Alessandro Laganà# 1,
Matteo Totaro# 1, Maria Laura Bisegna1, Loredana
Elia1, Stefania Intoppa1, Marco Beldinanzi1,
Mabel Matarazzo1, Mariangela di Trani1,
Alessandro Costa2, Raffaele Maglione1,
Biancamaria Mandelli1, Sabina Chiaretti1,
Maurizio Martelli1
and Maria Stefania De Propris1.
1 Hematology,
Department of Translational and Precision Medicine, Sapienza
University, 00161 Rome, Italy.
2 Hematology Unit, Businco Hospital, Department of Medical
Sciences and Public Health, University of Cagliari, Cagliari, Italy.
# Both authors contributed equally to this work.
Correspondence to:
Maria Stefania De Propris. Hematology Department of Translational and
Precision Medicine Sapienza University of Rome, Italy. Via Benevento 6,
00161 Rome, Italy. Tel. +39 0685795313, Fax +39 0644241984. E-mail:
depropris@bce.uniroma1.it
Published: September 01, 2024
Received: May 02, 2024
Accepted: August 02, 2024
Mediterr J Hematol Infect Dis 2024, 16(1): e2024064 DOI
10.4084/MJHID.2024.064
This is an Open Access article distributed
under the terms of the Creative Commons Attribution License
(https://creativecommons.org/licenses/by-nc/4.0),
which permits unrestricted use, distribution, and reproduction in any
medium, provided the original work is properly cited.
|
Abstract
Background: B-lineage
acute lymphoblastic leukemias (B-ALL) harboring the
t(9;22)(q34;q11)/BCR::ABL1 rearrangement represent a category with
previously dismal prognosis whose management and outcome dramatically
changed thanks to the use of tyrosine kinase inhibitors (TKIs) usage
and more recently full chemo-free approaches. The prompt identification
of these cases represents an important clinical need.
Objectives: We sought to
identify an optimized cytofluorimetric diagnostic panel to predict the
presence of Philadelphia chromosome (Ph) in B-ALL cases by the
introduction of CD146 in our multiparametric flow cytometry (MFC)
panels.
Methods: We prospectively
evaluated a total of 245 cases of newly diagnosed B-ALLs with a CD146
positivity threshold >10% referred to the Division of Hematology of
'Sapienza' University of Rome. We compared the results of CD146
expression percentage and its mean fluorescence intensity (MFI) between
Ph+ ALLs, Ph-like ALLs, and molecularly negative ALLs.
Results: Seventy-nine of
the 245 B-ALL cases (32%) did not present mutations at molecular
testing, with 144/245 (59%) resulting in Ph+ ALL and 19/245 (8%)
Ph-like ALLs. Comparing the 3 groups, we found that Ph+ B-ALLs were
characterized by higher expression percentage of myeloid markers such
as CD13, CD33, and CD66c and low expression of CD38; Ph+ B-ALL showed a
higher CD146 expression percentage and MFI when compared with both
molecular negative B-ALL and Ph-like ALLs; neither the mean percentage
of CD146 expression neither CD146 MFI were statically different between
molecular negative B-ALL and Ph-like ALLs.
Conclusions: Our data
demonstrate the association between CD146 expression and Ph+ ALLs.
CD146, along with myeloid markers, may help to identify a distinctive
immunophenotypic pattern, useful for rapid identification in the
diagnostic routine of this subtype of B-ALLs that benefits from a
specific therapeutic approach.
|
Introduction
Acute
lymphoblastic leukemia (ALL) is a malignancy characterized by the
uncontrolled proliferation of lymphoid B or T progenitor cells. A
prompt and accurate diagnostic process is of the utmost importance to
allow optimal risk-oriented therapy and maximize the chances of cure.[1]
Multiparametric flow cytometry (MCF) is a well-established and
user-friendly single-cell technology that simultaneously measures
multiple analyte expression patterns in individual cells.
Immunophenotype characterization performed by MFC is an essential step
for ALL diagnosis and has significant relevance in the evaluation of
minimal residual disease (MRD).[2] Indeed, leukemic
cells express surface and intracytoplasmic antigens whose
identification allows us to determine the line of belonging, the level
of differentiation and maturation, the lineage infidelity, and peculiar
aberrations.[3,4] In particular, B-ALLs EGIL
classification (European Group for the immunological classification of
leukemias),[5-6]
in addition to the expression of B lineage antigens, is based on the
detection of cytoplasmic IgM (cIgM) and CD10 on leukemic B cells. The
expression of TdT/CD19/CD22/cCD79a with or without CD20/CD34, in the
absence of cIgM and CD10, identifies the pro-B ALL subtype. The
presence of CD10 antigen (CALLA) without cIgM defines the B-common ALL,
while CD10+/– expression associated with cIgM identifies pre-B ALL.
Finally, the presence of surface Ig light chains defines mature B-ALL.[7]
The most important and prognostically and therapeutically relevant
distinction for B-ALL is between Ph-positive (Ph+) and Ph-negative
(Ph-) B-ALL. Ph+ ALLs are defined by the presence of the Philadelphia
chromosome (Ph) generated by the translocation between chromosomes 9
and 22, with t(9;22)(q34;q11) being the most common chromosomal
abnormality in adults.[8-10]
This aberration produces the BCR::ABL1 fusion gene, which encodes for a
constitutively activated tyrosine kinase signaling protein that
sustains leukemic cell genesis and proliferation.[8]
Ph chromosome has an overall incidence of roughly 20% to 25% in B-ALLs.[11-12]
This translocation was considered one of the worst prognostic factors
before the introduction of tyrosine kinase inhibitors (TKIs) in
clinical practice.[13]
TKIs revolutionized the outcome of ALL-Ph+ patients, with chemotherapy
and allogeneic stem cell transplant (allo-HSCT) questioned by the usage
of chemo-free approaches.[14]
In 2009, the term "Philadelphia–like" or "BCR::ABL1–like" ALL was first
used.14 Ph-like ALLs are characterized by a gene expression profile
(GEP) highly similar to that of BCR::ABL1-positive ALL but lacking the
BCR::ABL1 fusion protein derived from the t(9;22)(q34;q11)
translocation, with high frequency of deletions of IKZF1 gene, encoding
the lymphoid transcription factor IKAROS, and other lymphoid
transcription factor genes, such as PAX5 and EBF1.[15-19]
Ph-like ALLs also have a phenotypic expression profile similar to Ph+
ALL, presenting myeloid antigens such as CD13/CD33 and low expression
of CD38. Importantly, Ph-like ALLs have a poor outcome, and therefore,
their distinction from the other subgroups of ALL is fundamental.
Cluster of differentiation 146 (CD146, also known as Mel-CAM or MUC18)
is a cell transmembrane glycoprotein belonging to the immunoglobulin
family and represents an adhesion molecule first discovered on the
plasma membrane of human melanoma cells, and it was initially named
MCAM (melanoma cell adhesion molecule).[20] This
113-kDa glycoprotein is expressed in normal tissues, including smooth
muscles, mesenchymal cells, and vascular endothelium on the entire
vascular tree that exerts cation-independent adhesion through
interactions with an unidentified ligand.[21]
CD146 presents multifunctional activities in both physiological and
pathological conditions, including immunity, angiogenesis, and
development. CD146 is also expressed in several cancers. A growing
number of studies suggest that CD146 overexpression was significantly
correlated with the progression, angiogenesis, and metastasis of
different malignant tumors and, especially for solid neoplasms, was
associated with poor survival and might be considered as a useful
prognostic biomarker and promising therapeutic target.[22]
The role of CD146 in hematopoietic cells has yet to be thoroughly
understood, and few data are available: CD146 is rarely expressed in
acute myeloid leukemia (AML) cells, while it can be found frequently in
ALLs. In particular, an Italian study showed that only 3.3% of AML were
CD146 positive, and these cases were indeed classified as AML, which
was not otherwise specified. Conversely, 66% of T-ALLs and 36.8% of
B-ALLs, comprising Ph+ ALL cases, expressed CD146 on the blast cells.[23] Hence, CD146 antibody inclusion in MFC panels for
suspected acute leukemia (AL) may improve accurate diagnostic workup.
Therefore, the purpose of our study is to propose an optimized MFC
diagnostic panel of routine antigens to predict the presence of BCR
quickly: ABL1 rearrangement or Ph-like ALL, by evaluating CD146
expression as a possible marker that may aid in the prompt
identification of these specific subgroups of B-ALLs.
Materials and Methods
Patients. This
prospective single-institution study included a total of 245 cases of
newly diagnosed B-ALLs referred for a diagnostic purpose to our center
at the Division of Hematology of 'Sapienza' University of Rome since
the introduction of CD146 to our MFC panels for AL in 2022. Among the
245 B-ALL cases evaluated, 79 (32%) did not carry molecular aberrations
(i.e. TCF3/PBX1, KMT2A and BCR::ABL1 rearrangements), 144 (59%) were
Ph+ ALL, 19 (8%) were Ph-like ALL, defined according to the
BCR::ABL1-like predictor[24] and 3 (1%) had other
molecular abnormalities. Cytological diagnosis of B-ALL was made
according to the 2022 World Health Organization (WHO) classification[25] and EGIL
criteria.5-6 Median age was 56 years (range 17-90) years. Patients’
bone marrow (BM) samples were obtained with informed consent in
accordance with the Declaration of Helsinki.
B-ALL
diagnosis by MCF analysis.
B-ALL diagnosis was assessed by MFC using a combination of monoclonal
antibodies (mAbs) recommended by the EuroFlow Consortium.[26]
BM cells (0.5 × 106) were first stained using a combination of mAbs
directed against myeloid (MPO), B and T lymphoid (cCD79a and cCD3)
lineage antigens; after that, with a combination of mAbs against:
CD45/CD10/CD34/TdT/HLADR/CD19/CD22/CD20/CD38/CD58/CD123/Igκ/Igλ/cIgM/CD3/CD13/CD33/CD66c/NG2
(Becton Dickinson, San Jose, CA; Società Italiana Chimici, SIC, Life
Sciences, Rome, Italy, Beckman Coulter, Brea, CA).
In all B-ALL samples, CD146 (Società Italiana Chimici, SIC, Life
Sciences, Rome, Italy) analysis was optimized using additional
combinations of monoclonal antibodies as follows:
CD58FITC/CD146PE/CD19PECy7/CD34APC/CD20APC-Cy7/CD13R718/CD38V450/CD45V500/CD10
BV605/CD33 BV711/CD22 BV786).
Data on standardized 12 color staining combinations were acquired on
FACSLyric flow cytometers (Becton Dickinson) by collecting at least
50,000 ungated events and analyzed using the PAINT-A-GATE and FACSDIVA
softwares (Becton Dickinson). Cytometer setup and tracking beads (BD)
were used for daily cytometer optimization. Leukemic cells were gated
within the total CD45+ leukocyte population, considering that all cases
of B-lineage ALLs were positive for the pan-leukocyte antigen.
Representative plots of the flow gating strategy are reported in Figure 1.
![Figure 1](https://www.mjhid.org/mjhid/article/download/5714/version/2817/4580/46131/Mini%20figure%201.jpg) |
- Figure 1.
Representative plots of the flow gating strategies to detect CD146+in
Ph+ B common patient. Leukemic B cells were gated within the total
CD45+ leukocyte population, then CD146+/CD19+ leukemic cells were
identified in CD34+ population.
CD34+/CD146+/CD19+/CD10+/CD66c+/CD13+/CD33+/CD38- cells are depicted in
red, residual B lymphocytes are depicted in blue while residual
granuloblast in green.
|
The
presence of pathological cells was identified in comparison with the
known patterns of antigen expression by normal maturing lymphoid
precursors and was quantified as a percentage of total leukocytes. In
all cases, antigen expression was defined by the percentage of blast
cells that resulted in positive for the different markers in the
immunological gate.[5,27] CD146
expression was assessed on the blast immunological gate. Cell surface
antigen expression was quantified on the same flow cytometer and with
the same mAbs combination as the mean fluorescent intensity (MFI) of
values obtained with specific mAbs compared with values given by the
isotype controls. A sample was considered positive for surface antigens
if ≥20% of leukemic cells exhibited fluorescence compared with negative
control. A threshold ≥10% of gated blasts was used to define the
positivity of CD146 expression based on the results
of the Receiver Operating Characteristic (ROC) curve
for discrimination between ALL-Ph+ and ALL-Ph negative.
Molecular
testing.
Molecular analysis of BM samples was carried out using a nested
approach using a Multiplex RT-PCR system. As previously described,[28]
the screening with Multiplex-RT-PCR was designed to detect
simultaneously and in a quick time the most common fusion genes in
T-ALL rather than in B-ALL: TCF3::PBX1, ETV6::RUNX1, SIL::TAL1,
NUP98::RAP1GDS1, SET::NUP214, BCR::ABL1 p190 (e1a2) and p210 (e13a2,
e14a2), KMT2A::AFF1 and KMT2A::MLLT1, with two genes screened,
KMT2A::AFF1 and KMT2A::MLLT1.
Statistical
methods. Summary
statistics (mean and standard deviation, median, and range) were
reported by category groups. Mann-Whitney or Kruskal-Wallis test for
independent groups was used to compare categories. Differences in the
study groups were estimated using the chi-square test or the Fisher
exact test for categorical covariate. The optimal cut-off for CD146
expression was identified as the optimal threshold through ROC curve
analysis. Sensitivity, specificity, positive predictive value (PPV),
and negative predictive value (NPV), associated with the 10% cut-off of
CD146, were reported in supplementary
figure 1.
A multivariate logistic regression model was used to evaluate the
independent role of biomarkers. All tests were two-sided, accepting
p<0.05 as indicating a statistically significant difference. All
analyses were performed using R software.
Results
B-ALL
characterization.
Overall, within the 245 cases of B-ALLs, the mean blast percentage was
67%±22% (range 20-97). The mean percentage of CD146 expression is
23%±32% (range 0-97) with a mean MFI of 66 ± 123 (range 0-620). The
analysis was based on the stratification in 3 groups: Group A,
including molecularly negative B-ALL (B-neg ALL, n=79, 32%); Group B,
including Ph+ ALLs (n=144, 59%); Group C, including Ph-like ALLs (n=19,
8%). The remaining 3 ALLs cases that harbored other molecular
abnormalities were excluded from the analysis. Moreover, in order to
define the predictivity of CD146 and of the other antigens for the
BCR::ABL1 rearrangement, we considered in the analysis another
stratification of these 245 B-ALL cases: BCR::ABL1-positive (Ph+) ALL
(n=144, 59%), and BCR::ABL1-negative (Ph-neg) ALL (which included
molecularly negative B-ALLs and Ph-like ALLs) (n=98, 40%).
Within the 79 B-neg-ALLs, 62 (79%) were phenotypically classified as
B-common ALL, 12 (15%) as pro-B, 3 (4%) as pre-B, and 2 (2%) as
B-mature. The mean blast percentage was 66%±23% (range 20-96),
characterized by a mean percentage of CD146 expression of 15%±29%
(range 0-90), with a mean MFI of 32 ± 80 (range 0-550) on leukemic
cells.
Out of 144 Ph+ ALL cases, 135 were phenotypically subclassified cases
(94%) as B-common ALL, 7 as pre-B (5%), and 2 as pro-B (1%). The mean
blast percentage was found to be 67%±22% (range 23-97). In this
subcategory, the mean percentage of CD146 expression on blast cells was
29%±34% (range 0-97) with a mean MFI of 89±136 (range 1-548).
Within the 19 Ph-like ALL cases, all B-common ALL (100%) were
identified.23 The mean blast percentage was 75%±20% (range 26-90).
Ph-like leukemic B cells were characterized by a mean percentage of
CD146 expression of 11%±28% (range 0-88) with a mean MFI of 48±144
(range 2-620).
Considering the total of 98 Ph-neg ALL cases, the mean blast percentage
was 68%±23% (range 20-96), characterized by a mean percentage of CD146
expression of 14%±28% (range 0-90), with a mean MFI of 36 ± 95 (range
0-620) on leukemic cells.
CD146
differential antigen detection and surface expression intensity.
We evaluated the differential expression of various CDs in the 3 groups
to identify the potential association between CD146 expression and its
MFI with one or more B-ALL subtypes. By ROC analysis, a value of 10%
was pinpointed as the optimal cut-off in CD146 expression to maximize
the separation between the BCR::ABL1-positive and BCR::ABL1-negative
groups (Supplementary Figure 1).
Therefore, CD146 was considered positive for surface expression if ≥10%
of leukemic cells exhibited fluorescence compared with negative
control, while a classical threshold of ≥20% was considered for the
other surface antigens.
Firstly, we compared Ph+ ALL with B-neg ALL. As expected and previously
described in many studies,[29-30]
Ph+ B-ALLs were characterized by higher mean expression of myeloid
markers such as CD13 (32%±33% vs. 16%±29%, p<0.001), CD33 (30%±32%
vs 18%±31%, p<0.001), CD66c (29%±29% vs 21%±29%, p=0.004) and by
lower mean expression of CD38 (46%±36% vs 56±32%, p=0.043) (Table 1).
Such data was confirmed by higher positivity detection rate for CD13
[74/144 (51%) vs 16/79 (20%), p<0.001], CD33 [72/144 (50%) vs. 22/79
(28%), p=0.002], CD66c [74/144 (51%) vs. 28/79 (35%), p=0.025] and
lower number of cases positive for CD38 [94/144 (65%) vs 63/79 (80%),
p=0.024] (Supplementary Table 1).
Moreover, due to the higher percentage of B-common ALL in the Ph+
group, CD10 presented a higher mean expression (62%±25% vs. 48%±33%,
p=0.004) and was more expressed in such group [133/144 (92%) vs. 57/79
(72%), p<0.001]. As for CD146, Ph+ ALLs showed a higher mean
expression (29%±34% vs 15%±29%, p<0.001) as well as MIF (89±136 vs
32±80, p<0.001), and greater positivity detection rate [74/144 (51%)
vs. 22/79 (28%), p<0.001] of this antigen when compared with B-neg
ALLs (Table 1 and Supplementary Table 1).
![Table 1](https://www.mjhid.org/mjhid/article/download/5714/version/2817/4580/46146/Table%201.jpg) |
- Table 1. Comparison of expression of
the markers analyzed between Ph+ B-ALL (n=144) and B-ALLs negatives (n=
79).
|
Between
Ph-like ALL and B-neg ALL, statistically significant differences in the
mean expression of CD33 myeloid marker (38%±36% vs 18%±31%, p=0.014)
and in its positivity rate [12/19 (63%) vs 22/79 (28%), p=0.004] were
found. CD38 had a lower expression rate in Ph-like ALL cases compared
to B-neg ALL cases [10/19 (53%) vs 63/79 (80%), p=0.021]. Due to the
presence of only B-common ALL cases in the Ph-like group CD10 resulted
in a higher mean expression (75%±20% vs. 48%±33%, p=0.001) and more
detected [19/19 (100%) vs. 57/79 (72%), p=0.006] in this group. At
variance, no difference was found in CD13 and CD66c expression (p=0.7
and p=0.6, respectively); similarly, neither the mean percentage of
CD146 expression (11%±28% vs 15%±29%, p=0.3), neither CD146 MFI (48±144
vs 32±80, p=0.084) or CD146 positivity rate [3/19 (16%) vs 22/79 (28%),
p=0.39] were statically different between these two groups (Table 2 and Supplementary Table 2).
![Table 2](https://www.mjhid.org/mjhid/article/download/5714/version/2817/4580/46147/Table%202.jpg) |
- Table 2. Comparison of expression of
the markers analyzed between B-ALLs negatives (n=79) and Ph-like B-ALLs
(n= 19).
|
Comparing
Ph+ ALLs and Ph-like ALLs, CD33, and CD38 expressions were not found to
be statistically significant (p=0.4 and p=0.7, respectively). Indeed,
Ph+ blasts cells were characterized by a higher CD13 mean expression
(32%±33% vs 8%±14%, p=0.001) and positivity rate [74/144 (51%) vs. 3/19
(16%), p=0.004] as well as by higher CD66c positivity rate [74/144
(51%) vs 5/19 (26%), p=0.043]. Conversely, due to a higher percentage
of B-common ALL present in the Ph-like group, CD10 resulted in meanly
more expressed in such group (62%±25% vs. 75%±20%, p=0.012).
Statistically significant differences emerged in CD146 mean expression
between Ph+ and Ph-like B-ALLs (29%±34% vs 11%±28%, p=0.004) as well as
in the CD146 MFI (89±136 vs 48±144, p<0.001) and in CD146 positivity
rate [74/144 (51%) vs 3/19 (16%), p=0.003] (Table 3 and Supplementary
Table 3).
An overall comparison between antigens mean expressions in the
three groups is shown in supplementary Table 4, with Ph+ ALLs
characterized by a superior CD13, CD33, and CD66c expression, by lower
CD38 expression along with the higher mean percentage of CD146
expression and higher CD146 MFI.
Confronting Ph+ ALLs with all cases of Ph-neg ALLs (B-neg ALL + Ph-like
ALLs) a superior mean expression of myeloid markers such as CD13
(32%±33% vs. 14%±27%, p<0.001), CD33 (30%±32% vs 22%±32%, p=0.007)
and CD66c (29%±29% vs 22%±29%, p=0.003) was found (Table 4).
Such data was confirmed by a higher positivity detection rate for CD13
[74/144 (51%) vs 19/98 (19%), p<0.001], CD33 [72/144 (50%) vs. 34/98
(35%), p=0.025], CD66c [74/144 (51%) vs 33/98 (34%), p=0.008] (Table
5).
As for CD146, Ph+ An overall comparison between antigens mean
expressions in the three groups is shown in supplementary Table 4, with
Ph+ ALLs characterized by a superior CD13, CD33, and CD66c expression,
by lower CD38 expression along with the higher mean percentage of CD146
expression and higher CD146 MFI.
Confronting
Ph+ ALLs with all cases of Ph-neg ALLs (B-neg ALL + Ph-like
ALLs) a superior mean expression of myeloid markers such as CD13
(32%±33% vs. 14%±27%, p<0.001), CD33 (30%±32% vs 22%±32%, p=0.007)
and CD66c (29%±29% vs 22%±29%, p=0.003) was found (Table 4). Such data
was confirmed by a higher positivity detection rate for CD13 [74/144
(51%) vs 19/98 (19%), p<0.001], CD33 [72/144 (50%) vs. 34/98 (35%),
p=0.025], CD66c [74/144 (51%) vs 33/98 (34%), p=0.008] (Table 5). As
for CD146, Ph+ ALLs showed a higher CD146 mean expression (29%±34% vs
14%±28%, p<0.001) as well as CD146 MIF (89±136 vs. 36±95,
p<0.001) and CD146 positivity rate [74/144 (51%) vs 25/98 (26%),
p<0.001] when compared with Ph-neg ALLs (Table 4 and Table 5),
confirming the key role played by the presence of CD146 in the peculiar
phenotype of Ph+ B-ALL leukemic cells.
![Table 3](https://www.mjhid.org/mjhid/article/download/5714/version/2817/4580/46148/Table%203.jpg) |
- Table 3. Comparison of expression of
the markers analyzed between Ph+ B-ALL (n=144) and B- Ph-like ALLs (n=
19).
|
![Table 4](https://www.mjhid.org/mjhid/article/download/5714/version/2817/4580/46149/Table%204.jpg) |
- Table 4. Comparison of expression of
the markers analyzed between Ph+ B-ALL (n=144) and Ph-Neg ALLs (n= 98).
|
![Table 5](https://www.mjhid.org/mjhid/article/download/5714/version/2817/4580/46150/Table%205.jpg) |
- Table 5. Comparison of positivity
rate of the markers analyzed between Ph+ B-ALL (n=144) and Ph-Neg ALLs
(n= 98).
|
No
difference was evidenced in any of the groups in the expression of
B-cell precursors classical marker such as CD19, CD22, CD20 and TdT.
To confirm the relevance of CD146 expression as an independent
predictive factor for predicting the BCR::ABL1 rearrangement in B-ALLs.
We carried out a multivariate study that included all the
immunophenotypic variables considered important in Ph+ ALLs diagnosis.
In such multivariate analysis (including CD146, CD10, CD13, CD20, CD33,
CD66c, CD38) positivity of CD146 showed to be statistically associated
with BCR::ABL1 rearrangement detection in B-ALLs [OR 1.01 (95% Cl:
1.01–1.02) (p=0.021)], along with CD13 [OR 1.02 (95% Cl: 1.01–1.04)
(p<0.001)] and negative CD38 [OR 0.99 (95% Cl: 0.98–0.99) (p=0.005)].span
style="font-family: 'Open Sans',sans-serif;">
Discussion
Immunophenotypic
characterization of ALL is essential for diagnosis and
subclassification and also provides important prognostic information.
This study identifies a peculiar immunophenotypic marker profile that
can be useful for the rapid identification of Ph+ ALLs. Indeed, in our
series, the immunophenotypic features of leukemic blasts can help in
predicting the diagnosis of BCR::ABL1+ ALL, which were characterized by
the co-expression of myeloid markers CD13/CD33/CD66c, a low expression
of CD38 but above all by the expression of CD146 and by a higher CD146
MFI, suggesting a strong association of CD146 with the presence of the
BCR::ABL1 fusion protein. By contrast, this strong association did not
emerge in the Ph-like ALLs. It is of utmost importance to highlight
that certain diagnoses of BCR::ABL1+ ALL are possible just by
exploiting tests to pinpoint pathognomonic translocation, such as
multiplex RT-PCR.
Nevertheless, a peculiar immunophenotypic pattern might be useful in
case of limited access to molecular tests. Other studies have shown
CD146 expression in B-ALLs and, in particular, in Ph+ ALLs.
Nevertheless, in most of them, the sample size was small, or very
heterogeneous groups were considered. The possible strength of our
study relies on a homogeneous and relatively large cohort of newly
diagnosed B-ALL cases referred to a single institution. CD146
expression as a useful prognostic biomarker has also been investigated
by various reports and studies for different solid tumors.[21]
In literature, reports about CD146 expression on progenitor cells in
normal bone marrow or on hematological disease cells are limited.
Cavazzini et al. found that the expression of CD146+cells was detected
in 38.8% of B-cell ALL (14/38).[23] Interestingly,
all the seven cases of Ph+ ALL were CD146-positive. They also found
that the expression of CD13 and CD33 on adult B-ALL blasts was higher
in the CD146-positive group, and CD146 expression was strongly
associated with the presence of the Ph chromosome (p=0.001).[23]
In a Chinese study, Xie et al. compared CD146 expression rates in adult
and childhood B-ALL patients, which were 29.17% and 9.09%,
respectively, showing a statistically significant difference (p<
0.05), probably due to higher incidence of Ph+ ALL in adult patients.[31]
Another study involving 31 pediatric patients showed that for B-ALLs,
the mean expression for CD146+ blasts was 51.347 ± 24.133, with the
mean expression for CD146 on the blast cells was 51% ± 24%.
Nevertheless, in this study, Zahran AM et al. could not correlate the
percentage of CD146 expression with that of the Ph chromosome, possibly
because of a shortage of data collection. Even so, CD146 was
significantly associated with a lower response to induction therapy,
suggesting a possible correlation with a subgroup of B-ALLs with an
unfavorable prognosis.[32]
In summary, we can state that the evaluation in MFC panels of antigens
like CD146, CD13, CD33, CD38, and CD66c, along with classical B-ALLs
MFC markers (such as CD10, CD19, CD22, CD34, TdT), can aid the
diagnosis of ALL and can partially rapidly suggest a distinction
between molecularly negative ALL cases and Ph+ ALL cases.
In our cohort, Ph+ ALLs showed higher expression of CD146 in terms of
mean percentage, positivity rate, and in term of MFI. In addition,
myeloid markers such as CD33 and CD13, as already known,[26-27] are
highly expressed in Ph+ ALLs and Ph-like ALLs, helping in the
distinction from molecularly negative cases. Indeed, our data shows
that CD13 and CD66c have higher expressions in Ph+ ALLs compared both
with Ph-like ALLs and with molecularly negative ALLs. Hence, along with
the expression of myeloid markers, CD146 expression is likely to
represent an aberrant marker frequently associated with the
t(9;22)(q34;q11)/BCR::ABL1, making possible to suspect a Ph+ ALL and
prompting a “fast-track” for the detection of Ph chromosome. Indeed,
Ph+ ALL blasts are characterized by the co-expression of myeloid
markers CD13/CD33/CD66c with a low expression of CD38 and by the
expression of CD146, as shown in the literature and in our data. On the
other hand, this peculiar CD146 expression and MFI did not emerge in
Ph-like ALLs. In addition, in our study, CD146 was never expressed in
the entire blast population, so its role in minimal residual disease
(MRD) remains limited. Further studies are necessary to establish
CD146's role in the diagnosis, monitoring, and eventual relapse of
ALLs.
Conclusions
Our
data show that the CD146 antigen associated with the peculiar
immunophenotypic pattern observed in our study leads to a reliable
prediction of BCR::ABL1 fusion protein detection in adult B-ALL cases
and should always be included in the diagnostic MFC panel for the rapid
detection of this peculiar B-ALL subgroup that nowadays may benefit
from specific therapeutic approaches, even chemo-free treatment.
Acknowledgments
Stefania
Intoppa were supported by ROMAIL ONLUS; we would like to thank Matteo
Leoncin, Marco Cerrano, Bianca Serio, Caterina Alati, Daniele Mattei,
Elisa Mauro, Erika Borlenghi, Catello Califano, Patrizia Chiusolo, Lara
Pochintesta, Valeria Cardinali, Monia Lunghi, Matteo Piccin, Carmela
Gurrieri, Valentina Mancini, Federico Lussana, Antonella Cucca,
Massimiliano Bonifacio for providing samples.
Author Contributions
Alessandro
Laganà and Matteo Totaro: Writing – original draft in equal
contribution. Alessandro Laganà – corrected the paper and integrated
the required reviews. Maria Laura Bisegna: Data collection,
acquisition, analysis and interpretation, Loredana Elia: Molecular data
collection acquisition and interpretation, Stefania Intoppa: Flow
cytometry data collection, acquisition and analysis, Marco Beldinanzi:
Molecular data analysis, Mabel Marrazzo: Molecular data analysis,
Mariangela di Trani: Samples collection, Alessandro Costa, Raffaele
Maglione, Biancamaria Mandelli: Contribution to the lab work, Sabina
Chiaretti: supervision and manuscript editing, Maurizio Martelli:
Manuscript editing, Maria Stefania De Propris: Conceptualization,
Investigation, Funding acquisition, Formal analysis, Writing – original
draft.
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Supplementary Files
![Supp Figure 1](https://www.mjhid.org/mjhid/article/download/5714/version/2817/4580/46130/Figure%201.jpg)
|
- Supplementary Figure 1.
Receiver Operating Characteristic (ROC) curve for CD146 expression
aimed at discriminating between ALL-Ph+ and ALL-Ph-Neg. ROC curve
identified 10% as the optimal cut-off to considered CD146 expression
“positive”, which maximizes the separation between the
BCR::ABL1-positive ALLs and BCR::ABL1-negative ALLs. it would be
necessary to recalculate the sensitivity, specificity, and predictive
values. The 10% cut-off point on the ROC curve has a Sensitivity of
59%, Specificity of 72%, with Positive Predictive Value of 76% and
Negative Predictive Value of 55%.
|
![Supp Table 1](https://www.mjhid.org/mjhid/article/download/5714/version/2817/4580/46146/Table%201.jpg) |
- Supplementary Table 1. Comparison of
positivity rate of the markers analyzed between Ph+ B-ALL (n=144) and
B-ALLs negatives (n= 79).
|
![Supp Table 2](https://www.mjhid.org/mjhid/article/download/5714/version/2817/4580/46147/Table%202.jpg) |
- Supplementary
Table 2. Comparison of positivity rate of the markers analyzed
between
B-ALLs negatives (n= 79) and Ph-like B-ALLs (n=19).
|
![Supp Table 3](https://www.mjhid.org/mjhid/article/download/5714/version/2817/4580/46148/Table%203.jpg) |
- Supplementary Table 3. Comparison of
positivity rate of the markers
analyzed between Ph+ B-ALL (n=144) and Ph-like B-ALLs (n=19).
|
<
![supp Table 4](https://www.mjhid.org/mjhid/article/download/5714/version/2817/4580/46149/Table%204.jpg) |
- Supplementary Table 4. Comparison
of expression of the markers analyzed
between all the 3 groups: Ph+ B-ALL (n=144), Ph-like ALLs (n= 19) and
B-ALLs negatives (n= 79). For this statistical analysis based on 3
groups the Kruskal-Wallis test was used.
|