New Insights to Reshape the Management of Patients with Metastatic Breast Cancer-Focus on Overcoming Challenges in HER2 Status Interpretation

Approximately 20% of invasive Breast Cancers (BCs) are characterized by Human Epidermal growth factor Receptor 2 (HER2) protein overexpression or HER2 gene amplification. HER2 represents a standard diagnostic test and a predictive biomarker for the use of HER2-directed treatments in patients with BC. At present, the HER2 Immunohistochemistry (IHC) assay is applied for screening purposes, and the In Situ Hybridization (ISH) test serves as a confirmation, when HER2 IHC results are equivocal. However, an accurate assessment and interpretation of the HER2 status can be complicated in many women with BC. These difficulties can be attributed to various factors such as HER2 Intratumoral Heterogeneity (ITH) and changes of HER2 in the process of BC metastatic progression or post neoadjuvant Chemotherapy (CHT). In particular, the status of biomarkers (e.g., HER2 and co-expressed Hormone Receptor (HR)) can be altered in patients with metastatic BC and such receptor changes influence the therapeutic responses and clinical outcomes. The goal of this article is to present challenges in the assessment of HER2 expression and to underscore a need for the biomarker status reevaluation in patients with metastatic BC. This mini-review also provides some insights into the interpretation of equivocal HER2 status in women with metastatic BC and discusses the impact of HER2 and HR biomarker conversions on therapeutic decision-making and the patient prognosis in metastatic BC. It is crucial to correctly interpret the HER2 biomarker status and to assess conversions of HER2 and HR in the BC metastatic lesions since timely detection of such alterations is critical to management modifications of individual patients with metastatic BC.


INTRODUCTION
Breast Cancer (BC), which begins as a local tumor, can potentially metastasize, and thus, during an initial diagnostic workup, various biomarkers are checked to help evaluate the probability of the metastatic progression [1]. In approximately 20% of patients with BC, Human Epidermal growth factor Receptor 2 (HER2) (a proto-oncogene located on chromosome 17) is amplified or HER2 protein is overexpressed [1]. On the one hand, a positive HER2 status is related to the increased metastatic potential of BC and poor patients outcomes [2]. However, on the other hand, a positive HER2 biomarker predicts a favorable response to standard HER2-targeted therapies (e.g., anti-HER2 monoclonal antibodies, such as trastuzumab or pertuzumab, and Tyrosine Kinase Inhibitors (TKIs), such as lapatinib or neratinib) in women with BC [2].
Similarly, Hormone Receptors (HR), such as Estrogen Receptors (ER) and Progesterone Receptors (PR), which can be co-expressed together with HER2, are important prognostic and predictive factors in patients with BC, and thus, their assessment in both early and metastatic BC stages allows to more precisely guide the Endocrine Therapy (ET) [9].
Consequently, women with HR-positive breast tumors experience beneficial effects from adjuvant ET, in the form of tamoxifen (a Selective Estrogen Receptor Modulator (SERM)) or Aromatase Inhibitors (AI) (e.g., anastrozole, letrozole, and exemestane) [10]. In contrast, patients with HR-negative breast tumors have no advantage from ET and their prognoses are usually worse [10]. However, it should be highlighted that the response to systemic therapies, among breast tumors coexpressing HER2 and HR is very complicated, because of the bilateral cross-communication between the HER2 and HR signaling pathways, which can lead to a decreased sensitivity or resistance to the HER2-targeted therapy and ET [10]. According to a recent meta-analysis, the receptor features of the primary BC tumors are frequently preserved in metastatic lesions [11]. However, the HER2 and HR conversion can take place during the BC progression, and thus, a reevaluation of their status in BC metastases is critically important, since it affects the patient management [11]. A need for the repeated assessment of BC biomarkers (e.g., in samples from primary and metastatic BC lesions), in order to precisely inform treatment decision-making for patients with metastatic BC, was highlighted in the American Society of Clinical Oncology (ASCO) practice guidelines [12].
The goal of this article is to present challenges in the assessment of HER2 expression and to underscore a need for the reevaluation of biomarker status in patients with metastatic BC. This mini-review also provides some insights into the interpretation of equivocal HER2 status in women with metastatic BC and discusses the impact of HER2 and HR biomarker conversions on therapeutic decision-making and the patient prognosis in metastatic BC.

COMMON CHALLENGES TO HER2 STATUS ASSESSMENT AND INTERPRETATION
At present, HER2 Immunohistochemistry (IHC) is applied for screening purposes, and in situ Hybridization (ISH) serves as a confirmation, if the HER2 IHC test results are equivocal (this has usually been determined via HER2 Fluorescence In Situ Hybridization (FISH) or Silver In Situ Hybridization (SISH) probes) [12]. Furthermore, when there is a discrepancy between the results of HER2 status between primary and metastatic BC lesions, FISH or SISH tests should be conducted [12].
In response to these challenging problems, over the past years, the American Society of Clinical Oncology/College of American Pathologists (ASCO/CAP) guidelines have incorporated some modified recommendations for a precise interpretation of the HER2 status [12,13]. For instance, according to the ASCO/CAP guidelines (2018), it has been recommended that tumors with double-equivocal HER2 status (based on IHC and in ISH assays) should be considered as HER2-negative (since there is no evidence that the HER2targeted treatment is beneficial in such cases) [12]. Some important suggestions relevant to the assessment of HER2 status in metastatic BC are summarized in Table 1 [12, 13].

HER2 AND HR CONVERSIONS AND THEIR CLINICAL CONSEQUENCES FOR PATIENTS WITH BC
HER2 and HR (ER and PR) represent important biomarkers for the management of patients with metastatic BC, and thus, any changes in HER2 and HR expression need to be addressed [11]. "Receptor conversion", which means an alteration of the receptor status during BC metastatic progression, can take place in both HER2 and HR (with different incidence rates) [11]. For instance, the frequency of HER2 conversion from positive to negative was 21.3%, but in the opposite direction (negative to positive), it was only 9.5% [11].
According to a recent meta-analysis, different studies have reported various discordance rates in HER2 and HR expression between BC primary tumors and metastases [11]. For instance, changes in HER2 and HR (ER and PR) biomarker status have been estimated as approximately 21.9% and 34.2%, respectively. Such conversions merit careful diagnostic evaluations and should be followed by individualized therapeutic approaches in patients with metastatic BC [10]. It should be pointed out that a negative conversion basically implies that the present therapy is not effective anymore, and thus, another management strategy needs to be introduced. In contrast, a positive conversion implies that there is a possibility of some specific treatments that could be tried [11]. As an illustration of the HER2 and HR expression changes, another meta-analysis has indicated that the prevalence rate of negative conversion outnumbered that of positive conversion (13 vs. 5%) [14]. Similarly, it has been reported that the HER2 discordance rate was about 3%, and all the observed cases switched from a positive HER2 status in the primary tumors to a negative one in the metastatic lesions [15].
Concurrently, based on another review of discordant BC cases, it has been shown that changes in HER2 and HR status more often included negative conversions (e.g., from HER2positive and/or HR-positive to HER2-negative and/or HRnegative status), which are frequently associated with adverse BC outcomes [16]. The biomarker conversion that leads to a difference in the HER2 and HR expression has an impact on the selection of treatment options. It should be emphasized that in cases of the discordant HER2 expression, a conversion from HER2-negative primary BC tumors to HER2-positive metastases can also occur, and such a positive conversion opens a new therapeutic possibility that should not be overlooked. At this point, a prompt reevaluation of the receptor status in the metastatic BC lesions is critical to precisely adjust the therapeutic regimen and possibly improve the patient outcome [16]. In addition, it should be kept in mind that the discrepant findings between the HER2-negative primary BC tumors and the HER2-positive metastases may be due to some inaccurate procedural issues, which need be clarified [16]. With regard to the HR (ER and PR) status, it has been reported that its changes are associated with the prognosis of women with metastatic BC, and in particular, a negative conversion of HR status has been linked to adverse patient outcomes [17].

POSSIBLE MECHANISMS OF HER2 STATUS CHANGES DURING BC METASTATIC PROGRESSION
Although the exact reasons for the receptor conversion during BC metastatic progression are still unknown, there are some possible explanations of this phenomenon. For instance, according to a concept of Intratumoral Heterogeneity (ITH), BC cells in a given tumor mass may display heterogeneity (in terms of genotype and phenotype), and only a few of such cells can create a metastasis [1,11]. Consequently, when in the primary BC tumor, the biomarker's status is heterogeneous, there is a likelihood of their alterations during the metastatic progression [1,11]. A similar phenomenon can occur under the circumstances of selection pressure from therapy when a patient with BC is given HER2-targeted therapy (e.g., trastuzumab) or ET (depending on the BC receptor status). In consequence, only those BC cells, which were able to survive the treatment's selective pressure could subsequently create a metastatic lesion, which might reveal a changed biomarker status (compared to a primary BC tumor) [1,11,12].
Furthermore, heterogeneity of HER2 gene amplification that has been shown in some women with BC, contributed to inaccurate HER2 status evaluation, and in consequence, could have some influence on the effects of HER2-directed therapy [18]. In addition, it has been revealed that HER2 ITH is more common in women with HER2-positive metastatic BC, who initially had demonstrated HER2-positive BC, and then, were determined to have equivocal HER2 protein expression and low-level HER2 gene amplification [19]. Such patients have responded poorly to trastuzumab therapy and had worse outcomes [19]. In addition, it has been reported that BC cases with HER2 ITH displayed a reduced rate of the Disease-Free Survival (DFS), in comparison to the ones without HER2 ITH [20]. It was particularly evident in the HR-positive subset of women with BC, who received trastuzumab therapy in the adjuvant setting [20]. Similarly, it has been reported that HER2 ITH was a predictive factor for an incomplete therapeutic response to anti-HER2 agents (e.g., in the neoadjuvant setting) [21].
Even though the exact mechanism of HER2 status conversion during BC metastatic progression still remains unclear, it is conceivable that HER2 ITH and therapeutic selection pressure play a contributing role [22]. In the instance of positive to negative HER2 conversion, a lower degree of HER2 protein expression and heterogeneous HER2 gene amplification has been revealed, compared to the cases, which continuously displayed HER2-positive test results [22]. These findings suggest that the breast tumors, which are characterized by HER2 ITH, are able to present different HER2 status in metastatic lesions, since the HER2-directed therapy had eliminated susceptible clones [22]. Moreover, it should be highlighted that the timely detection of HER2 conversion is of utmost importance, since it may have a profound impact on future treatment choices. For instance, some studies have revealed a beneficial response to trastuzumab therapy, among women who switched to HER2-positive status in the metastatic sites [23,24] Furthermore, in patients with metastatic BC, in whom the HER2 status was changed from negative to positive, a longer Time To Progression (TTP) was reported (e.g., 10 months vs. 4 months) with trastuzumab treatment, compared to those who did not receive trastuzumab [24]. Therefore, it is crucial to reassess the HER2 status in BC metastatic lesions (particularly in patients, in whom the primary BC tumors display HR-positive expression) to be able to apply the most optimal targeted therapy and improve patient outcomes [12].

HER2 GENE CHANGES IN THE BC PRIMARY TUMOR AND METASTASES -THE INTERFERING ROLE OF CHEMOTHERAPY
The status of the HER2 gene in BC (that is usually determined at the time of BC diagnosis) can be changed in both the BC primary tumor and metastases (e.g., local lymph nodes and distant sites) [25]. Such alterations can be a result of the tumor evolution itself or the therapeutic intervention [25]. At present, no specific recommendations exist with regard to the modification of treatment, depending on changed biomarker status after applying neoadjuvant Chemotherapy (CHT) (standard therapy for locally advanced BC) [22]. However, a change of the HER2 status can affect the therapeutic approach in individual patients, and thus, a reassessment of the HER2 status post neoadjuvant CHT is advisable [22].
The mechanism of HER2 conversion after neoadjuvant CHT is not clear, but it may be related to the selection of HER2-positive or HER2-negative clones after neoadjuvant CHT, tumor heterogeneity, and some analytical or procedural errors [22].
For instance, it has been reported that women with breast tumors, who underwent HER2 negative conversion, after neoadjuvant CHT, had decreased Disease-Free Survival (DFS) compared to those who presented continuous HER2 positivity [26].
Furthermore, it should be underlined that an interpretation of HER2 ISH, after neoadjuvant CHT is really needed for correct distinguishing between a true HER2 amplification and an increase of HER2 copy number by chromosome 17 polysomy [25]. In fact, under these circumstances, the increase in the HER2 copy number might not be attributed to the true HER2 amplification, but it could rather reflect a polyploidization event due to CHT (that can happen in all of the chromosomes) [25]. At this point, an accurate HER2 status assessment, using dual-probe ISH with concurrent IHC assay has been recommended [25]. In addition, it should be underscored that the increase of centromere17 copy number is predominantly related to the amplification of the centromeric region, and thus, correcting the HER2 gene copy number with Centromere17 Enumeration Probe (CEP17) may lead to an inaccurate assessment of HER2 amplification [27].

THE ROLE OF BIOMARKERS IN INFORMING THE MANAGEMENT DECISIONS FOR PATIENTS WITH METASTATIC BC
The aim of the ASCO guidelines has been to present the concise evidence-based recommendations for the oncology community (e.g., medical, surgical, or radiation oncology specialists, pathologists, as well as primary care physicians, nursing staff and patients with BC) [12,28]. To keep abreast of the newest trends in this dynamically developing area, the focused ASCO recommendations highlight selected BC biomarkers and indications for their practical applications (e.g., using the updated HER2 and HR status to help decide whether to switch or to continue a given targeted therapy) ( Table 2) [12,28]. Consequently, in women with BC metastases, a biopsy for confirmation of the BC process and reevaluation of the HER2 and HR (ER and PR) status has been recommended. However, the available evidence is still not sufficient to decide about potential changes in BC treatment for such patients.
Nevertheless, in the face of the discordant results between BC primary tumor and metastatic lesions, it has been recommended to use preferentially the HER2 and HR status of the metastases to guide treatment. Moreover, such a decision needs to be convergent with the patient's clinical context and management goals (e.g., medical condition and personal preferences) ( Table 2) [12,28]. In addition, it should be kept in mind by medical practitioners (and clearly conveyed to their patients with BC) that evidence is still limited to determine if switching the oncology treatment, based on BC biomarker status alterations would influence patient outcomes. Furthermore, it needs to be underscored that the use of circulating tumor biomarkers depends on clinical experience and judgment, and thus, physicians should not apply such biomarkers only as additional therapeutic indications ( Table 2) [12, 28]. In practical terms, ( Table 3) [29 -45] briefly summarizes current and emerging treatment strategies for patients with HER2-positive metastatic BC, including anti-HER2 monoclonal antibodies, Antibody-Drug Conjugates (ADCs), and Tyrosine Kinase Inhibitors (TKIs). In addition, possible combinations of anti-HER2 agents and endocrine therapies (depending on the HR status) with different therapeutic modalities, such as Cyclin-Dependent Kinase (CDK) 4/6 inhibitors, phosphatidylinositol-3 kinase/ mammalian target of rapamycin signaling pathway (PI3K/mTOR) inhibitors, and immunotherapy, are also being considered. In this light, the right "points of attack", meaning molecular targets, related to HER2 expression can facilitate an adequate selection of candidates for these new and emerging treatment options. Since safe and effective therapeutic strategies for HER2-positive and/or HR-positive metastatic BC are urgently needed, the future clinical trials should investigate the use of innovative therapies, guided by biomarkers (e.g., related to gene expression or molecular targets) for metastatic BC, which is so difficult to manage.  T-DM1 prolonged PFS and OS, with less toxicity (compared to lapatinib plus capecitabine), in patients with HER2-positive mBC, previously treated with trastuzumab and a taxane [Verma et al. 2012;31] HER2 Trastuzumab deruxtecan (DS-8201) Anti-HER2 ADC, Topoisomerase I inhibitor DESTINY-Breast01 P 2 NCT03248492 Trastuzumab deruxtecan revealed durable antitumor activity in pretreated (with T-DM1) pts with HER2-positive mBC; in the trastuzumab deruxtecan arm: ORR = 60.3%; median PFS = 16.4 ms; interstitial lung disease is a serious AE that requires vigilant monitoring/intense therapy [Modi et al. 2020;32] HER2 Trastuzumab duocarmazine (SYD985); Anti-HER2 ADC Duocarmycin derivative -an alkylating agent P 1 (ongoing) NCT02277717 (SYD985 dose-escalation/doseexpansion) Trastuzumab duocarmazine has shown clinical activity in pretreated pts with HER2-positive mBC (HER2-positive, T-DM1-resistant, HER2-low expression BC), with manageable safety; ORR in HER2-positive BC = 33%; ORR in HER2-low, HR-positive BC = 27%; ORR in HER2-low, HRnegative BC = 40% [Banerji et al. 2019;33] HER2 Margetuximab novel Anti-HER2 mAB; (Fcengineered)

EFFECTIVE COMMUNICATION WITH PATIENTS ABOUT BC BIOMARKERS STATUS -A VALUABLE HELP IN THEIR CARE
To successfully distribute and practically implement the updated ASCO guidelines, it is necessary to raise awareness of these recommendations among both oncology treatment teams and BC patients/survivors (as well as their families and caregivers). In the face of a large number of patients with multiple chronic diseases, designing evidence-based recommendations to direct therapy for such patients is very difficult [12,28]. For instance, it should be underlined that in a population of patients with comorbidities (e.g., hepatic and renal insufficiency), the test results of some serum biomarkers of malignant tumors can be altered, and thus, their diagnostic value may be controversial [46]. In such cases, the oncology teams need to carefully consider the specific comorbidities (with their poly-pharmacotherapy) in preparing the therapeutic plans. For practical purposes, this could mean that many of the recommended treatment options should be modified or used only after a comprehensive analysis of individual patient cases. Moreover, to facilitate the BC management, the afflicted patients need to be educated about the results of pathology tests (including the main BC biomarkers), and their impact on the targeted treatment plans. This is especially important since women diagnosed with BC (e.g., advanced or metastatic stages) often experience severe emotional distress and are overwhelmed by confusing medical terminology. To help overcome these obstacles, effective communication and a trustworthy relationship with the treatment team members are essential (e.g., using easy to understand language, patient-friendly brochures, clear written notes, or reminders, and asking patients to repeat the main information about therapy plan) [12,28]. For instance, patients should understand that HER2 and HR status determines if certain medications (e.g., trastuzumab, pertuzumab, lapatinib, or tamoxifen) are recommended for them. In addition, the physician should review the test results with them, interpret the main data, clearly explain their impact on the patient's treatment course, inform about available clinical trials in this field, and answer any relevant questions, which the patient may have [12,28,47]. Such patient-centered care should augment adherence to medical advice. Some basic, most practical recommendations focused on biomarker application, to guide management decisions in patients with metastatic BC are summarized in Table 2 [12, 28].
As patients move along the trajectory of their metastatic BC course, no definite cure is available for them. Therefore, searching for therapies, which will not only control their malignancy for prolonged intervals of time but also extend their quality lifespan, is of utmost importance. Simultaneously, it is imperative to find biomarkers beyond HER2, which will accurately predict both the response and resistance to various treatment options and help identify which patients can particularly benefit from the specific therapies. Many challenges still remain with the treatment of patients with HER2-positive BC. For instance, availability, accuracy, and cost of genetic and molecular testing present serious limitations. Since this is not "a one size fits all" approach, it is critical to establish criteria, according to which the patients would be selected for the use of particular therapeutic agents. It is also crucial to spare a sufficient amount of time for a discussion with each patient about the pros and cons of the different proposed therapies, considering an individual patient's clinical context, characteristics of a given therapy, as well as personal preferences, goals, and needs.

CONCLUSION
HER2 overexpression/amplification in patients with BC is crucial for establishing a precise diagnosis and individualized HER2-targeted treatment plan. It is of utmost importance to accurately interpret the HER2 biomarker status, including assessment of conversions of HER2 and HR biomarkers in the BC primary tumor and metastatic lesions (because detection of these changes is necessary for the specific management modifications of individual patients with metastatic BC). If such alterations in the biomarker status are timely detected, there is a chance for selecting the most optimal, targeted therapeutic agents for the afflicted women with metastatic BC. To shed some more light on this area, large prospective studies, assessing the impact of HER2 and HR biomarker conversions on the treatment selection and efficacy of HER2-targeted therapy/ET and patient outcomes are warranted in the future.