Breast cancer is a leading cause of cancer mortality in the world. It is now the fifth leading cause of cancer-related fatalities. According to Global Cancer Statistics 2020, breast cancer in females is now the most commonly diagnosed cancer (11.7%), exceeding lung cancer (11.4%). In females, breast cancer is both the most commonly diagnosed malignancy and the leading cause of mortality due to cancer.¹

Breast cancer is known to be a heterogeneous disease, showing variable morphological and clinical features, with variable response to treatment. Assessment of breast cancer mainly comprises the evaluation of histologic type, grade, and stage of the tumor. Apart from these, assessment of estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2) expression is also done routinely in the evaluation of breast cancer.² The molecular subtyping emphasizes the biological heterogeneity of breast cancer, paving the way for the evolution of new therapeutic strategies for breast cancer.³

Treatment of patients with operable breast cancer is multidisciplinary and combines local treatment and systemic therapy. Local treatment includes surgical excision and radiation therapy. Systemic therapy in breast cancer includes chemotherapy, endocrine therapy, and anti-HER2 therapy. The molecular subtype of the tumor guides the appropriate choice of therapy for the patient. Systemic therapies aim to improve survival by controlling micro-metastasis. Based on the timing, systemic therapy can either be adjuvant, which is given after surgery, or neoadjuvant, which is given before surgery. Neoadjuvant chemotherapy (NACT) is mainly used in locally advanced breast cancer to downstage the disease and enable breast-conserving surgery.⁴

Several factors may cause alterations in the biomarker profile in surgical specimens of breast cancer. They include tumor heterogeneity, a limited amount of tissue evaluated in a biopsy being poorly representative of the tumor, and changes in tumor biology due to chemotherapy given to the patient.⁵

The Ki-67 proliferation index is also expected to change with the administration of NACT. As Ki-67 has been reported to have predictive and prognostic value in patients with invasive breast cancer who received NACT. The post-therapy Ki-67 proliferation index level could provide an additional prognostic value where the pathological complete response is not being achieved.

Substantial evidence from various studies has shown significant alterations in biomarker expression following neoadjuvant chemotherapy in breast cancer. Such a change in receptor status calls for evaluation, as the conversion of receptor status from negative to positive may warrant a change in the treatment plan. However, there is still only limited evidence on whether such a change in treatment plan benefits the patient and also on the impact of alterations in biomarker profile on the survival of the patient.

Still more controversial is whether targeted therapies should be stopped or continued if the receptor status becomes negative in a patient.⁶ Therefore, all of the above warrant evaluation of receptor status post-neoadjuvant chemotherapy in breast cancer. Only a few studies have been conducted in the Indian population evaluating changes in receptor status post-neoadjuvant chemotherapy. Through this study, we intend to evaluate the alterations in receptor status and Ki-67 index post-neoadjuvant chemotherapy in breast cancer.

The mean age of the participants was 43.839.40 years, with ages ranging from 26-65 years. There were 31.4% of the patients in the 36-45 and 46-55 year age groups. Twenty-four (68.6%) patients had lesions in the right breast, and the most common quadrant was central (20%), followed by upper outer and upper inner quadrants (17.1% & 14.3%), respectively. The distribution of the “T” stage of the tumor among study subjects included 34.3% of T1, followed by T3 (28.6%). Thirteen patients (37.1%) had no nodal involvement, while 13 patients (37.1%) belonged to the N1 stage. Nodal status could not be assessed in one patient as lymph node resection was not done. We observed no change in MBR scoring in 54.2% of patients following NACT. A decrease in MBR scoring was seen in 31.4%, whereas 14.2% showed an increased MBR score. In 4 patients, no tumor was left, and 1 had in situ carcinoma (nodal involvement present in both) post-chemotherapy; therefore, no comparison was done. Histological grade decreased post-chemotherapy in 17.1% of the patients, whereas 8.6% of the patients showed an increase in histological grading. However, the majority of the patients (74.3%) observed no change in their histological grade. Also, 51.4% of patients were ER-negative and 48.6% of patients were ER-positive before chemotherapy. Five patients had a change in ER status from positive to negative; however, this change was not statistically significant (P=0.06). Similar observations were made for PR expression, as 5 patients had a change in PR status after chemotherapy, although the change was not significant. Changes in HER2neu expression were also observed as two 3+ positive patients changed to 1+ and 2+ staining (Tables 1, 2). In addition, 100% of the participants had a Ki-67 index of >14% in their prechemotherapy biopsy samples, two of whom (5.7%) observed a change in the Ki-67 index to <14% post-neoadjuvant chemotherapy.

The most common subtype before chemotherapy was Luminal B (48.6%), whereas triple-negative breast cancer was the most common (42.9%) post-chemotherapy (Table 3).

When AR expression was compared before and after chemotherapy, we observed a statistically significant change (P=0.03). Six AR-positive patients changed to negative after receiving NACT and also showed a decrease in Allred score.

The discordance was highest in the Luminal B subtype, with a total of seven cases showing differences in pre- and post-chemotherapy, and most of them changed to TNBC (5 cases). (Table 3).

A significant change in the Allred mean score of ER expression (from 3.17±3.45 to 2.31±3.31, P=0.03) and AR expression (3.86±3.48 to 2.46±3.12, P=0.001) was observed between pre- and post-chemotherapy samples (Tables 4 and 5). A significant association was also observed between histological grade and change in HER2neu status (P=0.01).

Treatment for breast cancer is multidisciplinary and combines local and systemic therapy. Systemic therapy, essential for reducing tumor size and eradicating micrometastasis, encompasses hormonal therapy, anti-HER2 therapy, and chemotherapy.

When the change in receptor status was compared to TNM staging, no significant associations were observed (Tables 6 and 7).

Neoadjuvant systemic therapy, which is administered before surgical resection, is based on the hormone receptor expression and HER2 status of the patient, which is now routinely assessed in the core needle biopsies sent to confirm a histological diagnosis of breast carcinoma.^4,10 The existing literature regarding the alterations in receptor status due to neoadjuvant chemotherapy ranges from studies showing no alterations to ones reporting significant alterations.

Due to the inconsistency in the literature, there is uncertainty about whether re-evaluation of the receptor status is essential in the residual tumor and if the treatment options are to be modified according to the final molecular profile of the tumor. The pathogenesis behind the change in hormone receptor profile following neoadjuvant chemotherapy is multifactorial. Chemotherapy can induce changes at the molecular level that affect the expression of hormone receptors on cancer cells.

These changes may be due to direct cytotoxic effects on cancer cells, selective pressure exerted by chemotherapy, or alterations in the tumor microenvironment. Additionally, chemotherapy may lead to the clonal selection of subpopulations of cancer cells with different receptor profiles, contributing to the observed discordance in receptor status pre- and post-treatment.^10,11

The patient cohort in the present study exhibited a younger age profile with a mean age of 43.83 years when compared to similar studies by Gahlaut et al. and Avci et al., who all observed a mean age of 47 years. This may be due to the smaller sample size of our study and the patients belonging to a different geographical area.^10,11

Ramteke et al. and Gahlaut et al. reported that most of their patients exhibited histological grade 3 (57% and 48.6%, respectively). However, our observations aligned more closely with a previous study by Rey et al., in which most of the patients belonged to histological grade 2 (59.7%).^5,7,10

In this study, we observed a change in histological grade from higher to lower in 17.10% of cases and vice versa in 8.6% of the cases. The percentage of change is lower in comparison to a previous study by Gahlaut et al., who documented a change to a lower grade in 28.8% and an upgradation to a higher histological grade in 13.8%, which could possibly be due to a larger sample size.¹⁰ We found that five patients experienced a change in ER and PR expression from positive to negative, post-neoadjuvant chemotherapy. However, changes in both ER and PR (5/35 patients – 14.2%) were statistically not significant (P=0.06 for both ER and PR). Our findings are consistent with those of prior studies by Katayama et al. and Rey et al., who also documented insignificant changes in ER and PR expression post-chemotherapy. Katayama et al. reported a change in ER expression in 19.9% (4/143 patients) and a change in PR expression in 11.4% (16/140), both of which were not significant (P =0.13 and 0.56). Rey et al. evaluated 78 patients post-chemotherapy and observed a change in ER expression in 2.56% (2/78 patients) and a change in PR expression in 8.97% (7/78 patients), which were both statistically not significant (P= 0.82 and 0.71). In a study on 38 patients, Kinsella et al. found a statistically significant change only in the PR expression in 23.6% of the patients (P=0.03). However, a significant change in ER alone (P=0.0016) was observed by Ramteke et al. with alterations in ER expression in 17% (17/100 patients), with an insignificant change in PR expression (P=0.13) in 13% of the sample (13/100 patients). Contrary to our findings, significant changes in both ER and PR expression had been observed in an earlier study by Gahlaut et al., who observed changes in ER and PR expression in 12% and 14.5% of the patients (out of 133 patients) with p values of <0.001 in both.^5,7,10,12,13

Change in HER2 status was seen only in 8.57% (3/35) patients (P=0.22). Results discordant with our study were observed by Katayama et al., who found a significant change in the HER2 status in 19.9% (44/221) patients (P=0.01). However, their study included only HER2neu-positive patients, either by IHC or FISH, which creates a selection bias when compared to the present study. However, findings similar to the present study were observed by Rey et al., who reported a change in HER2 status in 11.5% (9/78) of patients. Changes in HER2 status have been attributed to the internalization of the HER2 protein and lysosomal degradation.^5,12

We observed a significant change in the androgen receptor expression in the post-chemotherapy surgical specimens (P=0.03). Six patients who had positive AR expression in the biopsy showed negative expression in the resection specimens. The data regarding the change in Androgen receptors after chemotherapy is limited and warrants evaluation in larger cohorts. In this study, only 2 cases out of 35 showed a change in the Ki-67 index from >14% to <14%. This is in contrast to the literature, as most of the studies have shown a significant change in the Ki-67 index post-chemotherapy, possibly due to a larger sample size. Rey et al. documented a change in the Ki-67 index in 18 out of 78 patients, which was statistically significant.⁵

The AR is not routinely assessed in breast cancer patients, despite emerging evidence suggesting its potential significance. AR expression has been found to play a role in the progression and prognosis of breast cancer, particularly in certain subtypes such as TNBC and ER+ cancers. Including AR assessment in the diagnostic and treatment planning process could provide a more comprehensive understanding of tumor biology and aid in the development of tailored treatment strategies

Incorporating AR evaluation into routine practice could offer several benefits for patient care. For instance, AR positivity in TNBC patients, who traditionally lack targeted therapies, might identify a subset of patients who could benefit from anti-androgen therapies. This could open new avenues for treatment in a group that otherwise has limited options. Additionally, for ER+ breast cancers, understanding AR status might help in predicting resistance to traditional endocrine therapies and in developing combination treatment approaches that could improve outcomes.

In the setting of metastatic breast cancer, where treatment options are often limited and the disease is more aggressive, incorporating AR-targeted therapies for AR+ patients might help in controlling disease progression and managing symptoms, ultimately improving the quality of life and survival rates.

Lee et al. observed that the most common molecular subtype to have changed in the post-chemotherapy resection specimens is the Luminal B subtype (14 out of 92 cases), which is in line with the findings of the current study. The resulting subtypes noted in the study were HER2-enriched (1 out of the 14), Triple negative (4 out of the 14 cases), and Luminal A (9 out of the 14 cases), which are also similar to the changes observed in the present study.¹⁴

The notable changes in the receptor status observed in this study were statistically not significant; however, they exhibited a trend toward significance, justifying the need for reassessment in post-chemotherapy specimens. While this study provides valuable insights into the dynamics of breast cancer biomarkers post-neoadjuvant chemotherapy (NACT), it is important to address certain limitations that may impact the interpretation and generalizability of the findings. One notable limitation is the relatively small sample size of 35 patients, which may limit the statistical power of the study and potentially influence the robustness of the observed changes in biomarker expression. The small cohort size might also affect the ability to detect subtle yet clinically significant alterations, thereby necessitating cautious interpretation of the results. Furthermore, the limited sample size can restrict the ability to perform subgroup analyses that could provide a more nuanced understanding of biomarker dynamics across different patient populations. Future studies with larger cohorts are essential to validate these findings and to explore the broader applicability of the results, ensuring that the observed trends are not artifacts of sample size limitations but rather reflective of true biological and clinical phenomena.

This study was done in accordance with the Declaration of Helsinki and after receiving approval from the Institutional Ethics Committee (IEC/VMMC/SJH/Thesis/06/2022/CC-229). Informed consent was taken from the patients.