Effect of neoadjuvant therapy on breast cancer biomarker profile

Breast cancer is the malignancy with the highest incidence (46.3 per 100.000) and mortality rates (13.0 per 100.000) in women worldwide. According to the Surveillance, Epidemiology and End Results Program (SEER), in 2018 breast cancer accounted for 15.3% of all new cancer cases and 6.7% of all cancer deaths in the United States (US) [1, 2].

Neoadjuvant therapy (NAT) has become an important strategy to reduce tumor size in locally advanced breast cancer and facilitate breast conservative surgery, along with monitoring treatment response and eliminating possible micrometastasis [3‐5]. In order to choose an appropriate NAT scheme according to tumor biology, a preoperative evaluation on core-needle biopsies from the primary tumor is performed, where histological type and grade are assessed. Additionally, immunohistochemistry (IHC) of biomarkers, such as: hormone receptors (estrogen receptor (ER) and progesterone receptor (PR)), human epidermal growth factor receptor 2 (HER2) and the cellular proliferation index (Ki67), is also analyzed to guide the therapy and predict survival [4, 6, 7].

These biomarkers have been used as surrogates for breast cancer classification into four main intrinsic subtypes: luminal A, luminal B, HER2-enriched and triple negative (TN) [8]. Both, luminal A and luminal B tumors express ER, therefore these patients are candidates for hormone therapy with ER modulators or aromatase inhibitors [8‐11], whilst HER2-enriched and TN subtypes lack the expression of hormone receptors, therefore are mainly treated with biological therapy agents such as trastuzumab or pertuzumab, and cytotoxic chemotherapy, respectively [5, 11, 12].

Standard clinical recommendations indicate the assessment of ER, PR, HER2 and Ki67 by IHC in biopsy samples [13, 14]. Nevertheless, many retrospective studies have reported changes in biomarker expression in surgical specimens after NAT administration [15‐21]. The main changes correspond to discordances in hormone receptors and HER2 status [22], along with decreases in the percentage of expression, especially for PR and Ki67 [23‐26]. Most of these studies end up suggesting the need to re-evaluate its expression, justifying its importance not only to assess tumor response to treatment but to adjust therapy according to these changes [3, 27]. However, other studies show that these changes are not statistically significant [28] and suggest that the re-evaluation of biomarker expression after NAT might not be necessary, especially for health care institutions with limited resources, as it is the case for many hospitals in Latin-America, including Colombia [29].

Given the prognostic value of biomarkers expression and its important role for deciding treatment scheme, the aim of this study was to compare the IHC expression of ER, PR, HER2, and Ki67 in core-needle biopsies and surgical excision specimens in NAT-treated and non-treated breast cancer samples from patients diagnosed in the Colombian National Cancer Institute (NCI), in order to evaluate NAT effect on biomarker expression profile.

In the current study, we aimed to analyze changes in IHC biomarker status and expression in paired biopsy and surgical samples in breast cancer cases treated and non-treated with NAT, given that changes in biomarker expression may have several clinical implications for disease outcome in breast cancer patients [31‐33]. It may also affect adjuvant therapy regimen, as variation in breast cancer intrinsic subtype after NAT could lead to the addition or discontinuation of therapy schemes [16, 34].

It has been well described that NAT treatment affects Ki67 index, as it targets mainly cycling cells and major cell proliferation pathways [35]. We observed a significant decrease in tumor size and in Ki67 expression values only in the NAT-treated group. Despite the fact that we did not analyze differences in outcome according to these changes, a decrease in Ki67 expression after NAT has been associated with a good clinical-pathological response, better disease-free survival and overall survival [36, 37], whereas no reduction in Ki67 expression after NAT have been associated with a significantly higher risk of breast cancer recurrence and death [38]. Interesting results reported by Dowsett et al. [35] show that Ki67 expression values after 2 weeks of NAT were more useful as prognostic markers for prediction of recurrence-free survival than baseline Ki67 expression before NAT. Our results showing a significant decrease in Ki67 expression after NAT, along with previously reported results, highlight the utility of assessing this biomarker in the surgical specimen after NAT, for prognosis and patients’ clinical outcomes evaluation.

It has been well reported that chemotherapy may have several effects on tumor biology, which could potentially alter biomarker expression [3, 39]. We found a statistically significant decrease in PR expression values between biopsy and surgical specimens after NAT, which is consistent with other reports where the PR, along with the Ki67 index, are the most commonly altered biomarkers after NAT administration [7, 15, 16, 31]. Contrary to what has been reported for Ki67, PR expression loss has been associated with worse tumor characteristics [15] and poor clinical outcomes [23, 32, 40]. It has also been shown that loss of PR expression may be an indicator of a decrease of hormone sensitivity in tumor cells, activation of alternate proliferation pathways such as PI3K/AKT/mTOR [41, 42], and also, the induction of a non-functional ER state which could lead to a diminished response to endocrine therapy, specifically in ER-positive/PR-negative tumors [18, 43].

Loss of ER expression has also been associated with bad clinical outcomes as it affects tumor response to endocrine therapy [44]. Nevertheless, as we reported here, variation in ER expression after NAT is much less frequent than for PR. We did not observe statistically significant changes in ER status nor its expression in neither of the NAT treated or not-treated cases, although we did observe two cases with status loss in the NAT-treated group. These results suggest that analyzing ER after NAT administration may not be as useful as a prognostic marker, as it could be PR and Ki67 status evaluation.

On the other hand, gain of hormone receptor expression after NAT is associated with significantly better outcomes, compared with patients with unchanged hormone receptor expression [45, 46]. It has even been shown that the improvement on survival rates for patients with ER and PR expression gain is dependent on the magnitude of change [47], however, gain of hormone receptor expression is much less frequently reported [22, 31, 32]. In our study, no gain in ER status was observed in neither of the NAT-treated nor non-treated cases, whilst for PR, we observed gain of status in only two cases from the NAT-treated group. Overall, these results suggest that the gain of hormone receptor may not be frequent enough for its implementation to assess treatment response and disease outcomes.

HER2 status variation between biopsy and surgical samples are reported to be less frequent than for hormone receptors and Ki67 index [32, 48]. We did not find statistically significant changes in HER2 biomarker status neither in the NAT-treated nor non-treated cases. Some reports have found important changes in HER2 expression, which seem to be driven not just by NAT, but by specific types of therapeutic agents [33, 40, 49]. Ignatov et al. [25] reported that trastuzumab administration was associated with a decrease in HER2 expression in 47.3% of cases, and interestingly, when pertuzumab was added to the trastuzumab-NAT scheme, the decrease in HER2 expression rise to 63.2%. In our data, when we assessed HER2 variations according to type of NAT regimen, no statistically significant changes were found in neither of the NAT-schemes groups, including the cytotoxic + trastuzumab group. Despite our results not being statistically significant, we did observe some cases in the cytotoxic + trastuzumab scheme group with a decrease in HER2 status. Hypotheses regarding HER2 downregulation after treatment includes the internalization in endosomal compartments and lysosomal degradation of HER2 receptor induced by anti-HER2 agents (pertuzumab, trastuzumab) [50]. Nonetheless, as have been shown, ERBB2 amplification when evaluated by FISH remains stable after NAT treatment [51].

Undoubtedly, cancer treatments may alter in some degree tumor gene expression [52], leading to possible modification of the IHC biomarker profile. However, tumor heterogeneity is also an important factor to take into account when considering changes in biomarker expression between tumor samples, especially when changes are observed in non-previously NAT treated cases [53], as we reported here. Tumor heterogeneity in breast cancer has been observed in multiple studies [17, 54‐56]. Rye et al. [56] evaluated tumor heterogeneity of ER and HER2 expression within individual breast tumors at different time points, and reported the presence of tumor cells within the same sample with both HER2+/ ER+ and HER2+/ER- expression profile, reveling a high rate of cell-to-cell variation. Tumor heterogeneity may have several clinical implications for patient’s outcome. For example, a heterogeneous expression of HER2 copy number in tumors have been reported to be associated with higher risks of relapse and breast cancer death [56]. Such findings are expected given that this kind of intra-tumoral heterogeneity is often the result of clonal evolution, which is highly correlated with metastatic events [57, 58].

External factors different from tumor heterogeneity may also account for changes in biomarkers expression between biopsy and surgical samples in cases not previously treated with NAT [17, 51, 54]. Among these are technical preparation of the IHC stain, fixation times, and inter- and intra-observer variability [17]. It has also been reported that this variability could be a result of the so-called dilution effect, which refers to a decrease of biomarker expression with increasing number of available tumor cells to evaluate at the surgical sample [59]. As have been shown before, larger tumors from surgical excision procedures are more likely to present variations of biomarkers expression between biopsy and surgical samples [15, 55]. This may indicate that at the initial biopsy only a small portion of a tumor is sampled for its evaluation, therefore large tumors could end up being poorly represented and present with IHC profile variations.

Our study certainly had limitations, mainly regarding the small sample size, which limited the statistical power of the analyses, especially when NAT-treated cases were grouped according to the therapy scheme. Small sample size in the hormonal, cytotoxic + trastuzumab and combined NAT-scheme groups may not have allowed us to observe statistically significant expression changes between biopsy and surgical samples. Additionally, all cases were recruited from a single institution and only pathological, but not clinical information was collected. However, our results regarding changes of biomarker expression in NAT-treated and non-treated cases are mostly consistent with what has been reported previously in other studies [17, 51, 54]. On the other hand, we did not have FISH confirmatory amplification results for some cases with HER2 ambiguous result by IHC, which did not allow us to give a more precise classification of these cases. Additionally, it is important to highlight that, unlike most studies evaluating changes of IHC biomarker expression after NAT treatment, we included a group of cases not previously treated with NAT in our analysis, which allowed us to determine if NAT administration could in fact induce changes in the IHC biomarker profile.

Overall, our results confirmed that NAT administration may cause changes in IHC biomarker profile, mainly in Ki67 and PR expression, and that patients not previously treated with NAT do not present significant changes in biomarker expression. Since it is not cost-effective for the health care system to reassess the expression of each biomarker, for every patient after NAT, we suggest that only PR and Ki67 biomarkers should be reassessed after NAT treatment, as it has been shown that changes in these two may have prognosis implications for breast cancer patients. The implementation of the PR and Ki67 biomarkers as prognosis tools, along with other clinical variables such as tumor stage and nodal status [60, 61], could provide enough information about treatment response and it could be used by physicians to readjust therapy.