Breast Cancer Res Treat (2015) 150:447–455 DOI 10.1007/s10549-015-3336-0

EPIDEMIOLOGY

Frequent alterations of HER2 through mutation, amplification, or overexpression in pleomorphic lobular carcinoma of the breast Huang-Chun Lien1,2 • Yu-Ling Chen2 • Yu-Lin Juang2 • Yung-Ming Jeng1,2

Received: 6 January 2015 / Accepted: 6 March 2015 / Published online: 14 March 2015 Ó Springer Science+Business Media New York 2015

Abstract Mutations in HER2 gene have been identified in a small subset of breast cancer cases. Identification of HER2 mutation has therapeutic implications for breast cancer, but whether a subgroup of breast cancer with a higher frequency of HER2 mutation exists, remains unknown. We analyzed HER2 mutation and pathologic factors on 73 formalin-fixed, paraffin-embedded samples, including 21 pleomorphic invasive lobular carcinoma (p-ILC) cases, 3 pleomorphic lobular carcinoma in situ (p-LCIS) cases, and 49 classic invasive lobular carcinoma (c-ILC) cases. Mutations were identified through direct sequencing. HER2 overexpression and amplification were determined through immunohistochemistry and fluorescent in situ hybridization. Six mutations were identified, including five in the 24 p-ILC or p-LCIS (p-ILC/p-LCIS) cases (20.8 %) and one in the 49 c-ILC cases (2.0 %), and the difference in frequency was significant (p = 0.013). Eight of the 24 (33.3 %) p-ILC/p-LCIS cases exhibited HER2 amplification or overexpression (amplification/ overexpression), which was significantly higher than in the c-ILC cases (1/49, 2 %). Mutation and amplification/ overexpression were mutually exclusive. HER2 mutations were identified more frequently in the p-ILC/p-LCIS cases

Electronic supplementary material The online version of this article (doi:10.1007/s10549-015-3336-0) contains supplementary material, which is available to authorized users. & Yung-Ming Jeng [email protected] 1

Department of Pathology, College of Medicine, National Taiwan University Hospital, No. 1 Jen-Ai Road, 1st Section, Taipei 100, Taiwan

2

Graduate Institute of Pathology, National Taiwan University, Taipei, Taiwan

with extensive apocrine change (p = 0.018). Combined HER2 alterations through mutation or amplification/overexpression were more frequently identified in p-ILC/pLCIS cases without estrogen receptor expression. The high frequency (54.1 %, 13/24) of combined HER2 alterations in the p-ILC/p-LCIS cases suggests a crucial role of HER2 in the pathogenesis of p-ILC/p-LCIS. Because of the reported responsiveness of HER2 mutation to anti-HER2 therapy, p-ILC patients without HER2 amplification/overexpression should receive HER2 mutation analysis to identify this therapeutically relevant target. Keywords Breast cancer
Lobular carcinoma
Pleomorphic lobular carcinoma
HER2 mutation

Introduction Invasive lobular carcinoma (ILC) is a subtype of breast cancer, representing 5–15 % of invasive breast tumors [1, 2]. The tumor cells are typically small and uniform with round nuclei and inconspicuous nucleoli. Cases with such low-grade nuclei are regarded as classic ILC (c-ILC). By contrast, some ILCs consist entirely or in part of cells that feature enlarged nuclei with greater nuclear irregularity, hyperchromasia, and one or more prominent nucleoli. Such variants with higher nuclear grade have been referred to as pleomorphic ILC (p-ILC) [3, 4]. c-ILCs are typically positive for estrogen receptor (ER) and progesterone receptor (PR), lack human epidermal growth factor receptor 2 (HER2) amplification or overexpression, and have low Ki-67 proliferation indexes. By comparison, a lower frequency of reactivity to ER and PR, higher chance of HER2 gene amplification, and higher Ki-67 index have been observed in p-ILC [5–7]. Consistent with the aggressive

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biological features, clinical data demonstrate that p-ILC displays an aggressive behavior and unfavorable prognosis compared with c-ILC [3, 8, 9]. Data to guide the management of patients diagnosed with p-ILC are currently lacking. Therefore, patients are managed in a similar manner to patients with ductal carcinomas [9]. A study comparing ductal and lobular breast cancer revealed that the complete pathological response rate of four cycles of AC chemotherapy was approximately 10 % for IDC, but less than 2 % for ILC [10]. Moreover, neoadjuvant chemotherapy did not appear to have substantial benefits for patients with ILC [11]. Therefore, an improved understanding of the biological nature of p-ILC is critical for identifying the most appropriate management strategy for this subgroup of patients. HER2 is a member of the epidermal growth factor receptor (EGFR) family of receptor tyrosine kinase. HER2 gene amplification and protein overexpression occur in approximately 20–30 % of breast cancer cases [12]. Recently, Bose et al. summarized eight breast cancer genome-sequencing projects and identified 25 HER2 somatic mutations in a total of 1499 patients, indicating an overall HER2 mutation rate of approximately 1.6 % [13]. Compared with ductal carcinoma cases, lobular carcinoma cases seemed to exhibit a higher frequency of HER2 mutation. Two studies have identified HER2 mutation in 7.7 % (3/39) and 2.7 % (3/113) of lobular carcinoma cases, respectively [14, 15]. In addition, Ross et al. identified a higher frequency of HER2 somatic mutation in relapsed classic CDH1-mutated ILC cases (18 %, 4/22), compared with non-CDH1-mutated breast cancer cases (2 %, 5/286) [16]. These finding are crucial for the clinical management of patients because the majority of reported HER2 mutations in breast cancer patients are sensitive to HER2 inhibitors [13]. Although the results suggest a potential treatment option for ILC, whether a subgroup of breast cancer with a higher frequency of HER2 mutation exists remains unclear. Because a higher frequency of HER2 amplification or overexpression (amplification/mutation) has been reported in p-ILC, implying a role of HER2 alteration in this cancer subtype [5], we hypothesized that p-ILC might be a subgroup of breast cancer with a higher frequency of HER2 mutation. In this study, we determined the frequency of HER2 mutations in cases of c-ILC and p-ILC or pleomorphic lobular carcinoma in situ (p-LCIS) (p-ILC/p-LCIS).

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defined as c-ILC when all the tumor cells were small and uniform with round nuclei and inconspicuous nucleoli. By comparison, p-ILC was defined by adopting the cytological criteria for p-ILC, according to Weidner et al. (i.e., tumor cells with nuclei four times larger than a lymphocyte nucleus) [17]. A total of 73 formalin-fixed, paraffin-embedded samples of lobular carcinoma were retrieved and used in this study, including 21 cases of p-ILC, three cases of p-LCIS, and 49 cases of c-ILC. The invasive tumors were graded according to the Elston and Ellis modification of the Nottingham grading system [18]. All slides were reviewed by 2 pathologists (H.C.L. and Y.M.J.), and cases were included only when they reached a consensus on the diagnosis. Because focal apocrine morphologic features were not uncommonly identified in ILC, we defined extensive apocrine change as more than 80 % of tumor cells with centrally located, round vesicular nuclei, prominent nucleoli, and eosinophilic granular cytoplasm. This study was approved by the Research Ethics Committee of National Taiwan University Hospital. Immunohistochemistry and fluorescence in situ hybridization Immunohistochemical studies were conducted using an autoimmunostainer (Ventana BenchMark system, Tucson, AZ, USA) with the iVIEW DAB Detection Kit (Ventana). The antibodies used were E-cadherin (NCH-38, Dako, Glostrup, Denmark), ER (SP1, Ventana), PR (1E2, Ventana), HER2 (c-erb-B2, Dako), and Ki-67 (MIB-1, Dako). Positive ER and PR results were defined as unequivocal nuclear staining in more than 1 % of tumor cells. HER2 was scored using the American Society of Clinical Oncology/ College of American Pathologists (ASCO/CAP) criteria [19]. All HER2-equivocal cases (2? by immunohistochemistry) were further tested by fluorescence in situ hybridization (FISH), using a PathVysion dual-color HER2 DNA Probe Kit (Vysis, Downers Grove, IL, USA) and scored in accordance with ASCO/CAP criteria [19]. The proliferation marker Ki-67 staining index of each case represented the average of the percentage of positively nuclearstained tumor cells of two representative tumor parts. The Ki-67 index was scored as follows: Score 1:\3 %; Score 2: 3 to \10 %; Score 3: 10 to \14 %; Score 4: C14 %. Mutation analysis

Materials and methods Tumor samples The cases were retrieved from the Department of Pathology, National Taiwan University Hospital. Cases were

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For each case, 3–5 representative formalin-fixed, and paraffin-embedded tumor sections were macrodissected to ensure a purity of at least 50 %. Genomic DNA was extracted using a QIAamp DNA Micro Kit (Qiagen, Hilden, Germany). Polymerase chain reaction (PCR) was performed for all DNA samples using the intron-based primers

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for seven exons of the entire tyrosine kinase domains and exon 8 of the extracellular domain of HER2. Primer sequences and PCR conditions are shown in supplementary Table 1. All PCR products were subjected to direct sequencing, using an autosequencer (Applied Biosystems, Model 3730, Foster City, CA, USA).

449 Table 1 Histological grading and biomarker status in 49 cases of classic ILC (c-ILC) and 24 cases of pleomorphic ILC or LCIS (p-ILC/ p-LCIS) c-ILC (n = 49) Gradea

Statistics analysis Statistical analyses were conducted using SPSS Version 11.0 software for Windows (SPSS Inc., Chicago, IL, USA). A Fisher’s exact test was used in the comparison for categorical variables. Statistical significance was set at the level of 0.05.

Results Tumor characteristics The tumor grading and biomarker status for c-ILC and p-ILC/p-LCIS are summarized in Table 1. The majority of c-ILC cases were grade 1 (77.6 %). By contrast, 61.9 % of the 21 p-ILC cases were grade 3. ER was positive in all cases of c-ILC, but negative in 54.2 % of the p-ILC/p-LCIS cases. Eight of the 24 cases (33.3 %) of p-ILC/p-LCIS were positive for HER2 overexpression (n = 6) or amplification (n = 2), a substantially higher frequency than that of c-ILC, in which only one of the 49 cases exhibited HER2 overexpression (p \ 0.001) (Fig. 1). Ki-67 proliferation indexes equal to or higher than 10 % were detected in 50.0 % (12/24) of the p-ILC/p-LCIS cases, compared with 10.2 % (5/49) of the c-ILC cases (p = 0.001).

p-ILC/p-LCIS (n = 24)

\0.001

1

38 (77.6)

2

11 (22.4)

3

0 (0) 8 (38.1)

0 (0)

13 (61.9)

?

49 (100)

24 (100)

-

0 (0)

0 (0)

E-cadherin

NA

\0.001

ER ?

49 (100)

11 (45.8)

-

0 (0)

13 (54.2) \0.001

PR ?

47 (95.9)

7 (29.2)

-

2 (4.1)

17 (70.8)

1 (2.0)

8 (33.3)

48 (98.0)

16 (66.7)

HER2b ? -

p value

\0.001

Ki-67c

0.001

Score 1

24 (49.0)

Score 2

20 (40.8)

5 (20.8) 7 (29.2)

Score 3

4 (8.2)

7 (29.2)

Score 4

1 (2.0)

5 (20.8)

NA not accessed a

Tumor grading (Elston and Ellis modification of the Nottingham grading system) was performed only on the 21 cases of invasive lobular carcinoma

b Positivity for HER2 was defined as 3? by immunohistochemistry or 2? by immunohistochemistry but positive on HER2 FISH test c

Frequency of HER2 mutation

Ki-67 index was scored as follows: the percentage of positively nuclear-stained tumor cells (Score 1: \3 %; Score 2: 3 to \10 %; Score 3: 10 to \14 %; Score 4: C14 %)

HER2 contains extracellular, transmembrane, and intracellular domains [20]. The majority of the reported HER2 mutations were clustered in two major areas, with 20 % of patients having extracellular domain mutations at residues 309–310 and 68 % of patients having kinase domain mutations between residues 755–781 [13]. Therefore, we sequenced the region of exon 8 that encodes residues including 309–310 and the seven exons of the entire HER2 tyrosine kinase domains in all 73 samples. A total of 6 HER2 mutations were identified in the 73 lobular carcinoma samples (8.2 %), including 5 of the 24 cases (20.8 %) of p-ILC/p-LCIS and 1 of the 49 cases (2 %) of c-ILC (Tables 2, 3; Fig. 2). Two of the five mutations in p-ILC/p-LCIS cases were identified in p-LCIS cases, whereas the remaining three mutations were identified in p-ILC cases. The higher frequency of HER2 mutations in the p-ILC/p-LCIS cases (20.8 %) compared with that in the

c-ILC cases (2 %) was statistically significant (p = 0.013). Of the five HER2 mutations identified in the p-ILC/p-LCIS cases, one was a missense mutation, S310F, involving extracellular domain, two were identical exon 19 deletion mutations, L755_T759del, and the remaining two were exon 20 missense mutations, A775V and T791I. The one mutation discovered in the c-ILC cases was an exon 20 mutation, A771V. To determine whether these mutations were somatic mutations or inherited/germline polymorphisms, we sequenced the non-tumor parts of the six cases of lobular carcinoma with HER2 gene mutation, including un-metastasized sentinel lymph node in three cases, uninvolved nipple and areolar part in two cases, and un-involved section margin in one case. We found wild-type sequence of HER2 gene in the non-tumor part in all the six cases (Supplementary Fig. 1), confirming that these

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Fig. 1 Histomorphology, immunohistochemistry (IHC), and fluorescent in situ hybridization (FISH) in classic and pleomorphic lobular carcinomas. Histomorphology (upper panel) and corresponding IHC staining for HER2 (lower panel). HER2 staining was negative in a

Table 2 HER2 alterations in 49 cases of c-ILC and 24 cases of p-ILC/p-LCIS p-ILC/p-LCIS (n = 24)

c-ILC (n = 49)

p value

\0.001

HER2 alterations ?

13 (54.2)

2 (4.1)

-

11 (45.8)

47 (95.9)

Amplification or overexpressiona ?

8 (33.3)

1 (2.0)

-

16 (66.7)

48 (98.0)

Mutationb ? -

5 (20.8)

1 (2.0)

19 (79.2)

48 (98.0)

\0.001

0.013

a

Positivity for HER2 was defined as 3? by immunohistochemistry or 2? by immunohistochemistry but positive by HER2 FISH test b

Mutation in the exon 8 of the extracellular domain and the seven exons (exons18–24) of the entire HER2 tyrosine kinase domain

mutations were somatic mutations instead of inherited/ germline polymorphisms. Correlation between HER2 alterations with histomorphology and biomarkers The frequency of HER2 mutation (20.8 %, 5/24), HER2 amplification/overexpression (33.3 %, 8/24) and combined HER2 alterations (52.2 %, 13/24), defined as the combined HER2 mutation or HER2 amplification/overexpression, was significantly higher in the p-ILC/p-LCIS cases than in the c-ILC cases. We therefore investigated if there were any factors correlated with HER2 mutation, HER2 amplification/ overexpression, or combined HER2 alterations in p-ILC/p-

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c-ILC case (LC-30) (left panel) but was strongly positive in a p-ILC case (LC-11) (middle panel). Equivocal HER2 staining was observed in a p-ILC case (LC-1) (right panel), but HER2 FISH demonstrated amplification, with a HER2/CEP17 ratio of 2.6 (inset)

LCIS. As shown in Table 4, tumor grading, which was performed on ILCs, was significantly correlated with HER2 amplification/overexpression (p = 0.018), but not with HER2 mutation. All the three p-LCIS cases showed nuclear grade 3. Two of them, which harbored HER2 mutation, were ER-/PR-/HER2-, whereas the remaining one p-LCIS, which did not harbor HER2 mutation, was ER-/PR? with HER2 amplification. Extensive apocrine change was identified in 4 of the p-ILC/p-LCIS cases (2 p-LCIS and 2 p-ILC cases) and three of these four cases exhibited HER2 mutation (2 p-LCIS and 1 p-ILC case). The association between extensive apocrine change and HER2 mutation was statistically significant (p = 0.018). ER negativity was significantly correlated with the combined HER2 alterations, with alterations observed in 66.7 % (8/12) of ER-negative cases versus 27.3 % (3/11) of ER-positive cases (p = 0.038). All five cases with HER2 mutation were negative for HER2 amplification/overexpression. The only case of c-ILC with HER2 mutation was histologically grade 2, positive for ER and PR, and negative for HER2 staining; the Ki-67 index was 2.3 %.

Discussion In this study, we demonstrated a substantially higher frequency of HER2 mutation in p-ILC/p-LCIS, compared with that in c-ILC (20.8 vs. 2 %). The frequency was considerably higher than the overall HER2 mutation rate of 1.6 %, identified in a total of 1499 patients with breast cancer, and also higher than the rates of 2.7 % (3/39) and 7.7 % (3/113) identified in two studies of lobular carcinoma cases [14, 15]. It is noteworthy that among the four classic ILC cases with HER2 mutation identified by

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Table 3 HER2 mutations identified in cases of c-ILC and p-ILC/LCIS Case

Tumor type

Histologic gradea

Extensive apocrine changeb

ER/PR/HER2/Ki-67 (%)

Mutation type

Nucleotide change

Predicted protein change

LC-2

p-ILC

Grade 2

?

ER-/PR-/HER2-/4.3 %

Deletion

c.2263_2277del

p.L755_759del

LC-6

p-ILC

Grade 3

-

ER-/PR-/HER2-/4.8 %

Missense

c.2372C [ T

p.T791I

LC-13

p-ILC

Grade 2

-

ER ?/PR ?/HER2-/1.2 %

Missense

c.2324C [ T

p.A775 V

LC-22

p-LCIS

Nuclear grade 3

?

ER-/PR-/HER2-/13 %

Deletion

c.2263_2277del

p.L755_759del

LC-38

c-ILC

Grade 2

-

ER ?/PR ?/HER2-/2.6 %

Missense

c.2312C [ T

p.A771 V

LC-73

p-LCIS

Nuclear grade 3

?

ER-/PR-/HER2-/8.1 %

Missense

c.929C [ T

p.S310F

a

Histologic grading using Elston and Ellis modification of the Nottingham grading system was performed only on the cases of invasive lobular carcinoma. Pleomorphic LCIS was graded based on nuclear grading

b

Extensive apocrine change was defined by apocrine change in more than 80 % of tumor cells

Fig. 2 Histomorphology and sequencing for HER2 gene in pleomorphic lobular carcinomas. Histomorphology (left panel) and corresponding sequencing (right panel) for HER2 gene in three cases of pleomorphic lobular carcinoma. Exon 19 in-frame deletion mutation, p.L755_T759del, was found in a p-ILC case (LC-2) with extensive apocrine change (upper panel). Exon 8 missense mutation, p.S310F, was identified in a p-LCIS case (LC-73) with extensive apocrine change (middle panel). Exon 20 missense mutation, p.A775 V, was found in a p-ILC case (LC-13) (lower panel)

Ross et al., one was histologically grade 3 and negative for ER and PR, whereas another was histologically grade 3, but had no available information regarding ER and PR. The grade-3 histology with negativity for ER and PR was highly suggestive of the pleomorphic variant of ILC. These results demonstrate a substantial enrichment of HER2 mutations in p-ILC/p-LCIS. However, because all these 73 patients were Taiwanese, testing HER2 mutation frequencies in other populations will need to be done to determine

whether HER2 mutation frequencies differ among ethnicities. In addition to HER2 mutation, we observed a considerably higher frequency of HER2 gene amplification/overexpression in p-ILC/p-LCIS compared with c-ILC (33.3 vs. 2.0 %, p \ 0.001). Prominently, the cases with HER2 mutation or HER2 amplification/overexpression were mutually exclusive, resulting in an overall 54.2 % (13/24) of combined HER2 alterations in the p-ILC/p-LCIS cases. This finding is clinically relevant because the

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123 8 (33.3) 16 (66.7) NS

NS

NS

NS

0.018

NS

2.6 ± 1.2

0 (0)

8 (33.3)

6 (25.0)

2 (8.3)

6 (25.0)

2 (8.3)

8 (33.3)

0 (0)

7 (33.3)

0 (0)

2.4 ± 1.0

16 (66.7)

0 (0)

11 (45.8)

5 (20.8)

7 (29.2)

9 (37.5)

12 (50.0)

4 (16.7)

6 (28.6)

8 (38.1)

16 (66.7)

8 (33.3)

17 (70.8)

7 (29.2)

13 (54.2)

11 (45.8)

20 (83.3)

4 (16.7)

13 (61.9)

8 (38.1)

Total

NS

\0.001

NS

NS

NS

0.018

p

2.4 ± 1.0

5 (20.8)

8 (33.3)

10 (41.7)

3 (12.5)

8 (33.3)

3 (12.5)

10 (41.7)

3 (12.5)

8 (38.1)

2 (9.5)

?

2.6 ± 1.1

11 (45.8)

0 (0)

7 (29.1)

4 (16.7)

3 (13.0)

8 (34.8)

3 (12.5)

8 (33.3)

5 (23.8)

6 (28.6)

-

Extensive apocrine change was defined by apocrine change in more than 80 % of tumor cells

Histologic grading (Elston and Ellis modification of the Nottingham grading system) was performed only on the 21 cases of invasive lobular carcinoma

Positivity for HER2 amplification/overexpression or HER2 mutation

Positivity for HER2 was defined as 3? by immunohistochemistry or 2? by immunohistochemistry, but positive on HER2 FISH test

2.6 ± 1.1

11 (45.9)

8 (33.3)

17 (72.8)

7 (29.2)

13 (54.2)

11 (45.8)

20 (83.3)

4 (16.7)

13 (61.9)

8 (38.1)

-

Combined alterationsb

16 (66.7)

8 (33.3)

17 (70.8)

7 (29.2)

12 (52.1)

11 (47.8)

13 (54.2)

11 (45.8)

13 (61.9)

8 (38.1)

Total

NS

0.002

NS

0.038

NS

NS

p

Ki-67 ratio was scored as follows: the percentage of positively nuclear-stained tumor cells (Score 1: \3 %; Score 2: 3 to \10 %; Score 3: 10 to \14 %; Score 4: C14 %). Ki-67 score was represented as mean ± standard deviation

e

d

c

b

a

NS not significant

2.0 ± 0.8

5 (20.8)

Ki-67 scoree

0 (0)

13 (54.1)

4 (16.7)

-

6 (25.0)

?

HER2

-

?

9 (37.5)

10 (41.7)

18 (75.0)

1 (4.2)

4 (16.7)

-

PR

1 (4.2)

2 (8.3)

1 (4.2)

12 (57.1)

1 (4.8)

3 (12.5)

6 (28.6)

2 (9.5)

?

ER

-

Extensive apocrine changed ?

3

2

Gradec

p

?

Total

?

-

Amplification/overexpressiona

Mutation

HER2

Table 4 Correlation between HER2 alterations (mutation or amplification/overexpression) and histomorphology and biomarkers in 24 cases of p-ILC/p-LCIS

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majority of the reported HER2 mutations in breast cancer patients have been shown to be activating mutations and sensitive to HER2 inhibitors [13]. Consistently, a recent report demonstrated response to anti-HER2 therapy, administered based on the identification of HER2 mutation, in a patient with inflammatory breast cancer who was initially refractory to multiple systemic chemotherapy [21]. Moreover, a preliminary report suggested a favorable response in 4 patients with p-ILC on the use of trastuzumab. Two of the patients were tested for HER2 through FISH and were positive for amplification [22]. Thus, our findings of combined HER2 alterations in over half of the cases of p-ILC/p-LCIS may implicate a potential therapeutic benefit of anti-HER2 therapy in p-ILC. Focal apocrine differentiation is frequently seen in p-ILC [4, 23, 24]. In this study, we identified 4 cases (2 p-LCIS and 2 p-ILC case) showing extensive apocrine change. Noticeably, three of them were positive for HER2 mutation. The association between apocrine features and HER2 expression in breast cancer has been suggested by the findings that IDC cases demonstrating apocrine features frequently overexpress HER2 [25, 26] and that HER2 amplification is more common in breast cancer cases with a molecular apocrine signature identified through gene expression array analysis [27–29]. Our finding, despite being preliminary, suggests that HER2 mutation might be an alternative mechanism associated with extensive apocrine change in p-ILC and implicate a role of HER2 dysregulation in apocrine differentiation. In the present study, we determined a substantially lower frequency of ER and PR expression in p-ILC/p-LCIS than in c-ILC. When analyzing the correlation between the expression of ER and the status of HER2 mutation, we observed a tendency of ER negativity in HER2 mutationpositive p-ILC/p-LCIS and HER2 amplification/overexpression-positive p-ILC/p-LCIS, but the correlation was not significant. However, ER negativity was significantly correlated with combined HER2 alterations (p = 0.038). Although the mechanism underlying the association between HER2 alterations and ER-negativity remains elusive, such an association in p-ILC suggests a potential therapeutic benefit from anti-HER2 therapy in patients who exhibit HER2 alterations and are less likely to express ER and benefit from hormone therapy. The L755_T759 deletion mutation is homologous to EGFR exon 19 deletion (ELREA) [30]. This mutation has been reported in breast cancer to cause increased phosphorylation of EGFR and HER3, the heterodimerization partners of HER2, and such mutation was responsive to tyrosine kinase inhibitor, neratinib [13]. The mutation of residues 309–310 of the HER2 extracellular domain have been reported as recurrent mutations in breast cancer and micropapillary carcinoma of the urinary bladder [13, 31]. The

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G309A missense mutation has been shown to be an activating mutation and is responsive to HER2 inhibitors [13]. Although the significance of S310F has not been evaluated in breast cells, it has been demonstrated to have activating properties in lung cancer [32]. The remaining three exon 20 missense mutations, namely A771V, A775V, and T791I, have not been reported in breast cancer. Exon 20 insertion mutation, A775_G776insYVMA, has been reported in ILC, and this mutation has been associated with HER2 activation and sensitivity to HER2 inhibitors [16, 33–35]. Furthermore, three exon 20 missense mutations, namely D769H, D769Y, and V777L, have been shown to be activating mutations and responded to an irreversible HER2 inhibitor. Although the relevance of the three missense mutations in the present study remains to be investigated, the mutation sites of A771V and A775V, which were located within the regions (codons 769–777) frequently harboring activating mutations with responsiveness to inhibitors, might suggest a biologic and therapeutic relevance of these two mutations. Finally, the chronological nature of HER2 mutation and its involvement in the early stage of mammary tumorigenesis or later, during tumor progression, remains elusive. Although future studies comparing HER2 mutation of primary or relapsed cases would address this question, the identification of HER2 mutation in two cases with pure p-LCIS provided evidence in support of the assertion that HER2 mutation occurs in the pre-invasive stage of p-ILC. In summary, we identified a substantial enrichment of HER2 mutation and HER2 amplification/overexpression in p-ILC/p-LCIS. The mutations and amplification/overexpression are mutually exclusive, suggesting alternative mechanisms in the activation of HER2 signaling in p-ILC/ p-LCIS. Considering the overall HER2 alterations in 54.1 % of p-ILC/p-LCIS, and the preliminary results showing responsiveness of HER2 mutations to a HER2 inhibitor and a favorable response of p-ILC patients with HER2 amplification to trastuzumab [13, 21, 22], our findings propose that p-ILC patients without HER2 amplification/overexpression should receive HER2 mutation analysis to identify this therapeutically relevant target. These data together also encourage larger-scale prospective trials that include anti-HER2 target therapy for p-ILC, which exhibits aggressive clinical course with an unfavorable response to existing chemotherapy. Acknowledgments This study was supported by National Taiwan University Hospital and National Science Council Grant NSC 102-2320-B-002-009 and 103-2320-B-002-030-MY2. The authors thank the second core laboratory of National Taiwan University Hospital for the technical support.

Conflict of interest The authors declare that they have no conflicts of interest and no financial relationship with the organization that sponsored the research.

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