Relationship between pathological response and molecular subtypes in locally advanced breast cancer patients receiving neoadjuvant chemotherapy

Abstract Majority of patients with breast cancer were diagnosed with locally advanced stages of the disease (54%). This study aimed to explain the pathological response received to neoadjuvant chemotherapy (NACT) according to the molecular classification of breast cancer in patients with locally advanced tumors. One hundred and one patients with locally advanced breast cancer treated with neoadjuvant chemotherapy were analyzed. Patients were classified into five molecular subtypes based on the profile of the estrogen receptor, progesterone receptor, HER2, and Ki-67. We determined associations between complete pathological response (no invasive tumor after neoadjuvant chemotherapy) and molecular subgroups. Most patients had luminal A tumors (n: 28, 27.7%). The overall rate of complete pathological response (pCR) was 34.7% (n:35). Tumors that presented with the highest rate of pCR were pure HER2-positive, at 60% (n:6; OR, 3.2; 95% CI, 0.8–12.2). According to logistic regression analysis, the factors affecting pCR were HER2 positivity and clinically positive axilla before NACT. Luminal A tumors had a significantly lower pCR rate. (7.1%,p: 0.001). Despite the low pCR rate, Luminal A tumor had the best survival rate in the subgroups (p < 0.001). However, there was no difference between EFS and OS according to pCR in any molecular subgroups. Pathological complete response is directly related to the subtypes of breast cancer. A high complete pathological response rate is observed in the pure HER2-positive group. However, EFS and OS were not statistically significant in patients with and without pCR.


Introduction
Neoadjuvant chemotherapy is an option for patients with early and advanced breast cancer when an indication for chemotherapy is given. The main goal of giving neoadjuvant chemotherapy (NACT) to patients is to provide a complete pathological response(pCR) [1]. The pathological response of breast cancer to neoadjuvant chemotherapy is a prognostic indicator of long-term disease-free and overall survival [2]. Pathological complete response means that invasive tumor does not remain microscopically in the breast tissue after neoadjuvant chemotherapy. Pathological complete response is observed in 10-40% of breast cancer patients [3]. Many factors affect the difference in the ratio. Incidence and prognostic impact of pCR vary among breast cancer subtypes. Molecular subtypes of breast cancer give different responses to neoadjuvant chemotherapy [4]. Breast cancer is divided into five distinct molecular subtypes based on the absence or presence of molecular markers for estrogen or progesterone receptors and human epidermal growth factor 2(ERGF2 or HER2): Luminal A, Luminal B-HER2 positive, Luminal B-HER2 negative, Pure HER2 positive and Triple-negative. Luminal A tumor; hormone receptorpositive, HER2 negative, and Ki-67 index is less than 15%. Luminal B tumor; hormone receptor-positive and a Ki-67 index of more than 15%. Luminal B tumor contains two subgroups as HER2 positive and negative. Pure HER2 positive tumor; hormone receptor-negative, HER2 positive subgroup. In the triple-negative subgroup, there is no hormone and HER2 receptor. A 70% of all breast cancers are hormone-positive, HER2 negative tumors, 10% are hormone-positive HER2 positive tumors, 5% are pure HER2 positive tumors, 15% are triple-negative tumors [5]. Molecular subgroups have different behavior from each other. Luminal A subtype responds poorly to chemotherapy, but its prognosis is the best among others. A triple-negative tumor has the worst prognosis. Fortunately, triple-negative breast cancer has a higher response to chemotherapy [6].
This study aimed to determine the pathological response and prognosis following neoadjuvant chemotherapy in the molecular subtypes of breast cancer in patients with locally advanced tumors treated within Istanbul University-Cerrahpaşa, Cerrahpaşa Medical Faculty Oncology Department.

Methods
Ethics approval was obtained from Istanbul University Cerrahpaşa-Cerrahpaşa Medical Faculty, Turkey, at the beginning of the study. We evaluated 101 patients diagnosed between 2016 and 2020 by true-cut biopsy and treated with NACT followed by surgical management. We excluded patients who progressed to clinical stage IV, those who, for some reason, did not receive surgical management after neoadjuvant therapy, and patients whose pathology reports cannot be reached ( Figure 1). We obtained information about the clinic and demographic data of all enrolled patients from the hospital database. The variables analyzed in our study were age, body mass index, menopausal status, histopathological characteristics of the biopsy, initial clinical stage of the disease, NACT regimen received, clinical response, type of surgical management, pathological response according to Miller-Payne grading, adjuvant treatment if offered, presence of recurrence and disease progression, overall survival.
The NACT regimens comprised of 5-fluorouracil, epirubicin, and cyclophosphamide and docetaxel (FEC-T); F 600 mg/m2 intravenously on day 1, E 60 mg/m 2 intravenously on day 1, and C 600 mg/m 2 intravenously on day 1, repeated every 21 days for 4 or 6 cycles. Patients who received four cycles of FEC received an additional four cycles of docetaxel 75 mg/ kg); adriamycin, cyclophosphamide, and docetaxel or weekly paclitaxel (ACT, A 60 mg/m 2 intravenously on day 1, C 600 mg/m 2 intravenously on day 1 for four cycles after that T 75 mg/m 2 intravenously on day 1 for four cycles of weekly paclitaxel for 12 weeks. Patients with HER2-positive received Trastuzumab(H) 6 mg/kg, following 8 mg/kg loading dose intravenously on day 1, repeated every 21 days). Trastuzumab was given as adjuvant therapy for four cycles to HER-2 positive cases (ACTH or FEC-TH). Thirteen cycles were given after the operation. Trastuzumab (H), a humanized anti-HER2 monoclonal antibody, was added to chemotherapy in all HER2-positive patient regimens. In recent years, pertuzumab(P) has been used in neoadjuvant therapy for HER2-positive patients, and six patients were able to receive pertuzumab in addition to trastuzumab (ACTHP). For some patients with the triple-negative subtype, Carboplatin AUC2 weekly treatment was added to the ACT regimen. Twenty-three patients underwent eight ACT cycles; five patients received ACT-H, two patients received ACT plus carboplatin, six patients received FEC, forty patients received FEC-T, and fourteen patients received FEC-TH chemotherapy regimens. Three patients, who were not eligible to take anthracycline, received docetaxel and trastuzumab, and two patients received only weekly paclitaxel.
Lumpectomy or mastectomy was performed in patients whose neoadjuvant chemotherapy was completed. The axillary approach was divided into two groups as sentinel lymph node biopsy and axillary curettage. The patients were divided into five groups by true-cut biopsy according to their molecular subtypes before the operation. Luminal A, Luminal B HER2-negative, Luminal B HER2-positive, Pure HER2-positive and Triple negative. Luminal A tumor; ER, PR positive, HER2 negative, Ki-67 less than 15%, and Luminal B; ER, PR positive, Ki-67 above 15% were defined. Luminal B itself was examined in two groups: HER2 positive and HER2 negative. Pure-HER2 is ER, PR negative, HER2 positive; triple-negative; ER, PR, HER2 negative subgroups. They defined the cut-off value as 1% as the definition of ER and PR positive. A positive Ki-67 finding was defined as >15%, and a negative finding was defined as 15%. HER2 scores of 0 and 1þ were considered harmful, 3þ meant HER2-positive. When a score of 2þ was found, additional fluorescent in situ hybridization testing was performed to determine the HER2 gene amplification status.
Several histopathological classifications categorize the tumor response to NACT [7]. We use the Miller-Payne grading (MPG) system to determine the pathological response to NACT. In our pathologist's opinion Miller-Payne histological grading system was the most useful. MPG provides a five-step scale based on tumor cellularity in the lumpectomy or mastectomy specimen compared to the pretreatment core biopsy. MPG is divided into five grades. Grade 1, pathological no response(pNR); grade 2, minor loss of cellularity (30%); grade 3, estimated 30-90% reduction in tumor cells; grade 4, 90% loss of tumor cells; and grade 5, complete pathological response(pCR) means no invasive carcinoma but ductal carcinoma in situ may be present [8].
Statistical analysis SPSS 15.0 for Windows program was used for statistical analysis. Descriptive statistics; numbers and percentages were given for categorical variables, and numerical variables were given as mean, standard deviation, minimum, maximum, median, interquartile range. The rates in the groups were compared with the Chi-Square Test. When the numeric variables did not meet the normal distribution condition, more than two independent groups' comparisons were made using Kruskal-Wallis Test. The Mann-Whitney U Test made two independent group comparisons. In more than two groups, subgroup analyses were performed with Mann-Whitney U Test and interpreted with Bonferroni correction. Two independent group comparisons of the numerical variable satisfying the normal distribution condition were compared by Student's t-test. Logistic Regression Analysis evaluated determining factors. Statistical alpha significance level was accepted as p < 0.05. The EFS and OS functions were determined by the Kaplan-Meier estimator.

Results
Our study included one hundred thirty-five patients who received neoadjuvant chemotherapy between January 2016 and January 2020. Of the 135 patients who received neoadjuvant treatment, 34 patients were excluded for the following reasons: 20 (14%) patients had missing data, 9 (8%) patients were restaged because of distant metastasis, and 5 (4%) patients were withdrawn for surgery.
In total, 101 patients were included (median age, 46.7 ± 10.9 years; range, 24-73); 55.4% (n: 56) of the patients were premenopausal, 33.7% (n:34) of the patients were postmenopausal. The tumor was located in the left breast in 59 (58.4%) patients, in 41 (40.6%) in the right breast, and in the bilateral breast in 1 (1%) patient. There was no significant correlation between age, menopausal status, tumor location, and complete pathological response.
We examined the patients in five groups according to their body mass index. Mean body mass index was 27.9 ± 5.4 (range 17-43). There was no difference between those with and without a complete pathological response (p: 0.23).
The most common histological grade 2 tumor was found in true-cut biopsies (60.4%, n ¼ 61). (table1) Patients were divided into five groups according to molecular subtypes. Luminal A tumors were 27.7% of cases, with 28 patients; luminal B HER2-negative was described in 25 patients (24.8%); 10 patients presented with pure HER2-positive tumors (9.9%); and the triple-negative tumors reached 18  The axilla was clinically positive in 88.6% of the 35 patients with the complete pathological response. A fine-needle aspiration biopsy confirmed the clinically positive axilla in each patient. According to the univariate regression analysis, the invasive tumor in the axilla positively affects the chemotherapy response. (p: 0.043, OR: 3.69; 95% CI 1.0-13.1). It was observed that 57% of the patients with clinically positive axilla were in the group with a pCR. The pathological complete response rates in the axillary lymph nodes of luminal A, luminal B/HER2-, luminal B/HER2þ, pure HER2-positive and triple-negative disease were 14.3%, 52.0%, 89.5%, 60.0% and 47.4%, respectively. There was a statistically significant difference in the axillary response rates in the molecular subtype groups (p < 0.001). The pathological axillary response was the best of the group of luminal B-HER2 positive. As expected, the pathological response of the primary tumor was similar in this group (57.7%). While only 5.3% of this patient group did not respond to chemotherapy, the axilla showed partial response in the remaining 5.3%. Luminal A was the group with the least axillary pathological response. In the luminal A subgroup, most of the patients (64.3%) had a partial response to chemotherapy in the axilla.
Nuclear grading of the patients was performed by true-cut biopsy before the neoadjuvant therapy. The most common tumors were Grade 2, while grade 1 tumors were the least. The number of patients with grade 2 tumors was 61 (60.4%), the number of patients with grade 3 tumors was 39 (38.6%), and the number of patients with grade 1 tumors was 1 (1%). When the complete pathological response was compared with the nuclear grade, It was found that grade 3 tumors had the highest pCR rate. 46.2% of the patients with grade 3 tumors had a complete pathological response to neoadjuvant therapy. In addition, the relationship between pathological complete response and grade was examined for each group in molecular subtypes. Again, there was no significant difference (p: 0.67).
The index Ki-67 in the entire patient population was 39.4 ± 24.9 (range 5-90). The median index of Ki-67 was 40 (range 30-60) in patients with the complete pathological response and 30 in (15-60) patients with no complete pathological response. There was no statistically significant difference between the two groups (p: 0.06).
Anthracycline-based chemotherapy was the most administered neoadjuvant therapy (n ¼ 96, 95%). Taxane-based chemotherapy was given to 5 (5%) patients who could not receive anthracycline. Four of these patients were HER2 positive and received trastuzumab in addition to taxane-based therapy. Every patient who was HER2 positive was able to receive trastuzumab (n:29, 28.7%). Pertuzumab þ trastuzumab was used in 6 patients. Carboplatin was added to 2 (2%) treatments with triple-negative tumors. The adriamycin þ cyclophosphamide þ taxane (ACT) regimen achieved the highest pCR rate, 29.9% (n:26), and ıt was similar to FEC and FETCH. Adjuvant chemotherapy was administered to 9 (8.9%) patients, and most of them had triple-negative tumors (n: 7, 77%) with the evidence of residual tumor in the surgical specimen. Capecitabine was the most preferred agent in adjuvant therapy (n: 5, 56%). In the adjuvant treatment, docetaxel was preferred for the patients who did not receive taxane-based therapy in neoadjuvant therapy (n: 4, 44%). All patients with positive hormone receptors received adjuvant hormone therapy (n: 73, 74.3%) and 91% (n:90) of the patients received adjuvant radiotherapy. The most preferred hormonotherapy was letrozole, as 34% of the patients were postmenopausal (table 3).
A breast examination was performed before and after neoadjuvant therapy. Patients who showed a clinical reduction in the primary breast mass and lymph nodes in the axilla were considered a clinical response. As a result, 88.1%(n: 89) of patients responded clinically to neoadjuvant chemotherapy. However, 39.2%(n: 35) clinically responding patients also had a complete pathological response.
Lumpectomy management was performed in 22.8% (n: 23) of patients, and mastectomy was performed in 78 (77.2%) patients. Eighty patients (79.2%) were taken to axillary lymphadenectomy since their tumors Tumor markers were examined before and after the neoadjuvant in the patients. The initial CEA level was 3.00 ± 1.94 (in the range of 0.5-9), and the Ca15.3 level was 28.3 ± 23.1 (6-184). After the neoadjuvant therapy, the CEA level was 1.81 ± 1.16 (range 0. [3][4][5], and the Ca15.3 level was 14.9 ± 8.7 (range 3.4-60). The mean initial CEA of the pure HER2positive group was significantly higher in the subgroup analysis than Luminal B HER2-positive groups (p: 0.003). However, there was no significant difference between the initial and post-neoadjuvant tumor markers in groups with and without complete pathological responses.
The overall recurrence rate (local and distant) was 14% (n: 14), and the median recurrence time of 24 months (6-44 months) with a follow-up of 60 months. Local or regional recurrence was n:3 (21%), and the number of distant metastases was n:11 (79%). Two of the patients (67%) with local recurrence were triple-negative. The other patient was Luminal B HER2-positive. None of the patients with local recurrence had a complete pathological response. The pathological response of all these patients was MPG 2. The molecular subtype that determined the most relapses was luminal B HER2-positive, at 36% (n: 5), followed by the triple-negative at 29% (n: 4), luminal B HER2-negative, 21% (n: 3), and pure HER2-positive presented two cases (14%). Of the 11 who developed distant metastases, 36% (n: 4) had a complete pathological response. Most recurrences (71.5%; n: 10) occurred in patients who did not obtain pathological response (table 4). The most common site of metastasis was bone (7%), the second lungs, (6%).
A total of 5 deaths were documented, describing 5% of the total patients, with a median follow-up time of 19 months (6-31 months). Among these, the number of patients who died despite a complete pathological response was n: 2 (40%). Two of these patients were HER2-positive. Luminal A subtype had significantly higher overall survival (p < 0.001). EFS and OS, according to pCR in molecular subgroups, were evaluated by a non-parametric Kaplan-Meier estimator. However, there was no difference between EFS and OS according to pCR in any molecular subgroups.

Discussion
In 2020, there were 2.3 million women diagnosed with breast cancer and 685.000 deaths globally. According to statistical data, approximately 54% of these patients are locally advanced [9]. Neoadjuvant chemotherapy is the standard treatment for locally advanced breast cancer. It is also recommended for breast-conserving surgery and minimal axillary lymph node dissection in early-stage breast cancer [10]. However, the first aim of neoadjuvant treatment is to downstage the disease and render in-operable tumors resectable. The pathological target of neoadjuvant therapy is the complete pathological response (pCR) [11]. pCR is a prognostic factor and linked to better overall survival (OS) and event-free survival (EFS) [12]. The most important factor affecting the complete pathological response is the affinity of the tumor to neoadjuvant therapy. Tumors' sensitivity to neoadjuvant chemotherapy differs in molecular subtypes [13]. Molecular subtypes are divided into five main groups depending on the presence or absence of estrogen, progesterone, HER2 receptor, and the percentage of Ki-67 [14]: Pathologic responses of primary breast tumor were evaluated according to the Miller-Payne grading system, which defines pCR as the absence of invasive and in situ carcinoma in the breast and axillary nodes (ypT0/ypN0) [15]. The clinical trials have shown that the complete pathological response is related to good treatment results [16]. In recent years, factors predicting complete pathological response were investigated. ER, PR levels, HER2 status, tumor grade, molecular subtype, perineural invasion, vascular invasion, Ki-67, and tumor size could not fully correlate with pathological response [17]. Grade 5 in the MPG system was considered to be complete pathological response. In our study, the number of patients with complete pathological responses was 35 (34.7%). In 101 patients, pathological complete response rates for primary tumor in luminal A, luminal B/HER2-, luminal B/HER2þ, pure HER2-positive, and triplenegative disease were 7.1%, 44.0%, 57.8%, 60.0%, and 21.0%, respectively. In other words, the group with the highest pathological response rate was pure HER2 positive, and the group with the lowest was luminal A (%7.1). pCR rate was significantly lower in luminal A tumors (p ¼ 0.001). Even if the pathological complete response rate of luminal A tumors is low, survival rates are high. No local recurrence or death was observed in any Luminal A tumors in our data.
Although luminal A tumors respond less to chemotherapy, the positivity of ER, PR, and low Ki-67 levels are associated with a good prognosis [18]. HER2-positive tumors are known for their aggressive behavior. In the study of Buzdar et al., the pCR rate for HER2-positive tumors was given as 66.7% [19]. Our study examined the HER2-positive tumors in 2 categories: Luminal B HER2-positive and pure HER2-positive. Pathological complete responses of these two subgroups were compatible with the literature, 57.8%, and 60%, respectively. When the patients with a pathologically complete response and those who did not were compared, a significantly higher HER2-positivity was found at complete responders (p ¼ 0.017). Anti-HER2 treatments contributed to this critical response in neoadjuvant therapies. A metaanalysis by Antonis et al., determined that the addition of trastuzumab in HER2-positive breast cancer in the neoadjuvant setting improves the probability of achieving higher pCR [20]. In addition, clinical studies have shown that using dual HER2 blockade (pertuzumab þ trastuzumab) in neoadjuvant therapy further increases the pCR rate [21]. Clinical studies have shown that neoadjuvant chemotherapy with trastuzumab plus pertuzumab increases the pCR rate compared with trastuzumab alone, resulting in about 50-70% pCR rate [22]. In our data, pCR was observed in all six patients who received dual HER2 blockade. The low number of patients given pertuzumab is due to the fact that it was not paid by health insurance before 2019.
Triple-negative tumors are also tumors with an aggressive course. However, they have a high sensitivity to chemotherapy. Neoadjuvant chemotherapy is seen as the standard treatment for triple-negative and HER2-positive tumors. In the study of Sharma et al., 35-55% pCR was seen with neoadjuvant cytotoxic chemotherapy in triple-negative tumors [23,24]. In the GeparSixto study, 53.2% pCR was obtained by adding carboplatin to neoadjuvant therapy in triplenegative tumors [25]. In our study, the rate of pCR in triple-negative tumors was much lower than in the literature (21%, n: 5). The neoadjuvant treatment of patients with a complete response was combinedbased therapy with anthracycline and a taxane (FEC, ACT or FECT). Carboplatin was added to the treatment of only one of these patients. When all triplenegative patients were examined, there were already only two patients to whom carboplatin was added to their treatment. In other words, 50% of the patients who added carboplatin in our study gave a complete response.
Luminal A tumor is the least chemosensitive; our study achieved pCR rates of 7.1% (n:2). In the study of Collins et al., 114 patients with Luminal A tumor had a pCR rate of 7.9% [26]. In Collins et al. study, patients with complete pathological responses were the patients with high-grade tumors and weak PR expression. In our study, the tumor grade of 2 patients with complete pathological response was two, and the PR expression percentage was below 10%. Similar results were obtained for luminal A tumors in the 14000 diseases neoadjuvant study of Haque et al. The rate of pCR in patients with luminal A tumor was found to be 0.03%. However, in the 5-year overall survival analysis, Luminal A tumors are much better than other subtypes (98.2%) [27]. This shows that the survival of luminal A tumors is quite good, although they do not give a complete pathological response. In our study, no recurrence was observed in the patients with luminal A tumor despite the low pCR rate.
In a review by Mikhail et al. it was stated that luminal tumors have low chemosensitivity. In addition, it has been emphasized that intra-and intertumor heterogeneity may affect treatment responses to adjuvant therapy in luminal tumors. Luminal breast cancer is a highly heterogeneous disease, including different gene-expression profiles and mutational patterns, with various clinical courses and responses to neoadjuvant treatment. With explicit knowledge of the genomic data of patients with luminal tumors, we can understand chemotherapy resistance. This situation affects neoadjuvant chemotherapy responses. Genomic maps of patients can predict treatment response. [28] In our data, the second-highest pCR rate was in Luminal B HER2-positive tumor (%57.8). In the study of Wang et al., the rate of pCR was 13.4% in the patients with Luminal B tumors. However, Wang et al. showed that patients with a high Ki-67 index above 40% had a better pathological complete response and recurrence-free survival [29]. A metaanalysis of 6793 patients by Tao et al. showed that Ki-67 labeling index is a predictive marker for a complete pathological response to neoadjuvant chemotherapy in breast cancer [30]. In our data, the median index of Ki-67 of all patients with a complete pathological response was 40% , and the median index of Ki-67 for those who did not have a complete pathological response was 30 . There was no statistically significant difference between the two groups (p: 0.06). However, when patients with luminal B tumors were examined separately, the median Ki-67 was 41.7 (range 10-80). The high Ki-67 value of our patients with luminal B tumors may have contributed to a better response to neoadjuvant chemotherapy.
Axillary lymph involvement is the most critical factor in breast cancer survival. [31]. In our data, patients with complete pathological response had a high rate of clinical axilla positivity before neoadjuvant chemotherapy. (88.6%, p: 0.043, OR: 3.69; 95% CI 1.0-13.1) On the contrary, Samiei et al. showed that patients with clinically negative axilla achieved a higher complete pathological response rate in ER þ HER2þ, ER-HER2þ and triple-negative subtypes [32]. Furthermore, in our study, all patients with clinically positive axilla were in the luminal B HER2-positive and pure HER2-positive groups.
Patients with grade 3 tumor differentiation constituted 38.6% of the entire population (n: 39). The grade 3 group had the highest rate of pCR (n: 18, 46.2%). Diaz-Casas et al. reported the pCR rate as 21.3% in grade 3 tumors [33]. In the study of Jarzab et al., the pCR rate was reported as 30.9% in the patients with grade 3 nuclear differentiation [34].
Pathological complete response is associated with the neoadjuvant chemotherapy regimen. Therefore, anthracycline-based chemotherapy regimens form the basis of treatment. The ACT regimen achieved the highest pCR rate, 29.9% (n ¼ 26), similar to FEC and FETCH. Recently, pertuzumab has been added to the treatment of HER2-positive tumors along with trastuzumab. In addition, carboplatin was added to the anthracycline and taxane regimen in triple-negative tumors. However, since the number of these patients was small in our study, the effects of pertuzumab and carboplatin on pCR could not be reported.
There was a significantly higher EFS and OS difference in the Luminal A subtype compared to the other molecular subtypes (p < 0.001). However, the complete pathological response did not affect the survival in Luminal A tumor. This can be explained by the low chemosensitivity of Luminal A tumors. Fortunately, long survivals have been achieved by the increasing hormone therapy options over the years. The HER2 positive group had a high pathological complete response. However, this situation was not reflected in EFS and OS in our patient population. OS analysis for the HER2 positive group was not significant as p: 019 for the complete pathological responder and non-pathological group. Regardless of the subgroup, when the patients were divided into two groups as pathological complete response and nonresponder, and OS analysis was performed, there was no significant difference in survival between the two groups (p: 0.06). However, the short follow-up period in our study may have prevented us from seeing a significant difference. We hope that we will distinguish between the pathologically responding and nonpathological subgroups with a larger patient group, over an extended follow-up period and by the use of new neoadjuvant treatment.

Disclosure statement
No potential conflict of interest was reported by the authors.