Dramatic Response to Pyrotinib and T-DM1 in HER2-Negative Metastatic Breast Cancer With 2 Activating HER2 Mutations

Abstract HER2 signaling is activated in response to somatic HER2 mutations, which are often found in invasive lobular breast cancer (ILC) and are associated with poor prognosis. Tyrosine kinase inhibitors (TKIs) have demonstrated considerable antitumor activity in patients with HER2-mutated advanced breast cancer (BC). Further, several clinical trials have indicated that HER2-targeted antibody-drug conjugates (ADCs) exhibit promising efficacy in lung cancer with HER2 mutations, and the efficacy of ADCs against HER2-mutated BC is currently being evaluated. Several preclinical studies have demonstrated that the therapeutic efficacy of ADCs in HER2-mutated cancer can be enhanced by the addition of irreversible TKIs, but the potential of such a combined treatment regimen for the treatment of HER2-mutated BC has not been reported. Herein, we describe a case in which a patient with estrogen receptor-positive/HER2-negative metastatic ILC with 2 activating HER2 mutations (D769H and V777L) exhibited a significant and durable response to anti-HER2 treatment with pyrotinib (an irreversible TKI) in combination with ado-trastuzumab emtansine, which was administered after multiple lines of therapy that had resulted in disease progression. Further, based on the evidence from the present case, TKI plus ADC seems to be a promising combination anti-HER2 regimen for patients with HER2-negative/HER2-mutated advanced BC, although further rigorous studies are warranted to confirm these findings.


Introduction
In patients with advanced breast cancer (BC), the posterior-line treatment stage is particularly challenging. However, with the increase in studies on genomic profiling and the clinical application of NGS, the management of advanced BC has already entered the era of precision treatment. As a result, it is possible to explore and design personalized treatment strategies according to the gene alteration patterns of individual patients.
The HER2 gene was first discovered and cloned in 1983. 1 Amplification of the HER2 gene results in activation of the HER2 signaling pathway, which is closely related to the occurrence and poor prognosis of breast cancer. 2,3 HER2 mutations were preliminarily described as oncogenic drivers based on single-strand conformational polymorphism assay and NGS in 2006 and 2009, respectively. 4,5 HER2 somatic mutations can drive tumor progression by activating the HER2 signaling pathway, but the frequency of somatic HER2 mutations is lower than that of HER2 amplification, and the incidence of HER2 somatic mutations is 2%-4% in breast cancer. 4,6,7 Somatic HER2 mutations are more likely to occur in patients e535 with HER2-negative BC or BC with low HER2 expression, and these mutations are associated with a lower survival rate in patients. [8][9][10] Ross et al found that the co-occurrence of HER2 amplification and HER2 mutation in recurrent and metastatic BC was 0.7%. 11 Interestingly, HER2 positivity and HER2 mutation are probably mutually exclusive in primary tumors and co-occur more often in metastatic lesions. 12,13 HER2 mutations have been shown to be associated with invasive lobular breast cancer (ILC), which has a HER2 mutation rate of 5.1%. 14 Furthermore, in patients with CDH1-mutated ILC, somatic HER2 mutation co-occurs more frequently, particularly in metastases (15% in ILC versus 5% in invasive BC of no specific type). 8,15,16 HER2-targeted compounds for HER2-positive BC consist of monoclonal antibodies (trastuzumab and pertuzumab), tyrosine kinase inhibitors (TKIs), and antibody-drug conjugates (ADCs), of which trastuzumab is usually considered a cornerstone and first-line drug. However, trastuzumab usually fails to effectively suppress HER2-negative/HER2-mutant BC, since the expression level of HER2 in HER2-negative BC is relatively low and 70%-90% of HER2 mutations occur in the kinase domain. 7,8,11 T-KIs are well-established anti-HER2 agents for the treatment of metastatic BC with HER2 overexpression or HER2 gene amplification. 17 Similarly, TKIs, especially one of the most studied irreversible TKI neratinib, also demonstrated considerable activity against advanced BC with HER2 mutations in in vitro and clinical studies, but the response rate and clinical benefit rate were still lower than those of anti-HER2 therapy in HER2-amplified BC. 8,[18][19][20] An ongoing phase II clinical trial has been designed to explore the efficacy of TKI tucatinib plus trastuzumab ±fulvestrant in HER2-mutated metastatic BC and other malignant solid tumors (NCT04579380).
The use of ADCs, including T-DM1 and trastuzumab deruxtecan (T-DXd), has been described in the treatment of HER2-mutated cancer, especially lung cancer. In a study involving 18 patients with HER2-mutated advanced lung adenocarcinomas, a response rate of 44% and a median progression-free survival (PFS) of 5 months was observed for T-DM1. 21 Furthermore, the DESTINY-Lung 01 trial enrolled 91 metastatic HER2-mutated non-small cell lung cancer (NSCLC) patients whose tumors were refractory to standard therapy, and the results demonstrated that T-DXd showed durable anticancer activity with a median PFS of 9.3 months. 22 Up to now, no clinical trial has been designed to specifically examine the efficacy of ADCs in treating HER2-mutant BC. However, Li and team, who initiated the DESTINY-Lung 01 trial, have included HER2-mutated/HER2-negative metastatic BC in their ongoing clinical trial on the efficacy and safety of T-DXd for the treatment of advanced solid tumors with activating HER2 mutations (DESTINY-PanTumor01 trial number NCT04639219).
Pyrotinib, an irreversible pan-HER tyrosine kinase inhibitor against HER1, HER2, and HER4, has demonstrated promising anticancer activity in HER2-positive metastatic BC, and the results of a multicenter phase III trial (PHOBE) indicated that pyrotinib plus capecitabine significantly improved PFS in HER2-positive metastatic BC, as compared to lapatinib plus capecitabine. 23 In HER2-mutated cancer settings, pyrotinib has shown stronger antitumor activity than afatinib and T-DM1 in HER2-mutated NSCLC patient-derived organoid or xenograft models, and pyrotinib monotherapy exhibited promising activity in 60 unresectable stages III or IV NSCLC patients with an objective response rate of 30% and a median overall survival of 14.4 months. 24,25 Most recently, Kaplan et al reported a patient with metastatic ER-positive, HER2-nonamplified ILC with an activating HER2 mutation (HER2 p780_y781insGSP) whose tumor became resistant to neratinib as well as capecitabine, but whose subsequent leptomeningeal disease exhibited a dramatic response to tucatinib plus capecitabine with a PFS of 10 months. 26 The past, future, and immense potential of treating HER2-mutated BC with TKI alone or in combination with ADCs have also been thoroughly reviewed and discussed in their case report. Based on the available evidence, it appears that the treatment of HER2-mutant advanced BC with various TKIs has been previously evaluated with promising outcomes. In addition, ADCs may have potential for the treatment of HER2-mutated BC based on their robust activity in lung cancer. However, the efficacy of irreversible TKIs in combination with ADCs for the treatment of advanced BC with HER2-activating mutations has not been explored yet. Herein, we try to shed light on this question by presenting a case of an estrogen receptor (ER)-positive/HER2-negative ILC with 2 HER2 activating mutations that showed a significant and durable response to treatment with pyrotinib/T-DM1/ exemestane/zoledronic acid.
However, 2 metastases were detected in the lungs on a chest CT scan in March 2019 (Fig. 2). Treatment with a CDK4/6 inhibitor was recommended, but the patient was unable to undergo the treatment because she could not afford it. Therefore, letrozole was replaced by fulvestrant for further maintenance treatment. Three months later, she developed dyspnea and bone pain, and more metastases were detected in the liver and bones (Fig. 2). In addition, the lung metastases detected earlier had increased in size, and right pleural effusion was observed. Liver biopsy and IHC confirmed that the metastases had originated in the BC and were ER-positive (60%+), PR-negative, HER2-negative (1+), and Ki67-positive (30%+). Based on the progression of systemic disease and obvious clinical symptoms, the patient received six cycles of salvage chemotherapy (paclitaxel plus lobaplatin) and 4 doses of zoledronic acid between July and October 2019, but she did not respond to the treatment (stable disease).
After salvage chemotherapy, capecitabine/toremifene/zoledronic acid treatment was administered between November e536 The Oncologist, 2023, Vol. 28,No. 7 2019 and March 2020. In March 2020, CT surveillance and bone scan revealed that the metastases in the lungs and liver had decreased in size, but the bone metastases had progressed (with new metastases detected). The patient developed intolerable hand-foot syndrome, so capecitabine was replaced by tegafur. She was started on tegafur/toremifene/ zoledronic acid treatment in March 2020, but disease progression was observed in July 2020. Between July 2020 and January 2021, the treatment regimen was adjusted to tegafur/exemestane/zoledronic acid, but she still exhibited systemic disease progression over this 7-month treatment period (with newly detected metastases in the lungs and vertebrae). In January 2021, disease progression was confirmed through imaging examinations, and the patient developed dyspnea, abdominal distention, and exacerbation of bone pain.

Molecular Tumor Board
Somatic profiling was conducted after discussion with a multidisciplinary team. NGS-based genetic testing (425gene panel) of the patient's liver metastatic tissue identified 2 HER2 missense mutations (D769H and V777L) and a CDH1 splicing mutation (c.2164 + 1G>C). After careful evaluation and with the informed consent of the patient, pyrotinib/T-DM1/exemestane/zoledronic acid treatment was started in January 2021. Three months after this regimen was started, dyspnea, abdominal distention, and ostalgia almost  The Oncologist, 2023, Vol. 28, No. 7 e537 disappeared, and imaging indicated a decrease in the size of the lung metastases and bilateral pleural effusion (stable disease) (Fig. 3A).

Patient Update
Seven months after the treatment was started, partial response of the lung metastases and almost complete disappearance of the pleural effusion were observed (Fig. 3  A). Twelve months later, the lung metastases were invisible (complete response) (Fig. 3 B), and the liver/bone metastases showed partial remission (Fig. 4). No central nervous system metastases were detected. As of October 2022, the patient had received pyrotinib/T-DM1/exemestane/zoledronic acid therapy for approximately 21 months with tolerable toxicity (grade 2 abdominal distension; grade 1 fatigue, headache, and paresthesia; no myelosuppression occurred), and the disease remained stable after previously achieving partial response.

Discussion
To the best of the authors' knowledge, the present report is the first to describe the treatment of ILC carrying HER2 mutations with combined treatment involving the TKI pyrotinib and the ADC ado-trastuzumab emtansine. The regimen was promising, as the patient achieved stable disease with partial response at 21 months after the treatment was started.
Activating mutations in HER2, as well-established oncogenic drivers, have been identified in a vast array of solid tumors. 6,27 In clinical practice, the efficacy of molecular therapy targeting actionable HER2 mutations has been mainly investigated in breast and lung cancer. 20, 28 Bose et al functionally characterized 13 HER2 mutations, and they found that the G309A, D769H, D769Y, V777L, P780 ins, V842I, and R896C mutations are activating mutations that may agonize breast cancer development. 8 Patients with HER2-activating mutations may benefit from anti-HER2 therapy, especially irreversible TKIs. The V777L and D769H mutations are 2 common activating mutations located in the kinase domain, which were identified and considered therapeutic targets for this patient.
The V777L mutation results in an amino acid substitution at position 777 in HER2-from a valine (V) to a leucine (L). 29 Several studies have reported reduced tumor growth and signaling activity in V777L-mutated tumors treated with neratinib and lapatinib. 8,30 Clinical activities were also observed in HER2 V777L-mutated cancer treated with neratinib. 20 Croessmann et al reported that neratinib reverses estrogen-deprivation resistance and fulvestrant resistance in tumors with HER2 variants and ER-positive tumors with the HER2 missense mutations G309A, L755S, and V777L. 31 In addition, in a case reported by Lei et al, a patient with HER2-negative advanced BC harboring the V777L mutation was treated with trastuzumab in combination with vinorelbine and had favorable results. 32 However, a preliminary study indicated that the HER2 V777L mutation may mediate resistance to trastuzumab in the context of HER2-positive  The Oncologist, 2023, Vol. 28,No. 7 metastatic breast cancer. 33 The patient in this report harbored not only the V777L mutation but also the D769H mutation, which resulted in an asparagine to histidine substitution at position 769 in the alpha-helix within the kinase domain of HER2, affecting ATP binding to HER2 and increasing HER2 dimerization. 8,34 A preclinical study demonstrated that patients harboring the D769H mutation were sensitive to neratinib as well. 8 Accordingly, these 2 mutations, together or individually, have been considered as biomarkers in several clinical studies evaluating the efficacy of TKIs or ADCs in BC and other solid tumors (NCT04579380, NCT04209465, NCT04172597, NCT04644237).
Pyrotinib is considered to have potential therapeutic benefits in patients with HER2 mutations. 35 A single-arm, prospective, phase-II study on the therapeutic efficacy of pyrotinib in metastatic BC with HER2 mutations in 10 patients 13 reported that 1 patient experienced complete response and 3 patients experienced partial response. Further, the median PFS was 4.9 months (95% CI: 3.8-6.0 months), and the clinical benefit rate was 60% (CR+PR+stable disease over 24 weeks). Further, at present, there are many relevant ongoing studies about TKIs for the treatment of HER2-mutated BC. For example, there is a phase II, open-label, multicenter study whose principal objectives are to evaluate the efficacy and safety/tolerability of poziotinib in 5 cohorts, including 30 pretreated patients with HER2-positive or HER2-negative BC with HER2-activating mutations (NCT04172597). However, although recent research studies and clinical trials have shown that TKI monotherapy may have promising outcomes for the treatment of HER2-mutated/HER2-negative advanced BC, TKIs have not yet been approved for the treatment of HER2-mutated breast cancer because their clinical activity is limited in terms of the clinical benefit rate, duration of response, PFS, and overall survival. 8,13,19,36 Moreover, acquired gatekeeper mutations, PI3K/AKT/mTOR pathway activation, and co-occurring mutations in HER2 and HER3 confer primary or de novo resistance, so these mutations are potential obstacles to the therapeutic efficacy of TKIs. 31,[37][38][39] Data from the MutHER and SUMMIT trials indicate that patients with ER-positive BC with HER2 mutations can benefit from dual-blockade therapies, such as neratinib plus fulvestrant, which have demonstrated prolonged PFS and duration of response, as well as a higher clinical benefit rate than neratinib monotherapy (38% versus 30%). 36,40

e539
T-DM1 is a HER2-targeted ADC in which trastuzumab is linked with the microtubule-inhibitory agent DM1, and it is an approved treatment agent for patients with HER2-amplified or overexpressing metastatic BC. 41 Mukohara et al reported a case of BC with HER2 amplification and L755S mutation in which the patients exhibited an excellent response to T-DXd, after initial progression with trastuzumab and lapatinib therapy. 42 Both T-DM1 and T-DXd have been demonstrated to be promising agents for the treatment of lung cancer with HER2 mutations. 22,42 At present, the potential of these ADCs for the treatment of HER2-mutated metastatic BC is unclear, but there is an ongoing clinical trial on the efficacy of T-DXd for the treatment of HER2-mutated BC (NCT04639219).
Combined treatment with TKIs and ADCs in patients with HER2 mutations is worthy of further exploration. The safety of the T-DM1 plus neratinib regimen has been evaluated in patients with metastatic HER2-positive BC, and 63% (12/19) of patients achieved an objective response with tolerable toxicity. 43 Further, it has been demonstrated that the irreversible pan-HER TKI poziotinib upregulated HER2 cell-surface expression by decreasing the ubiquitination of mutant HER2 and, consequently, potentiated the antitumor activity of T-DM1. 44 Notably, in a HER-mutated patient-derived tumor xenograft mouse model, combined treatment with poziotinib and T-DM1 resulted in complete tumor regression. 44 However, Li et al reported that HER2 ubiquitination and internalization, rather than HER2 overexpression, are key mechanisms that mediate endocytosis and the efficacy of ADCs (T-DM1 and T-DXd). 45 Furthermore, in the context of combination therapy, the irreversible TKIs neratinib and afatinib, instead of reversible TKIs, enhanced receptor ubiquitination, and consequent ADC internalization and efficacy, respectively. 45 Interestingly, there is a convergence between these 2 studies in terms of phenotypic outcome. Further research is warranted to explain this phenomenon.
In the present case, pyrotinib was chosen as an anti-HER2 agent. This decision was based on the 2 activating mutations (D769H and V777L) in HER2 identified in this patient, the promising efficacy of TKIs reported in patients with HER2 activating mutations, the lack of response to previous multiline therapies in this patient, and practical concerns such as drug accessibility (T-DXd was unavailable in China). Although ADCs have not currently been approved for the treatment of HER2-mutated metastatic BC, based on the previously reported clinical activity of ADCs alone and ADCs in combination with irreversible TKIs in HER2-mutated lung cancer and the findings of ongoing trials evaluating ADC efficacy in metastatic BC with HER2 mutations, we speculated that our patient would benefit from the addition of T-DM1 to her regimen. Further, exemestane was also added to her regimen because previous studies have shown that simultaneous blockage of ER signaling can improve the prognosis of patients with HER2-mutated metastatic BC. This patient responded well to combined therapy with pyrotinib/T-DM1/ exemestane/zoledronic acid, with durable PFS. Thus, based on the available evidence and our experience with this patient, we believe that this is currently an ideal combination therapy for patients who have metastatic BC with HER2-activating mutations.
The findings of the present case indicate that combined treatment with TKI and ADC is a potent strategy for the treatment of HER2-mutated advanced BC. Ongoing trials on this treatment strategy are expected to provide further evidence on the application of this combination regimen and how it can be tailored to patients' needs based on detected mutations or responses to other lines of treatment. Further, this report also highlights the need to use NGS in challenging cases to detect target mutations, as this can help clinicians make the right treatment decision and lead to better outcomes.