Calreticulin Immunohistochemistry in Myeloproliferative Neoplasms - Evolution of a New Cost-Effective Diagnostic Tool: A Retrospective Study with Histological and Molecular Correlation
Sanjeet ROY1, Marie Therese MANIPADAM1, Poonkuzhali BALASUBRAMANIAN2
1Department of Pathology, Christian Medical College, Tamil Nadu, INDIA
2Department of Haematology, Christian Medical College, Tamil Nadu, INDIA
Keywords: Immunohistochemistry, CAL2, Calreticulin, Myeloproliferative neoplasm, CALR
Recent WHO 2017 guidelines mandates mutational analysis for the diagnosis of myeloproliferative disorders (MPN). JAK2V617F has
been found in only 50-60% of Primary myelofibrosis (PMF) and Essential thrombocythaemia (ET). A recently discovered somatic Calreticulin
(CALR) mutation has been linked to MPN. This mutation leads to a common 36 amino acid C-terminus that can be detected accurately by
immunohistochemistry (IHC). Limited published literature exists on the utility of CAL2IHC as a diagnostic tool. The study aimed to validate
the sensitivity and specificity of CAL2IHC for its use as a cost effective and rapid diagnostic tool.
Material and Method: Subjects included 23 patients of MPN (15 PMF, 6 ET, 2 PV (Polycythaemia Vera)), diagnosed between January 2014
to November 2016 with adequate available tissue for histopathological and mutational analysis. Mutational analysis had been performed with
Bidirectional Sanger sequencing. CAL2IHC was performed in all cases and the sensitivity and specificity of CAL2 IHC to identify the Calreticulin
mutation was evaluated with respect to comparison with the gold standard mutation analysis.
Results: In the 23 MPN patients, CAL2 IHC detected CALR mutation with a sensitivity of 95% and a specificity of 100%. Both cases of PV were
negative for CAL2IHC. CAL2IHC showed cytoplasmic positivity in ET (2-3+) and PMF (1-3+) with (62-69%) positive megakaryocyte staining.
All 6 ET cases and all 14/15 PMF cases were CAL2IHC positive, and these results were concordant with CALR mutational analysis.
Conclusion: Anti-CAL2 immunohistochemistry is a specific and a sensitive marker to detect CALR mutation. Its’ cost effectiveness and fast
results are quite advantageous as compared to molecular analysis.
Myeloproliferative neoplasms (MPNs) are a heterogeneous
group of diseases that have a diverse clinical presentation
and a myriad of morphologies. These intriguing disorders
have always proven to be a challenge for haematologists and
hematopathologists. Recent studies have necessarily aimed
at classifying MPNs based on molecular alterations as there
is increasing evidence that molecular or chromosomal
alterations have a better correlation with clinical
presentation, response to therapies, and prognosis rather
than conventional morphological classification 1,2
significant number of gene mutations have been identified
in MPNs with JAK2 and MPL being the major ones by
the World Health Organization (WHO) 2007 3
JAK2V617F mutation has been found to occur in almost
95% of Polycythemia Vera (PV) 4
. However, JAK2V617F
has been found in only 50-60% of Primary myelofibrosis
(PMF) and Essential thrombocythaemia (ET) 5
A significant gap was present that comprised many
cases of MPN that do not harbor any of these mutations,
but was recently filled by the discovery of Calreticulin
(CALR) mutation in MPNs 6,7. CALR gene mutations
are predominantly found in patients with essential
thrombocythemia or primary myelofibrosis and are
considered to be mutually exclusive with JAK2 and MPL.
In spite of the mutational diversity, all the respective
mutations have been shown to function via activation of
the JAK-STAT pathway 6,7. With regards to diagnostics,
the identification of CALR mutations is confirmatory for
a diagnosis of MPN in JAK2 and MPL wild type patients,
presenting with thrombocytosis. Furthermore, its presence
has also shown to carry significant prognostic implications
in patients with confirmed MPN.
CALR-mutated PMF patients were younger than their
JAK2-mutated counterparts and displayed higher platelet
count, lower leukocyte count and longer survival 8. The haemoglobin levels in PMF with CALR mutations were less
likely to display anaemia or require transfusion 8.
Many studies demonstrated that ET with CALR mutations
had higher platelet count than that with JAK2 mutation and
a lower incidence of thrombosis 9. Also, JAK2-mutated
ET has a 29% cumulative risk of progressing to PV whereas
polycythemic transformation was not observed in CALRmutated
The CALR gene is located in the short arm of chromosome
19 10. The most commonly reported pathogenic
mutations in CALR occur in exon 9 and include 52 base pair
(bp), 34 bp, and 19 bp deletions and a 5 bp insertion 6,7.
All these insertions or deletions finally result in a frame
shift mutation. As a result, the new reading frame codes
for a characteristic protein C-terminus that is the same
across the last 36 amino acids irrespective of the underlying
mutation 11. It becomes important to understand that
irrespective of the presence of all the different pathogenic
mutations, the final end result is a common protein
epitope consisting of a similar sequence of amino acids.
If mutation-specific immunohistochemistry is directed
to the characteristic C-terminus of mutated Calreticulin,
it would prove to be a diagnostic screening tool which is
cost effective and provide faster results. Vannucchi et al.
11, using a rabbit polyclonal antibody, and Stein et al.
12, using a mouse monoclonal antibody, have previously
demonstrated excellent sensitivity and specificity for the
diagnosis of all the different types of Calreticulin mutations.
The mouse monoclonal anti-mutant Calreticulin
antibody (clone CAL2), the same as that was used by
Stein et al., has recently become commercially available.
Till date there are limited studies 11-16 on the use of
Calreticulin immunohistochemistry as a diagnostic tool for
According to the current WHO Update 17, the gold
standard for mutational analysis in MPN is prioritised with
initial analysis of JAK2V617F mutation and if negative
followed by CALR mutation. A bone marrow (BM) biopsy
is mandatory for diagnosis of MPN. It has been proposed
that if immunostaining for CAL2IHC in BM biopsies is
validated, it can be conveniently used for identifying patients
harbouring CALR mutations. Furthermore, considering its
feasibility in any routine histopathology laboratory and the
lower cost compared with molecular tests, an initial testing
for CAL2IHC may supervene an unnecessary molecular
JAK2V617F analysis thereby reducing the healthcare
charges for a patient.
Therefore, we aimed to test the sensitivity and specificity of
CAL2IHC in a diagnostic surgical pathology laboratory that
would aid in the identification of pathogenic Calreticulin
mutations in the routine clinical setting.
Subjects of myeloproliferative neoplasms fulfilling the
inclusion criteria from January 2014 till November 2016
were recovered with the use of key word search of the
electronic data bases of the Pathology and Haematology
departments of our institution. The inclusion criteria for
the study were: a) subjects with biopsy-proven diagnosis of
myeloproliferative neoplasm, b) the availability of adequate
bone marrow trephine biopsies for immunohistochemistry,
c) blood/tissue available and subjected to CALR/JAK2
V617F/ JAK Exon 12 mutational analysis.
Following the selection, all formalin-fixed and paraffinembedded
sections were stained in the automated
immunostainer in the General Pathology department with
CAL2 antibody (clone CAL2, catalogue DIA-CAL100;
Dianova, Germany) at a dilution of 1:20, and according
to the protocol T40 in the Ventana Benchmark automatic
immunostainer, keeping in accordance with the steps
mentioned as per the protocol. The investigators were
blinded to the mutation status when examining the slides.
To confirm the pathological evaluation, all biopsies were
reviewed by 2 pathologists (SR and MTM).
Positive immunohistochemical staining of CAL2IHC was
defined by the presence of any intensity (grade 1-3+) of
cytoplasmic staining of megakaryocytes. If a tissue section
contained more than 50 megakaryocytes, then a total of 50
megakaryocytes were counted. From this count, the number
of megakaryocytes staining positively for CAL2IHC (1+ to
3+ staining intensity) were counted separately. Following
this, the total percentage of CAL2 positive megakaryocytes
was calculated from it (CAL2 positive megakaryocytes / 50)
X100. If a section contained less than 50 megakaryocytes,
then the total numbers of megakaryocytes present in a
section were counted, and from among them the percentage
of CAL2 positive megakaryocytes (CAL2 positive
megakaryocytes / total megakaryocytes counted) X100
was calculated accordingly. The intensity of cytoplasmic
staining for CAL2IHC in megakaryocytes was graded from
1+ weak positivity to 3+ strong positivity.
CALR gene deletions and insertions were tested by capillary
electrophoresis (gene scan analysis) and the positive cases
were confirmed by bidirectional Sanger Sequencing to
identify the type of mutation using published protocols
6,7. The sensitivity and specificity of CAL2IHC positivity in patients with MPN were calculated and the results
compared with the gold standard molecular analysis for
validation as a rapid diagnostic tool.
Clinical information of the patients was collected and
histological evaluation and review of CAL2 positive bone
marrow trephine biopsies were also done for confirmation
of the diagnosis. The parameters evaluated were as follows:
a) Cellularity, b) Erythroid hypoplasia, c) Megakaryocyte
hyperplasia, d) Presence of giant hyperlobulated cells, e)
Presence of small to intermediate size megakaryocytes, f)
Nuclear abnormality of megakaryocytes, g) Clustering and
paratrabecular location of megakaryocytes, h) Reticulin
fibrosis (WHO 2008 grading), i) Vascular proliferation,
and j) Osteosclerosis.
All procedures performed in the current study were
approved by the Institutional review board (IRB Min
no.10587, date 29/3/17) in accordance with the 1964
Helsinki Declaration and its later amendments. Formal
written informed consent was not required with a waiver
by the institutional review board committee.
A total of 23 subjects with adequate bone marrow
trephine biopsies and peripheral blood sample available
for mutational analysis were included in the study. There
were 19 males and 4 female patients, aged (30-65) years.
The cohort included 15 patients with diagnosis of primary
myelofibrosis, 6 with essential thrombocythaemia, and
2 with Polycythemia Vera. Detailed clinicopathological
features were described in Table I
Click Here to Zoom
|Table I: Clinicopathological characteristics of myeloproliferative neoplasm cases with JAK2 and CALR Mutational status with CAL2IHC.
Analysis of CAL2 Immunohistochemistry (IHC)
The histopathological diagnosis, CAL2 IHC results, and
correlation with the mutational analysis were described in
Click Here to Zoom
|Table II: Pathological analysis of CAL2IHC and comparison with mutational analysis.
All the patients in our cohort had undergone mutational
All 6 cases of ET (Figure 1A-D) in our study showed strong
cytoplasmic (2-3+) staining of CAL2IHC, displaying 69%
(20-100%) positive megakaryocyte staining and showing
complete concordance with molecular analysis. Only 1 case
showed 1+ positivity, whereas 3 cases showed 2+ positivity,
and 2 cases showed 3+ positivity.
Click Here to Zoom
|Figure 1: A-D) Bone marrow trephine biopsy in essential thrombocythaemia; Patient 1: A) Increased number of giant hyperlobulated
megakaryocytes, Haematoxylin and Eosin, 200x magnification. B) CAL2IHC (2-3+ intensity) diffuse positive cytoplasmic staining
of megakaryocytes in essential thrombocythaemia, 200x magnification; Patient 2: C) Aggregate of hyperlobulated megakaryocytes,
Haematoxylin and Eosin, 200x magnification, D) Diffuse cytoplasmic staining of megakaryocytes for CAL2IHC(2-3+ intensity), 100x
14/15 cases of PMF (Figure 2A-D
) showed (1-3+)
staining of CAL2 IHC (Figure 2B
), with 62% (25-90%)
of megakaryocytes showing positive staining. 5 cases showed 1+ positivity, 6 cases showed 2+ positivity, and
3 cases showed 3+ positivity. One case was negative
for CAL2 IHC but was positive for the Calreticulin
mutation. This continued to be negative even on repeated
Click Here to Zoom
|Figure 2: A-D) Bone marrow trephine biopsy in primary myelofibrosis A-D) Patient 1: A) Clustering of dysplastic megakaryocytes,
Haematoxylin and Eosin, 100x magnification, B) Cytoplasmic staining of megakaryocytes (1-2+ intensity) by CAL2IHC. Inset shows
cytoplasmic staining of a dysplastic megakaryocyte, 200x magnification; Patient 2: C) Increased number of dysplastic megakaryocytes,
demonstrating clustering, abnormal clumped nuclear chromatin, anisocytosis and high N:C ratio, Haematoxylin and Eosin, 200x
magnification. D) CAL2IHC (1-2+ intensity) positive cytoplasmic staining in megakaryocytes in primary myelofibrosis, occasional
megakaryocytes are negative for CAL2IHC, 200x magnification.
Two cases of Polycythemia Vera (Figure 3A,B) were
negative for CAL2IHC, and were also concordant with
negative Calreticulin mutation. One of these two patients
was positive for the JAK2 Exon 12 mutation, and the other
one was positive for the JAK2V617F mutation.
Click Here to Zoom
|Figure 3: A,B) Bone marrow trephine biopsy in Polycythemia Vera: A) Panmyelosis with erythroid and megakaryocyte lineage
predominance along with absence of stromal fibrosis, Haematoxylin and Eosin, 200x magnification, B) The megakaryocytes are negative
for CAL2IHC, 200x magnification.
CAL2IHC had a sensitivity of 95.2% and specificity of
100% for effective diagnosis of Calreticulin positive MPN.
Histological findings in the bone marrow trephine
biopsies of 21 CAL2 IHC positive cases (15 PMF/6 ET)
were reviewed. Upon evaluation of 15 cases with PMF,
9/15 (60%) cases showed increased cellularity, 10/15
(66.7%) had granulocytic hyperplasia, 11/15 (73%) had
megakaryocyte hyperplasia, with predominantly small
megakaryocytes in 12/15 (80%) patients. All cases showed
clustering, paratrabecular location with Grade 3 reticulin
fibrosis. 13/15 (86%) showed vascular proliferation and
14/15 (93.3%) cases showed osteosclerosis.
All the 6 cases with ET showed increased bone marrow
cellularity and megakaryocyte hyperplasia with giant
hyperlobulated megakaryocytes. 4/6 (66.7%) cases had 1+
reticulin fibrosis and the remaining 2 cases showed Grade
1 to focal grade 2+ reticulin fibrosis. None of the cases
demonstrated any evidence of vascular proliferation or
The detection of the Calreticulin mutation has been time
proven to carry prognostic value. The importance of its
detection also lies in the confirmation of a diagnosis of
MPN. On many occasions both clinically and histologically,
it becomes extremely difficult even to separate a benign
reactive phenomenon from MPN.
Recently, a number of reports have come up highlighting
the concurrent presence of multiple MPN related
mutations (JAK2V617F, MPL Exon 10 or CALR) with
concurrent BCR-ABL positive Chronic Myeloid Leukemia
18,19. These reports show that a complex admixture of
different clonal population of cells with varying mutations
can possibly exist together in the same patient.
The postulations for such a phenomenon are as follows.
Firstly, a particular clone by gradual evolution progressing
to show distinct different mutations 20. Secondly, the
presence of two independent clones in different proportions
right from the beginning of disease manifestation. Targeted
therapy driven towards a particular clonal population (mostly Ph +v CML) may suppress the former population
and facilitate the emergence of the other relatively masked
clonal (CALR mutated) population 19,20. There are
few reports showing that further research with detailed
molecular studies is warranted to uncover such hidden
anomalies in patients with atypical presentations of MPN.
Considering such complex situations, the mutational
analysis often serves as an important confirmatory marker
in the diagnosis of a neoplasm 13. Currently molecular
testing is the gold standard for identification of CALR
Recently, the use of CAL2IHC has been reported to serve as
an effective marker for detection of Calreticulin mutations.
Currently, there is only limited reported data on the
sensitivity/specificity of this novel antibody and its
effectiveness as a diagnostic tool 11-16 (Table III). Our
study aimed to validate the utility of CAL2IHC in routine
diagnostics, which in the long run could possibly serve as a
surrogate diagnostic tool for molecular studies.
Click Here to Zoom
|Table III: Comparative analysis of sensitivity and specificity of
Among the 6 reported studies, the first study was done by
Vannucchi et al. 11 where a novel polyclonal antibody
was developed against all different CALR mutations.
The antibody was found to be extremely effective with
100% sensitivity and specificity. There was predominant
cytoplasmic staining of megakaryocytes and weaker faint
staining of erythroids. This was postulated to be due to over
expression of CALR mutant protein in megakaryocytes. Our
study did not show any positive staining of the erythroids
or myeloids, and demonstrated a crisp cytoplasmic positive
staining of megakaryocytes, therefore concurring with
the proposed postulation of over expression of mutant
Calreticulin in megakaryocytes.
Subsequently the largest study was performed by Stein
et al 12 where a monoclonal antibody was tried on 173
subjects. The subjects included 155 patients with MPN and
the results of immunohistochemistry were compared to
the gold standard Sanger sequencing for CALR mutation.
A high sensitivity and specificity of 100% was quoted in
the study. A study on 38 subjects by Nomani et al 14 also
showed a sensitivity and specificity of 100%. A recent study
by Andrici et al. 13 showed a mildly lower sensitivity of
91%. Our study similarly showed a sensitivity of 95.2%, and
specificity of 100%.
One patient in our study with a diagnosis of PMF was
consistently negative for CAL2IHC even after repeated
immunohistochemical staining. This observation was
also noted by Andrici et al. 13 where a case of PMF was
persistently negative for CAL2IHC. Although a possibility
of true negative could not be predicted accurately, it was postulated that in end stage cases of PMF, the extensive
fibrosis could mask the neoplastic clone population
(CAL2 IHC positive staining) of megakaryocytes. In such
a situation, only the non-neoplastic population of (CAL2
IHC negative) megakaryocytes may remain more relatively
exposed and visible. Hence, based on this observation,
the biopsy could be falsely interpreted to be negative for
This observation could certainly apply for our case as there
was extensive fibrosis with paucity of megakaryocytes in the
trephine biopsy. The patient was eventually lost to follow
up and a repeat biopsy could not be performed. The other
possibility was of a false positive result on the mutational
analysis. This was difficult for us to evaluate further as
there was insufficient tissue for a repeat molecular analysis.
If we consider this to be a true negative, it becomes
imperative to realise that a negative CAL2IHC may not
always predict negativity for CALR mutation. This fact is
justified well by Andrici et al. 13 and also by our study.
The next part of our study focused on the morphometric
assessment of CAL2IHC positivity on megakaryocytes.
Our study showed 69% positive megakaryocyte staining
both in ET and PMF cases. Both the cases of PV were
negative for CAL2IHC. Mózes et al. 15 in their study had
also performed a manual and automated morphometric
analysis and correlated it with the CALR mutation load.
45.7% (±2.6) of the megakaryocytes had demonstrated a
moderate to strong CALR expression manually, and 68.5%
(±1.28) of the megakaryocytes by automated analysis. It
was also shown that the percentage of megakaryocytes with moderate to strong staining had a positive correlation with
higher CALR mutation loads. Our study demonstrates a
higher proportion of megakaryocytes (83% (2-3+ intensity)
in cases of ET, and 60% (2-3+ intensity) in cases of PMF)
with moderate to strong CAL2IHC staining. We could
not do a detailed mutational load analysis due to financial
constraints. It remains to be discovered on a larger scale
study whether or not the proportion/ staining intensity
of megakaryocyte staining could indeed indicate a higher
mutation load and therefore be prognostically significant.
Molecular analysis from peripheral blood is non-invasive
and indeed provides more accurate results than IHC on
bone marrow trephine biopsies. However, the cost of
molecular detection via bidirectional Sanger sequencing
is higher than the cost of a single immunohistochemical
marker and most importantly requires a high level of
technical expertise. Therefore, the need of the hour is a cost
effective, sensitive and specific diagnostic test that may aid
in substituting the need for molecular diagnostics. This
situation becomes extremely important in centers where a
set up for extensive molecular testing is not available for
A novel approach to the step wise diagnosis of MPN has
been recently proposed by Vanucchi et al 11 where
instead of the step wise mutational analysis starting with
JAK2 mutation, CAL2IHC can be done. If the CAL2IHC
is positive, it essentially excludes the positivity of JAK2,
MPL and other mutations 7. It also becomes important
to understand from a different perspective that the current
WHO update 17 mandates the histopathological analysis
of bone marrow trephine biopsy, as a major criterion for
diagnosis of MPN. So needless to say, it becomes feasible,
time saving and cost effective for both patient and clinician
to perform immunohistochemistry with faster accurate
results. Hence in small health care centers, the role for
molecular mutational analysis can be considered as a
secondary supporting diagnostic test for discrepant cases
instead of a mandatory primary test. Whether it stands
the test of time to completely substitute the present gold
standard of molecular testing is yet to be seen.
In summary, we conclude that CAL2IHC is rapid, cost
effective and highly specific for detecting CALR mutation,
and is an effective diagnostic tool for diagnosis of MPN.
Our study had limitations that could not be eliminated due
to financial constraints. Firstly, our sample size was limited
to 23 patients with a selection bias (primarily based on
cases which had a sample available for molecular analysis).
A larger sample size with varying population could have
highlighted the specificity more accurately.
Secondly, CAL2IHC was not performed on normal/non
MPN subjects. Due to limited resources and infrequent
molecular testing of patients, our study was primarily
focused on JAK2 negative and CALR positive MPN.
Thirdly, our cohort of PMF did not include cases of
prefibrotic stage of- PMF that histologically can very often
be a close mimicker of ET. Finally, a detailed gene sequence
analysis could not be performed to locate the exact base
pair deletion in CALR mutation. This could have helped
us to understand the specificity of CAL2IHC better as it is
reported to be positive in all the different types of CALR
CONFLICT of INTEREST
The authors declare no conflicts of interest.
Concept: SR, MTM, Design: SR, MTM, Data collection
or processing: SR, MTM, PB, Analysis or Interpretation:
SR, MTM, Literature search: SR, Writing: SR, MTM, PB,
Approval: SR, MTM, PB.
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