Abstract
Purpose
Patients with HER2-positive breast cancer commonly receive anti-HER2 neoadjuvant chemotherapy and pathologic complete response (pCR) can be achieved in up to half of the patients. HER2 protein expression detected by immunohistochemistry (IHC) can be quantified using digital imaging analysis (DIA) as a value of membranous connectivity. We aimed to investigate the association HER2 IHC DIA quantitative results with response to anti-HER2 neoadjuvant chemotherapy.
Methods
Digitized HER2 IHC whole slide images were analyzed using Visiopharm HER2-CONNECT to obtain quantitative HER2 membranous connectivity from a cohort of 153 HER2+ invasive breast carcinoma cases treated with anti-HER2 neoadjuvant chemotherapy (NAC). HER2 connectivity and other factors including age, histologic grade, ER, PR and HER2 FISH were analyzed for association with the response to anti-HER2 NAC.
Results
Eight-three cases (54.2%) had pCR, while 70 (45.8%) showed residual tumor. Younger age, negative ER/PR, higher HER2 DIA connectivity, higher HER2 FISH ratio and copy number were significantly associated with pCR in univariate analysis. Multivariate analysis demonstrated only age, HER2 DIA connectivity, PR negativity, and HER2 copy number was significantly associated with pCR; whereas HER2 DIA connectivity had the strongest association.
Conclusions
HER2 IHC DIA connectivity is the most important factor predicting pCR to anti-HER2 neoadjuvant chemotherapy in patients with HER2-positive breast cancer.
Keywords: HER2 immunohistochemistry, quantitative digital imaging analysis, breast carcinoma, anti-HER2 neoadjuvant chemotherapy, pathologic complete response
Introduction
Approximately up to 20% of breast cancers harbor human epidermal growth factor receptor 2 (HER2; ERBB2) gene amplification and/or protein overexpression. [1–4] Anti-HER2 targeted drugs, such as trastuzumab and pertuzumab, are effective to treat HER2-positive breast cancers, but not HER2-negative breast cancers. [5–8] Given anti-HER2 drugs’ side effects and significant cost, accurate determination of HER2 positive status is mandatory before offering them to any breast cancer patient. [9]
HER2 status is usually assessed by immunohistochemistry (IHC) for HER2 protein expression and/or by in situ hybridization (ISH) for HER2 gene amplification. [9] HER2 IHCs are evaluated non-quantitatively by pathologists to give a score from 0 to 3+ based on membranous staining of HER2 protein and the American Society of Clinical Oncology and the College of American Pathologists (ASCO/CAP) guidelines; however, interobserver variability commonly occurs among pathologists. [9–11] Quantitative digital image analysis (QDIA) has emerged as a new method to assess HER2 IHC since whole slide imaging (WSI) has been widespread adopted. [12–16] Recent studies have demonstrated QDIA showed good concordance with pathologists’ read and could reduce HER2 IHC equivocal (2+) cases. [12,14,17,18] Besides giving a score from 0 to 3+, QDIA can objectively evaluate HER2 membranous connectivity in a quantitative manner and render an absolute continuous value from 0 to 1, correlating with HER2 protein expression level. [12,19,20]
Currently, HER2 FISH serves as a gold standard to determine HER2 status because it can provide relatively objective evaluation of HER2 gene amplification in a quantitative manner. It has been demonstrated to correlate with response to anti-HER2 targeted therapy. [21] However, anti-HER2 therapy targets HER2 protein instead of HER2 gene; therefore, it is reasonable to hypothesize that QDIA of HER2 protein will provide better correlation with response to anti-HER2 therapy than HER2 gene assessment by FISH. In this study, we used a QDIA algorithm to quantitatively evaluate HER2 protein levels and further investigated the correlation of HER2 protein QDIA results with response to anti-HER2 targeted neoadjuvant chemotherapy (NAC) in breast cancer patients.
Materials and Methods
Case selection
The study cohort included 153 HER2-positive invasive breast carcinoma patients treated with anti-HER2 neoadjuvant chemotherapy (NAC) and follow-up surgical resection (91 lumpectomy specimens and 62 mastectomy specimens) within a study period between January 2014 and January 2018 from The Ohio State University (n=82) and Emory University (n=71). This study was approved by the Ohio State University and Emory University institutional Research Board. For NAC, the majority of patients received four cycles of AC (doxorubicin + cyclophosphamide) together with paclitaxel or docetaxel followed by trastuzumab. Alternatively, the remaining patients received four cycles of AC together with PTD (pertuzumab + trastuzumab + docetaxel).
HER2 immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH)
HER2 status was assessed on pre-treatment core biopsy specimens. At The Ohio State University, HER2 IHC was performed using PATHWAY anti-HER2 (4B5) on Benchmark XT automated slide Stainer according to the manufacturer’s protocol (Roche Ventana Medical Systems, Tucson, AZ). At Emory University, HER2 IHC was performed using HercepTest™ on Dako Autostainer according to the manufacturer’s protocol (Dako, Carpinteria, CA). HER2 IHC assay for each case had been evaluated and reported in accordance with the HER2 ASCO/CAP guidelines by subspecialized breast pathologists. At both institutions, HER2 FISH assay was performed using the dual-color Vysis FDA-approved PathVysion HER2 DNA Probe Kit (Abbott Molecular, Des Plaines, IL). HER2 FISH results were interpreted by molecular pathologists according to the HER2 ASCO/CAP updated guidelines. [9]
Evaluating pathologic response to anti-HER2 neoadjuvant chemotherapy
Pathologic response was evaluated on resected specimens from the cohort patients. The resected specimens were carefully examined grossly by experienced pathologist assistants and tumor beds were entirely submitted for histologic examination. Pathologic complete response (pCR) was defined as no detectable residual invasive tumor in breast tissue and absence of lymph nodal metastasis.
Image Acquisition and digital imaging analysis
HER2 IHC glass slides were scanned using Philips UltraFast Scanner (Philips, the Netherlands) at 40x magnification with a single-focus plane. Whole slide images (WSIs) were stored in a centralized server located at The Ohio State University’s campus. HER2 IHCs were evaluated using the HER2-CONNECT algorithm in the Visiopharm Integrator System (VIS) (Visiopharm, Hørsholm, Denmark) and recorded as a value from 0 to 1. [12] (Figure 1) In order to compare with Pathologists’ read, HER2 DIA connectivity was categorized into four scoring categories with the following cut-off values: 1)0: connectivity = 0; 2) 1+: 0 < connectivity ≤ 0.12; 3) 2+: 0.12 < connectivity ≤ 0.49; 4) 3+: connectivity > 0.49. Similar to the HER2 scores used by pathologists, a HER2 DIA score 0 and 1+ were defined as negative, 2+ as equivocal, and 3+ as positive based on our previous validation study, which included a cohort of 612 invasive breast carcinomas with 432 cases of HER2 IHC scores of 0 or 1+ (negative), 101 cases of score of 2+ (equivocal) and 79 cases of score of 3+ (positive) by pathologists’ manual scoring. Each HER2 IHC WSI slide was analyzed using a HER2 membrane algorithm in Visiopharm and the HER2 connectivity value was recorded. The abovementioned cut off value was established using classification and regression trees classifiers to finds optimal binary splits in that validation study. [22]
Fig. 1.
HER2 IHC and membranous connectivity analyzed by Visiopharm HER2 IHC digital imaging analysis (DIA) algorithm. A, B) one case with HER2 IHC 2+; C, D) one case with HER2 IHC 3+. A, C) HER2 IHCs; B, D) HER2 connectivity (green color line) detected by Visiopharm DIA.
Statistical analyses
Statistical analysis was conducted using SAS Version 9.4. Descriptive statistics for each variable were reported. For numeric covariates, the mean and standard deviation were calculated and presented. Frequency and percentage were also shown for category variables. One-way ANOVA test and Kruskal-Wallis test were performed for numerical covariates univariate analysis if appropriate. Chi-square test or Fisher’s exact test was employed for categorical covariates if appropriate. A multivariable Logistic Regression model was fit by a backward variable selection method with an alpha =.20 removal criteria. Significant level was set at 0.05.
Results
Clinical and pathologic characteristics of the study cohort
The clinical and pathologic characteristics from the study cohort of 153 primary HER2-positive invasive breast carcinomas treated with anti-HER2 NAC and excision were summarized in table 1. The median age of the cohort was 55 years with a range of from 26 to 86 years. All cases except two were invasive ductal carcinoma while those two cases were invasive lobular carcinoma. More than half of carcinomas (84/153, 55.6%) had Nottingham grade 3. Seventy-five carcinomas (49%) were estrogen receptor (ER) positive and 53 carcinomas (34.6%) were progesterone receptor (PR) positive. Thirty cases had a pathologist’s manual score of 2+ on IHC, and the remaining cases had a score of 3+. One hundred twenty-two cases had HER2 FISH results with the median HER2 copy number per cell as 17.8 (range 3.6-35.5) and the median HER2/CEN17 ratio as 6.78 (range 0.96-22.98) (Table 1).
Table 1.
Clinical and pathological results in all HER2-positive cases with neoadjuvant chemotherapy
| Variable | Level | N (%) = 153 |
|---|---|---|
| pCR | No | 70 (45.8) |
| Yes | 83 (54.2) | |
| Nottingham grade | 1-2 | 67 (44.4) |
| 3 | 84 (55.6) | |
| Missing | 2 | |
| Nuclear grade | 2 | 54 (35.8) |
| 3 | 97 (64.2) | |
| Missing | 2 | |
| ER positivity | No | 78 (51.0) |
| yes | 75 (49.0) | |
| PR positivity | No | 100 (65.4) |
| yes | 53 (34.6) | |
| HER2 IHC manual score | 2 | 30 (19.6) |
| 3 | 123 (80.4) | |
| Age | Median | 55 |
| Range | 26-86 | |
| HER2 DIA Connectivity | Median | 0.83 |
| Range | 0.12-0.98 | |
| Missing | 2 | |
| HER2 FISH Copy number | Median | 17.81 |
| Range | 3.60-35.50 | |
| Missing | 31 | |
| HER2 FISH Ratio | Median | 6.78 |
| Range | 0.96-22.98 | |
| Missing | 31 |
ER estrogen receptor, PR progesterone receptor, IHC immunohistochemistry, DIA digital imaging analysis
HER2 IHC WSI slides were analyzed using HER2 membranous connectivity algorithm in Visiopharm and the HER2 DIA connectivity value (from 0 to 1) was recorded with the median as 0.83 (range 0.12-0.98). (Table 1) One case had HER2 IHC DIA score less than 0.12 (0.11942), which was classified as negative by the algorithm. This patient showed residual tumor with a residual cancer burden (RCB) of 3.084 (RCB II). Additionally, The HER2 DIA connectivity (mean ± SD: 0.48 ± 0.27) of HER2 IHC equivocal cases (n=30) was significantly lower than the DIA connectivity (mean ± SD: 0.82 ±0.13) of HER2 IHC 3+ cases with pCR (n=123) (p<0.001).
HER2 IHC DIA values are moderately associated with HER2 FISH copy numbers/ratios
First, we analyzed the correlation between HER2 IHC DIA connectivity values with HER2 FISH copy numbers and ratios using Pearson correlation in 122 cases in which FISH results were available. The Pearson correlation coefficient (r) for HER2 IHC DIA connectivity and HER2 FISH copy number was 0.5421 (n = 122; P < .0001) with a mean coefficient of determination (r2) of 0.2938 (y = 19.069x + 3.3432) (Figure 2). The Pearson correlation coefficient (r) for HER2 IHC DIA connectivity and HER2 FISH ratio was 0.5118 (n = 122; P < .0001) with a mean coefficient of determination (r2) of 0.2619 (y = 8.3636x + 0. 7472) (Figure 3).
Fig 2.
Correlation between HER2 IHC DIA connectivity and HER2 FISH copy number. The Pearson correlation coefficient (r) for HER2 IHC DIA connectivity and HER2 FISH copy number was 0.542054925 (n= 122; P < .0001). (y = 19.069x + 3.3432, R2 = 0.2938)
Fig. 3.
Correlation between HER2 IHC DIA connectivity and HER2 FISH ratio. The Pearson correlation coefficient (r) for HER2 IHC DIA connectivity and HER2 FISH ratio was 0.511776265 (n = 122; P < .0001) (y = 8.3636x + 0.7472; R2 = 0.2619).
The Pearson correlation coefficient (r) for HER2 IHC DIA connectivity and HER2 FISH copy number in HER2 equivocal cases was 0.394968353 (n = 30; P < .0001) (y = 9.6949x + 5.4593; R2 = 0.156). The Pearson correlation coefficient (r) for HER2 IHC DIA connectivity and HER2 FISH ratio in HER2 equivocal cases was 0.293257566 (n = 30; P < .0001) (y = 2.5778x + 2.2578; R2 = 0.086). The correlation coefficients in HER2 equivocal cases were lower than the correlation coefficients in all cases.
HER2 DIA value independently predicts pCR to anti-HER2 NAC
In our cohort, 83 (54.2%) patients had pCR and 70 (45.8%) had residual tumor. First, the associations of NAC response with clinical and pathologic variables and HER2 DIA connectivity were examined using univariate analysis (Table 2). No significant difference was detected in respect to Nottingham grade or nuclear grade between the pCR cases and the cases with residual tumor. There were significantly more cases with HER2 IHC manual score of 3+ (61.8% vs. 38.2%, p < 0.001), ER negativity (64.1% vs. 35.9%, p = 0.013) and PR negativity (64% vs. 36%, p < 0.001) in pCR group than in the residual tumor group. Additionally, the pCR cases showed significantly greater median HER2 signal (19.9 vs. 12.46, p < 0.001) and HER2/CEN17 ratio (7.51 vs. 4.19, p < 0.001) than the cases with residual tumor. Furthermore, the pCR cases also showed significantly greater median HER2 DIA connectivity than the cases with residual tumor (0.88 vs. 0.75, p < 0.001) (Table 2).
Table 2.
Univariate analysis of factors associated with incomplete response to anti-HER2 neoadjuvant chemotherapy
| pCR |
||||
|---|---|---|---|---|
| Covariate | Level | No N=70 | Yes N=83 | P-value |
| Age | Median | 57 | 53 | 0.006 |
| Range | 30-86 | 26-76 | ||
| Nottingham grade | 1-2 | 29 (43.28) | 38 (56.72) | 0.595 |
| 3 | 40 (47.62) | 44 (52.38) | ||
| Nuclear grade | 2 | 23 (42.59) | 31 (57.41) | 0.568 |
| 3 | 46 (47.42) | 51 (52.58) | ||
| ER positivity | No | 28 (35.9) | 50 (64.1) | 0.013 |
| Yes | 42 (56) | 33 (44) | ||
| PR positivity | No | 36 (36) | 64 (64) | <.001 |
| Yes | 34 (64.15) | 19 (35.85) | ||
| HER2 manual IHC score | 2 | 23 (76.67) | 7 (23.33) | <.001 |
| 3 | 47 (38.21) | 76 (61.79) | ||
| HER2 DIA connectivity | Case # | 70 | 81 | <.001 |
| Median | 0.75 | 0.88 | ||
| Range | 0.12-0.98 | 0.5-0.97 | ||
| HER2 FISH copy number | Case # | 56 | 66 | <.001 |
| Median | 12.46 | 19.9 | ||
| Range | 4-35.5 | 3.6-34.36 | ||
| HER2 FISH ratio | Case # | 56 | 66 | <.001 |
| Median | 4.19 | 7.51 | ||
| Range | 0.96-15.5 | 2.3-22.98 | ||
pCR pathologic complete response, ER estrogen receptor, PR progesterone receptor, DIA digital imaging analysis
Next, a multivariable logistic regression model was fit by a backward variable selection method with an alpha =.20 removal criteria to further analyze all variables with p < 0.05 in the univariate analysis. The results demonstrated only age (OR = 0.96, p = 0.035), PR negativity (OR = 3.20, p = 0.019), HER2 copy number (OR = 1.07, p = 0.014), and HER2 DIA connectivity (OR = 136.08, p = 0.002) being significantly associated with the pCR to anti-HER2 targeted therapy with HER2 DIA connectivity as the most powerful factor (Table 3). In order to assure the results were not driven by outliers or other data artifacts, we further analyzed the distribution of HER2 DIA connectivity based on patients’ outcome (pCR vs no pCR). The median and range of the two groups were 0.75 (0.12 - 0.98) (no pCR group), and 0.88 (0.50-0.97) (pCR group), respectively. As shown in figure 4, the values of HER2 DIA connectivity in no pCR groups were much wider than the values in pCR group, resulting in high OR and large CI for HER2 DIA connectivity in table 3. They should be improved if the sample size was larger.
Table 3.
Multivariate analysis of factors associated with incomplete response to anti-HER2 neoadjuvant chemotherapy
| pCR | Incomplete response | OR (95% CI) | p value | |||
|---|---|---|---|---|---|---|
| # / median | % / range | # / median | % / range | |||
| Case # | 83 | 70 | ||||
| Age (yr) | 53 | 26‐76 | 57 | 30‐86 | 0.96 (0.91‐1.00) | 0.035 |
| PR negativity | 64 | 64% | 36 | 36% | 3.20 (1.24‐8.59) | 0.019 |
| HER2 FISH copy number | 19.9 | 3.6‐34.36 | 12.46 | 4‐35.5 | 1.07 (1.01‐1.15) | 0.040 |
| HER2 DIA connectivity | 0.88 | 0.5‐0.97 | 0.75 | 0.12‐0.98 | 136.08 (8.21‐3965.94) | 0.002 |
pCR pathologic complete response, OR odds ratio, PR progesterone receptor, DIA digital imaging analysis
Fig. 4.
Box plot on HER2 DIA Connectivity between cases without pathologic complete response (pCR) and with pCR.
Additionally, we investigated the ability of HER2 DIA connectivity in predicting pCR for patients with HER2 IHC equivocal (2+) results. The HER2 DIA connectivity (mean ± SD: 0.36 ± 0.17) of HER2 IHC equivocal cases without pCR (n=23) was significantly lower than the DIA connectivity (mean ± SD: 0.87 ± 0.06) of cases with pCR (n=7) (p<0.001), suggesting its predictive ability in HER2 IHC equivocal cases.
The comparision of HER2 DIA connectivity and its correlation with FISH results between two different HER2 IHC assays
Since HerceptTest and PATHWAY 4B5 were used in current study, we compared HER2 DIA connectivity and its correlation with HER2 FISH results between these two platforms. Since only 42 cases from Emory University (HercepTest) had HER2 FISH results, we analyzed HER2 DIA connectivity and its correlation with HER2 FISH results in these 42 cases in comparison with all 79 cases from OSU (PATHWAY 4B5). The descriptive statistics of DIA connectivity were 0.70 ± 0.24 for cases using PATHWAY 4B5, and 0.74 ± 0.20 for cases using HercepTest. No significant statistical difference was detected (p=0.3347). (Table 4) The Pearson correlation coefficients between HER2 DIA connectivity and FISH results were comparable between these two platforms. The Pearson correlation coefficient (r) for HER2 IHC DIA connectivity and HER2 FISH copy number was 0.526877595 (n = 79; P < .0001) (y = 17.707x + 5.3314; R2 = 0.2776) in the group of cases using PATHWAY 4B5. The Pearson correlation coefficient (r) for HER2 IHC DIA connectivity and HER2 FISH copy number was 0.668655367 (n = 43; P < .0001) (y = 25.76x - 3.7013; R2 = 0.4471) in the group of cases using HercepTest. The Pearson correlation coefficient (r) for HER2 IHC DIA connectivity and HER2 FISH ratio was 0.506260802 (n = 79; P < .0001) (y = 8.0722x + 1.3415; R2 = 0.2563) in the group of cases using PATHWAY 4B5. The Pearson correlation coefficient (r) for HER2 IHC DIA connectivity and HER2 FISH ratio was 0.595734840 (n = 43; P< .0001). (y = 10.254x - 1.4428; R2 = 0.3549) in the group of cases using HercepTest. Based on manual IHC results, there were 12 manually equivocal cases from the group of cases using PATHWAY 4B5 (15%), and 18 manually equivocal cases from the group of cases using HercepTest (24%). Based on DIA connectivity, there were 15 DIA equivocal cases from the group of cases using PATHWAY 4B5, but only 4 DIA equivocal cases from the group of cases using HercepTest.
Table 4.
Univariate analysis of DIA results between cases using PATHWAY 4B5 and HercepTest assays
| Assays | ||||
|---|---|---|---|---|
| Covariate | Statistics | PATHWAY 4B5 N=79 |
HercepTest N=42 |
P-value |
| Digital HER2 Connectivity |
N | 79 | 42 | 0.3347 |
| Mean | 0.7 | 0.74 | ||
| Median | 0.8 | 0.85 | ||
| Min | 0.12 | 0.34 | ||
| Max | 0.97 | 0.96 | ||
| Std Dev | 0.24 | 0.20 |
HER2 DIA connectivity and pathologic complete rates among patients with different chemotherapy regimens
Lastly, pCR rates and DIA scores were investigated in patients with different chemotherapy regimens. There were 101 patients who received AC plus paclitaxel or docetaxel followed by trastuzumab and 52 patients who received AC together pertuzumab, trastuzumab, and docetaxel. The pCR rate was slightly higher in group of patients who received AC together pertuzumab, trastuzumab, and docetaxel than in group of patients who received AC plus paclitaxel or docetaxel followed by trastuzumab [59.6% (31/52) vs 51.5% (52/101)], but the difference was not statistically significant. The descriptive statistics of DIA were 0.81 ±0.15 in group of patients treated with AC plus paclitaxel or docetaxel followed by trastuzumab, and 0.84 ±0.11 in group of patients treated with AC together pertuzumab, trastuzumab, and docetaxel. No statistically significant difference was identified. The numbers of patients with equivocal IHC were 19 (18.8%) in group of patients treated with AC plus paclitaxel or docetaxel followed by trastuzumab, and 11 (21.1%) in group of patients treated with AC together pertuzumab, trastuzumab, and docetaxel. No statistically significant difference was identified.
Discussion
To our knowledge, our study is the first to investigate the correlation between the response to anti-HER2 NAC and HER2 protein expression assessed by digital imaging analysis using HER2 IHC WSIs. Our data have demonstrated an excellent correlation between HER2 DIA connectivity and clinical outcome of HER2-positive breast cancer patients treated with anti-HER2 NAC.
Since the widespread implementation of WSI, quantitative DIA has emerged as an objective tool to assess HER2 IHC [12–16]. DIA has been demonstrated to be equal or better than pathologists’ manual scoring and could reduce HER2 IHC equivocal (2+) cases. [12,14,17,18] Besides giving a score from 0 to 3+, DIA can objectively evaluate HER2 membranous connectivity in a quantitative manner and render an absolute continuous value from 0 to 1, correlating with HER2 protein expression level. [12,19,20] Currently, HER2 FISH serves as a gold standard to determine HER2 status because it can provide relatively objective evaluation of HER2 gene amplification in a quantitative manner. It has been demonstrated to correlate with response to anti-HER2 targeted therapy. [21] However, anti-HER2 therapy targets HER2 protein instead of HER2 gene. In this study, we used a DIA algorithm to quantitatively evaluate HER2 protein levels and correlated DIA results with the response to anti-HER2 targeted therapy.
The pCR to anti-HER2 targeted NAC has served as an important surrogate factor for long-term survival in HER2-positive breast cancer patients. Several large clinical trials have reported pCR rates range from 40% to 50% in HER2-positive breast cancer patients who received anti-HER2 NAC.[23–25] The overall pCR rate of 54.2% in our cohort is comparable to the previously reported range. Consistent with several previous studies, our data have demonstrated that hormone receptor negativity, especially PR negativity, is an independent predictive factor for pCR to anti-HER2 NAC. [26,27,23,21] Other factors including younger age, HER2 IHC 3+, HER2 FISH ratio and copy number are also found in our study to be potentially predictive for pCR, consistent with previously reported results. [28–32,21] In current study, we have also discovered HER2 IHC DIA connectivity as a novel predictive factor for pCR to anti-HER2 NAC, suggesting it may be necessary to objectively assess HER2 IHCs using digital imaging analysis tools, especially with current widespread availability of WSIs.
Additionally, our data have demonstrated HER2 IHC DIA values are moderately associated with HER2 FISH copy numbers/ratios, consistent with our previous results. [22] The correlation coefficients in HER2 equivocal cases were lower than the correlation coefficients in all cases. Our data have revealed HER2 IHC connectivity has better correlation with HER2 copy number than HER2/CEP11 ratio, suggesting HER2 copy number may be more accurate to predict HER2 protein expression than HER2 ratio.
Our study has some limitations. The study cohort consisted of clinical cases from two institutions and HER2 IHCs were performed using two different methods [PATHWAY anti-HER2 (4B5) and HercepTest™). The staining intensity slightly varies between these methods and might affect HER2 DIA membranous connectivity. Future study may be necessary to establish different algorithms for each method. Another limitation is the relatively small sample size of our cohort; however, we were able to detect the significant association of HER2 DIA connectivity with pCR.
In summary, our data have demonstrated age, HER2 DIA connectivity, PR negativity, and HER2 copy number independently predict pCR to anti-HER2 NAC in HER2-positive breast cancer patients and, more importantly, HER2 IHC DIA connectivity is the most powerful factor.
Acknowledgements
Funding
None.
Footnotes
Publisher's Disclaimer: This Author Accepted Manuscript is a PDF file of an unedited peer-reviewed manuscript that has been accepted for publication but has not been copyedited or corrected. The official version of record that is published in the journal is kept up to date and so may therefore differ from this version.
Conflict of interest
All authors have no financial relationship to disclose.
Ethical approval
All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards.
This article does not contain any studies with animals performed by any of the authors.
Informed consent
Informed consent was obtained from all individual patients included in the study.
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