Breast Cancer Res Treat (2014) 145:615–623 DOI 10.1007/s10549-014-2983-x
PRECLINICAL STUDY
Two histopathologically different diseases: hormone receptor-positive and hormone receptor-negative tumors in HER2-positive breast cancer Hee Jin Lee • In Ah Park • So Yeon Park • An Na Seo • Bora Lim Yun Chai • In Hye Song • Na Eun Kim • Joo Young Kim • Jong Han Yu • Jin-Hee Ahn • Gyungyub Gong
•
Received: 13 March 2014 / Accepted: 23 April 2014 / Published online: 13 May 2014 Ó Springer Science+Business Media New York 2014
Abstract The clinical behavior of human epidermal growth factor 2 (HER2)-positive breast cancer, including pathologic complete response rate and pattern of relapse and metastasis, differs substantially according to hormone receptor (HR) status. We investigated various histopathologic features of HER2-positive breast cancer and their correlation with HR status. We retrospectively analyzed tumors of 450 HER2-positive breast cancer patients treated with chemotherapy and 1 year of trastuzumab. HR-/ HER2? tumors showed higher nuclear grade, less tubule Hee Jin Lee and In Ah Park have contributed equally to this work. H. J. Lee I. A. Park Y. Chai I. H. Song N. E. Kim J. Y. Kim G. Gong (&) Department of Pathology, Asan Medical Center, University of Ulsan College of Medicine, 88 Olympic-ro 43-gil, Songpa-gu, Seoul 138-736, South Korea e-mail: [email protected] S. Y. Park A. N. Seo Department of Pathology, Seoul National University Bundang Hospital, Seongnam, Gyeonggi, South Korea S. Y. Park Department of Pathology, Seoul National University College of Medicine, Seoul, South Korea B. Lim Department of Hematology-Oncology, Penn State Hershey Cancer Institute, Penn State College of Medicine, Hershey, PA, USA J. H. Yu Department of Surgery, Asan Medical Center, University of Ulsan College of Medicine, Seoul, South Korea J.-H. Ahn Department of Oncology, Asan Medical Center, University of Ulsan College of Medicine, Seoul, South Korea
formation, higher histologic grade, frequent apocrine features, diffuse and abundant lymphocytic infiltration, strong HER2 immunohistochemical staining (3?), higher average HER2 copy number and HER2/CEP17 ratio, the absence of HER2 genetic heterogeneity, and greater p53 expression than HR?/HER2? tumors. An inverse correlation was observed between estrogen receptor or progesterone receptor Allred score and average HER2 copy number or HER2/CEP17 ratio. The percentage of ductal carcinoma in situ (DCIS) within the tumor was negatively correlated with ER Allred score, but positively correlated with average HER2 copy number and HER2/CEP17 ratio. Pathologic tumor size and DCIS percentage also showed a significant inverse correlation. Ratio of metastatic to total examined lymph node number was significantly correlated with average HER2 copy number and HER2/CEP17 ratio. High pT stage (hazard ratio, 2.370; p = 0.027), the presence of lymphovascular invasion (hazard ratio, 2.806; p = 0.005), and HR negativity (hazard ratio, 2.202; 1.074–4.513; p = 0.031) were found to be independent prognostic indicators of poor disease-free survival. In conclusion, HR?/HER2? and HR-/HER2? breast cancer showed distinct histopathologic features that may be relevant to their distinct clinical behavior. Keywords Breast carcinoma Gene amplification HER2 Hormone receptor
Introduction With the advance of genomic technology, the heterogeneous nature of breast cancer has been well recognized [1]. Recently, the Cancer Genome Atlas (TCGA) Network determined the existence of at least two subtypes of human
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epidermal growth factor 2 (HER2)-positive breast cancers based on comprehensive analysis [2]. One of the two subtypes is the HER2-enriched mRNA subtype, characterized by significantly higher expression of receptor tyrosine kinases including EGFR, HER2, and FGFR4. Another group belongs to the luminal mRNA subtype that displays higher expression of a luminal cluster of genes: GATA3, BCL2, and ESR1. These two subgroups of HER2positive tumors are, therefore, genetically distinct from each other. The clinical behaviors of hormone receptor-positive/ HER2-positive (HR?/HER2?) and hormone receptornegative/HER2-positive (HR-/HER2?) tumors are quite different. In neoadjuvant settings, HR-/HER2? tumors show higher pathologic complete response rate than HR?/ HER2? tumors [3, 4]. Patterns of relapse, metastatic spread, and survival are also different [5, 6]. ER-/HER2? patients showed earlier and higher level of relapse than ER?/HER2? patients [5]. HR-/HER2? tumors tended to have liver as the first metastatic site than HR?/HER2? tumors [6]. HR-/HER2? tumors were found to be of higher histologic grade and stage compared to HR?/ HER2? tumors [7]. However, the detailed histologic features of HR- and HR? HER2-positive breast cancer remain to be explored. We, therefore, performed the first comprehensive histologic analysis of surgically resected HER2-positive breast cancer from a large series of Asian patients treated with adjuvant chemotherapy and trastuzumab. We compared the clinicopathologic factors between HR?/HER2? and HR-/HER2? tumors, and analyzed the correlation among the various factors. In addition, we found prognostic significance of clinicopathologic factors in HER2-positive breast cancer.
Materials and methods Patients and tissue specimens A total of 450 HER2-positive breast cancer patients who underwent surgery for primary breast cancer between 2006 and 2011 at the Asan Medical Center, who had available formalin-fixed paraffin-embedded tissue samples for analysis, were included in this study. All patients were preoperatively chemo- and radiotherapy naı¨ve, and all underwent adjuvant treatment. Of 450 patients, 161 patients with node-negative breast cancer were treated with four cycles of adjuvant anthracycline and cyclophosphamide (AC, adriamycin 60 mg/m2 and cyclophosphamide 600 mg/m2) and 1 year of trastuzumab. The remaining 289 patients had node-positive breast cancer, and were treated with either four cycles of AC followed by four cycles of
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paclitaxel (175 mg/m2) or four cycles of AC followed by four cycles of docetaxel (75 mg/m2) and 1 year of trastuzumab. Trastuzumab (6 mg/kg) was given every 3 weeks. Clinicopathologic information was obtained from the patient’s medical records and surgical pathologic reports. Expressions of standard biomarkers including estrogen receptor (ER), progesterone receptor (PR), HER2, and p53 were reviewed in full sections that were immunohistochemically stained at the time of diagnosis. ER and PR levels were regarded as positive if there was at least 1 % positive tumor nuclei staining [8]. HR? group was defined as ER- or PR-positive tumors. Additionally, Allred score was calculated for ER and PR [3]. HER2-overexpressing tumors were defined as those with scores of 3? by immunohistochemistry or gene amplification by fluorescence in situ hybridization (FISH) or silver in situ hybridization (SISH) [9]. Among 450 patients, 386 patients had an immunohistochemical score of 3; 61 patients had a score of 2; and three patients had a score of 1. FISH or SISH was performed for samples with immunohistochemical score of 1 or 2, and HER2 amplification was observed in all cases. Samples with positive staining of 10 % or more cells were considered p53 positive [10]. Exemption from informed consent after de-identification of information was approved by the Institutional Review Board of Asan Medical Center. Histologic evaluation Hematoxylin and eosin (H&E)-stained sections were histopathologically analyzed for the number of invasive tumors, histologic subtype and grade, nuclear grade, tubule formation, apocrine features, lymphocytic infiltration (location: absent/minimal, border of tumor, or diffuse; abundance: absent/minimal, moderate, or high), ductal carcinoma in situ (DCIS) percentage, the presence or absence of extensive intraductal component and Paget disease, tumor size, pT stage, pN stage, ratio of metastatic to total examined lymph nodes, and lymphovascular invasion [11]. Histologic type was defined based on the 2012 WHO classification criteria, and histologic grade was assessed employing the modified Bloom–Richardson classification [12]. Apocrine feature of tumor cells included abundant granular eosinophilic cytoplasm, cytoplasmic vacuolization/clearing, round vesicular nuclei, and prominent eosinophilic nucleoli [13]. Tissue microarray construction Formalin-fixed, paraffin-embedded tissue samples were arrayed by a tissue-arraying instrument. After the review of HER2 expression in immunostained whole tumor section slides, three areas were selected and arrayed in 1-mm-
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diameter cores. If the tumor showed heterogeneous staining for HER2, representative areas of different stainings were chosen.
617 Table 1 Comparison between HR status and histopathologic factors in HER2-positive breast cancer HR negative (%)
HR positive (%)
p
Invasive carcinoma of no special type
235 (96.7)
185 (89.4)
\0.001
Carcinoma with micropapillary differentiation
6 (2.5)
14 (6.8)
Carcinoma with mucinous differentiation
0 (0.0)
8 (3.9)
Metaplastic carcinoma
2 (0.8)
0 (0.0)
2
65 (26.7)
103 (49.8)
3
178 (73.3)
104 (50.2)
SISH assays for HER2 gene amplification Histologic type
Automated SISH assays on tissue microarray sections were performed with INFORM HER2 DNA and Chromosome 17 (CEP17) probes (Ventana Medical Systems, Tucson, AZ, USA) using an ultraView SISH Detection Kit (Ventana Medical Systems) according to the manufacturer’s protocols [14]. HER2 gene amplification status was separately assessed in the three tissue microarray cores from each sample. Fifty tumor cells per core were evaluated; consequently, a total of 150 cells were counted for each case where possible. HER2/CEP17 ratio, the average HER2 gene copy number per cell, and the average CEP17 copy number per cell were evaluated. In cases with distinct subpopulations of amplified and non-amplified cells, counting was weighted by the percentage of each population in the entire tumor. According to American Society of Clinical Oncology (ASCO)/College of American Pathologists (CAP) criteria, HER2 gene amplification was determined as follows: (1) the average ratio of HER2 to CEP17 signals was C2.0, or (2) the ratio was \2.0 but an average HER2 copy number of C6 [9]. HER2 amplification status was classified into two groups: a high-level amplification group defined as having a HER2/CEP17 ratio C4.0, and a low-level amplification group with 2.0 B HER2/CEP17 ratio \4.0. Tumors with a HER2/CEP17 ratio\2.0 and average HER2 copy number C4 and \6 were considered equivocal for amplification. The CEP17 copy number alteration was evaluated using a modification described by Ma et al. [15]. Cases with average CEP17 copy numbers between 1.25 and 2.25 were defined as having disomy 17. The remaining cases were aneusomic for chromosome 17, i.e., they had either monosomy 17 (\1.25 signals per cells), low polysomy 17 ([2.25 but B3.75 signals per cell), or high polysomy 17 ([3.75 signals per cell). HER2 genetic heterogeneity was defined, according to the CAP guidelines, as the existence of tumor cells with a HER2/CEP17 ratio [2.2 in 5–50 % of all the examined tumor cells [16].
Nuclear grade \0.001
Tubule formation 1
2 (0.8)
2 (1.0)
2
56 (23.2)
72 (35.0)
3
183 (75.9)
132 (64.1)
64 (26.3)
104 (50.2)
179 (73.7)
103 (49.8)
Absent
50 (20.7)
91 (44.2)
Present
191 (79.3)
115 (55.8)
Histologic grade 2 3
0.014
\0.001
Apocrine feature \0.001
Extensive intraductal component Absent
141 (58.8)
138 (66.7)
Present
99 (41.3)
69 (33.3)
27.9 ± 30.5
22.4 ± 25.1
0.037
229 (94.2) 14 (5.8)
203 (98.1) 4 (1.9)
0.052
Absent/minimal
84 (34.6)
115 (55.6)
\0.001
Border, moderate
36 (14.8)
28 (13.5)
Border, high
24 (9.9)
13 (6.3)
Diffuse, moderate
19 (7.8)
21 (10.1)
Diffuse, high
77 (31.7)
27 (13.0)
DCIS percentage (average ± SD)
0.096
Paget disease Absent Present Lymphocyte infiltration
Number of invasive tumor foci 1
163 (67.1)
149 (72.0)
C2
77 (31.7)
58 (28.0)
0.233
1
122 (50.6)
86 (41.5)
2
113 (46.9)
112 (54.1)
3
6 (2.5)
9 (4.3)
2.3 ± 1.1
2.4 ± 1.2
0.165
88 (36.2)
73 (35.3)
0.832
pT
Statistical analysis All statistical analyses were performed using SPSS statistical software (version 18; SPSS, Chicago, IL, USA). Unpaired Student’s t test, Fisher’s exact test, chi square test, Spearman’s correlation coefficients, log rank test, and COX proportional hazards regression model were used as appropriate. Disease-free survival was defined as time from
Invasive tumor size (average ± SD)
0.128
pN 0
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was found in 99 cases (22.2 %). HER2 genetic heterogeneity was identified in 30 out of 446 cases (6.7 %).
Table 1 continued HR negative (%)
HR positive (%)
1 mi
7 (2.9)
7 (3.4)
1
84 (34.6)
80 (38.6)
2
37 (15.2)
25 (12.1)
3 Ratio of metastatic to total lymph nodes (average ± SD)
p
Correlation between HR status and histopathologic characteristics
27 (11.1)
22 (10.6)
0.18 ± 0.23
0.17 ± 0.22
0.828
0.723
Stage 1
58 (23.9)
47 (22.7)
2
120 (49.4)
110 (53.1)
3
65 (26.7)
50 (24.2)
Absent
142 (59.4)
117 (56.5)
Present
97 (40.6)
90 (43.5)
Lymphovascular invasion 0.564
SD standard deviation
trastuzumab treatment to recurrence of breast cancer at any site. All tests were two-sided, and statistical significance was set at 5 %.
Results Clinicopathological characteristics of the study population All 450 patients were women, and their median age at diagnosis was 49 years (range 22–79 years). Tumor sizes ranged from 0.1 to 8.2 cm (median: 2.2 cm). Two hundred and eight cases were pT1 tumors, 229 were pT2, and 11 were pT3. There was one invasive tumor in 312 cases, two in 39 cases, three in nine cases, and more than three in 87 cases. In lymph node analysis, 161 tumors were pN0 stage, 14 were pN1mi, 164 were pN1, 62 were pN2, and 49 were pN3. Paget disease was identified in 18 out of 450 (4.0 %) cases. Higher score between ER and PR Allred scores was 8 in 82 cases, 7 in 54 cases, 6 in 28 cases, 5 in 23 cases, 4 in 17 cases, 3 in 16 cases, and 2 in three cases. Of the original 450 cases included in the analysis, only 446 cases had HER2 status determined, because SISH assays failed to identify HER2 signals in four cases due to poor sample quality. Of the 446 cases that had assessable HER2 status, 409 cases had three assessable cores, 29 cases had two, and eight cases had only one assessable core for SISH. High levels of HER2 gene amplification were observed in 347 out of 446 cases (77.8 %), whereas low-level amplification
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The relationship between HR status and various clinicopathological parameters is summarized in Table 1. Carcinomas with micropapillary or mucinous differentiation were more frequently found in the HR?/HER2? group (p \ 0.001). HR-/HER2? tumors showed higher nuclear grade, less tubule formation, and higher histologic grade than HR?/HER2? tumors (p \ 0.001, p = 0.014, and p \ 0.001, respectively). Apocrine features were highly apparent in HR-/HER2? tumors (p \ 0.001). Although the presence of extensive intraductal components was not significantly different between the two groups (p = 0.096), the overall DCIS percentage was significantly higher in HR-/HER2? tumors (p = 0.037). Lymphocytic infiltration within and around the invasive tumor was significantly higher in HR-/HER2? tumors (p \ 0.001). In particular, 31.7 % of HR-/HER2? tumors had diffuse and high lymphocytic infiltration. While the incidence of Paget disease was higher in HR-/HER2? tumors (5.8 %) than in HR?/HER2? tumors (1.9 %), the difference was not statistically significant (p = 0.052). Tumor size, pT, lymph node metastasis, stage, and the presence of lymphovascular invasion were not different between the two groups. Correlation between HR status and HER2 status Equivocal results for HER2 immunohistochemistry were more frequent in HR?/HER2? tumors (Table 2, p \ 0.001). Furthermore, average HER2 copy number and HER2/CEP17 ratio were significantly lower in HR?/ HER2? tumors (p \ 0.001 for both). HR?/HER2? tumors more frequently showed HER2 genetic heterogeneity (p = 0.001), but p53 expression was significantly correlated with HR-/HER2? tumors (p = 0.042). The presence of CEP17 polysomy and the average CEP17 copy number were not different between the two groups. Correlation between continuous variables ER Allred score was significantly correlated with PR Allred score (Table 3, q = 0.748, p \ 0.001). Both ER and PR Allred scores showed inverse correlations with HER2/ CEP17 ratio (ER: q = -0.287, p \ 0.001; PR: q = -0.209, p \ 0.001). Similarly, ER or PR Allred score and the average HER2 copy number also showed negative correlations (ER: q = -0.296, p \ 0.001; PR: q = -0.223, p \ 0.001).
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Table 2 Comparison between HR status and HER2 status in HER2positive breast cancer HR negative (%)
HR positive (%)
p
Prognostic significance of clinicopathologic factors
HER2 immunohistochemistry \0.001
1 ? or 2?
20 (8.2)
44 (21.3)
3?
223 (91.8)
163 (78.7)
Absent
203 (86.0)
178 (88.1)
Present
33 (14.0)
24 (11.9)
Average HER2 copy number (average ± SD)
12.1 ± 3.8
10.0 ± 4.7 \0.001
Average CEP17 number (average ± SD)
1.7 ± 0.5
1.7 ± 0.5
0.453
HER2/CEP17 ratio (average ± SD)
7.2 ± 2.8
6.0 ± 3.0
\0.001
\0.001
CEP17 polysomy 0.57
HER2 SISH status Low-level amplification
34 (14.0)
65 (31.4)
High-level amplification
207 (85.2)
140 (67.6)
Absent
234 (97.1)
182 (88.8)
Present
7 (2.9)
23 (11.2)
94 (40.3) 139 (59.7)
101 (50.2) 100 (49.8)
HER2 genetic heterogeneity 0.001
p53 Negative Positive
scores (q = 0.009, p = 0.849 and r = 0.046, p = 0.328, respectively).
0.042
SD standard deviation
DCIS percentage was not correlated with ER Allred score (q = -0.069, p = 0.144), but was positively correlated with the average HER2 copy number (q = 0.105, p = 0.027) and HER2/CEP17 ratio (q = 0.160, p = 0.001). Pathologic tumor size and overall DCIS percentage also showed a significant inverse correlation (q = -0.313, p \ 0.001). Although the ratio of metastatic to total examined lymph nodes was positively correlated with the average HER2 copy number (q = 0.158, p = 0.001) and HER2/CEP17 ratio (q = 0.140, p = 0.003), it showed no correlation with ER or PR Allred
The median follow-up for patients was 42.1 months (range 21–105 months). There were 35 recurrences. As shown in Table 4, pT stage and lymphovascular invasion were the prognostic factors for disease-free survival in all the patients. Lymph node metastasis and HR status also showed marginal prognostic significance (p = 0.056 and p = 0.077, respectively). In multivariate analysis including pT stage, lymphovascular invasion, HR status, high pT stage (pT2 *3 vs. pT1; hazard ratio, 2.370; 95 % confidence interval, 1.102–5.096; p = 0.027), the presence of lymphovascular invasion (positive vs. negative; hazard ratio, 2.806; 95 % confidence interval, 1.367–5.761; p = 0.005), and HR negativity (negative vs. positive; hazard ratio, 2.202; 95 % confidence interval, 1.074–4.513; p = 0.031) were found to be independent prognostic indicators of poor disease-free survival. We further investigated the prognostic significance of clinicopathologic factors in each subtype according to the HR status. In HR- tumors, pT stage, lymphovascular invasion, and lymphocytic infiltration were the prognostic factors for disease-free survival. In HR? tumors, only the presence of lymphovascular invasion was a worse prognostic factor for disease-free survival.
Discussion In the present study, we examined whether HR status is associated with significant differences in the histopathologic features of HER2-positive breast cancer. We observed that HR-/HER2? tumors had higher nuclear grade, less tubule formation, higher histologic grade, more frequent apocrine features, more diffuse and high lymphocytic infiltration, stronger HER2 immunohistochemical staining, higher average HER2 copy number and HER2/
Table 3 Correlation coefficients between each continuous variable
ER Allred score (p value)
PR allred score
HER2 copy number
HER2/CEP17 ratio
DCIS percentage
0.748 (\0.001)
-0.296 (\0.001)
-0.287 (\0.001)
-0.069 (0.144)
0.009 (0.849)
-0.223 (\0.001)
-0.209 (\0.001)
-0.032 (0.503)
0.046 (0.328)
0.857 (\0.001)
0.105 (0.027)
0.158 (0.001)
PR Allred score (p value)
–
HER2 copy number (p value)
–
–
HER2/CEP17 ratio (p value)
–
–
–
DCIS percentage (p value)
–
–
–
0.160 (0.001) –
Metastatic/total lymph nodes ratio
0.140 (0.003) -0.035 (0.455)
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Table 4 Disease-free survival analysis in HER2-positive breast cancer Variables
All cases Hazard ratio
HR negative 95 % CI
p
Hazard ratio
HR positive 95 % CI
p
Hazard ratio
95 % CI
p
pT stage T1
–
T2–T3
2.516
– 1.179–5.372
0.017
0.978–5.746
0.056
0.669–2.788
0.393
0.409–1.655
0.584
1.469–6.138
0.003
0.399–1.665
0.575
0.756–2.862
0.256
3.1
– 1.230–7.815
0.016
1.931
0.730–5.286
0.181
0.449–2.855
0.792
0.209–1.146
0.1
1.061–5.570
0.036
5.861
0.141–2.566
0.492
20.753
0.675–3.349
0.318
0.512–7.279
0.331
0.631–39.123
0.128
0.384–4.126
0.704
0.382–4.480
0.669
1.265–27.149
0.024
\0.001 to [100
0.746
0.349–4.078
0.779
0.082–5.031
0.675
0.200–2.851
0.679
0.161–1.740
0.295
0.168–1.961
0.376
Lymph node metastasis Negative
–
Positive
2.371
Histologic grade 2 3
–
– 1.365
1.965
–
– 1.133
4.97 – 1.259
Apocrine feature Negative
–
Positive
0.823
– 0.49
– 1.308
Lymphovascular invasion Negative
–
Positive
3.003
– 2.431
–
Paget disease Negative
–
Positive
0.815
– 0.601
–
Extensive intraductal component Negative
–
Positive
1.471
Lymphocyte infiltration Other Diffuse, high
–
– 0.417
1.503
–
– 0.147–1.183
0.1
0.293
0.352–2.048
0.717
0.685
0.399–1.948
0.755
0.678–2.707
0.389
0.257–1.072
0.077
1.193 –
0.087–0.983
0.047
0.644
0.204–2.306
0.541
0.756
0.322–3.653
0.895
0.836–5.424
0.113
HER2 immunohistochemistry 1? or 2?
–
3?
0.85
–
–
HER2 SISH status Low-level amplification
–
High-level amplification
0.881
– 1.085
– 0.53
p53 expression Negative
–
Positive
1.355
– 2.129
– 0.574
HR status Negative
–
Positive
0.525
–
–
–
–
CI confidence interval
CEP17 ratio, the absence of HER2 genetic heterogeneity, and more p53 expression than HR?/HER2? tumors. HR negativity was an independent prognostic indicator of poor disease-free survival. Distinct histopathologic features may reflect the significant differences in clinical behavior between HR?/HER2? and HR-/HER2? tumors [3–6]. To the best of our knowledge, this is the first large, comprehensive analysis of histopathologic characteristics in
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HER2-positive breast cancer according to HR status in East Asian patients. Interestingly, we found significant inverse correlations between ER or PR Allred score and average HER2 copy number or HER2/CEP17 ratio. These findings are consistent with those obtained by a previous study, which reported an inverse correlation between HER2 amplification and ER expression [7]. Additionally, equivocal HER2
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immunohistochemistry results (2?) were more frequent in HR?/HER2? tumors. We also analyzed the association between clinicopathologic variables across the two groups, which were dichotomized according to HR status, as this could suggest substantial differences in clinical outcome, and found statistically significant differences in histologic grade, apocrine features, and the presence of tumor-infiltrating lymphocytes. Currently all HER2-positive breast cancer patients are treated in a standardized manner. Given the histologic differences in HR-/HER2? and HR?/ HER2? tumors, development of personalized treatment strategies might be warranted for effective treatment of HER2-positive breast cancer, rather than a single, standardized, ‘‘one size fits all’’ approach. The importance of HER2 heterogeneity was recently recognized [10, 17–19]. In our study, HER2 genetic heterogeneity was more frequently observed in HR?/HER2? tumors, which had relatively lower levels of HER2 gene amplification compared to the HR-/HER2? group. In our study population, all but one case with HER2 genetic heterogeneity showed overall low levels of HER2 gene amplification. HER2 heterogeneity may contribute to inaccurate assessment of HER2 status. This is clinically important, given the benefit that patients with low-level HER2 amplification receive from trastuzumab treatment [20, 21]. Pathologists should pay close attention to accurate assessment of HER2 copy number in HER2 in situ hybridization analyses, especially when evaluating cases with low-level gene amplification. Intraductal components were not significantly different between HR?/HER2? and HR-/HER2? tumors, but DCIS percentage was positively correlated with average HER2 copy number and HER2/CEP17 ratio. This suggests that HER2 status itself is the important factor in correlation with the extent of DCIS. These results are comparable to previous reports from the Memorial SloanKettering Cancer Center; in which HER2-positive breast cancer was significantly associated with the presence of an extensive intraductal component (p \ 0.0001) [4]. We also showed that pathologic tumor size and DCIS percentage had a significant inverse correlation. In addition, several studies suggested that multifocal or multicentric cancers were more frequently present in HER2-positive breast cancer than in HR ?/HER2-negative breast cancer [4, 11, 22]. In the present study, we found that 31.7 % of patients with HR-/HER2? cancer had multifocal or multicentric cancers. In the light of these findings, the presence of diffuse DCIS with multifocal invasive tumors of small pathologic size can be considered as a characteristic histopathologic feature of HR-/HER2? cancer. This morphologic feature can adversely affect the accurate assessment of radiologic and pathologic tumor size and determination of pT stage.
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Variation of the presence and the extent of nodal involvement in different subtypes of breast cancer is also reported. HER2-positive tumors are more likely to have nodal involvement [22, 23]. However, the association between HR status and nodal involvement in HER2-positive breast cancer has not been clarified. In our study, pN stage was not significantly different between HR?/HER2? and HR-/HER2? tumors, but the ratio of metastatic to total examined lymph nodes was significantly correlated with average HER2 copy number and HER2/CEP17 ratio. We confirmed a high frequency of apocrine differentiation in HER2-positive breast cancer, and showed a strong association between apocrine features and HR-/HER2? tumors. While apocrine differentiation of breast cancer can be seen in all subtypes, its association is strongest with the HER2-positive subtype [13]. Lehmann-Che et al. reported that molecular apocrine breast cancer showed frequent 3? HER2 expression (67 %) by gene expression array analysis, and was negative for ER (93 %) by immunohistochemistry. We also found significantly higher expression of p53 in HR-/HER2? breast cancer. Similarly, in TCGA data analysis, TP53 mutation was much higher in HER2enriched subtypes (72 %) than in luminal A (12 %) or luminal B (29 %) subtypes [2]. In addition, a significant inverse correlation between ER and p53 expression in breast cancer was reported [24]. Paget disease comprises 0.5–5 % of all breast cancers, and underlying carcinoma is present in most cases [25]. Since the underlying carcinoma usually shows the same immunohistochemical staining pattern, Paget disease appears to originate from the underlying ducts and then subsequently migrates to the epidermis [25]. In the present study, Paget disease was more frequently observed in HR-/HER2? tumors, even though the overall incidence of Paget disease was low. Therefore, close follow-up with examination of the nipple is warranted in patients treated with nipple-sparing surgery, especially in those with HR-/ HER2? tumors. Recently, the prognostic and predictive relevance of tumor-infiltrating lymphocytes in adjuvant and neoadjuvant settings was determined in breast cancer [26–29]. The significance of tumor-infiltrating lymphocytes in breast cancer was largely related to the triple-negative subtype; however, several studies indicate the additional importance of tumor-infiltrating lymphocytes in HER2-positive breast cancer. In this study, patients with diffuse and high lymphocytic infiltration showed better disease-free survival than other patients in HR- tumors. However, survival difference was not found in patients with HR? tumors. In general, the level of tumor-infiltrating lymphocytes is inversely correlated with ER and/or PR expression, but positively correlated with pathologic complete response rate [27, 28, 30]. We, therefore, analyzed the level of
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lymphocytic infiltration according to HR status in HER2positive breast cancer, and found an association between diffuse and high lymphocytic infiltration and negative HR status. Our findings could provide an additional rationale to explain the higher rates of pathologic complete response in HR-/HER2? tumors compared to HR?/HER2? tumors in neoadjuvant settings [3, 4]. The retrospective design of this study represents a limitation. Nevertheless, this study provides a relevant basis for the apparent differences in clinical behaviors between HR?/HER2? and HR-/HER2? breast cancers. Further studies are necessary to confirm our results in HER2positive breast cancer. In conclusion, HR-/HER2? tumors had higher nuclear grade, less tubule formation, higher histologic grade, more frequent apocrine features, more diffuse and abundant lymphocytic infiltration, stronger HER2 immunohistochemical staining, higher average HER2 copy number and HER2/CEP17 ratio, the absence of HER2 genetic heterogeneity, and more p53 expression than HR?/HER2? tumors. These distinct histopathologic features may be relevant to the significant differences in clinical behavior between HR?/HER2? and HR-/HER2? tumors. Acknowledgments This study was supported by 2013 ‘‘Moon-Ho Yang’’ research fund from The Korean Society of Pathologists. Conflict of interest
The authors declare no conflict of interest.
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9. Wolff AC, Hammond ME, Hicks DG et al (2013) Recommendations for human epidermal growth factor receptor 2 testing in breast cancer: American Society of Clinical Oncology/College of American Pathologists clinical practice guideline update. Arch Pathol Lab Med 138(2):241–256. doi:10.5858/arpa.2013-0953SA 10. Seol H, Lee HJ, Choi Y et al (2012) Intratumoral heterogeneity of HER2 gene amplification in breast cancer: its clinicopathological significance. Mod Pathol 25:938–948 11. Pekar G, Hofmeyer S, Tabar L et al (2013) Multifocal breast cancer documented in large-format histology sections: long-term follow-up results by molecular phenotypes. Cancer 119:1132–1139 12. Lakhani SR, Eliis IO, Schnitt SJ, Tan PH, van de Vijver MJ (eds) (2012) WHO classification of tumours of the breast, 4th edn. International Agency for Research on Cancer, Lyon 13. Bhargava R, Beriwal S, Striebel JM, Dabbs DJ (2010) Breast cancer molecular class ERBB2: preponderance of tumors with apocrine differentiation and expression of basal phenotype markers CK5, CK5/6, and EGFR. Appl Immunohistochem Mol Morphol 18:113–118 14. Nitta H, Hauss-Wegrzyniak B, Lehrkamp M et al (2008) Development of automated brightfield double in situ hybridization (BDISH) application for HER2 gene and chromosome 17 centromere (CEN 17) for breast carcinomas and an assay performance comparison to manual dual color HER2 fluorescence in situ hybridization (FISH). Diagn Pathol 3:41 15. Ma Y, Lespagnard L, Durbecq V et al (2005) Polysomy 17 in HER-2/neu status elaboration in breast cancer: effect on daily practice. Clin Cancer Res 11:4393–4399 16. Vance GH, Barry TS, Bloom KJ et al (2009) Genetic heterogeneity in HER2 testing in breast cancer: panel summary and guidelines. Arch Pathol Lab Med 133:611–612 17. Lewis JT, Ketterling RP, Halling KC et al (2005) Analysis of intratumoral heterogeneity and amplification status in breast carcinomas with equivocal (2?) HER-2 immunostaining. Am J Clin Pathol 124:273–281 18. Striebel JM, Bhargava R, Horbinski C, Surti U, Dabbs DJ (2008) The equivocally amplified HER2 FISH result on breast core biopsy: indications for further sampling do affect patient management. Am J Clin Pathol 129:383–390 19. Brunelli M, Manfrin E, Martignoni G et al (2009) Genotypic intratumoral heterogeneity in breast carcinoma with HER2/neu amplification: evaluation according to ASCO/CAP criteria. Am J Clin Pathol 131:678–682 20. Paik S, Kim C, Wolmark N (2008) HER2 status and benefit from adjuvant trastuzumab in breast cancer. N Engl J Med 358:1409–1411 21. Dowsett M, Procter M, McCaskill-Stevens W et al (2009) Disease-free survival according to degree of HER2 amplification for patients treated with adjuvant chemotherapy with or without 1 year of trastuzumab: the HERA Trial. J Clin Oncol 27:2962–2969 22. Wiechmann L, Sampson M, Stempel M et al (2009) Presenting features of breast cancer differ by molecular subtype. Ann Surg Oncol 16:2705–2710 23. Viani GA, Afonso SL, Stefano EJ, De Fendi LI, Soares FV (2007) Adjuvant trastuzumab in the treatment of her-2-positive early breast cancer: a meta-analysis of published randomized trials. BMC Cancer 7:153 24. Yamashita H, Nishio M, Toyama T et al (2004) Coexistence of HER2 over-expression and p53 protein accumulation is a strong prognostic molecular marker in breast cancer. Breast Cancer Res 6:R24–R30 25. Sek P, Zawrocki A, Biernat W, Piekarski JH (2010) HER2 molecular subtype is a dominant subtype of mammary Paget’s
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PRECLINICAL STUDY
Two histopathologically different diseases: hormone receptor-positive and hormone receptor-negative tumors in HER2-positive breast cancer Hee Jin Lee • In Ah Park • So Yeon Park • An Na Seo • Bora Lim Yun Chai • In Hye Song • Na Eun Kim • Joo Young Kim • Jong Han Yu • Jin-Hee Ahn • Gyungyub Gong
•
Received: 13 March 2014 / Accepted: 23 April 2014 / Published online: 13 May 2014 Ó Springer Science+Business Media New York 2014
Abstract The clinical behavior of human epidermal growth factor 2 (HER2)-positive breast cancer, including pathologic complete response rate and pattern of relapse and metastasis, differs substantially according to hormone receptor (HR) status. We investigated various histopathologic features of HER2-positive breast cancer and their correlation with HR status. We retrospectively analyzed tumors of 450 HER2-positive breast cancer patients treated with chemotherapy and 1 year of trastuzumab. HR-/ HER2? tumors showed higher nuclear grade, less tubule Hee Jin Lee and In Ah Park have contributed equally to this work. H. J. Lee I. A. Park Y. Chai I. H. Song N. E. Kim J. Y. Kim G. Gong (&) Department of Pathology, Asan Medical Center, University of Ulsan College of Medicine, 88 Olympic-ro 43-gil, Songpa-gu, Seoul 138-736, South Korea e-mail: [email protected] S. Y. Park A. N. Seo Department of Pathology, Seoul National University Bundang Hospital, Seongnam, Gyeonggi, South Korea S. Y. Park Department of Pathology, Seoul National University College of Medicine, Seoul, South Korea B. Lim Department of Hematology-Oncology, Penn State Hershey Cancer Institute, Penn State College of Medicine, Hershey, PA, USA J. H. Yu Department of Surgery, Asan Medical Center, University of Ulsan College of Medicine, Seoul, South Korea J.-H. Ahn Department of Oncology, Asan Medical Center, University of Ulsan College of Medicine, Seoul, South Korea
formation, higher histologic grade, frequent apocrine features, diffuse and abundant lymphocytic infiltration, strong HER2 immunohistochemical staining (3?), higher average HER2 copy number and HER2/CEP17 ratio, the absence of HER2 genetic heterogeneity, and greater p53 expression than HR?/HER2? tumors. An inverse correlation was observed between estrogen receptor or progesterone receptor Allred score and average HER2 copy number or HER2/CEP17 ratio. The percentage of ductal carcinoma in situ (DCIS) within the tumor was negatively correlated with ER Allred score, but positively correlated with average HER2 copy number and HER2/CEP17 ratio. Pathologic tumor size and DCIS percentage also showed a significant inverse correlation. Ratio of metastatic to total examined lymph node number was significantly correlated with average HER2 copy number and HER2/CEP17 ratio. High pT stage (hazard ratio, 2.370; p = 0.027), the presence of lymphovascular invasion (hazard ratio, 2.806; p = 0.005), and HR negativity (hazard ratio, 2.202; 1.074–4.513; p = 0.031) were found to be independent prognostic indicators of poor disease-free survival. In conclusion, HR?/HER2? and HR-/HER2? breast cancer showed distinct histopathologic features that may be relevant to their distinct clinical behavior. Keywords Breast carcinoma Gene amplification HER2 Hormone receptor
Introduction With the advance of genomic technology, the heterogeneous nature of breast cancer has been well recognized [1]. Recently, the Cancer Genome Atlas (TCGA) Network determined the existence of at least two subtypes of human
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epidermal growth factor 2 (HER2)-positive breast cancers based on comprehensive analysis [2]. One of the two subtypes is the HER2-enriched mRNA subtype, characterized by significantly higher expression of receptor tyrosine kinases including EGFR, HER2, and FGFR4. Another group belongs to the luminal mRNA subtype that displays higher expression of a luminal cluster of genes: GATA3, BCL2, and ESR1. These two subgroups of HER2positive tumors are, therefore, genetically distinct from each other. The clinical behaviors of hormone receptor-positive/ HER2-positive (HR?/HER2?) and hormone receptornegative/HER2-positive (HR-/HER2?) tumors are quite different. In neoadjuvant settings, HR-/HER2? tumors show higher pathologic complete response rate than HR?/ HER2? tumors [3, 4]. Patterns of relapse, metastatic spread, and survival are also different [5, 6]. ER-/HER2? patients showed earlier and higher level of relapse than ER?/HER2? patients [5]. HR-/HER2? tumors tended to have liver as the first metastatic site than HR?/HER2? tumors [6]. HR-/HER2? tumors were found to be of higher histologic grade and stage compared to HR?/ HER2? tumors [7]. However, the detailed histologic features of HR- and HR? HER2-positive breast cancer remain to be explored. We, therefore, performed the first comprehensive histologic analysis of surgically resected HER2-positive breast cancer from a large series of Asian patients treated with adjuvant chemotherapy and trastuzumab. We compared the clinicopathologic factors between HR?/HER2? and HR-/HER2? tumors, and analyzed the correlation among the various factors. In addition, we found prognostic significance of clinicopathologic factors in HER2-positive breast cancer.
Materials and methods Patients and tissue specimens A total of 450 HER2-positive breast cancer patients who underwent surgery for primary breast cancer between 2006 and 2011 at the Asan Medical Center, who had available formalin-fixed paraffin-embedded tissue samples for analysis, were included in this study. All patients were preoperatively chemo- and radiotherapy naı¨ve, and all underwent adjuvant treatment. Of 450 patients, 161 patients with node-negative breast cancer were treated with four cycles of adjuvant anthracycline and cyclophosphamide (AC, adriamycin 60 mg/m2 and cyclophosphamide 600 mg/m2) and 1 year of trastuzumab. The remaining 289 patients had node-positive breast cancer, and were treated with either four cycles of AC followed by four cycles of
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paclitaxel (175 mg/m2) or four cycles of AC followed by four cycles of docetaxel (75 mg/m2) and 1 year of trastuzumab. Trastuzumab (6 mg/kg) was given every 3 weeks. Clinicopathologic information was obtained from the patient’s medical records and surgical pathologic reports. Expressions of standard biomarkers including estrogen receptor (ER), progesterone receptor (PR), HER2, and p53 were reviewed in full sections that were immunohistochemically stained at the time of diagnosis. ER and PR levels were regarded as positive if there was at least 1 % positive tumor nuclei staining [8]. HR? group was defined as ER- or PR-positive tumors. Additionally, Allred score was calculated for ER and PR [3]. HER2-overexpressing tumors were defined as those with scores of 3? by immunohistochemistry or gene amplification by fluorescence in situ hybridization (FISH) or silver in situ hybridization (SISH) [9]. Among 450 patients, 386 patients had an immunohistochemical score of 3; 61 patients had a score of 2; and three patients had a score of 1. FISH or SISH was performed for samples with immunohistochemical score of 1 or 2, and HER2 amplification was observed in all cases. Samples with positive staining of 10 % or more cells were considered p53 positive [10]. Exemption from informed consent after de-identification of information was approved by the Institutional Review Board of Asan Medical Center. Histologic evaluation Hematoxylin and eosin (H&E)-stained sections were histopathologically analyzed for the number of invasive tumors, histologic subtype and grade, nuclear grade, tubule formation, apocrine features, lymphocytic infiltration (location: absent/minimal, border of tumor, or diffuse; abundance: absent/minimal, moderate, or high), ductal carcinoma in situ (DCIS) percentage, the presence or absence of extensive intraductal component and Paget disease, tumor size, pT stage, pN stage, ratio of metastatic to total examined lymph nodes, and lymphovascular invasion [11]. Histologic type was defined based on the 2012 WHO classification criteria, and histologic grade was assessed employing the modified Bloom–Richardson classification [12]. Apocrine feature of tumor cells included abundant granular eosinophilic cytoplasm, cytoplasmic vacuolization/clearing, round vesicular nuclei, and prominent eosinophilic nucleoli [13]. Tissue microarray construction Formalin-fixed, paraffin-embedded tissue samples were arrayed by a tissue-arraying instrument. After the review of HER2 expression in immunostained whole tumor section slides, three areas were selected and arrayed in 1-mm-
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diameter cores. If the tumor showed heterogeneous staining for HER2, representative areas of different stainings were chosen.
617 Table 1 Comparison between HR status and histopathologic factors in HER2-positive breast cancer HR negative (%)
HR positive (%)
p
Invasive carcinoma of no special type
235 (96.7)
185 (89.4)
\0.001
Carcinoma with micropapillary differentiation
6 (2.5)
14 (6.8)
Carcinoma with mucinous differentiation
0 (0.0)
8 (3.9)
Metaplastic carcinoma
2 (0.8)
0 (0.0)
2
65 (26.7)
103 (49.8)
3
178 (73.3)
104 (50.2)
SISH assays for HER2 gene amplification Histologic type
Automated SISH assays on tissue microarray sections were performed with INFORM HER2 DNA and Chromosome 17 (CEP17) probes (Ventana Medical Systems, Tucson, AZ, USA) using an ultraView SISH Detection Kit (Ventana Medical Systems) according to the manufacturer’s protocols [14]. HER2 gene amplification status was separately assessed in the three tissue microarray cores from each sample. Fifty tumor cells per core were evaluated; consequently, a total of 150 cells were counted for each case where possible. HER2/CEP17 ratio, the average HER2 gene copy number per cell, and the average CEP17 copy number per cell were evaluated. In cases with distinct subpopulations of amplified and non-amplified cells, counting was weighted by the percentage of each population in the entire tumor. According to American Society of Clinical Oncology (ASCO)/College of American Pathologists (CAP) criteria, HER2 gene amplification was determined as follows: (1) the average ratio of HER2 to CEP17 signals was C2.0, or (2) the ratio was \2.0 but an average HER2 copy number of C6 [9]. HER2 amplification status was classified into two groups: a high-level amplification group defined as having a HER2/CEP17 ratio C4.0, and a low-level amplification group with 2.0 B HER2/CEP17 ratio \4.0. Tumors with a HER2/CEP17 ratio\2.0 and average HER2 copy number C4 and \6 were considered equivocal for amplification. The CEP17 copy number alteration was evaluated using a modification described by Ma et al. [15]. Cases with average CEP17 copy numbers between 1.25 and 2.25 were defined as having disomy 17. The remaining cases were aneusomic for chromosome 17, i.e., they had either monosomy 17 (\1.25 signals per cells), low polysomy 17 ([2.25 but B3.75 signals per cell), or high polysomy 17 ([3.75 signals per cell). HER2 genetic heterogeneity was defined, according to the CAP guidelines, as the existence of tumor cells with a HER2/CEP17 ratio [2.2 in 5–50 % of all the examined tumor cells [16].
Nuclear grade \0.001
Tubule formation 1
2 (0.8)
2 (1.0)
2
56 (23.2)
72 (35.0)
3
183 (75.9)
132 (64.1)
64 (26.3)
104 (50.2)
179 (73.7)
103 (49.8)
Absent
50 (20.7)
91 (44.2)
Present
191 (79.3)
115 (55.8)
Histologic grade 2 3
0.014
\0.001
Apocrine feature \0.001
Extensive intraductal component Absent
141 (58.8)
138 (66.7)
Present
99 (41.3)
69 (33.3)
27.9 ± 30.5
22.4 ± 25.1
0.037
229 (94.2) 14 (5.8)
203 (98.1) 4 (1.9)
0.052
Absent/minimal
84 (34.6)
115 (55.6)
\0.001
Border, moderate
36 (14.8)
28 (13.5)
Border, high
24 (9.9)
13 (6.3)
Diffuse, moderate
19 (7.8)
21 (10.1)
Diffuse, high
77 (31.7)
27 (13.0)
DCIS percentage (average ± SD)
0.096
Paget disease Absent Present Lymphocyte infiltration
Number of invasive tumor foci 1
163 (67.1)
149 (72.0)
C2
77 (31.7)
58 (28.0)
0.233
1
122 (50.6)
86 (41.5)
2
113 (46.9)
112 (54.1)
3
6 (2.5)
9 (4.3)
2.3 ± 1.1
2.4 ± 1.2
0.165
88 (36.2)
73 (35.3)
0.832
pT
Statistical analysis All statistical analyses were performed using SPSS statistical software (version 18; SPSS, Chicago, IL, USA). Unpaired Student’s t test, Fisher’s exact test, chi square test, Spearman’s correlation coefficients, log rank test, and COX proportional hazards regression model were used as appropriate. Disease-free survival was defined as time from
Invasive tumor size (average ± SD)
0.128
pN 0
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was found in 99 cases (22.2 %). HER2 genetic heterogeneity was identified in 30 out of 446 cases (6.7 %).
Table 1 continued HR negative (%)
HR positive (%)
1 mi
7 (2.9)
7 (3.4)
1
84 (34.6)
80 (38.6)
2
37 (15.2)
25 (12.1)
3 Ratio of metastatic to total lymph nodes (average ± SD)
p
Correlation between HR status and histopathologic characteristics
27 (11.1)
22 (10.6)
0.18 ± 0.23
0.17 ± 0.22
0.828
0.723
Stage 1
58 (23.9)
47 (22.7)
2
120 (49.4)
110 (53.1)
3
65 (26.7)
50 (24.2)
Absent
142 (59.4)
117 (56.5)
Present
97 (40.6)
90 (43.5)
Lymphovascular invasion 0.564
SD standard deviation
trastuzumab treatment to recurrence of breast cancer at any site. All tests were two-sided, and statistical significance was set at 5 %.
Results Clinicopathological characteristics of the study population All 450 patients were women, and their median age at diagnosis was 49 years (range 22–79 years). Tumor sizes ranged from 0.1 to 8.2 cm (median: 2.2 cm). Two hundred and eight cases were pT1 tumors, 229 were pT2, and 11 were pT3. There was one invasive tumor in 312 cases, two in 39 cases, three in nine cases, and more than three in 87 cases. In lymph node analysis, 161 tumors were pN0 stage, 14 were pN1mi, 164 were pN1, 62 were pN2, and 49 were pN3. Paget disease was identified in 18 out of 450 (4.0 %) cases. Higher score between ER and PR Allred scores was 8 in 82 cases, 7 in 54 cases, 6 in 28 cases, 5 in 23 cases, 4 in 17 cases, 3 in 16 cases, and 2 in three cases. Of the original 450 cases included in the analysis, only 446 cases had HER2 status determined, because SISH assays failed to identify HER2 signals in four cases due to poor sample quality. Of the 446 cases that had assessable HER2 status, 409 cases had three assessable cores, 29 cases had two, and eight cases had only one assessable core for SISH. High levels of HER2 gene amplification were observed in 347 out of 446 cases (77.8 %), whereas low-level amplification
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The relationship between HR status and various clinicopathological parameters is summarized in Table 1. Carcinomas with micropapillary or mucinous differentiation were more frequently found in the HR?/HER2? group (p \ 0.001). HR-/HER2? tumors showed higher nuclear grade, less tubule formation, and higher histologic grade than HR?/HER2? tumors (p \ 0.001, p = 0.014, and p \ 0.001, respectively). Apocrine features were highly apparent in HR-/HER2? tumors (p \ 0.001). Although the presence of extensive intraductal components was not significantly different between the two groups (p = 0.096), the overall DCIS percentage was significantly higher in HR-/HER2? tumors (p = 0.037). Lymphocytic infiltration within and around the invasive tumor was significantly higher in HR-/HER2? tumors (p \ 0.001). In particular, 31.7 % of HR-/HER2? tumors had diffuse and high lymphocytic infiltration. While the incidence of Paget disease was higher in HR-/HER2? tumors (5.8 %) than in HR?/HER2? tumors (1.9 %), the difference was not statistically significant (p = 0.052). Tumor size, pT, lymph node metastasis, stage, and the presence of lymphovascular invasion were not different between the two groups. Correlation between HR status and HER2 status Equivocal results for HER2 immunohistochemistry were more frequent in HR?/HER2? tumors (Table 2, p \ 0.001). Furthermore, average HER2 copy number and HER2/CEP17 ratio were significantly lower in HR?/ HER2? tumors (p \ 0.001 for both). HR?/HER2? tumors more frequently showed HER2 genetic heterogeneity (p = 0.001), but p53 expression was significantly correlated with HR-/HER2? tumors (p = 0.042). The presence of CEP17 polysomy and the average CEP17 copy number were not different between the two groups. Correlation between continuous variables ER Allred score was significantly correlated with PR Allred score (Table 3, q = 0.748, p \ 0.001). Both ER and PR Allred scores showed inverse correlations with HER2/ CEP17 ratio (ER: q = -0.287, p \ 0.001; PR: q = -0.209, p \ 0.001). Similarly, ER or PR Allred score and the average HER2 copy number also showed negative correlations (ER: q = -0.296, p \ 0.001; PR: q = -0.223, p \ 0.001).
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619
Table 2 Comparison between HR status and HER2 status in HER2positive breast cancer HR negative (%)
HR positive (%)
p
Prognostic significance of clinicopathologic factors
HER2 immunohistochemistry \0.001
1 ? or 2?
20 (8.2)
44 (21.3)
3?
223 (91.8)
163 (78.7)
Absent
203 (86.0)
178 (88.1)
Present
33 (14.0)
24 (11.9)
Average HER2 copy number (average ± SD)
12.1 ± 3.8
10.0 ± 4.7 \0.001
Average CEP17 number (average ± SD)
1.7 ± 0.5
1.7 ± 0.5
0.453
HER2/CEP17 ratio (average ± SD)
7.2 ± 2.8
6.0 ± 3.0
\0.001
\0.001
CEP17 polysomy 0.57
HER2 SISH status Low-level amplification
34 (14.0)
65 (31.4)
High-level amplification
207 (85.2)
140 (67.6)
Absent
234 (97.1)
182 (88.8)
Present
7 (2.9)
23 (11.2)
94 (40.3) 139 (59.7)
101 (50.2) 100 (49.8)
HER2 genetic heterogeneity 0.001
p53 Negative Positive
scores (q = 0.009, p = 0.849 and r = 0.046, p = 0.328, respectively).
0.042
SD standard deviation
DCIS percentage was not correlated with ER Allred score (q = -0.069, p = 0.144), but was positively correlated with the average HER2 copy number (q = 0.105, p = 0.027) and HER2/CEP17 ratio (q = 0.160, p = 0.001). Pathologic tumor size and overall DCIS percentage also showed a significant inverse correlation (q = -0.313, p \ 0.001). Although the ratio of metastatic to total examined lymph nodes was positively correlated with the average HER2 copy number (q = 0.158, p = 0.001) and HER2/CEP17 ratio (q = 0.140, p = 0.003), it showed no correlation with ER or PR Allred
The median follow-up for patients was 42.1 months (range 21–105 months). There were 35 recurrences. As shown in Table 4, pT stage and lymphovascular invasion were the prognostic factors for disease-free survival in all the patients. Lymph node metastasis and HR status also showed marginal prognostic significance (p = 0.056 and p = 0.077, respectively). In multivariate analysis including pT stage, lymphovascular invasion, HR status, high pT stage (pT2 *3 vs. pT1; hazard ratio, 2.370; 95 % confidence interval, 1.102–5.096; p = 0.027), the presence of lymphovascular invasion (positive vs. negative; hazard ratio, 2.806; 95 % confidence interval, 1.367–5.761; p = 0.005), and HR negativity (negative vs. positive; hazard ratio, 2.202; 95 % confidence interval, 1.074–4.513; p = 0.031) were found to be independent prognostic indicators of poor disease-free survival. We further investigated the prognostic significance of clinicopathologic factors in each subtype according to the HR status. In HR- tumors, pT stage, lymphovascular invasion, and lymphocytic infiltration were the prognostic factors for disease-free survival. In HR? tumors, only the presence of lymphovascular invasion was a worse prognostic factor for disease-free survival.
Discussion In the present study, we examined whether HR status is associated with significant differences in the histopathologic features of HER2-positive breast cancer. We observed that HR-/HER2? tumors had higher nuclear grade, less tubule formation, higher histologic grade, more frequent apocrine features, more diffuse and high lymphocytic infiltration, stronger HER2 immunohistochemical staining, higher average HER2 copy number and HER2/
Table 3 Correlation coefficients between each continuous variable
ER Allred score (p value)
PR allred score
HER2 copy number
HER2/CEP17 ratio
DCIS percentage
0.748 (\0.001)
-0.296 (\0.001)
-0.287 (\0.001)
-0.069 (0.144)
0.009 (0.849)
-0.223 (\0.001)
-0.209 (\0.001)
-0.032 (0.503)
0.046 (0.328)
0.857 (\0.001)
0.105 (0.027)
0.158 (0.001)
PR Allred score (p value)
–
HER2 copy number (p value)
–
–
HER2/CEP17 ratio (p value)
–
–
–
DCIS percentage (p value)
–
–
–
0.160 (0.001) –
Metastatic/total lymph nodes ratio
0.140 (0.003) -0.035 (0.455)
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Table 4 Disease-free survival analysis in HER2-positive breast cancer Variables
All cases Hazard ratio
HR negative 95 % CI
p
Hazard ratio
HR positive 95 % CI
p
Hazard ratio
95 % CI
p
pT stage T1
–
T2–T3
2.516
– 1.179–5.372
0.017
0.978–5.746
0.056
0.669–2.788
0.393
0.409–1.655
0.584
1.469–6.138
0.003
0.399–1.665
0.575
0.756–2.862
0.256
3.1
– 1.230–7.815
0.016
1.931
0.730–5.286
0.181
0.449–2.855
0.792
0.209–1.146
0.1
1.061–5.570
0.036
5.861
0.141–2.566
0.492
20.753
0.675–3.349
0.318
0.512–7.279
0.331
0.631–39.123
0.128
0.384–4.126
0.704
0.382–4.480
0.669
1.265–27.149
0.024
\0.001 to [100
0.746
0.349–4.078
0.779
0.082–5.031
0.675
0.200–2.851
0.679
0.161–1.740
0.295
0.168–1.961
0.376
Lymph node metastasis Negative
–
Positive
2.371
Histologic grade 2 3
–
– 1.365
1.965
–
– 1.133
4.97 – 1.259
Apocrine feature Negative
–
Positive
0.823
– 0.49
– 1.308
Lymphovascular invasion Negative
–
Positive
3.003
– 2.431
–
Paget disease Negative
–
Positive
0.815
– 0.601
–
Extensive intraductal component Negative
–
Positive
1.471
Lymphocyte infiltration Other Diffuse, high
–
– 0.417
1.503
–
– 0.147–1.183
0.1
0.293
0.352–2.048
0.717
0.685
0.399–1.948
0.755
0.678–2.707
0.389
0.257–1.072
0.077
1.193 –
0.087–0.983
0.047
0.644
0.204–2.306
0.541
0.756
0.322–3.653
0.895
0.836–5.424
0.113
HER2 immunohistochemistry 1? or 2?
–
3?
0.85
–
–
HER2 SISH status Low-level amplification
–
High-level amplification
0.881
– 1.085
– 0.53
p53 expression Negative
–
Positive
1.355
– 2.129
– 0.574
HR status Negative
–
Positive
0.525
–
–
–
–
CI confidence interval
CEP17 ratio, the absence of HER2 genetic heterogeneity, and more p53 expression than HR?/HER2? tumors. HR negativity was an independent prognostic indicator of poor disease-free survival. Distinct histopathologic features may reflect the significant differences in clinical behavior between HR?/HER2? and HR-/HER2? tumors [3–6]. To the best of our knowledge, this is the first large, comprehensive analysis of histopathologic characteristics in
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HER2-positive breast cancer according to HR status in East Asian patients. Interestingly, we found significant inverse correlations between ER or PR Allred score and average HER2 copy number or HER2/CEP17 ratio. These findings are consistent with those obtained by a previous study, which reported an inverse correlation between HER2 amplification and ER expression [7]. Additionally, equivocal HER2
Breast Cancer Res Treat (2014) 145:615–623
immunohistochemistry results (2?) were more frequent in HR?/HER2? tumors. We also analyzed the association between clinicopathologic variables across the two groups, which were dichotomized according to HR status, as this could suggest substantial differences in clinical outcome, and found statistically significant differences in histologic grade, apocrine features, and the presence of tumor-infiltrating lymphocytes. Currently all HER2-positive breast cancer patients are treated in a standardized manner. Given the histologic differences in HR-/HER2? and HR?/ HER2? tumors, development of personalized treatment strategies might be warranted for effective treatment of HER2-positive breast cancer, rather than a single, standardized, ‘‘one size fits all’’ approach. The importance of HER2 heterogeneity was recently recognized [10, 17–19]. In our study, HER2 genetic heterogeneity was more frequently observed in HR?/HER2? tumors, which had relatively lower levels of HER2 gene amplification compared to the HR-/HER2? group. In our study population, all but one case with HER2 genetic heterogeneity showed overall low levels of HER2 gene amplification. HER2 heterogeneity may contribute to inaccurate assessment of HER2 status. This is clinically important, given the benefit that patients with low-level HER2 amplification receive from trastuzumab treatment [20, 21]. Pathologists should pay close attention to accurate assessment of HER2 copy number in HER2 in situ hybridization analyses, especially when evaluating cases with low-level gene amplification. Intraductal components were not significantly different between HR?/HER2? and HR-/HER2? tumors, but DCIS percentage was positively correlated with average HER2 copy number and HER2/CEP17 ratio. This suggests that HER2 status itself is the important factor in correlation with the extent of DCIS. These results are comparable to previous reports from the Memorial SloanKettering Cancer Center; in which HER2-positive breast cancer was significantly associated with the presence of an extensive intraductal component (p \ 0.0001) [4]. We also showed that pathologic tumor size and DCIS percentage had a significant inverse correlation. In addition, several studies suggested that multifocal or multicentric cancers were more frequently present in HER2-positive breast cancer than in HR ?/HER2-negative breast cancer [4, 11, 22]. In the present study, we found that 31.7 % of patients with HR-/HER2? cancer had multifocal or multicentric cancers. In the light of these findings, the presence of diffuse DCIS with multifocal invasive tumors of small pathologic size can be considered as a characteristic histopathologic feature of HR-/HER2? cancer. This morphologic feature can adversely affect the accurate assessment of radiologic and pathologic tumor size and determination of pT stage.
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Variation of the presence and the extent of nodal involvement in different subtypes of breast cancer is also reported. HER2-positive tumors are more likely to have nodal involvement [22, 23]. However, the association between HR status and nodal involvement in HER2-positive breast cancer has not been clarified. In our study, pN stage was not significantly different between HR?/HER2? and HR-/HER2? tumors, but the ratio of metastatic to total examined lymph nodes was significantly correlated with average HER2 copy number and HER2/CEP17 ratio. We confirmed a high frequency of apocrine differentiation in HER2-positive breast cancer, and showed a strong association between apocrine features and HR-/HER2? tumors. While apocrine differentiation of breast cancer can be seen in all subtypes, its association is strongest with the HER2-positive subtype [13]. Lehmann-Che et al. reported that molecular apocrine breast cancer showed frequent 3? HER2 expression (67 %) by gene expression array analysis, and was negative for ER (93 %) by immunohistochemistry. We also found significantly higher expression of p53 in HR-/HER2? breast cancer. Similarly, in TCGA data analysis, TP53 mutation was much higher in HER2enriched subtypes (72 %) than in luminal A (12 %) or luminal B (29 %) subtypes [2]. In addition, a significant inverse correlation between ER and p53 expression in breast cancer was reported [24]. Paget disease comprises 0.5–5 % of all breast cancers, and underlying carcinoma is present in most cases [25]. Since the underlying carcinoma usually shows the same immunohistochemical staining pattern, Paget disease appears to originate from the underlying ducts and then subsequently migrates to the epidermis [25]. In the present study, Paget disease was more frequently observed in HR-/HER2? tumors, even though the overall incidence of Paget disease was low. Therefore, close follow-up with examination of the nipple is warranted in patients treated with nipple-sparing surgery, especially in those with HR-/ HER2? tumors. Recently, the prognostic and predictive relevance of tumor-infiltrating lymphocytes in adjuvant and neoadjuvant settings was determined in breast cancer [26–29]. The significance of tumor-infiltrating lymphocytes in breast cancer was largely related to the triple-negative subtype; however, several studies indicate the additional importance of tumor-infiltrating lymphocytes in HER2-positive breast cancer. In this study, patients with diffuse and high lymphocytic infiltration showed better disease-free survival than other patients in HR- tumors. However, survival difference was not found in patients with HR? tumors. In general, the level of tumor-infiltrating lymphocytes is inversely correlated with ER and/or PR expression, but positively correlated with pathologic complete response rate [27, 28, 30]. We, therefore, analyzed the level of
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lymphocytic infiltration according to HR status in HER2positive breast cancer, and found an association between diffuse and high lymphocytic infiltration and negative HR status. Our findings could provide an additional rationale to explain the higher rates of pathologic complete response in HR-/HER2? tumors compared to HR?/HER2? tumors in neoadjuvant settings [3, 4]. The retrospective design of this study represents a limitation. Nevertheless, this study provides a relevant basis for the apparent differences in clinical behaviors between HR?/HER2? and HR-/HER2? breast cancers. Further studies are necessary to confirm our results in HER2positive breast cancer. In conclusion, HR-/HER2? tumors had higher nuclear grade, less tubule formation, higher histologic grade, more frequent apocrine features, more diffuse and abundant lymphocytic infiltration, stronger HER2 immunohistochemical staining, higher average HER2 copy number and HER2/CEP17 ratio, the absence of HER2 genetic heterogeneity, and more p53 expression than HR?/HER2? tumors. These distinct histopathologic features may be relevant to the significant differences in clinical behavior between HR?/HER2? and HR-/HER2? tumors. Acknowledgments This study was supported by 2013 ‘‘Moon-Ho Yang’’ research fund from The Korean Society of Pathologists. Conflict of interest
The authors declare no conflict of interest.
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