RESEARCH ARTICLE

The Diagnostic Utility of IDH2 R172
Immunohistochemistry in Tall Cell Carcinoma
With Reversed Polarity of the Breast
Eiichi Sasaki, MD, PhD,*† Akari Iwakoshi, MD, PhD,†‡ Tatsunari Satake, MD,§
Kosei Nakajima, MD, PhD,∥ Mizuho Kobayashi, MD, PhD,¶ Yoshiaki Ando, MD, PhD,#
Takashi Koshikawa, MD, PhD,*** Katsuhiro Masago, MD, PhD,* Naoko Honma, MD, PhD,††
Rieko Nishimura, MD, PhD,‡ Yukinari Kato, MD, PhD,‡‡§§ and Shu Ichihara, MD, PhD‡

Abstract: Tall cell carcinoma with reversed polarity (TCCRP) is arare histologic type of low-grade breast cancer, consisting of tallcolumnar cells with reversed nuclear polarity and characterized
by frequent IDH2 mutations. We herein report 3 cases of TCCRP
with sequencing analyses of the IDH2 gene and immunohistochemical examination using monoclonal antibodies(11C8B1) against IDH2 R172. IDH2 R172 mutations were detected in all 3 resected tumors (R172S in 2 tumors and R172T in 1
tumor), and the presence of these mutations was conﬁrmed by
IDH2 R172 immunohistochemistry. Tumor cells of TCCRP
showed strong and diffuse staining for the antibody against IDH2
R172. In 1 case, tumor tissue from 2 core needle biopsy samples
collected on different days were also immunohistochemically
positive for IDH2 R172. These results indicate that IDH2 R172
immunohistochemistry is suitable for the detection of TCCRP in
both resection and biopsy samples. In addition, a literature review
revealed that R172S and R172T account for 76% of IDH2 mutations in TCCRP, suggesting that 11C8B1, which reacts with
R172S and R172T, was likely most sensitive for IDH2-mutated
Received for publication June 9, 2022; accepted September 15, 2022.
From the *Department of Pathology and Molecular Diagnostics, Aichi
Cancer Center Hospital; †Clinical Research Center; ‡Department of
Pathology, National Hospital Organization Nagoya Medical Center;§Department of Pathology, Nagoya Ekisaikai Hospital; ¶Departmentof Pathology, Nagoya City University West Medical Center; #AndoBreast Clinic, Nagoya; ∥Department of Surgical Pathology, Ichinomiya Municipal Hospital; **Department of Medical Technology,
Shubun University Faculty of Medical Sciences, Ichinomiya; ††Department of Pathology, Toho University Faculty of Medicine, Tokyo;
Departments of ‡‡Antibody Drug Development; and §§Molecular
Pharmacology, Tohoku University Graduate School of Medicine,
Sendai, Japan.
Supported by Nagoya Medical Center and by the Aichi Cancer Research
Foundation.
E.S. and S.I. conceived and designed the study. E.S., A.I., T.S., K.N.,
M.K., Y.A., T.K., R.N., and S.I. contributed to the materials andpatients. E.S., R.N., and S.I. provided a pathology review. E.S.,
K.M., and S.I. analyzed and interpreted the data. E.S. and S.I. wrote
the paper. E.S. generated tables and ﬁgures. K.M., N.H., R.N., andY.K. reviewed and edited the paper.
The authors declare no conﬂict of interest.
Reprints: Eiichi Sasaki, MD, PhD, Department of Pathology and MolecularDiagnostics, Aichi Cancer Center Hospital, 1-1 Kanokoden, Chikusa-ku,
Nagoya, Aichi 464-8681, Japan (e-mail: sasakia1es@gmail.com).
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TCCRP among many available antibodies for IDH2 R172.
Furthermore, the combination of 2 or more antibodies against
IDH2 R172 could be more effective for detecting TCCRP mutation. However, it is important to note that IDH2 R172
immunohistochemistry is not absolute, because IDH2 wild type isfound in a small proportion (10%) of cases, and a few cases of
IDH2-mutated TCCRP may harbor rare subtypes of R172 thatare not covered by available antibodies.
Key Words: tall cell carcinoma with reversed polarity, IDH2,
R172, breast, 11C8B1(Appl Immunohistochem Mol Morphol 2022;00:000–000)T

all cell carcinoma with reversed polarity (TCCRP) of thebreast is a distinct subtype of triple-negative breast
cancer with a favorable prognosis. Histologically, TCCRP
is a low-grade carcinoma characterized by tall columnar
cells with reversed nuclear polarity, arranged in solid andsolid papillary patterns.1 The tumor was ﬁrst described by
Eusebi et al2 as a breast tumor resembling the tall cell variant of papillary thyroid carcinoma. Recently, this tumor
has been classiﬁed as a distinct tumor entity in the ﬁfth
edition of the World Health Organization Classiﬁcation ofbreast tumors.3 To date, more than 70 cases of TCCRP
have been reported in the literature.
In 2016, Chiang et al4 found that TCCRP is a breast
cancer subtype characterized by frequent IDH2 mutations.
IDH2 R172 mutations have not been reported in breast
cancer cases other than TCCRP, whereas IDH2 mutations,which did not target the R172 hotspot locus, have only been
described in a few cases.4,5 Therefore, the detection of IDH2
R172 mutations in breast cancer tissue can be helpful inmaking a correct diagnosis of TCCRP. An alternative
method to the molecular analysis of the IDH2 gene is immunohistochemistry using mutation-speciﬁc antibodies.
Many commercially available immunohistochemical monospeciﬁc and multispeciﬁc antibodies against IDH2 mutations
at codon 172 have been developed,6–11 but there are no antibodies that cover all the subtypes of IDH2 R172 mutations.
The selection of antibodies can inﬂuence the diagnostic utility

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Appl Immunohistochem Mol Morphol  Volume 00, Number 00, ’’ 2022

Sasaki et alof IDH2 R172 immunohistochemistry. However, this point
has not been discussed in previous studies of TCCRP.
In this study, we report 3 cases of TCCRP and review
the relevant literature to clarify the best immunohistochemical
panel for the diagnosis of TCCRP. Furthermore, we assessed
the programmed death-ligand 1 (PD-L1) expression in
TCCRP to consider the possibility of immune checkpoint inhibitor (ICI) treatment for this tumor.

MATERIALS AND METHODSPatients and Histologic Evaluation
We selected 3 cases of TCCRP of the breast from theconsultation archive of one of the authors (S.I.). Resected
specimens from all the 3 cases were evaluated. For 1 case, 2
core needle biopsy samples collected on different days werealso evaluated. All diagnoses were conﬁrmed by 3 pathologists (E.S., R.N., and S.I.). TCCRP was diagnosed according
to the most recent version of the World Health Organization
classiﬁcation.3 We also selected and retrieved 6 cases of
non-TCCRP breast carcinoma to compare the IDH2 R172
expression with its expression in TCCRP. These 6 tumors
included 5 triple-negative breast carcinomas (2 of secretory
carcinoma, 1 of carcinoma with apocrine differentiation, 1 ofmetaplastic carcinoma, and 1 of invasive carcinoma of no
special type) and 1 hormone receptor-positive invasive lobular carcinoma. All tissues were ﬁxed in 10% formalin andembedded in parafﬁn. All procedures used in this researchwere approved by the Ethical Committee of Nagoya Medical
Center (No. 2006-15). This study was conducted in accordance with the Declaration of Helsinki.

Immunohistochemistry
Immunohistochemistry was performed on 4-μmthick formalin-ﬁxed parafﬁn-embedded (FFPE) sections.
The following antibodies were used: ER [SP1, readyto-use (RTU); Roche], PgR (E12, RTU; Roche), HER2
(4B5, RTU; Roche), ki67 (MIB-1, 1:50; Dako), p63 (4A4,
RTU; Roche), CK5/6 (D5/16B4, RTU; Roche), S100
(Polyclonal Rabbit, RTU; Dako), CD31 (JC70A, RTU;
Dako), GATA3 (HG3-31, 1:50), SOX10 (E6B6I, 1:50;
Cell Signaling), PAX8 (Polyclonal Rabbit, 1:1500; Proteintech), Calretinin (DAK-Calret1, 1:100; Dako), mammaglobin (304-1A5, RTU; Dako), GCDFP15 (EP95,
RTU; BioGenex), MUC4 (8G7, 1:200; Santa Cruz),
BRAF V600E (VE1, RTU; Roche), epidermal growth
factor receptor (EGFR) (31G7, RTU; Nichirei Biosciences), pan-TRK (EPR17341, RTU; Roche), TTF-1(8G7G3/1, 1:200; Dako), PD-L1 (SP142, RTU; Roche,and 22C3, RTU; Dako), and IDH2 R172 (11C8B1,

1:2000; NewEast Biosciences). PD-L1 status was assessedwith clone SP142 using the OptiView DAB IHC Detection
Kit (Ventana Medical Systems) and with clone 22C3 using
the Dako pharmDx Kit (Dako, Agilent Technologies).
SP142-stained slides were scored for PD-L1 immune cell(IC) positivity,12 whereas 22C3-stained slides were readfor the PD-L1 combined positivity score (CPS).13

Mutation Analysis
DNA was extracted from the tumor areas of each unstained FFPE section with reference to hematoxylin and eosin–
stained specimens. Sample DNA was ampliﬁed with the following primer set encompassing the IDH2 R172 hotspot locus:
forward primer, TCAAGCTGAAGAAGATGTGGAA; andreverse primer, CAAAGTCTGTGGCCTTGTACTG. The
products were directly sequenced using an ABI PRISM 310
Genetic Analyzer (Applied Biosystems).

RESULTSClinical Findings
The patients were 3 women, with an average age of
57 years (44, 55, and 71 y, respectively) (Table 1). All the 3patients were Japanese, an ethnic group in which TCCRP has
not been reported thus far. The average tumor size at excision
was 9 mm (4, 9, and 14 mm, respectively). The laterality wasleft (n = 2) and right (n = 1). For 1 patient, follow-up
observation before excision was performed for 3 years, as it
was diagnosed as a benign lesion, likely intraductal papilloma,
based on the pathologic examination of a biopsy specimen
obtained at a previous institute (Case 3). In Case 3, the tumor
size increased slightly in the follow-up observation period. All
patients underwent surgical excision with sentinel lymph node
resection. In all the 3 cases, the surgical margin was free and no
sentinel lymph node metastasis was seen. The 3 patients were
followed for 19, 15, and 8 months after resection, respectively.
No patients developed local recurrence or metastasis.

Pathologic and Immunohistochemical Findings
All the 3 tumors showed similar pathologic features.
These tumors were composed of multilobulated nests withsolid and solid papillary structure separated by dense ﬁbrous stroma (Fig. 1A). The tumor cells were tall andcolumnar, with abundant eosinophilic cytoplasm andround to ovoid nuclei. The presence of nuclei at theapical sides, a striking histologic feature of TCCRP, wasfound in the periphery of the tumor nests (Fig. 1B).
Follicular-like structures containing eosinophilic material
were seen in all the 3 cases, at least focally (Fig. 1C).
Mitotic ﬁgures, if present, were rare (< 1 per 10 highpower ﬁelds). Necrosis and vascular invasion were absent

TABLE 1. Clinicopathologic Features of Tall Cell Carcinoma With Reversed Polarity
Case No.

Age (y)Sex

Laterality

1
2
3

55
44
71

F
F
F

Left
Right
Left

Size (mm)Presentation

Lymph Nodes

Treatment

Follow-up

4914

Palpable nodule
Palpable nodule
Palpable nodule present 3y

SN negative
SN negative
SN negative

Wide excision
Wide excision
Wide excision

NED (19 mo)
NED (15 mo)
NED (8 mo)

F indicates female; NED, no evidence of disease; SN, sentinel lymph node.

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Appl Immunohistochem Mol Morphol  Volume 00, Number 00, ’’ 2022

in all the 3 cases. Tumor-inﬁltrating lymphocytes weresparse, and the percentage of stromal tumor-inﬁltrating
lymphocytes was 0% to 1% in all the 3 tumors.
In Case 3, 2 specimens biopsied on 2 different days
(3 y and 1 mo before surgical excision, respectively) showed
identical histologic ﬁndings to the surgical specimen.
Therefore, the former tumor sampled 3 years previously wasreclassiﬁed as TCCRP, although the original diagnosis was
benign papilloma.
The key results from the immunohistochemical analysis
are summarized in Table 2. One tumor had a triple-negative
phenotype, and the remaining 2 tumors showed focal
hormone receptor expression. The Ki67 proliferation index
was low (up to 3%). All the 3 tumors were positive for CK5/6(2 cases, diffuse; 1 case, focal), calretinin (diffuse), GCDFP15(focal), and EGFR (focal, weak) (Figs. 1D–F). Occasional
positivity was seen for GATA3 (2/3, focal), mammaglobin

DH2 R172 Immunohistochemistry in TCCRP(2/3, focal), and S100 (2/3, focal and weak), whereas all the 3tumors were negative for TTF-1, PAX8, SOX10, and
MUC4. PD-L1 SP142 IC and 22C3 combined positivity
score were <1% in all the 3 tumors (Fig. 1G). CD31 staining
highlighted numerous capillaries in the basal lamina(Fig. 1H). Tumor islands surrounded by a layer of
p63-positive myoepithelial cells were not found in any
tumor areas. Flat epithelial atypia, atypical ductal
hyperplasia, ductal carcinoma in situ, and microglandular
adenosis, all of which can be precursors of invasive breast
cancer, were not found in the background breast tissue.

Genetic Findings by Sanger Sequencing and
Immunohistochemistry Using a Mutationspecific Antibody
IDH2 mutations were detected in all the 3 resected
tumors by Sanger sequencing. Two types of IDH2 mutation,

FIGURE 1. The histologic and immunohistochemical findings of tall cell carcinoma with reversed polarity. A, At low-power
magnification, the tumor shows a pushing margin. B, Tumor cells have abundant eosinophilic cytoplasm and reverse polarity,
characterized by apical localization of the nuclei. C, Follicle-like structures with colloid-like secretion are found. D, Tumor cells are
positive for CK5/6. E, Diffuse and strong positivity for calretinin is seen in both the nuclei and cytoplasm of tumor cells. F,
Membranous staining of epidermal growth factor receptor is focally present. G, Tumor cells and immune cells are negative for
22C3, and the programmed death-ligand 1 22C3 combined positive score is <1%. H, CD31 highlights the capillary network along
to the basal lamina.
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Appl Immunohistochem Mol Morphol  Volume 00, Number 00, ’’ 2022

—
—
—
—
—
—
*In Case 3, diffuse positive reactivity for IDH2 R172 was observed in 2 core needle biopsy samples collected on different days and an excision sample.
CPS, combined positive score; EGFR, epidermal growth factor receptor IC, immune cells.

+, diffuse+, diffuse+, diffuse*
< 1%
< 1%
< 1%
< 1%
< 1%
< 1%
—
—
—
—
—
—
—
—
—
+, focal +, focal+, focal +, focal+, focal +, focal+, diffuse +, diffuse+, diffuse +, diffuse+, focal +, diffuse
2
3
2
0
1+
0
2
5
0
10
2
0
1
2
3

Calretinin EGFR GCDFP15 PAX8 SOX10 MUC4
CK5/6
Case No. ER % PgR % HER2 Ki67%

TABLE 2. Immunohistochemical Findings of Tall Cell Carcinoma With Reversed Polarity

PD-L1 22C3
CPS

PD-L1 SP142
IC

IDH2
R172

BRAF
V600E

PanTRK

Sasaki et al

4 | www.appliedimmunohist.com

R172S and R172T, were found in 2 cases (Cases 1 and 2) and1 case (Case 3), respectively (Fig. 2A). To validate thepresence of these mutations and the distribution of mutatedtumor cells, we performed an immunohistochemical analysis
using monoclonal antibodies (11C8B1) against the IDH2
R172 mutation. All 3 tumors, irrespective of the mutation
types of R172S or R172T, were positive for the antibodies,with diffuse and strong positivity for tumor cells (Figs. 2B,
C). In Case 3, an IDH2 R172T mutation that was identical to
the resected sample was detected in a sample biopsied
1 month before excision. In another sample biopsied 3 years
previously, PCR products were not ampliﬁed because of the
low quality of extracted DNA and the mutation status could
not be analyzed. However, diffuse and strong positivity for
IDH2 R172 was observed in tumor cells in both the 2 coreneedle biopsy samples, irrespective of the DNA quality.
In addition, we performed an immunohistochemical
analysis using a BRAF V600E mutation-speciﬁc antibody,
because the mutations are most common in papillary
thyroid carcinoma, a tumor with similar histologic features
to TCCRP. All the 3 tumors were immunohistochemicallynegative for BRAF V600E mutation-speciﬁc antibody,which is highly speciﬁc and sensitive for the V600E
mutation.14 Furthermore, all the 3 tumors were negative
for pan-TRK, a surrogate marker for the identiﬁcation of
NTRK fusion.15
Immunohistochemically, IDH2 R172 was negativein all 6 non-TCCRP tumors.

DISCUSSION
Mutations in the IDH2 gene result in the simultaneous loss of normal catalytic activity, the productionof a-ketoglutarate (α-KG), and gain of a new function,
the production of 2-hydroxyglutarate (2-HG), which causes
altered stem cell differentiation and eventual tumorigenesis.16 IDH2 mutation has been reported in relatively
restricted tumor types, including glioma, acute myeloid leukemia, cartilaginous tumor, cholangiocarcinoma, and sinonasal undifferentiated carcinoma and is assumed to play animportant role in the carcinogenesis of these tumors.16 In thisstudy, we conducted a mutational analysis of 3 cases of
TCCRP and found that all the 3 tumors harbored IDH2
R172 mutations. The IDH2 mutations were also conﬁrmed
by immunohistochemistry using monoclonal antibodies tothe IDH2 R172 mutation and almost all tumor cells of thesecases were positive for the antibodies. In addition, in 1 case,the results of the immunohistochemistry using IDH2 R172
antibodies were in agreement among 3 matched samples(2 core needle biopsy samples and an excision sample)
collected on different days during the clinical course of
3 years. These ﬁndings suggest that the mutation is an early
oncogenic event during the tumorigenesis of TCCRP.
11C8B1, which was used in this study, was originallydesignated as an antibody against IDH2 R172S, but ourstudy and a previous study17 have shown that this antibody is positive not only for TCCRP with R172S mutations but also the tumors with R172T mutations. No
reaction with other variants of R172 (R172K/G/W/M/I)
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Appl Immunohistochem Mol Morphol  Volume 00, Number 00, ’’ 2022

A

DH2 R172 Immunohistochemistry in TCCRP

B

Case #1

AGG>AGC

Case #2

C

AGG>AGT

Case #3

AGG>ACGFIGURE 2. Detection of IDH2 R172 mutations by Sanger sequencing and immunohistochemistry. A, Sanger sequencing revealed
IDH2 R172 mutations in all 3 cases: c.516G > C (p.R172S) in Case 1, c.516G > T (p.R172S) in Case 2, and c.515G > C (p.R172T) in
Case 3. B, The tumor cells of Case 2 show strong and diffuse immunostaining for 11C8B1, whereas normal breast tissue at thelower side of the picture is negative for this marker. C, At high-power magnification, the tumor cells of Case 3 are positive in thecytoplasm, with a granular pattern.

was found in previous studies that examined 11C8B1 immunohistochemistry in various tumor types with IDH2
mutations.7,17
The distribution of IDH2 mutation types in a totalof 60 TCCRP cases with sequencing analyses for the IDH2
gene reported thus far are summarized in Table 3.4,17–26
IDH2 mutation was detected in 90% of the cases (54/60),
and the mutational regions were limited to codon 172.
Five subtypes of IDH2 R172 mutation [R172S, R172T,
R172G, R172W, and R172I (in descending order)] havebeen reported. No antibodies cover all 5 subtypes of IDH2
R172 mutations reported in TCCRP, although there aremany immunohistochemical antibodies against IDH2
R172 available on FFPE sections. Importantly, 11C8B1
seems to have the highest detection rate for IDH2-mutated
TCCRP; R172S and R172T, which are detectable by
11C8B1, account for 76% (41/54) of the R172 mutations in
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these tumors (Table 3). An alternative marker is MsMab1,
a multispeciﬁc antibody that reacts with R172S, R172G,
and R172M, but shows negativity for R172T, R172K,
R172W, and R172I.27 The estimated rate of MsMab1
positivity in IDH2-mutated TCCRP is 63% (34/54). IDH2
R172W, with which neither 11C8B1 nor MsMab1 react,can be detected by a monospeciﬁc antibody, WMab1.9 To
the best of our knowledge, no available antibody against
IDH2 R172I has been developed. Therefore, thecombination of 11C8B1 and MsMab1, or plus WMab1,
could be expected to detect almost all IDH2-mutated
TCCRPs [94% (51/54) or 98% (53/54), respectively].
Our study, like a previous study,17 showed that IDH2
R172 immunohistochemistry of core needle biopsy specimens of TCCRP is feasible. The evaluation for IDH2 R172
staining is unlikely affected by the site sampled via biopsy or
ﬁne needle aspiration, because of the homogeneous andwww.appliedimmunohist.com | 5

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Appl Immunohistochem Mol Morphol  Volume 00, Number 00, ’’ 2022

Sasaki et al

TABLE 3. Distribution of IDH2 Mutations in Previously Reported Cases of Tall Cell Carcinoma With Reversed Polarity With Genetic
Analyses
ReferencesnMethodology

R172S*

R172T*

R172G*

R172W†

R172I

Wild Type

Chiang et al4

13
3
6
7
1
9
14
2
1
1
3
60

NGS
NGS
Sanger sequencing
NGS
NGS
NGS
Sanger sequencing
NGS
Sanger sequencing
NGS
Sanger sequencing
—

3
1
3
4
—
3
7
1
—
—
2
24

4
1
2
2
—
1
6
—
—
—
1
17

3

—
—
—
1
—
—
—
1
—
—
—
2

—
—
—
—
—
—
1
—
—
—
—
1

3
1
—
—
—
2
—
—
—
—
—
6

Bhargava et al18
Lozada et al19
Zhong et al20
Haeﬂiger et al21
Alsadoun et al22
Pareja et al17
Wei et al23
Jassim et al24
Zhang et al25,26
This study
Total

1
—
1
3
—
—
1
1‡
—
10

NGS indicates next generation sequencing.
*Among multispeciﬁc antibodies against IDH2 mutations at codon 172, 11C8B1 reacts with R172S and R172T, whereas MsMab1 reacts with R172S and R172G.
†WMab1, monospeciﬁc antibody reacts with R172W.
‡The mutation was originally reported as IDH2 R120G, a novel mutation, rather than IDH2 R172,25 but we realized that the mutation was actually R172G.26

robust staining. Notably, in 1 case, tumor tissue from anarchived core needle biopsy sample was inadequate for a
genetic analysis because of low DNA quality but was immunohistochemically positive for IDH2 R172. The superiority of immunohistochemistry to genetic sequencing
for the detection of mutation-speciﬁc antibodies may
be remarkable when archived tumor tissue samples withdecreasing DNA quality are used, or when using specimens
with a small number of tumor cells or low-tumor purity areused.28
Pareja et al17 reported that none of 226 triple-negative
breast cancers or 34 benign or low-grade histologic mimics of
TCCRP, including 13 intraductal papillomas, 16 solid papillary carcinomas, and 5 encapsulated papillary carcinomas,
expressed IDH2 R172 by immunohistochemistry using
monoclonal antibodies (11C8B1). Although our study had asmall sample size, it included histologic types such as
secretory carcinoma, for which the IDH2 R172 immunohistochemistry has not been examined (or described) in
previous studies. Our study further conﬁrmed the speciﬁcityof IDH2 R172 immunohistochemistry in TCCRP among
various histologic types of breast tumors.
Our TCCRPs were invariably positive for calretinin.
Calretinin seems to be a useful marker for the differential
diagnosis of TCCRP among low-grade breast lesions,because the expression of calretinin has been reported to
be rare in low-grade breast cancer.29,30 Contrary to lowgrade cancer, high-grade breast cancers are frequently
positive for calretinin. Interestingly, among high-grade
breast cancers, the expression of calretinin is more frequently seen in association with the expression of CK5/
6.30,31 TCCRP is also positive for CK5/6. In addition, ourcases of TCCRP showed positivity—albeit weak positivity—for EGFR. Which intrinsic subtype of breast cancer
TCCRP falls into is unclear. A previous study using RNA
sequencing showed that 5 of the 9 tumors were categorized
as luminal-A type and 4 others were classiﬁed as basal
type.22 However, these shared immunohistochemical features of TCCRP and TNBC with a basal-like phenotype

6 | www.appliedimmunohist.com(CK5/6 and EGFR positive) may facilitate the understanding of TCCRP, in terms of the tumor origin anddifferentiation.
TCCRP is assumed to be an invasive cancer becauseof the absence of myoepithelial cells around tumor nests
and the presence of metastatic lesions in a small subset ofcases, but the precursor lesion of TCCRP remains unknown. Two of 3 tumors in our study were small(< 10 mm in size), and, to our knowledge, the tumor sizeof Case 1 (4 mm) is the smallest ever reported,3 suggestingthat these tumors may represent lesions at an early stage ofinvasive cancer. However, in situ carcinoma components
surrounded by a layer of myoepithelial cells were notfound in any tumor areas of our 3 TCCRPs, although apreexisting precursor lesion may be completely overtaken
by carcinoma with progression. Therefore, the multistep
progression model of invasive breast cancer via ductal
carcinoma in situ seems not to be applicable for TCCRP.
These histologic ﬁndings of TCCRP may reﬂect the biological nature as basal type rather than luminal type.
Follicular-like structures with colloid-like secretionare frequently seen in TCCRP cases, as shown in
Figure 1C. Despite its morphological characteristics, which
resemble thyroid cancer, variable staining for GCDFP15,
mammaglobin, and GATA3 combined with negativity for
TTF-1 and PAX8 suggest a tumor of breast—not thyroid—origin.32,33 Colloid-like secretions of TCCRP also may
mimic the extracellular secretions of a secretory carcinoma,a different tumor entity from TCCRP, which is frequently
associated with ETV6-NTRK3 fusion.34 Therefore, thedistinction between TCCRP and secretory carcinoma may
be difﬁcult in small biopsy specimens, and an immunohistochemical analysis may be needed to make acorrect diagnosis. Tumor cells of secretory carcinoma arefrequently positive for SOX10, MUC4, and panTRK,15,35,36 while our study showed that TCCRPs were
negative for these 3 markers.
TCCRP is a low-grade breast cancer and has anindolent clinical course, without disease recurrence in the
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Appl Immunohistochem Mol Morphol  Volume 00, Number 00, ’’ 2022

majority of patients. In this study, no local recurrence ormetastasis were found in any of the 3 TCCRP cases, including a case that was followed for 3 years follow-up
period before resection. However, an exceptional casewith distant metastasis of the bone was reported.37 Recently, IDH1/2 inhibitors were shown to be effective
against IDH2-mutated cancer.38 When a TCCRP shows
aggressive clinical behavior, IDH1/2 inhibitors may be atherapeutic option. In contrast, ICIs might be rarely indicated for the treatment of TCCRP, a tumor that usuallyexhibits a triple-negative phenotype. Our study showedthat the PD-L1 status in TCCRP was lower than thepositive cutoff value for ICI therapy, although the efﬁcacyof the combination ICIs with chemotherapy for advanced
triple-negative breast cancer has been shown in a subgroup of patients with PD-L1-positive tumors.12,13
To the best of our knowledge, there is no literature regarding the cytologic ﬁndings in TCCRP. Furthermore, noneof our 3 cases had any cytology specimens obtained via ﬁneneedle aspiration or touch imprint. Further studies will beneeded to clarify whether or not the diagnostic utility of R172
immunohistochemistry in TCCRP applies not only to FFPE
sections but also to cytology specimens among breast tumors.
In summary, we described 3 cases of TCCRP with
an immunohistochemical analysis of IDH2 R172 hotspot
mutations. Immunohistochemistry using antibodies
against IDH2 R172 is a sensitive and speciﬁc test for theidentiﬁcation of TCCRP harboring IDH2 R172 mutations. However, there are no antibodies that cover all
subtypes of IDH2 R172 mutations reported in TCCRP,
including multispeciﬁc antibodies such as 11C8B1 andMsMab1. Therefore, the combination of 2 or more antibodies against R172 could be more effective for the diagnosis of TCCRP. In addition, IDH2 R172 mutations
have not been detected in a minority of cases of TCCRP(~10%) (Table 3). In such cases, a detailed histologic
evaluation combined with an immunohistochemical
analysis using other markers, including calretinin andCK5/6 can help make a correct diagnosis of TCCRP.
ACKNOWLEDGMENTS
The authors thank Yukiko Takahashi and Kyoko
Kato for secretarial assistance.
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