3.3 Analysis of changes in tumor protein markers before and after NAC
The analysis of changes in individual tumor protein marker is shown in Table 4 and revealed that before and after NAC, tumor protein markers changed at different degrees. The smallest and largest changes occurred in HER2 (20.5%) and Ki-67 (40.2%), respectively. The highest positive-to-negative change rate occurred for Ki-67 (37.5%), while the smallest change was for HER2 (15.2%). In addition, the negative-to-positive changes were just the opposite that is the largest change occurred in P53 (13.3%), while the smallest occurred in Ki-67 (2.7%).
Before and after NAC, only the average expression rate of Ki-67 were decreased (from 28.6 ± 19.2% to 19.7 ± 18.5%, P < .001). There were no significant differences in ER (from 39.0 ± 33.7 to 42.2 ± 37.3%, P = .30), PR (from 27.0 ± 31.3% to 24.1 ± 31.7%, P = .27), or P53 (from 29.6 ± 28.0% to 31.4 ± 29.7%, P = .51).
3.4 Analysis of BC subtypes before and after NAC
The proportions of luminal A-like, HER2-positive, and triple-negative BC subtypes increased after NAC, while that of luminal B-like decreased. The proportion of luminal A-like BC increased from 16.1% to 30.4% after NAC. The largest change occurred in luminal B-like (HER2-negative), which reduced from 26.8% to 5.3% (P < .001) (Table 5).
3.5 Multivariate regression analysis to determine the predictor of markers change after NAC.
In the subsequent multivariate regression analysis, changes of markers were defined as the dependent variables. Lateral-superior Quadrant (OR = 0.586, P = .035) was observed to be independently associated with change in ER (Negative→Positive) (Table 6). Increased number of lymph nodes (OR = 0.237, P = .006) and BMI (OR = 0.305, P = .028) seemed to be related to conversion of PR (Positive→Negative). And, there was statistical association between the Ki-67 (Positive→Negative) and the age≥50 (OR = 2.702, P = .015). The BMI ≥ 24 (OR = 4.422, P = .021), age ≥ 50 (OR = 3.245, P = .047) and blood type A (OR = 0.183, P = .038) were independently associated with conversion of P53 (Positive→Negative). The BMI ≥ 24 (OR = 8.691, P = .004), number of lymph nodes ≥1 (OR = 6.137, P = .029) and TNM 1-2 (OR = 8.537, P = .008) were statistically associated with changes in HER2 (Positive→Negative). All other tested variables were not associated with the conversion of markers (P > .05)
Conversion of the HR status and HER2 status after NAC can be used to predict the prognosis of BC patients, but there is a lack of data about these changes in patients without pCR since most studies examined patients with pCR.[14–21] Changes in markers may benefit patients with some subtypes of BC. According to the 2013 St Gallen Consensus Conference, if IHC results of patients who undergo core biopsy are negative for both ER and PR, while the postoperative IHC results are positive for ER or (and) PR, these patients will be able to receive treatment with tamoxifen (premenopausal) or aromatase inhibitors (postmenopausal). If the IHC result after core biopsy is negative for Her2, but after surgery, the IHC result is positive for Her2, or FISH result is amplification of CerBb2, these patients will be able to receive treatment with Herceptin, which will greatly improve the overall survival of patients. Therefore, a close observation of the changes in markers will bring very great benefits to the treatment and prognosis of patients. These results imply that the optimal course of treatments for BC should be based on tumor characteristics before and after NAC.[33,34]
Van De Ven et al pointed out in a meta-analysis that HR may change in 8 to 33% of patients after NAC. Hirata et al reported that changes in ER and PR occurred in 23% of patients after NAC. Furthermore, the positive-to-negative rate of change in HR and HER2 were 8.2% and 6%, respectively; and the negative-to-positive rate of change was 7.9% and 3.5%, respectively. Nevertheless, direct comparisons among studies cannot be made because pCR has to be considered. Indeed, in the present study, ER changed in 22.3% of patients, PR changed in 28.6% of patients, and the positive-to-negative and negative-to-positive rates of change for HER2 were 15.2% and 5.3%, respectively. Van De Ven et al revealed that after NAC that included trastuzumab, negative-to-positive change for HER2 was observed in 5.3% of patients. For patients who require targeted therapy, since the rate of change seems to be higher, IHC should be carried out again on the specimens after surgical resection in order to avoid missing HER2-positive patients. The amplification of the HER2 gene is an important factor of prognosis. The HER2 positive patients can achieve a clinical response (CR) or pCR after NAC combined with trastuzumab treatment.[36,37]
An important source of bias is the correlation of IHC results between coarse needle biopsy (CNB) and surgical specimens. Nevertheless, among patients who did not undergo NAC, Arnedos et al reported that the accordance rates of ER, PR, and HER2 were 98.2%, 85.0%, and 98.8%, respectively. The changes observed after NAC in the present study are all higher than the non-accordance rate observed by Arnedos et al, suggesting that the changes observed in the present study were probably caused by NAC, as observed in previous studies.[14–21] Nevertheless, source of biases include tumor heterogeneity, the time interval between biopsy and surgery, technical issues such as the fixation delay, and differences in the subjective evaluation from different pathologists.
The present study revealed that ER, Ki-67, and HER2 were significantly changed after NAC. According to the literatures,[17,35] HRs either change with HER2, or both do not change. In samples in which PR and ER expression rates increased, HER2 expression would be downregulated accordingly; while in samples in which HER2 expression increased, ER and PR expression rates would be reduced accordingly. Similar results were also obtained with the use of NAC combined with trastuzumab. For BC that has a positive HER2 result in CNB only or surgery only, the heterogeneity of HER2 expression does not need to be considered and anti-HER2 treatment should be given or less.
Many different polygene analysis techniques have provided prognostic information for BC, and this information is mainly derived from proliferation-related genes. A study proposed that moderate or strong PR expression should act as an additional condition for the definition of the luminal alternative classification. As a marker of cell proliferation, Ki-67 expression levels are also important in the definition of luminal A. There is evidence that strongly positive PR (>20%) is helpful for improving the accuracy of distinguishing between luminal A and B BC. Due to the addition of this condition, the number of patients classified as luminal A BC should be reduced and the number of patients who are suggested to undergo chemotherapy would increase. In the present study, PR > 20% was used as the threshold for BC subtype classification. Luminal A-like BC increased from 16.1% to 30.4% and luminal B-like (HER2 negative) BC decreased from 26.8% to 5.3%, supporting that luminal B-like (HER2 negative) BC was sensitive to chemotherapy, and luminal A-like is less sensitive to chemotherapy.
The high expression of Ki-67 indicates poor prognosis, but the highly proliferative tumor cells are more sensitive to anthracycline chemotherapy. Studies have confirmed that the expression of Ki-67 was reduced after NAC, endocrine therapy,[46,47] or chemotherapy combined with endocrine therapy. Burcombe et al reported that the median value of the expression rate of Ki-67 decreased from 24.9% before chemotherapy to 18.1% after chemotherapy. These results support the results of the present study.
The P53 is a cancer suppressor gene. The P53 mutations can result in a variety of tumors and are closely correlated with anthracycline resistance. However, the average value of the P53 expression rate was not statistically significant before and after NAC in the present study. In addition, positive-to-negative conversion of P53 all occurred in BMI ≥ 24 (OR = 4.422, P = .021), age ≥ 50 (OR = 3.245, P = .047), and blood type A (OR = 0.183, P = .038). These suggest that when the patients are overweight or older, mutant P53 cells actively proliferate. Highly proliferating cells are sensitive to cytotoxic chemotherapy drugs, which could lead to a decrease in P53- and Ki-67-positive cells.
From the perspective of recurrence and poor prognosis of BC, obesity is widely considered as a risk factor. There is evidence that suggests that pluripotent stem cells in adipose tissues may affect tumor angiogenesis. In preclinical studies, this kind of cells has been proven to promote the occurrence and development of BC. In the present study, obese patients more easily presented a positive-to-negative conversion of PR (P = .028), HER2 (P = .004) and P53 (P = .021). Lymph node metastasis is also a very important prognostic factor. In this study, patients with axillary cavity lymph node metastasis after NAC are more prone to a positive-to-negative conversion of HER2 (P = .029) and PR (P = .006).
The determination of tumor markers is a useful tool for clinical management in cancer patients, assisting in diagnosis, staging, evaluation of therapeutic response, detection of recurrence and metastasis, and development of new treatment modalities. For example, after NAC, the number of patients whose ER and PR becoming positive is 7 and 10 respectively (Table 4). The percentage of patients luminal A subtype increased from 18 (16.1%) to 34 (30.4%), 16 new luminal A patients would be thought to be treated with hormonal therapy (Table 5). Luminal A patients are sensitive to hormonal manipulations, but less sensitive to chemotherapy, their prognosis is favorable. So new treatment modalities should be developed for these patients who can benefit from the new treatment of breast cancer.
The present study is not without limitations. The sample size was small and from a single center. The small sample size also prevented multivariable analyses. The IHC analysis is somewhat subjective and differences among pathologists could lead to some bias. The retrospective nature of the study prevented us from analyzing factors that were not reported in the medical charts. Finally, no molecular mechanisms could be explored.
In conclusion, our observational study demonstrated the existence of discordance in the HR status and markers’ status after NAC and the predictors of the conversion. These findings might help optimize the choice of sequential adjuvant therapy and improve treatment and prognosis. The administration of NAC might be the main reason for the change in receptor status, but the mechanism needs to be elucidated. In the future, further studies are required to identify the mechanism for this switch in receptor status after NAC and to validate the prognostic impact associated with this switch.
The authors acknowledge the help of Dr. Yanlin Chen, et al and the committee of pathologists, from the Pathology Department of Chongqing Medical University, for kindly providing pathological service and suggestions.
Conceptualization: Jian-Heng Peng, Hong-Yuan Li, Yong-Hong Wang.
Data curation: Jian-Heng Peng, Xiang Zhang, Jun-Long Song, Liang Ran, Rong Luo.
Formal analysis: Jian-Heng Peng, Xiang Zhang, Jun-Long Song, Liang Ran, Rong Luo.
Investigation: Jian-Heng Peng, Xiang Zhang, Jun-Long Song, Liang Ran, Rong Luo, Yong-Hong Wang.
Methodology: Jian-Heng Peng, Hong-Yuan Li, Yong-Hong Wang.
Project administration: Hong-Yuan Li, Yong-Hong Wang.
Resources: Jian-Heng Peng, Xiang Zhang, Jun-Long Song, Liang Ran, Rong Luo.
Software: Xiang Zhang, Jun-Long Song, Liang Ran, Rong Luo.
Supervision: Yong-Hong Wang.
Writing – original draft: Jian-Heng Peng.
Writing – review & editing: Xiang Zhang, Jun-Long Song, Liang Ran, Rong Luo, Hong-Yuan Li, Yong-Hong Wang.
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Keywords:Copyright © 2019 The Authors. Published by Wolters Kluwer Health, Inc. All rights reserved.
breast cancer subtype; IDC; markers; neoadjuvant chemotherapy