small population of MPO+ CD34+ blasts (less than 5\%). B: The
repeated flow cytometry on bone marrow aspirate revealed cytoplasmic CD3
dim+ (with intensity equal to normal T cells) and cytoplasmic MPO in a
sub-population of blasts (5-10\%). C: Representative blast on bone
marrow aspirate (originally 1000x Gimsa stain). D: The FISH study
revealed a copy gain of \emph{BCL11B} in 58 out of 100 cells (green for
BCL11B probe; red for TLX3 probe.). CD3 (E, 200x) and MPO (F, 200X)
immunostains are positive on a subset of blasts on core biopsy, which
also support the diagnosis of T/My MPAL.\selectlanguage{english}
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