Human Pathology (2023) 136, 75e83
www.elsevier.com/locate/humpathOriginal contributionMucin 4 protein is expressed in B-acute
lymphoblastic leukemia and is restricted to
BCR::ABL1-positive and BCR::ABL-like subtypes*
Catherine K. Gestrich DO a,1, Shanelle J. De Lancy MD a,
Adam Kresak MS a, Mohamad G. Sinno MD b,2, Akua Yalley PhD c,
Irina Pateva MD b, Howard Meyerson MD a, Shashirekha Shetty PhD a,
Kwadwo A. Oduro Jr MD, PhD a,*
aDepartment of Pathology, University Hospitals Cleveland Medical Center & Rainbow Children’s
Hospital & Case Western Reserve University, Cleveland, OH, 44106, USA
bDepartment of Pediatrics, Division of Hematology and Oncology, University Hospitals Rainbow Babies
and Children’s Hospital & Case Western Reserve University, Cleveland, OH, 44106, USA
cDepartment of Medical Laboratory Sciences, School of Biomedical and Allied Health Sciences,
University of Ghana, Accra, P.O. Box 143, GhanaReceived 30 January 2023; revised 25 March 2023; accepted 30 March 2023
Available online 5 April 2023Keywords:
MUC4;
Immunohistochemistry;
B-acute lymphoblastic
leukemia;
BCR::ABL1;
BCR::ABL1-like;
CRLF2* Disclosures: The authors did no
* Corresponding author. Departmen
E-mail addresses: Kwadwo.Odur
1 Current address: Department of P
2 Current address: Department of P
https://doi.org/10.1016/j.humpath.2023.0
0046-8177/© 2023 Elsevier Inc. All rigSummary Mucin 4 (MUC4) is a transmembrane mucin that, like most mucins, is not expressed in
normal hematopoietic cells, but little is known about its expression in malignant hematopoiesis. B-acute
lymphoblastic leukemia (B-ALL) consists of genetically distinct disease subtypes with similarities and
differences in gene expression most frequently studied at the mRNA level, which is less amenable to
widespread routine clinical use. Here, we demonstrate using immunohistochemistry (IHC) that
MUC4 protein is expressed in less than 10% of B-ALL, with expression restricted to BCR::ABL1þ
and BCR::ABL1-like (CRLF2 rearranged) subtypes of B-ALL (4/13, 31%). None (0/36, 0%) of the re-
maining B-ALL subtypes expressed MUC4. We compare clinical and pathologic features of MUC4þ
and MUC4 BCR::ABL1þ/like cases and most significantly report a possible shorter time to relapse
for MUC4þ BCR::ABL1 B-ALL that would need to be validated in larger studies. In conclusion,
MUC4 is a specific, albeit insensitive, marker for these high-risk subtypes of B-ALL. We propose that
MUC4 IHC may be used diagnostically to rapidly identify these B-ALL subtypes, particularly in
resource-limited settings or when an aspirate sample is not available for ancillary genetic studies.
© 2023 Elsevier Inc. All rights reserved.t report any potential conflicts of interest.
t of Pathology, University Hospitals Cleveland Medical Center, 11100 Euclid Avenue, Cleveland, OH, 44106, USA.
o@uhhospitals.org, kaoduroj@gmail.com (K.A. Oduro).
athology, University of Pittsburgh Medical Center, Pittsburgh, PA, 15213, USA.
ediatrics, Center for Cancer and Blood Disorders, Phoenix Children’s Hospital, Phoenix AZ, USA.
3.018
hts reserved.
76 C.K. Gestrich et al.1. Introduction
Mucin 4 (MUC4) is a transmembrane glycoprotein and a
member of the large family of secreted or transmembrane
mucins, characterized structurally by tandem repeat extra-
cellular PTS (proline, threonine, serine) domains, which
undergo extensive O-glycosylation [1,2]. Like other mem-
bers in this protein family, MUC4 is normally expressed on
the luminal surface of epithelial cells, such as respiratory,
colonic, gastric, and cervical epithelium, where the protein
provides a barrier function, protecting against cellular
injury. However, transmembrane mucins like MUC4 can
also be involved in downstream signaling, particularly in
pathologic states. With the exception of MUC1 (recognized
by the epithelial membrane antigen/EMA and other anti-
bodies) [3], physiologic protein expression of mucins,
including MUC4, has not been reported in hematopoietic
cells or hematopoietic organs.
Despite their generally protective function, upregulation
or aberrant expression of certain mucins has been reported
in some human malignancies and neoplastic proliferations.
MUC4 is known to be overexpressed in a subset of
pancreatic, breast, colonic, and lung carcinomas, and
MUC4 immunohistochemistry (IHC) has recently emerged
as a sensitive and specific marker for low-grade fibro-
myxoid sarcoma [4,5]. IHC for other mucinsdMUC1,
MUC2, MUC5AC, and MUC6dis routinely used in diag-
nostic pathology to clarify the cell of origin or differenti-
ation of neoplasms such as intraductal papillary mucinous
neoplasm or gastric carcinoma. MUC1 (EMA) detection is
also relevant in hematopathology as this protein is
expressed in neoplastic cells in a subset of Hodgkin lym-
phoma (more likely nodular lymphocyte predominant type)
as well as in some non-Hodgkin lymphoma (such as
anaplastic large cell lymphoma). Little is known about
MUC4 protein expression in hematologic malignancies.
B-acute lymphoblastic leukemia (B-ALL) is the most
prevalent malignancy in the pediatric population, although
about 40% of total cases occur in adults. It is characterized
by the proliferation of immature B lymphoid blasts, most
frequently replacing the marrow and circulating in pe-
ripheral blood (“leukemia”); less frequently, this disease
may manifest as a tissue-based mass lesion without
appreciable bone marrow involvement (“lymphoblastic
lymphoma”). Until recently, the classification of B-ALL
was based on the identification of recurrent genetic ab-
normalities: BCR::ABL1 (Philadelphia chromosome; Phþ),
ETV6::RUNX1, TCF3::PBX1, IGH::IL3, MLL rearrange-
ment, internal amplification of chromosome 21 (iAMP21),
hyperdiploidy and hypodiploidy. The 2017 World Health
Organization (WHO) classification introduced a new sub-
type BCR::ABL1-like (Ph-like B-ALL), previously
included in the B-ALL not otherwise specified (NOS)
category, that was identified based on the presence of a
gene expression profile similar to BCR::ABL1þ B-ALL butlacking the BCR::ABL1 translocation [6,7]. Instead, about
90% of BCR::ABL1-like B-ALL cases harbor other genetic
alterations resulting in the activation of kinase and cytokine
receptor signaling, most commonly rearrangements
involving CRLF2 [8]. BCR::ABL1þ and BCR::ABL1-like
B-ALL are considered high-risk subsets of B-ALL and
together comprise about 10e20% of pediatric and about
50% of adult B-ALL [6,9,10].
Gene expression profiling has previously identified
MUC4 overexpression at the RNA level in BCR::ABL1þ
and BCR::ABL1-like B-ALL compared with other subtypes
of B-ALL [7,11,12]. MUC4 mRNA overexpression is
included in the 8-gene low-density array (LDA) predictor
used in the identification of BCR::ABL1-like B-ALL cases
in Children’s Oncology Group clinical trials [13]. We
sought to evaluate whether MUC4 mucin is indeed
expressed at the protein level in B-ALL, to determine its
differential expression in subtypes of B-ALL and the po-
tential significance of such differential expression.
2. Materials and methods
2.1. Patient selection
Patients diagnosed with B-ALL between 2015 and
2019 at University Hospitals (UH) Cleveland Medical
Center (UHCMC) and UH Rainbow Children’s Hospital
were identified from the UH pathology information system
and a clinical database maintained by the Department of
Pediatric Hematology/Oncology at Rainbow Children’s
Hospital. Initial diagnostic bone marrow samples were used
for staining in the majority of cases. For 2 cases, the initial
bone marrow biopsy was not available, and relapse bone
marrow biopsy specimens from these patients were stained
instead. Patients with inadequate or unavailable B-ALL
positive bone marrow archival blocks for immunohisto-
chemical evaluation were excluded. Only de novo B-ALL
cases were included; cases with lymphoid blast phase of a
preceding chronic myeloid leukemia were excluded.
In total, there were 49 patients, who ranged from 1 to 82
years old at the time of initial diagnosis. A total of 11
normal bone marrow samples were used as controls. These
consisted of negative staging bone marrow biopsy speci-
mens from patients with Hodgkin lymphoma (n Z 4),
neuroblastoma (n Z 3), and diffuse large B cell lymphoma
(n Z 4). The ages of the patients ranged from 4 months to
64 years, with a mean age of 27 years and a median of 17
years. The study was approved by the UH Institutional
Review Board.
2.2. B-ALL diagnosis and classification
All B-ALL diagnosis and classification was made by
board-certified hematopathologists based on morphologic
evaluation, flow cytometry, and genetic studies and
Mucin 4 protein is expressed in B-acute lymphoblastic leukemia 77conformed to WHO 2017 criteria. Standard karyotype and
fluorescence in situ hybridization (FISH) testing to identify
B-ALL with BCR::ABL1, hyperdiploidy (FISH probes for
þ4, þ10, þ17), ETV6::RUNX1, TCF3::PBX1, hypodip-
loidy, and MLL rearrangement (KMT2A break apart probe)
were performed at UHCMC. BCR::ABL1-like cases were
identified by BCR::ABL1-like FISH panel testing at Cin-
cinnati Children’s Hospital (https://www.testmenu.com/
cincinnatichildrens/Tests/540531) for the commonest
BCR::ABL1-like associated rearrangements (breakapart
probes for CRLF2, ABL2, PDGFRB, CSF1R, JAK2, ABL1,
EPOR) or by BCR::ABL1-like LDA testing followed by
confirmatory genetic testing for positive cases performed at
UNM/Tricore Laboratories [13] for patients enrolled in
COG trials. All BCR::ABL1-like cases in our cohort had a
CRLF2 rearrangement and overexpressed CRLF2 by flow
cytometry performed at UHCMC (phycoerythrin conju-
gated anti-CRLF2/TSLP-R antibody, clone 1B4, Bio-
legend). Intrachromosomal amplification of chromosome
21 (iAMP21) is detectable using the RUNX1 probe used for
ETV6::RUNX1 testing. B-ALL with t(5;14) is detectable by
standard karyotyping. However, no cases fulfilling this
criterion were identified in our cohort. Patients for whom
any defining cytogenetic abnormalities above were not
identified were designated NOS. In 8 of 11 NOS cases,
BCR::ABL1-like testing is unknown because testing was
either not performed or was unsatisfactory. Karyotype on 2
of these NOS cases yielded a culture failure, but FISH
testing was negative for recurrent abnormalities. Never-
theless, the possibility of hypodiploidy in these cases
cannot be completely excluded. B-ALL with t(5;14) is
unlikely based on the lack of characteristic morphologic
findings (increased eosinophils) in these patients’ samples.
2.3. MUC4 IHC
The IHC was performed by the Tissue Resources divi-
sion of the Human Tissue Procurement Facility (HTPF) at
Case Western Reserve University. Unstained 5 mm sections
of bone marrow cores (after B-plus fixation, decalcification,
and paraffin embedding) were baked for 75 min at 60 C.
Deparaffinization (using xylene), antigen retrieval (using
pH6.0 citrate buffer, 125 C), and blocking of endogenous
peroxidase and nonspecific antibody binding were per-
formed prior to antibody staining. Prepared slides were
stained with MUC4 mouse monoclonal antibody (8G7
clone from Cell Marque, 1:50 dilution, 1 hr at room tem-
perature) and subsequently with horseradish peroxidase-
based reagents (BioCare Medical M4U534). Pancreatic
ductal adenocarcinoma (PDAC) specimens were used for
antibody validation, titration, and optimization of staining
conditions, and also as a positive control when B-ALL
samples were evaluated. MUC4 stained B-ALL slides were
reviewed in a blinded manner by a board-certified hema-
topathologist and a pathologist-in-training. Staining was
cellular, and nonspecific stromal staining was not seen. Theintensity of staining was scored as no staining (0), weak
(1þ), or strong (2þ). Cases with both weakly staining and
strongly staining blasts were scored as 2þ. The specificity
of MUC4 staining was confirmed by negative staining
when the primary antibody was omitted from the staining
protocol.
3. Results
3.1. MUC4 expression in B-ALL
To determine MUC4 protein expression in B-ALL, we
selected a total of 49 B-ALL cases (24 pediatric and 25
adult) diagnosed from a bone marrow sample at our insti-
tution. This included 5 cases of BCR::ABL1-like B-ALL,
all with a CRLF2 rearrangement and overexpression by
flow cytometry, 8 de novo BCR::ABL1þ B-ALL, and 33
cases representing the other most commonly recognized
subtypes of B-ALL, determined based on cytogenetic
studies performed at the time of diagnosis: hyperdiploidy
(n Z 10), ETV6::RUNX1 (n Z 6), TCF3::PBX1 (n Z 4),
hypodiploidy (n Z 2), MLL rearranged (n Z 3), NOS
(n Z 11). No cases of B-ALL with t(5;14) and B-ALL with
iAMP21 were represented.
Only 4 of these 50 B-ALL cases (8%) demonstrated
positive staining for MUC4 in lymphoblasts. The MUC4
staining was diffusely positive in over 50% of blasts in all 4
cases. No MUC4 staining was seen in 11 normal pediatric
and adult bone marrow cases, consistent with prior reports
that MUC4 mucin is not normally expressed in the he-
matopoietic system. All 4 MUC4þ B-ALL cases were
either BCR::ABL1þ (3/8, 38%) or BCR::ABL-like, CRLF2
rearranged (1/5, 20% of cases). All 3 BCR::ABL1þ cases
had strong (2þ) staining while the positive BCR::ABL1-
like case demonstrated weak (1þ) staining (Fig. 1).
3.2. Pathologic features of MUC4þ and MUC4-
BCR::ABL1þ/BCR::ABL1-like B-ALL
We subsequently compared the clinical and pathologic
features of MUC4þ BCR::ABL1þ/BCR::ABL1-like B-
ALL with MUC4 B-ALL in our cohort, which are sum-
marized in Tables 1 and 2. All the BCR::ABL1þ/
BCR::ABL1-like cases showed extensive marrow involve-
ment by lymphoblasts independent of MUC4 expression.
Marrow blast frequency determined by aspirate or touch
prep enumeration ranged from 70 to 92% (mean 82%) in
the MUC4þ group and from 65 to 95% (mean 83%) in the
MUC4 group. In about half of the cases (7/13), the blasts
demonstrated typical B-ALL lymphoblast cytology of
small cells with very scant cytoplasm, round nuclei, and
dense to open chromatin. Variant morphology occurred
similarly in both MUC4þ (2/4) and MUC4 (4/9) and
included increased cell size, nuclear clefting/irregularities,
abundant cytoplasm, and/or presence of cytoplasmic
vacuolation or granules (Table 1; Fig. 2).
78 C.K. Gestrich et al.
Fig. 1 MUC4 immunohistochemistry in B-ALL. A, Representative hematoxylin-eosin image of bone marrow core biopsy showing
extensive marrow involvement by lymphoblasts. B, Representative negative staining of case #4 (score 0). C, Weak staining blasts of case #1
(score 1þ). D, Strong staining blasts in a background of weaker staining blasts of case #13 (score 2þ). E, Strong staining blasts of case #6
(score 2þ). F, Strong staining blasts of case #9 (score 2þ). All images are at 400x magnification (40x objective).Flow cytometric analysis was possible in 12 cases (for 1
case, case #2, biopsy was performed at an outside institu-
tion). The lymphoblasts were all positive for CD19, CD10,
and Tdt in 12 of 12 cases. In all cases, all or a majority of
blasts were also positive for CD22 and CD34; 1 case each
(cases #12 and 8, both MUC4 cases) had a CD22 or
CD34 subset comprising 20% and 40% of blasts, respec-
tively. Most cases in both groups showed aberrant expres-
sion of CD13/33 and CD304 (3/4 MUC4þ, 6/8 MUC4
for both analyses). Notably, CD20þ B-ALL, defined based
on the clinically relevant threshold of >20% of blasts
positive for this antigen [14], were all MUC4 (0/4
MUC4þ and 4/8 MUC4 B-ALL were CD20þ). Also, all
the MUC4þ cases (4/4) showed aberrantly decreased or
absent CD38 expression in blasts, whereas this was noted in
only 4 of 8 MUC4 cases. There was aberrant T-cell
marker expression, mostly aberrant CD7 expression, in 3 of
4 MUC4þ cases but only 2 of 8 MUC4 cases.
3.3. Clinical features of MUC4þ and MUC4
BCR::ABL1þ/BCR::ABL1-like B-ALL
All MUC4þ patients were adults (age at diagnosis Z
22, 49, 53, and 67 years), and their age at diagnosis
(mean Z 48 years) was not statistically different from the
MUC4 cases (mean Z 37 years, P Z .35). Actually, with
the exception of 1 patient (BCR::ABL1-like patient, 4 years
of age at diagnosis), all BCR::ABL1þ or BCR::ABL1-like
cases in our cohort were over 18 years old at diagnosis
(range Z 22e67 years), which is consistent with the
known predilection of these subtypes of B-ALL for olderindividuals. Notably, none of the 13 adult non-
BCR::ABL1þ/BCR::ABL1-like B-ALL patients in our
cohort (age range Z 19e88 years, mean Z 51 years) were
positive for MUC4 versus 4 of 12 adult BCR::ABL1þ/
BCR::ABL1-like B-ALL (age range Z 22e67 years, mean
Z 43 years, P Z .32), indicating that MUC4 expression is
not simply a function of age (P Z .039).
Pediatric and adolescents and young adults (AYA) pa-
tients (patients #2, #3, #8, #10, and #13 who were aged 31,
4, 24, 24, and 22 years, respectively, at diagnosis) were
treated with COG AALL1131 protocols. All the remaining
patients, except patient #12, were initially treated with
hyperCVAD (Cyclophosphamide, Vincristine, Adriamycin,
Dexamethasone), and BCR::ABL1þ patients in addition
also received dasatinib (an ABL kinase inhibitor). Patient
#12 had a modified treatment due to comorbidities that
omitted anthracyclines (Adriamycin), and cyclophospha-
mide was renal dose adjusted. Vincristine and dasatinib
administration was also inconsistent due to drug side ef-
fects. This patient rapidly relapsed and died from the dis-
ease 7 months after diagnosis.
All 5 BCR::ABL1-like patients were refractory to treat-
ment or subsequently relapsed after achieving remission.
The only MUC4þ case (patient #1, BCR::ABL1-like) was
the only one of the 13 BCR::ABL1þ/like patients with
CSFþ disease. After treatment, the patient initially showed
partial response, with leukemic lymphoblast frequency
decreasing from 76% to 12% and then to 5%. However,
overt disease returned with 54% blasts, and despite multi-
ple attempts at salvage therapy, the patient died 14 months
after initial diagnosis. Patient #5 (MUC4) similarly
Mucin 4 protein is expressed in B-acute lymphoblastic leukemia 79
Table 1 Pathologic features of MUC4þ and MUC4- BCR::ABL1þ/BCR::ABL1-like B-ALL.
Case # Genetics Age MUC4 Blast Blast morphology CD19, CD22 CD34 CD13 or CD304 CD20 CD38 Aberrant T-cell
(y) (%) CD10, Tdt CD33 marker expression
1 CRLF2-R 49 1þ 76 Small, round þ þ þ e e e Y e
nuclei, very scant
cytoplasm
2 CRLF2-R 31 0 73 Small-medium, N/A N/A N/A N/A N/A N/A N/A N/A
round nuclei, scant
cytoplasm with
vacuoles
3 CRLF2-R 4 0 90 Small, round þ þ þ e þ þ þ Partial dim CD7
nuclei, very scant (5%)
cytoplasm
4 CRLF2-R 44 0 65 Small, round þ þ þ þ þ þ þ e
nuclei, very scant
cytoplasm
5 CRLF2-R 37 0 89 Small, round þ þ þ þ e e þ e
nuclei, very scant
cytoplasm
6 BCR::ABL1 67 2þ 92 Small, round þ þ þ þ þ e e Partial dim CD2
nuclei, very scant (50%)
cytoplasm
7 BCR::ABL1 63 0 95 Small, round þ þ þ þ þ e Y e
nuclei, very scant
cytoplasm
8 BCR::ABL1 24 0 80 Small, round þ þ þ/ (60%) e e þ þ e
nuclei, very scant
cytoplasm
9 BCR::ABL1 53 2þ 88 Small-medium, þ þ þ þ þ e Y Partial dim CD7
irregular/clefted (7%)
nuclei, scant
cytoplasm
10 BCR::ABL1 24 0 95 Small-medium, þ þ þ bi-modal þ þ þ Y Partial dim CD4
irregular/clefted (20%)
nuclei, scant
cytoplasm
11 BCR::ABL1 59 0 73 Medium, round þ þ þ þ þ e Y e
nuclei, moderately
abundant
cytoplasm
(continued on next page)
80 C.K. Gestrich et al.
Table 1 (continued )
Case # Genetics Age MUC4 Blast Blast morphology CD19, CD22 CD34 CD13 or CD304 CD20 CD38 Aberrant T-cell
(y) (%) CD10, Tdt CD33 marker expression
12 BCR::ABL1 46 0 87 Medium-large, þ þ/ (80%) þ þ þ e þ e
irregular nuclei,
moderately
abundant
cytoplasm,
prominent nucleoli
13 BCR::ABL1 22 2þ 70 Medium-large, þ þ þ þ þ e Y Partial dim CD7
round nuclei, (22%)
abundant
cytoplasm,
azurophilic
granules
NOTE. MUC4þ cases in bold font. N/A: flow cytometry at diagnosis is not available for case #2. CD22þ/ & CD34þ/ cases indicate partial staining with % of blasts positive in parenthesis. CD13/
CD33: þ indicates at least partial or dim expression of CD13 or CD33. CD304: þ indicates at least partial or dim expression of CD304. CD20: þ indicates 20% or more of blasts expressing CD20.showed a limited response to treatment, with marrow blast
frequency only decreasing from 89% to 27% after 1e2
months of treatment and subsequently fluctuating between
17% and 92% before the patient succumbed to the disease
11 months after diagnosis. The remaining 3 patients,
MUC4 BCR::ABL1þ/like patients (patients #2, #3, and
#4), achieved remission but then subsequently relapsed
several months later (46, 43, and 27, respectively). The
adult patients (patients #2 and #4) have both died.
For BCR::ABL1þ patients (excluding patient #12 with
compromised treatment), those with MUC4þ blasts had a
shorter time to relapse than MUC4 patients. The 3
MUC4þ patients (#6, #9, and #13) relapsed at 11, 18, and
15 months after diagnosis, respectively, despite initially
achieving morphologic remission. One of these patients
(patient #9) died 19 months after diagnosis. In contrast, the
remaining 4 MUC4 patients have either not relapsed
(patients #7, #8, and #11) after more than 5 years of follow-
up or took a longer time to relapse (43 months for patient
#10), compared with the MUC4þ patients. None of the
MUC4 patients have died.
Taken together, in BCR::ABL1þ/like patients who ach-
ieved morphologic remission with optimal initial induction
therapy, MUC4 protein expression by blasts was associated
with a shorter time to relapse (mean of 15 months versus 40
months, P < .01) and a trend toward a higher rate of relapse
(100% versus 57%, not significant).
4. Discussion
MUC4 overexpression has been increasingly identified
in a variety of malignancies in the last several years. Here,
by performing IHC on 49 de novo B-ALL bone marrows of
different subtypes, we show that MUC4, which is not
expressed in normal hematopoietic cells, is aberrantly
expressed at the protein level in a small subset of B-ALL.
We demonstrate that positive is specific to BCR::ABL1þ/
like case. By comparing morphologic and flow cytometric
features of MUC4þ and MUC BCR::ABL1þ/like cases,
we found the 2 groups to be largely similar, although there
was a tendency toward some immunophenotypic differ-
ences (CD38 down-regulation and aberrant T-cell marker
expression in the MUC4þ group, CD20 expression in the
MUC4 group). Lastly, we found that MUC4þ
BCR::ABL1þ cases tended to have a shorter time to
relapse.
Our study and a recent study by Kaumeyer et al. [15]
corroborate each other by showing the remarkable speci-
ficity but limited sensitivity of MUC4 IHC for
BCR::ABL1þ/like. In that study, the authors studied 54 B-
ALL cases and found that 3/7 BCR::ABL1þ, 9 of 25
BCR::ABL1-like and 2 of 22 non-BCR::ABLþ/like cases
were MUC4 IHC positive. For most of their positive cases,
only a minority of the blasts stained positively for MUC4
and when only cases with 50% of blasts staining were
considered, the positivity rate (2/7 BCR::ABL1þ, 3/25
Mucin 4 protein is expressed in B-acute lymphoblastic leukemia 81
Table 2 Clinical characteristics of MUC4þ and MUC4- BCR::ABL1þ/BCR::ABL1-like B-ALL.
Case # Genetics Age (y) MUC4 Treatment Disease after Relapse? Time to Dead? Survival/Follow-up Additional comments
induction?a relapse time (months)
(blast %) (months)
1 CRLF2-R 49 1þ HyperCVAD Yes (12%) Refractory e Y 14 CSFþ at diagnosis
2 CRLF2-R 31 0 COG AALL1131 MRD (0.01%) Y 46 Y 76 Down syndrome
3 CRLF2-R 4 0 COG AALL1131 No Y 43 N 71
4 CRLF2-R 44 0 HyperCVAD No Y 27 Y 28
5 CRLF2-R 37 0 HyperCVAD Yes (27%) Refractory e Y 11
6 BCR::ABL1 67 2þ HyperCVAD, Dasatinib No Y 11 N 59
7 BCR::ABL1 63 0 HyperCVAD, Dasatinib No N e N 64
8 BCR::ABL1 24 0 COG AALL1131 No N e N 78
9 BCR::ABL1 53 2þ HyperCVAD, Dasatinib No Y 18 Y 19
10 BCR::ABL1 24 0 COG AALL1131 No Y 43 N 65
11 BCR::ABL1 59 0 HyperCVAD, Dasatinib No N e N 75
12 BCR::ABL1 46 0 Cyclophosphamide, No Y 4 Y 7 Anthracycline,
Vincristine, methotrexate
Dexamethasone, omitted due to
Dasatinib comorbidities
13 BCR::ABL1 22 2þ COG AALL1131 MRD (0.1%) Y 15 N 48
NOTE. MUC4þ cases in dark bold font.
a Treatment response was evaluated 1e3 months after the start of induction chemotherapy.
82 C.K. Gestrich et al.
Fig. 2 Variable blast morphology in BCR::ABL1 and BCR::ABL1-like cases. Giemsa-stained bone marrow aspirate smears. A, Case #1;
B, case #2; C, case #9; D, case #11; E, case #12; and F, case #13. See Table 1 for a description of blast morphology. All images are at
500x magnification (50x objective, oil immersion).BCR::ABL1-like, 0/22 non-BCR::ABL1þ/like) was very
similar to what we have observed in our cohort (3/8
BCR::ABL1þ, 1/5 BCR::ABL1-like, 0/36 non-
BCR::ABL1þ/like). Differences between antibody vendor
and staining protocols may in part explain the presence of
minority partial staining in a subset of B-ALL cases
(including non-BCR::ABL1þ/like cases) in the prior study
but not in ours. However, more significantly, the apparent
difference between the overall BCR::ABL1-like positivity
rate (9/25, 36%) in the prior study versus what we observed
(1/5, 25%) may be attributable to the specific subtypes of
BCR::ABL1-like used in both studies. Specifically, all cases
in our study were CRLF2 rearranged (JAK-STAT pathway
activated group of BCR::ABL-like [16]), while Kaumeyer
et al.’s study included BCR::ABL1-like cases with JAK
class fusions as well as those with ABL class fusions and
demonstrated a significant higher frequency of MUC4
expression in the latter versus the former (5/6 or 83% in the
ABL group and 4/16 or 25% in the JAK group). In our
study, the intensity of MUC4 staining was noticeably
weaker in the CRLF2-rearranged positive case compared
with the strong staining of all 3 BCR::ABL1þ cases. This
raises the possibility of intensity differences in MUC4
staining between ABL class and JAK class BCR::ABL1-like
B-ALL, which could be explored in future studies.Nevertheless, results of our current study and Kaumeyer
et al.’s study indicate that: (1) MUC4 protein expression,
especially when a majority of blasts are IHCþ, occurs in
less than 10% of B-ALL in about a third of BCR::ABL1 B-
ALL and 10e20% of BCR::ABL1-like cases, (2) MUC4
protein expression may be at least 3-fold more prevalent in
ABL class versus JAK2 class BCR::ABL1-like B-ALL, and
(3) optimized staining protocols may be important in
realizing the full potential of the specificity of this stain in
clinical practice.
Our observations suggest that MUC4 expression may
not simply be an epiphenomenon since MUC4þ
BCR::ABL1þ patients appeared to have a worse outcome
(shorter time to relapse) than MUC4 BCR::ABL1þ pa-
tients, although this conclusion is limited by the relatively
small sample size. Nevertheless, this raises the question of
what potential mechanistic role MUC4 may play in the
pathogenesis of B-ALL. In solid tumors such as PDAC and
cholangiocarcinoma [17], where MUC4 overexpression has
been associated with a worse outcome, MUC4 is thought to
promote tumorigenesis and cancer metastasis by modu-
lating the interaction of tumor cells with their microenvi-
ronment, leading to downstream effects such as inhibition
of tumor cell apoptosis [18]. Its expression can also
contribute to the resistance of tumor cells to chemotherapy,
Mucin 4 protein is expressed in B-acute lymphoblastic leukemia 83such as gemcitabine resistance in PDAC [19]. In some
myeloid leukemias, myelomas, and cutaneous T cell lym-
phomas, another membrane-bound mucin, MUC1, has been
shown to be expressed and promote malignancy by also
inhibiting tumor cell death and decreasing sensitivity of
tumor cells to chemotherapy [20e23]. In fact, MUC1 is
capable of binding to and promoting the stability of
BCR::ABL1 protein and promoting resistance of chronic
myeloid leukemia cells to tyrosine kinase inhibition treat-
ment [23]. Additional mechanistic studies are warranted to
determine whether MUC4 has similar biochemical and cell
biological effects in BCR::ABL1þ B-ALL and whether
these effects are translatable to BCR::ABL1-like B-ALLs,
especially those with ABL rearrangements without a BCR
partner. Future studies using a larger sample, a prospective
design and/or involve uniformly treated cohorts of patients
would be important to confirm the prognostic significance
of MUC4 expression in BCR::ABL1þ/like B-ALL and
whether MUC4 expression may be used to select patients
who might benefit from MUC4-targeted therapy [24] or
more aggressive treatment or monitoring protocols.
Acknowledgments
The authors thank Gregory Maclennan, Ebenezer Osei,
Athena Sowell, and James Metcalf. The authors also
acknowledge the UHCMC Pathology Department Research
Funding and UHCMC Minority Faculty Development
Award given to K.A.O.
The results of this study were presented at the 2020
USCAP conference.
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