Chemosensitivity based on pathologic complete response in very young patients (<35 years) with ER-positive breast cancer: a large retrospective cohort study

Background In (ER)-positive is associated suggested mechanism as respond better to and is effective in than The to evaluate the chemoinsensitivity of very with excluding the of to the

Trial registration: retrospectively registered Background The incidence of breast cancer in very young women aged < 35 years is higher in Korea (~ 9.5%) than in Western countries (< 6%). (1,2) Young age at diagnosis is associated with poor prognosis. When compared with older women, young women are at a higher risk of having poorly differentiated and highly proliferative breast cancer. (3) Tumors in young women more often showed higher S-phase fractions and abnormal p53 expression. (4) In addition, these women were at higher risk of having more positive lymph nodes and larger tumors. (5) Young women are typically diagnosed with more advanced disease, which can be attributed to delayed diagnosis owing to the expected low incidence in young women, lower sensitivity of mammography in dense breasts, pregnancy, and lactation. (6)(7)(8) Young women are at a signi cantly increased risk of dying after adjustment for known prognostic factors and expected mortality. (9) Breast cancer is less ER-positive in younger women than in older women. It has been reported that the proportion of ER-positive tumor is 48-52% in young patients. (3,4,10) Survival outcomes are signi cantly worse in young patients than in older patients with ER-positive tumors. However, the prognosis did not differ between young patients and older patients with ER-negative tumors. (11)(12)(13) Even if positive ER is a good prognostic factor, it would affect different age groups differently. Several reports have suggested endocrine mechanisms as the possible underlying reason. Worse outcomes of young patients with ER positive tumors would be incompletely explained by a lower chance of chemotherapy-induced amenorrhea and the resistance to tamoxifen in adjuvant treatment in ER-positive tumor. (12,14) Conversely, it has been reported that adjuvant chemotherapy is less effective in ER-positive tumors than in ER-negative tumors. A trend towards an advantage for younger women under 35 years was observed. (15) In addition, the effect of age on response to chemotherapy according to the ER status has not been su ciently assessed. Herein, the authors hypothesized that response to chemotherapy is a cause of poor prognosis in young women with ER-positive breast cancer and tried to evaluate the chemoresistance to and bene ts from neoadjuvant chemotherapy in young patients with ER-positive breast cancer while excluding the effect of endocrine treatment as much as possible.

Patient population
In this study, all patients with primary breast cancer who received neoadjuvant chemotherapy followed by surgery between August, 1991 and November, 2013 were included for analysis. Patients with distant metastasis at diagnosis or unknown ER status and male patients were excluded (Figure 1). This study was approved by the institutional review board of the Ewha Clinical Trial Center at Ewha Womans University Medical Center, Korea.

Retrospective Data Collection
We collected individual patient data from the Korean Breast Cancer Society (KBCS). Korean Breast Cancer Registry (KBCR) is a collection of medical information relating to breast cancer patients. Nationwide, breast surgeons in a total of 102 general hospitals (41 university hospitals and 61 teaching hospitals) with 400 or more beds participate voluntarily in this registry since 1996. (14) Individual hospitals recruited new patients who were diagnosed with breast cancer by pathologic examination of breast tissue and sent prospectively collected data of these patients to KBCS in the form of a lled-up recorded cancer registration sheet. In 2001, KBCS developed an online registration system, which made it more convenient for surgeons to enter patient enrollment data themselves. This web-based database has been managed by KBCS. The patients who were newly diagnosed as having breast cancer between 1992 and 1996, which is before the registration system was introduced, were recruited retrospectively. Data of these patients were collected from the database of each individual hospital and added to the KBCS database. This database comprises essential patient information, including unique registration number, the date of diagnosis and surgery, age at surgery, gender, the surgical method used, and clinical and pathologic tumor stage according the American Joint Committee on Cancer classi cation. It contains optional information, such as information on clinical and laboratory ndings before surgery, tumor size, nodal status, histologic grade and type, Ki-67 proliferation index after surgery and before chemotherapy in case of neoadjuvant chemotherapy, and nuclear grade and biologic markers, including the expression status of estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor 2 (HER2). The goals of chemotherapy, such as neoadjuvant, adjuvant, or palliative therapy, and information about the administered chemotherapy regimen, radiotherapy, and endocrine therapy are recorded as treatment details. Even currently, these items are prospectively recorded starting from diagnosis of breast cancer, and overall survival data and the cause of death have been updated consistently through the Korean Central Cancer Registry, Ministry of Health and Welfare, Korea. Enrolled patients in this registry had given written informed consent for data collection at diagnosis. The database was reviewed to identify patients who met the criteria.

Variables
Patients with no evidence of invasive residual tumor in breast or nodes irrespective of noninvasive components in the nal surgical specimen were considered to have obtained pCR. Because not all cases could be con rmed histologically for lymph node metastasis before neoadjuvant chemotherapy was started, in these cases, total pCR and only-breast pCR statuses could be obtained by pathologic stage after surgery.
The status of ER, PR, and HER2 was classi ed as negative or positive through immunohistochemistry (IHC) in individual hospitals. For the classi cation of the HER2 status, if the IHC result was a score of 2+, which was considered as equivocal, the result of FISH was re ected. TNM (tumor size, node status, and metastasis) stages determined before 2010 were re-classi ed according to the seventh edition of the TNM classi cation.
The Ki-67 index was categorized into low and high according to the 2013 St. Gallen consensus(16). If the Ki-67 index information was not available, histologic grade III was considered as highly proliferative tumor and histologic grades I or II were considered as low proliferative tumors. The chemotherapy regimen administered was classi ed as anthracycline-based or not. Younger patient group comprised patients below the age of 35 years and the older group comprised those aged 35-49 years.
Statistical Analysis SPSS software version 20 (SPSS, Chicago, IL, USA) was used for all statistical analyses. Basic data on the characteristics of study subject groups were compared and analyzed using Chi-squared tests. The mean values of the subgroups were compared with each other using independent sample t-tests (Student t-test) to examine statistical signi cance. The association between clinicopathologic parameters and pCR rate within each subgroup was evaluated using the Chi-squared test and univariate Cox regression analysis. Furthermore, multivariate Cox regression analysis was used to determine the independent prognostic value within the strati ed cohorts. A p-value of <0.05 for a two-sided test was considered statistically signi cant.

Results
Clinicopathological characteristics of the study population As authors expected, young patients aged < 35 years were much fewer than older patients aged 35-49 years in both ER-positive and -negative groups (14.0% and 16.8%). The proportion of young patients was comparable in the two groups (P = 0.099).
Most baseline characteristics were comparable irrespective of age in each group (Table 1). However, in the ER-negative group, there were signi cantly more HER2-negative tumors in young patients than in older patients (P = 0.001). In addition, the proportion of patients with high proliferative index was higher in young patients in both ER-positive and -negative groups (35.4% and 67.9%); however, the proportion of patients with high proliferative index was smaller than that with low proliferative index in the ER-positive group irrespective of age.

Surgery and neoadjuvant chemotherapy
More than 70% of patients in both young and older patients received neoadjuvant chemotherapy with anthracycline-based regimen. The proportion of patients who were administered concurrent trastuzumab as neoadjuvant treatment varied and were less than 9% ( Table 2). The proportion of HER2-positive patients treated with trastuzumab was comparable between young and older patients in both ER-positive andnegative groups ( Table 2).

ER estrogen receptor
With regard to surgical treatment, the rate of breast-conserving surgery was not different between young and older patients in both ER-positive and -negative groups. Although the axillary lymph node status by pathological con rmation before neoadjuvant chemotherapy was uncertain, approximately > 80% patients in all groups received axillary lymph node dissection.

Meaningful factor for pCR
In univariate analysis, pCR was achieved in young patients with nonpalpable tumors. It was also achieved in patients with ER-and PR-negative tumors and high proliferative index. Odds ratio of ER status was higher than that of age. When patients were strati ed by their ER status, independent prognostic factors of pCR changed in young and older patients. Negative PR and high proliferative index were positive factors for pCR in the ER-positive group, and age < 35 years was the only one in the ER-negative group. Patients with nonpalpable tumors had a higher tendency to achieve pCR in both groups (Table 4).  Table 4).
The pCR rate of young patients was higher than that of older patients in the ER-negative group but was comparable in the ER-positive group. Even after adjusting for confounding variables, young patients maintained the higher probability of pCR than older patients in ER-negative tumors. However, the pCR rate did not differ according to age in ER-positive tumors. In addition, the odds of pCR rate changed from greater than 1 into less than 1 after adjustment (Table 5).

Discussion
This large retrospective study showed the chemosensitivity of young patients aged < 35 years having ERpositive breast cancer with minimal endocrine effect. When other confounding factors were controlled, young patients were not found to respond to chemotherapy better than older patients in the ER-positive group.
Although there was no change in odds ratio in the ER-negative group after adjustment, it was notable that odds ratio changed in the ER-positive group, albeit not statistically signi cantly. In other words, there was no difference of chemosensitivity beween young and older patients in ER-positive group while chemosensitivity of young patients was better than that of older pateints in ER-negative group. This suggests that bene t of chemotherapy in young patients with ER-positive breast cancers cannot be big, relatively.
Neoadjuvant chemotherapy is generally performed in patients with locally advanced breast cancers Physicians tend to give patients with advanced disease more aggressive treatment because of their high tumor burden and increased risk of recurrence and death. (17) In addition, as chemotherapy is considered more effective in younger patients with poor prognosis and shorter disease-free survival regardless of the type of treatment, tumor size, grade or progesterone receptor status, these patients are administered neoadjuvant chemotherapy for breast-conserving surgery or a lower threshold when determining chemotherapy. (1,2,18) A large study of patients aged < 50 years reported that among patients with lower risk disease, those who were not treated with adjuvant systemic therapy and were aged < 35 years had the worst prognosis. This age-related trend was not seen in high-risk patients with a greater number of metastatic nodes or a larger primary tumor size.They suggested that young women with breast cancer should be regarded as high risk patients and be given adjuvant cytotoxic treatment on the basis of age alone.(9) This recommendation is under the assumption that young patients would respond better to cytotoxic treatment even in lower risk disease. (19) However, steroid hormone status was not considered in this analysis. (9) The association of young age with response to chemotherapy has not been clearly de ned, particularly according to ER status. (20) In this study, young age was not an independent predictor of response to chemotherapy in ER-positive tumors. Patient age of < 35 years was found to be associated with pCR in ERnegative tumors but not in ER-positive tumors. Better response to chemotherapy was related to aggressiveness of tumors in ER-positive breast cancers. High proliferative index was an independent factor only in ER-positive tumors. Therefore, in ER-positive tumors, it is hard to expect that complete pathologic response would be achieved in low proliferative tumors, even though they developed in young patients. Conversely, Early Breast Cancer Trialists' Collaborative Group reported that adding adjuvant polychemotherapy to tamoxifen was associated with 7.6% of 5-year survival gain in patients aged < 50 years with ER-positive tumors. (21) The survival bene t of chemotherapy in young patients was greater than in older patients. However, because survival bene t may not come from a cytotoxic effect of chemotherapy and the criteria for age were different, response to polychemotherapy in young women cannot be interpreted clearly. Furthermore as tamoxifen is the only endocrine therapy, the endocrine effect of chemotherapy would play an important role in bene t of chemotherapy for patients less than 50 years with ER-positive tumors as in most other studies.
The evidence of the bene t of adding chemotherapy to endocrine therapy in hormone-responsive breast cancer of young women is not strong. (22,23) A pooled analysis of patients aged ≤ 40 years enrolled in EORTC trials showed that prolonged adjuvant CMF chemotherapy did not bring survival advantage to hormone receptor-positive patients compared with hormone receptor-negative patients.(23) Several studies suggest very good prognosis with optimal endocrine therapy alone, particularly in low-risk young patients. (24) Treatment advances with endocrine agents, such as ovarian function suppression or aromatase inhibitor, to overcome resistance to endocrine therapy in young breast cancer patients could improve patient outcomes without chemotherapy in young women with ER-positive breast cancers.(25, 26) Improvement of outcome in these patients is attributed to the endocrine effect. Particularly in ER-positive early breast cancer, the role of chemotherapy can be limited to endocrine effect, not cytotoxic effect. A STEPP analysis for patients with ERpositive tumors who received dose-intensive epirubicin and cyclophosphamide suggested a correlation between the achievement of ovarian function suppression and e cacy of chemotherapy despite no interaction of age and chemotherapy. (27) The bigger reason for the less bene t in these patients may be that ER-positive breast tumors resist chemotherapy. This study showed that the effect of ER status was greater than the effect of age. Some reports explain that the lower frequency of receptor positivity in premenopausal women may account for their increased response to cytotoxic chemotherapy. Predictive value for response to chemotherapy of young agein ER-positive breast cancers should be considered differently from that in ER-negative breast cancers. Most studies have focused on the endocrine effect of chemotherapy regarding the prognosis of young patients with ER-positive tumors. Although indirectly, this study shows the cytotoxic effect of chemotherapy in young patients with ER-positive tumors while excluding the endocrine effects as much as possible. Age was an important predictor of pCR in ERnegative tumors but not in ER-positive tumors in this study. In previous studies, it has been reported that young breast cancer patients had better response to chemotherapy than older breast cancer patients. (30) However, this result does not take ER status into consideration even though the proportion of ER-positive tumors was signi cantly different between both groups. A recent German study by Loibl et al. also reported that pCR was signi cantly higher following neoadjuvant treatment in patients aged < 35 years than in those aged over 35 years, and the outcomes were similar only for the triple-negative breast cancer subtype. The conventional perception that young women respond better to chemotherapy needs to be accepted as limited to ER-negative breast cancers.
In ER-positive tumors, the effect of endocrine therapy following adjuvant chemotherapy makes it di cult to determine the cytotoxic effect of chemotherapy only on the outcome unclear in an adjuvant setting. Conversely, in a neoadjuvant setting, low rate of complete response to neoadjuvant chemotherapy may suggest resistance to chemotherapy because neoadjuvant chemotherapy is an in vivo measure of chemosensitivity.(31) However, chemotherapy inevitably has an endocrine effect, and response to chemotherapy may have less impact on prognosis relatively in ER-positive tumors when compared to ERnegative tumors. This may be a cause of a weak relationship between pCR and outcome of ER-positive tumors when it is compared to ER-negative tumors.
In this study, there are some limitations because it is a retrospective cohort study using multicenter database. Even though TNM stage at diagnosis was recorded, the exact tumor size or axillary lymph node status before neoadjuvant chemotherapy was uncertain. Some patients did not receive ne needle aspiration or sentinel lymph node biopsy for axillary lymph nodes. Therefore, only patients with pathologic complete response were included. In addition, pCR indicates high tumor chemosensitivity and is a meaningful factor to investigate the relationship between response to chemotherapy and disease outcome.(32) In addition, the neoadjuvant chemotherapy regimen including trastuzumab administration was not the standard treatment during the study period. For HER2-positive tumors, this study does not re ect the current treatment.
In an adjuvant setting, only the cytotoxic effect of chemotherapy could not be compared between young and older patients with ER-positive tumors due to the effect of adjuvant endocrine therapy. Therefore, despite these limitations, this study suggests that young age alone is not an immediate factor in determining a chemotherapy escalation or neoadjuvant chemotherapy in ER-positive breast cancers. If other predictive factors for response to chemotherapy are not found, even increasing breast-conserving rate after neoadjuvant chemotherapy may be hard to expect As in ER-positive breast cancers, response to neoadjuvant chemotherapy was not correlated with long-term outcomes, and poor response to chemotherapy based on pCR would not mean survival bene t of chemotherapy.(33)

Conclusions
For improving overall survival young patients with advanced ER-positive tumors, more effective endocrine therapy would be considered rst, and then, a speci c chemotherapy regimen or combination of drugs to target a different biology of young women may be required.