The force of HER2 – A druggable target in NSCLC?

Journal Pre-proofs Anti-tumour Treatment The force of HER2 – a druggable target in NSCLC? M. Jebbink, A.J. de Langen, M.C. Boelens, K. Monkhorst, E.F. Smit PII: DOI: Reference:

S0305-7372(20)30034-7 https://doi.org/10.1016/j.ctrv.2020.101996 YCTRV 101996

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Cancer Treatment Reviews Cancer Treatment Reviews

Received Date: Revised Date: Accepted Date:

18 November 2019 20 February 2020 25 February 2020

Please cite this article as: Jebbink, M., de Langen, A.J., Boelens, M.C., Monkhorst, K., Smit, E.F., The force of HER2 – a druggable target in NSCLC?, Cancer Treatment Reviews Cancer Treatment Reviews (2020), doi: https://doi.org/10.1016/j.ctrv.2020.101996

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Title: The force of HER2 – a druggable target in NSCLC? Authors: Jebbink, M.; de Langen, A. J.; Boelens, M.C.; Monkhorst, K.; Smit, E.F. Authors names and affiliations: M. Jebbink, MD PhD-student Department of Thoracic Oncology Netherlands Cancer Institute Plesmanlaan 121 1066 CX Amsterdam The Netherlands Tel: +31-20-5122958 Email: [email protected] A.J. de Langen, MD, PhD Pulmonologist Department of Thoracic Oncology Netherlands Cancer Institute Plesmanlaan 121 1066 CX Amsterdam The Netherlands Tel: +31-20-5122958 Email: [email protected] M.C. Boelens, PhD Clinical molecular scientist Department of Pathology Netherlands Cancer Institute Plesmanlaan 121 1066 CX Amsterdam The Netherlands Tel: +31-20-5122766 Email: [email protected] K. Monkhorst, MD, PhD Pathologist Department of Pathology Netherlands Cancer Institute Plesmanlaan 121 1066 CX Amsterdam The Netherlands Tel: +31-20-5122958 Email: [email protected] E.F. Smit, MD, PhD Pulmonologist Department of Thoracic Oncology Netherlands Cancer Institute Plesmanlaan 121 1066 CX Amsterdam

The Netherlands Tel: +31-20-5122958 Email: [email protected] Corresponding author: E.F. Smit, MD, PhD Pulmonologist Department of Thoracic Oncology Netherlands Cancer Institute Plesmanlaan 121 1066 CX Amsterdam The Netherlands Tel: +31-20-5122958 Email: [email protected]

Title: The force of HER2 – a druggable target in NSCLC?

The force of HER2 – a druggable target in NSCLC? Highlights Three principal mechanisms of HER2 alterations can be identified: protein overexpression, gene amplification and gene mutations. No consensus about HER2 subgroup definitions has been obtained, complicating the interpretation of the results of the many trials studying possible treatment strategies. Future research should focus on the definition of HER2 alteration subgroups and test therapeutic agents in these different subgroups. Abstract Since several years targeted therapy has been part of treatment in NSCLC in subsets of patients with specific genetic alterations. One of these alterations involves HER2, a member of the ERBB family of tyrosine kinase receptors. Despite that HER2 alterations in NSCLC have been studied for years, there is still no consensus about subgroup definitions. In this review HER2 alterations in NSCLC are discussed, including diagnostic challenges and treatment strategies. Three principal mechanisms of HER2 alterations can be identified: HER2 protein overexpression, HER2 gene amplification and HER2 gene mutations. There are several methods for the detection of HER2 “positivity” in NSCLC, but no gold standard has been established. Laboratory methods for assessment of HER2 positivity in NSCLC include immunohistochemistry (IHC) for protein overexpression and fluorescent in situ hybridization (FISH) and next generation sequencing (NGS) for genetic alterations. Many trials testing HER2 targeted therapy in HER2 altered NSCLC has not lead to a renewed standard of care for this group of patients. Therefore, today the (re)search on how to analyse, define and treat HER2 alterations in NSCLC continues. Still there is no consensus about HER2 subgroup definitions and results of the many trials studying possible treatment strategies are inconclusive. Future research should focus on the most important missing link, whether all HER2 alterations are relevant oncogenic drivers and whether it should be considered as a therapeutic target in NSCLC. Keywords: HER2; NSCLC; driver; resistance; targeted therapy Introduction The past decade, systemic treatment of advanced non-small cell lung cancer (NSCLC) has undergone major changes. Several targeted therapies have been approved and recommended for use in subsets of patients who have specific genetic alterations (1). One of these alterations involves the human epidermal growth factor 2 (HER2), a member of the ERBB family of tyrosine kinase receptors. Each receptor is composed of three segments: an extracellular ligand binding domain, an α-helical trans membrane segment and an intracellular tyrosine kinase domain. There is no natural ligand identified for the HER2 receptor. Ligand binding promotes receptor dimerization and auto-phosphorylation of the kinase domain of the cytoplasmic part of the HER2 receptor and unleashes HER2 kinase activity. This results in initiation of a variety of signaling pathways including MAPK, PI3K/AKT, PKC and STAT. In HER2 altered cancer cells, genetic alterations result in constitutive dimerization and activation of the pathways, thereby promoting uncontrolled cell growth (2, 3). In lung cancer, HER2 alterations can be identified in small subsets of patients. These alterations can be identified as oncogenic drivers, but also as a mechanism of acquired resistance after targeted therapy. Similar alterations have been reported in various other tumors, including breast and gastric cancer, and are associated with poor disease prognosis and shorter overall survival (OS) (4-10). In breast cancer and gastric or gastro-esophageal junction cancer, HER2 targeted therapy has expanded OS in those patients who express HER2 and is considered standard of care (11, 12). Also, a shorter OS

has been observed in HER2 altered NSCLC in comparison with the general population of stage IV NSCLC, possibly due to intrinsic resistance to chemotherapy (13, 14). Definitions of HER2 alterations in NSCLC are still unclear and the question remains whether all these alterations are relevant oncogenic drivers and therefore therapeutic targets. Three subsets of HER2 alterations can be classified in NSCLC: mutation, amplification and protein overexpression (15, 16). There are several methods for the detection of HER2 “positivity” in NSCLC. Laboratory methods for assessment of HER2 positivity in NSCLC include, but are not limited to, immunohistochemistry (IHC) for protein overexpression and fluorescent in situ hybridization (FISH) and next generation sequencing (NGS) for genetic alterations. The reported prevalence of HER2 alterations varies in NSCLC, most likely caused by lack of definitions and standardized testing methods. Due to these differences, conclusions cannot be drawn easily. In this review, we will discuss HER2 alterations in NSCLC, including diagnostic challenges and treatment strategies. HER2 alterations as a primary driver HER2 mutations The HER2 gene (also known as ERBB2 or neu) is localised on the long arm of chromosome 17 (17q21) and known as a proto-oncogene. HER2 mutations have a frequency of 1-4% in NSCLC (17-21) and are predominantly found in patients who are nonsmokers (22, 23). HER2 mutations as primary driver are reported to be mutually exclusive with other oncogenic drivers (24, 25) like EGFR, KRAS, NRAS, ALK, PI3KCA and BRAF (18, 26, 27). The genetic diversity of HER2 mutations is low and most occur in exon 18-21 of the tyrosine kinase domain (28). In-frame exon 20 insertions occur in 83% of all cases and the most common mutation is a recurrent 12 base-pair insertion causing duplication of amino acids YVMA at codon 75, the Y772_A775dup insertion (27, 29). Moreover, there are more rare point mutations affecting the kinase domain. These mutations alter the intracellular ATP-binding pocket of the HER2 receptor (30), leading to increased HER2 kinase activity and enhanced signaling through downstream pathways, resulting in increased cancer cell survival, invasiveness and tumorgenicity (19, 24, 26). Other more rare mutations affect the extracellular domain, resulting in constitutively dimerization and activation of the HER2 receptor (30). An overview of the prevalence of HER2 cancer hotspot mutations in NSCLC is presented in Figure 1. Seldom, HER2 mutations are found in germline. Matsuda et al (2013) identified a germ line mutation (G660D) in the transmembrane domain of HER2 in a Japanese family with multiple cases of lung cancer. In an additional analysis of 253 sporadic lung adenomas another novel somatic mutation (V659E) was identified. Both mutations resulted in a more stable than wild-type protein and activation of the AKT pathway, suggesting that transmembrane domain mutations of HER2 are oncogenic and may cause hereditary and sporadic lung cancer (31). HER2 mutations are detected by sequencing methods like next-generation sequencing (NGS), a relative time-consuming and expensive method. Therefore, research has been done to identify a surrogate marker for HER2 mutations. HER2 protein expression was found to be insufficient as a surrogate marker for HER2 mutations. In a series of four HER2-mutant cases none were amplified (FISH: HER2-to-chromosome enumeration probe 17 [CEP17] ratio of at least 2.0) or tested positive on IHC staining (2+ or 3+), suggesting that gain of HER2 protein expression in general cannot serve as a surrogate marker for HER2 mutations (26). Later on, this was confirmed in a larger set of 21 cases of NSCLC with a HER2 mutation and 20 cases (3%) with HER2 amplification; only one case showed concurrent mutation and amplification and IHC analysis of 18 mutant cases ranged from 0 to 2+ (32). Co-occurrence of HER2 amplification and mutation was explored as well. Chen et al (2017) analyzed 54 samples (tumor and liquid biopsies) from 48 patients with HER2 mutation or amplification with NGS. Thirty-six samples carried HER2 mutations and 23 samples carried HER2 amplification with only 5 samples having concurrent HER2 mutation and amplification (22). Recently, detection of HER2 mutations in liquid biopsies has been further explored by Lee et al (2019) and they reported detection of HER2 exon 20 insertions in liquid biopsies in a cohort of lung cancer patients (33).

In another study by Mazieres et al (2013) 8 out of 34 HER2-mutated samples had an increase of HER2 gene copy number in the context of polysomy (> 2 HER2 copies in > 40% of cells) (23%) and 3 out of 34 had true HER2 amplification (HER2/CEP17 ratio per cell > 2 or regions with > 15 copies in > 10% of the cells) (24). Li et al (2012) found by FISH analysis HER2 copy number gains in 7 out of 8 HER2mutated tumors. They used FISH to define low polysomy (≤4 copies of EGFR in >40% of cells), high polysomy (≥4 copies of EGFR in >40% of cells), and gene amplification (homogenously staining regions with ≥15 copies in ≥10% of cells or a HER2/CEP17 ratio of ≥2) (34). Interestingly, all the HER2mutated samples showed either an increase of HER2 or EGFR GCNs, concluding that these patients could potentially benefit from combination therapy with EGFR and HER2 targeted therapy (24, 27, 34). As outlined above, HER2 mutations are not strictly associated with HER2 amplification and expression, suggesting a distinct mechanism of origin with different prognostic and predictive values (26). HER2 amplification Amplification of HER2 is less frequently observed in NSCLC compared to breast cancer (35). HER2 amplification has been strongly associated with pleural invasion (7), female sex, never smoking (36) and adenocarcinoma histology (37). Reported prevalence varies from 2 to 5% in lung adenocarcinomas (35, 38, 39). The prognostic impact of HER2 amplification has been evaluated by several studies and outcomes were inconsistent (5). In a meta-analysis (5) the combined hazard ratio (HR) for OS was 1.14 (95%CI: 0.2-1.83) for all patients and 0.89 (95% CI: 0.63–1.27) for stage III and IV NSCLC. These aggregated data suggest that HER2 amplification determined by FISH is not of prognostic value in NSCLC. Different methods have been described to detect HER2 amplification. FISH is used as a standard method to classify HER2 amplification with high specificity, standardization and consistency in breast cancer (23, 40). By FISH analysis, HER2 amplification is found in 2-4% of NSCLC tumors (18, 24). Although there is still no official definition for HER2 amplification in lung cancer, generally HER2 amplification is defined as HER2/CEP ratio ≥2. However, other cut-offs and parameters have been used as well, causing variation among different studies. The definition of HER2 amplification should be critically evaluated because using the ratio adopted from breast cancer may not be adequate for NSCLC (5). Also in some non-amplified tumors, polysomy can be found, usually defined by an absolute HER2 gene copy number higher than five or six while the HER2/CEP ratio is less than two. Whether HER2 polysomy has prognostic or predictive value, remains unclear but for now polysomy is not supposed to drive oncogenesis (26, 41). Ideally, future studies should discriminate between individuals with HER2 amplification and individuals with high levels of HER2 copy number gain, but no amplification (36, 42). HER2 amplification can also be detected in liquid biopsies from lung cancer patients. In the earlier mentioned study by Lee et al (2019) in 3.9% (9/231)HER2 amplification was detected in liquid biopsy from lung cancer patients (33). HER2 overexpression In approximately 10-15% of NSCLC cases, HER2 overexpression has been reported (43, 44). HER2 overexpression without amplification or mutation was found driving de novo tumorgenesis in NSCLC in mouse models (45). In previous studies, HER2 overexpression has been found to be associated with papillary predominant histology (7), poor disease prognosis and shorter OS (4-6). There is no consensus how to score and classify HER2 protein overexpression in lung cancer. Still, there is an unmet need for a universal definition and test. HER2 protein overexpression can be classified in two different ways. The first is an IHC scoring system ranging between 0 and 3+. HER2 0 and 1+ is defined as negative. IHC2+ is defined as weak to moderate staining in ≥10% of tumor cells. IHC3+ is defined as strong complete, basolateral, or lateral membranous staining in ≥10% of tumor cells. HER2 positivity defined

by IHC≥2+ staining is reported in 24% of NSCLC patients, IHC3+ is more rare and reported in 3-10% of NSCLC patients (21, 46). The second scoring system is the H-score, a semi-quantitative assessment performed by multiplying staining intensity (0, none; 1, weak; 2, moderate; 3, strong) with the percentage of positive tumor cells. The H-score takes membranous staining into account. The score ranges from 0 to 300 and can be classified as high (≥200), intermediate (≥100-200) and negative (Hscore <100) (47). The different scoring methods, together with the low concordance in HER2 expression assessment by pathologists (7, 48, 49) result in heterogeneous study populations and difficulties to compare individual studies. Hotta et al (2019) evaluated the characteristics of HER2 overexpression patterns using IHC and FISH in 15 patients with HER2-positive NSCLC. In this study in 79% of the patients the immunostaining pattern demonstrated incomplete or mixed-type membranous immunoreactivity with heterogeneity. These findings resemble earlier observations in gastric cancer rather than in breast cancer. There was a concordance of 87.5% between IHC-positivity and FISH positivity according to the criteria used in breast cancer. Classifying the NSCLC-specimens with the scoring system used in gastric cancer, the IHC score increased in 43.8% of the specimens and the concordance between IHC positivity and FISH positivity rose to 93.8%. These observations suggest that the pattern of IHC reactivity closely resembled that observed in gastric cancer rather than in breast cancer (50). In malignancies most cases of high protein overexpression (IHC3+) are due to HER2 amplification, while moderate expression (IHC2+) can be seen with and without HER2 gene amplification. Bunn et al (2001) found a strong correlation in 19 NSCLC cell lines between HER2 protein expression on the cell surface assessed by IHC and the HER2 copy number by FISH. Thirty-two percent of the cell lines tested HER2 IHC positive (26%, 2+; 5%, 3+). Polysomy determined by FISH was often found, rather than true amplification (51). Therefore, in case of HER2 IHC 2+ or 3+ additional ISH analysis needs to be performed to discriminate whether this is due to amplification, polysomy or that there is an absence of an increase in HER2 gene copy number. HER2 gene overexpression (mRNA levels) can be quantified by the use of other methods than IHC and ISH, such as quantitative polymerase chain reaction (qPCR) and real-time quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) (52). High HER2 mRNA expression levels have been associated with poor prognosis (52). Another study concluded that high mRNA expression levels of HER2 were correlated with worse survival, but only in women. The underlying mechanism or explanation for these surprising differences between both sexes has not been proposed (53). HER2 alterations as a secondary driver HER2 mutations HER2 mutations are identified as a primary driver in NSCLC, but can also be found as a mechanism of acquired resistance during EGFR TKI treatment. In vitro experiments showed that HER2 kinase domain mutations (Y772_A775dup) are able to cause resistance to EGFR TKIs by phosphorylation and activation of HER2 and EGFR, inducing survival, invasiveness and tumorgenicity in cell lines (19). A novel mechanism of osimertinib resistance HER2D16 was found by Hsu et al (2020). They found that HER2D16 could be an osimertinib resistance mechanism through an Src-independent pathway (54). In vivo, HER2 mutations have been found as a possible mechanism causing primary and acquired resistance to EGFR TKI treatment. First, Chen et al (2017) found two patients with EGFR+ NSCLC together with an HER2 mutation and/or HER2 amplification with primary resistance to EGFR-TKI (22). In the AURA study, a study where the effect of osimertinib was tested in 60 patients with EGFR mutation-positive NSCLC, one patient was shown to acquire the HER2 (Y772_A775dup) mutation after receiving osimertinib (55). HER2 amplification It has been suggested that HER2 amplification is one of the most frequent acquired resistance mechanism following the EGFR T790M mutation in EGFR+ NSCLC treated with 1st or 2nd generation EGFR TKIs (56)(56, 57). Preclinical studies identified HER2 amplification in 12% of TKI-resistant NSCLC

cell lines and all cases were EGFR T790M-negative (15). Two in vivo studies detected HER2amplification in 13% of NSCLC-patients with acquired resistance to EGFR TKI treatment (16, 58). Moreover, in a group of 9 EGFR-T790M NSCLC patients with primary resistance to osimertinib, HER2 amplification was identified as a mechanism of primary resistance in 1 of the 9 patients (59). In the FLAURA study, HER2 amplification has been reported as a mechanism of acquired resistence after first-line osimertinib treatment (60). Although true amplification can be observed as a driver of resistance to therapy, polysomy has been observed to be more frequent. Available data suggest that only amplification drives resistance, coherent with earlier findings with mechanisms of acquired resistance such as MET amplification (61). HER2 overexpression Protein overexpression of HER2 is often found at the time of resistance to EGFR TKI therapy, and could therefore be a potential secondary driver causing acquired resistance. De Langen et al (2018) found an increase of HER2 expression levels in paired biopsies of 17 patients over time during EGFR TKI treatment (pre-treatment median HER2 IHC 2+, H score 100; post-treatment median HER2 IHC 3+, H score 240). By H-score almost all patients showed an increase in HER2 protein overexpression after EGFR TKI treatment. This finding is suggestive of treatment induced selection of an HER2 driven resistant cell population (62). The selective pressure imposed by the chronic exposure to EGFR TKIs can lead to the expansion of pre-existing clones carrying specific (epi)genetic alterations, which may ultimately become dominant and cause resistance to targeted therapy (63). HER2 targeted therapy Targeting HER2 as a primary driver HER2 mutations Chemotherapy Wang et al (2018) found that patients with advanced HER2-mutant NSCLC had inferior outcome to first line pemetrexed-based chemotherapy (ORR 36.0%; median PFS 5.1 months) compared to patients with an ALK or ROS1 rearrangement (ORR 41.3%; median PFS 9.2 months, p=0.004) and a non-significant trend compared to EGFR mutation (ORR 33.8%; median PFS 6.5 months, p=0.247). Additionally, patients with the most common HER2 Y772_A775dup mutation had even inferior outcome, albeit non-significant, compared with other HER2 mutation variants (4.2 vs. 7.2 months, p = 0.085) (64). Tyrosine kinase inhibitors In HER2-mutated NSCLC several pan-HER TKI have been investigated with limited clinical efficacy (ORR 0-19%) (65-69). De Greve et al (2012) were the first to report efficacy of the pan-HER TKI afatinib in patients with HER2 mutation positive NSCLC (70, 71). Table 1 provides an overview of the clinical efficacy of (pan-HER) TKI’s. The response rates vary between 3.8 and 53.3% with PFS ranging from 3.7 to 6.4 months. Several salient observations can be made: first, the response rates and PFS are not within the scope of those obtained with targeted agents in the setting of other oncogenic drivers in NSCLC (e.g. EGFR, ALK, ROS1). Second, there is a lack of prospective studies of acceptable size. Third, there is some evidence pan-HER TKIs, afatinib in particular, have incidental efficacy in HER2-mutated NSCLC. In a named patient use (NPU) program, 28 patients with HER2-mutated NSCLC were treated with afatinib and safety and efficacy were retrospectively assessed. Despite a time to treatment failure (TTF) for all HER2-mutated patients being only 2.9 months, 4 out of 10 patients with the HER2 Y772_A775dup insertion in exon 20 remained on afatinib for more than one year, with a TTF of 9.6 months. These results suggest that afatinib may be effective in the subgroup of the Y772_A775dup insertion in exon 20 (72). In another retrospective study, Lai et al (2019) reported minor efficacy (ORR 13%) of afatinib in heavily pretreated patients with HER2-mutant lung cancer. Of the three patients that had a partial response on afatinib, two had an YVMA insertion (73). On the

contrary, Fang et al (2019) found no efficacy of afatinib in 14 patients with an Y772_A775dup insertion. Nevertheless, they found a potential benefit (ORR 40%) in the subgroup of G778_P780dup and G776delinsVC mutations (74). Neratinib is an irreversible pan-HER TKI that binds EGFR, HER2 and HER4. Nagano et al (2018) reported efficacy of both afatinib and neratinib against the HER2 Y772_A775dup mutation in cell lines, while L775P and L775S mutations were resistant (29). In short-term cultures of patient derived tumor organoids, it was found that neratinib and afatinib monotherapy induced a high degree of cell death. The combinations of neratinib/trastuzumab, and neratinib/temsirolimus enhanced the therapeutic benefit compared to neratinib alone, with the most effect on neratinib/temserolimus combination therapy. This study suggests that these combination therapies could be effective in patients with HER2-mutant NSCLC (75). According to the prospective SUMMIT study, neratinib showed limited efficacy in HER2-mutated lung cancer with an ORR of only 4% (1/26). Despite the low response rate, six patients remained on therapy for more than 1 year, resulting in a PFS of 5.5 months (66). Recently, new pan-HER TKIs showed encouraging activity at first glance. Poziotinib has the size and shape potent to overcome the hindered binding pocket of EGFR and HER2 exon 20 mutations. Therefore, it could be an effective inhibitor of HER2 exon 20 mutations. In vitro and in patientderived xenograft models of HER2 exon 20 mutant NSCLC, poziotinib seemed more effective than other pan-HER TKIs (76). Currently, poziotinib is being investigated in a phase II trial with HER2 exon 20 mutant NSCLC patients and in the first 12 evaluable patients some efficacy was observed (medium PFS of 5.6 months, (77)ORR 42%) (78). In the same study, a NSCLC mouse model harboring the Y772_A775dup insertion received a combination of poziotinib and T-DM1 had an average reduction of 47% of tumor burden (78). Pyrotinib, another small sized molecule showed superior anti-tumor efficacy compared with afatinib or trastuzumab-emtansine (T-DM1) in preclinical HER2 exon 20 mutation models. Mice treated with pyrotinib displayed profound tumor burden reduction (mean tumor volume, -52.2%). In a prospective clinical phase II study, pyrotinib showed promising efficacy with an ORR of 53.3% (79, 80). Osimertinib, in origin designed as a third generation EGFR TKI, could have affinity for HER2 and might be effective in HER2-mutated NSCLC as a single agent or combination treatment. Nagano et al (2018) found efficacy of osimertinib in vitro against the relatively rare L775P and L775S mutations, while lesser efficacy was found against the Y772_A775dup mutation as compared to afatinib and neratinib (29). Liu et al (2018) reported in vitro results of osimertinib showing lack of efficacy in HER2-mutated NSCLC, but by combining osimertinib with the BET inhibitor JQ1 tumor inhibition was seen (45). Antibody and antibody drug conjugates Many antibodies and antibody drug conjugates (ADCs) have been tested in HER2-mutated NSCLC. An overview is provided in Table 2. First, Trastuzumab is a monocolonal antibody that binds to the extracellular domain of the HER2 receptor (81). In 2006, a case report of a female with a HER2 exon 20 mutation described a long-lasting response to the combination of trastuzumab and paclitaxel (82). Next, a retrospective cohort study in European centers confirmed that patients with HER2-mutant NSCLC could be particularly sensitive to the combination of trastuzumab and chemotherapy with response rates up to 50% (14). However, how this compares to platinum-doublet chemotherapy without trastuzumab remains questionable as randomized data are not available. A different HER2 targeting monoclonal antibody is pertuzumab, developed with structural specificity that binds a site on HER2 where it interferes with dimerization and thus disrupt signaling function. In 2018, Hainsworth et al (2018) found limited efficacy in a phase II basket study where 14 patients with HER2-mutated NSCLC received treatment with trastuzumab plus pertuzumab (ORR 21%) (83). Next, the potential role of HER2 targeting ADCs in NSCLC was explored. Trastuzumab-emtansine (TDM1) is a HER2 antibody-drug conjugate that releases a cytotoxic anti-microtubule agent within HER2 positive tumor cells upon degradation of the HER2-T-DM1 complex in lysosomes. In a phase II

trial by Hotta et al (2018) limited efficacy was seen after treatment with T-DM1 monotherapy in relapsed HER2 positive NSCLC (ORR 6.7%). In the subgroup of HER2 mutation positive NSCLC patients an ORR of 14.3% was found (84). A higher response rate on T-DM1 was reported by Li et al (2018) in a phase II basket trial in which 18 patients with HER2-mutant NSCLC were treated with T-DM1. An ORR of 44% (95% 22-69%), a median duration of response (DOR) of 4 months (range 2-9 months) and a median PFS of 5 months (95%CI: 3-9 months) were reported with acceptable toxicity. Of the 8 patients with a PR, 6 patients received prior systemic therapy of which 4 with HER2 targeted regimens (85). Trastuzumab-deruxtecan is a novel ADC with trastuzumab coupled to MAAA-1181, a topoisomerase I inhibitor (86). Preliminary results from the phase I study (DS8201-A-J101) show potent antitumor activity (ORR 72.7%) and acceptable toxicity. Recently a multicenter, open-label phase 2 trial (NCT03505710) was initiated to study the safety and efficacy of trastuzumab deruxtecan in HER2mutated NSCLC (87). HER2 amplification Tyrosine kinase inhibitors In a randomized phase II trial two patients with HER2 amplified NSCLC were treated with lapatinib, one of these two patients had a 51% decrease in tumor size, however, this response was unconfirmed (88). Antibody and antibody conjugated drugs Limited effect of trastuzumab pertuzumab combination treatment (ORR 13%) was seen in patients with HER2 positive (defined as HER2 overexpression IHC 3+ and/or amplification defined as HER2/CEP17 ratio >2.0 or copy number >6 or increased copy numbers by NGS) NSCLC in a basket study by Hainsworth et al (2018) (83). Also the phase II basket trial by Li et al (2018) showed limited efficacy of T-DM1 with a DCR of 11% in 18 patients with HER2-amplification (defined as fold change ≥2 on MSK-IMPACT or another NGS platform or HER2/CEP17 ratio >2.0) NSCLC (Table 3) (85). HER2 overexpression Antibody and antibody drug conjugates Although in vitro activity was found in HER2 overexpressing NSCLC (35), trastuzumab failed to demonstrate clinical benefit as a single agent (Table 4) (89). Trastuzumab in combination with pertuzumab also failed to show efficacy with no responses seen in a group of 38 patients with detectable HER2 expression. Also, no relationship between the level of HER2 expression and PFS was found (90, 91). Hotta et al (2018) reported limited efficacy (ORR 6.7%, PFS 2.0 months) of T-DM1 in NSCLC with HER2 positivity (IHC3+ and IHC2+), using the IHC scoring system applied to gastric cancer (84). Peters et al (2019) also studied the efficacy of T-DM1 in HER2 overexpressing (IHC ≥2+) NSCLC. No responses were observed in the IHC 2+ cohort. However, in the IHC 3+ cohort an ORR of 20% was found. Surprisingly, the median PFS and OS were similar in both groups. Although this study suggests activity of T-DM1 in HER2-overexpressing (IHC 3+) advanced NSCLC, 3/4 responders had HER2 gene amplification. Therefore, according to this study, HER2 overexpression determined by IHC is an insufficient predictive biomarker for T-DM1 efficacy (92). Recently, profound antitumor activity of trastuzumab-deruxtecan was found in HER2 overexpressing NSCLC mouse models (86). After a phase I study showed antitumor activity with acceptable toxicity, in patients with advanced breast and gastric or gastro-esophageal tumors (86), a multicenter, openlabel phase 2 trial (NCT03505710) was initiated to study the safety and efficacy of trastuzumab deruxtecan in HER2-overexpressing (IHC+ ≥2+) or -mutated NSCLC. Preliminary results confirm efficacy of this ADC with an ORR of 58.8% (10/17) with acceptable toxicity (86, 87). Targeting HER2 in the secondary driver setting

HER2 mutations No evidence has been found in literature of studies exploring treatment of HER2 mutation positive NSCLC as a secondary driver. HER2 amplification and overexpression Antibody and antibody drug conjugates Targeting HER2 with the combination of trastuzumab-paclitaxel could be effective in targeting HER2 overexpressed NSCLC found after EGFR targeted treatment in patients with EGFR+ NSCLC, reported by De Langen et al (2018) (62). Twenty-four patients with EGFR+ NSCLC that showed tumor membrane HER2 expression (IHC≥2+) in a tumor biopsy after progression on EGFR TKI treatment, were treated with trastuzumab and paclitaxel. Responses were seen in 46% of all patients (PR 11/24) with a median DoR of 5.6 months. Treatment was well tolerated. HER2 expression and GCN were correlated with response rate and the highest response rates were seen for patients with HER2 IHC 3+ (67%) or HER2 copy number ≥10 (100%). Upon PD, initially responding patients were rebiopsied with 4/6 samples being negative for HER2, indicating that HER2 was effectively targeted in these patients (62). In vitro experiments suggest that T-DM1 might overcome HER2 bypass track resistance in EGFR mutated NSCLC (93). In the EGFR TKI resistance setting with HER2 in the role of acquired resistance, the possibility of combination treatment, combining HER2 and EGFR targeted treatment, covering the different growth pathways of the tumor has also been studied. La Monica et al (2017)(94) described HER2 overexpression as a mechanism of acquired resistance after treatment with osimertinib (94). Combination treatment with osimertinib and T-DM1 improved the efficacy of osimertinib by delaying resistance in NSCLC cell lines with EGFR activating mutation. The role of HER2 amplification was also investigated in cell lines and in a PC9/HER2c1 xenograft model. T-DM1 combined with osimertinib had an additive growth inhibitory effect and resistance to osimertinib treatment was delayed and even prevented in some cell lines (94). These data suggest that concomitant treatment with an EGFR TKI combined with a HER2 targeted ADC like T-DM1 may be a promising therapeutic strategy for EGFR-mutant NSCLC with HER2 activation as the mechanism of (acquired) resistance. Conclusion Today the (re)search on how to analyse, define and treat HER2 alterations in NSCLC continues. The definition of and relation between HER2 overexpression, amplification and mutation has been variable and therefore difficult to interpret. HER2 mutations are identified as a primary driver in NSCLC, but can also be found as a possible mechanism causing primary and acquired resistance to EGFR TKI treatment (18-20) and are detected by gene sequencing methods like NGS. HER2 amplification is less frequently observed as a possible oncogenic driver in NSCLC compared to breast cancer (35, 38, 39). As a mechanism for acquired resistance it has been suggested that HER2 amplification is one of the most frequent mechanisms of acquired resistance after the EGFR T790M mutation in EGFR+ NSCLC treated with 1st or 2nd generation EGFR TKIs (56, 57). FISH is used as a standard method to classify HER2 amplification with high specificity, early standardization and consistency (23, 40). However, the definition of HER2 amplification should be critically evaluated because the classical ratio adopted from breast cancer could not be adequate in NSCLC (5, 50). Although polysomy has been observed more frequently, especially co-existing with EGFR mutations, available data suggest that only amplification drives NSCLC tumors (26, 41, 61). HER2 overexpression has been reported in NSCLC with a wide range up to 30% (43, 44). This variation can be explained by the use of different definitions, testing methods and patient selection. It is suspected that in most studies, HER2 IHC positivity might have been overestimated because of the use of the scoring system for IHC status that is applied in gastric cancer, which is less strict than that

is used for breast cancer which is most commonly used for the classification of HER2 IHC in NSCLC (11, 46, 84). At the time of resistance to EGFR TKI therapy, protein overexpression is often found and could therefore be a potential secondary driver causing acquired resistance. In several NSCLC trials using a broad spectrum of HER2-targeted therapeutic agents, often with promising efficacy in other HER2 altered malignancies, reported results in NSCLC are inconclusive. The general observation is that in studies with HER2 targeting agents, including pan-HER TKIs, monoclonal antibodies and ADC, a median PFS of around 4 months can be found, comparable with the PFS in this population treated with chemotherapy. There are three possible explanations for this phenomenon. First, HER2-targeted regimens do not sufficiently suppress the altered HER2 signal in NSCLC. Second, due to tumor heterogeneity, merely strong HER2-positive cells are suppressed and killed by HER2 targeted treatment, but weakly positive cells escape cell death and continue to expand (84). Thirdly, there is still the possibility that HER2 is not a forceful oncogenic driver in NSCLC. Therefore we conclude that the force of HER2 in NSCLC driving tumorigenesis is still unclear and therefore it remains the question if HER2 in NSCLC is a druggable target. Future research should focus on this important missing link. An unambiguous answer to the question, whether and which HER2 alterations are relevant oncogenic drivers is urgently needed. Next, should be evaluated whether HER2 alterations should be considered as a therapeutic target in NSCLC. The development of HER2 targeted therapy is a broad galaxy still to be explored.

Table 1. Clinical trials targeting HER2 mutations as a primary driver with tyrosine kinase inhibitors. Author (ref) Trial Drug No DCR ORR PFS OS patients Kris et al Phase II trial dacomitinib 26 12%, 3 9 months, (65) 95%CI: months, 95%CI, 72-30% 95%CI 2- 21 4 months months De Greve Phase II trial afatinib 33 53% (71) Mazieres et afatinib 3 100% 33.3% 5.1 22.9 al (24) months months Mazieres et Retrospective afatinib 11 63.7% 18.2% 3.9 al (14) EURHER months study Dziadziusko NICHE trial afatinib 13 53.8% 7.7% 3.7 56.0 et al (95) months weeks (95%CI: (95%CI: 1.4-8.1) 16.3upper limit not estimable) Peters et al NPU program afatinib 28 4/10 TTF 9.6 (72) patients months treated >1 year Lai et al (73) Retrospective afatinib 23 70% 13%, 23 95%CI: months 4-33% (95%CI: 18-52) Fang et al Retrospective afatinib 32 69% 16% 3.2 (74) months (95% CI: 2.0-4.5 months) Hyman et al SUMMIT neratinib 26 3.8% 5.5 (66) study months Robichaux Phase II trial poziotinib 12 42% 5.6 et al (78) months Wang et al Phase II trial pyrotinib 15 73.3% 53.3% 6.4 12.9 (79, 80) months months (95%CI: (95%CI: 1.602.0511.20) 23.75) Table 2. Clinical trials targeting HER2 mutations as a primary driver with antibody and antibody drug conjugates. Author (ref) Trial Drug No DCR ORR PFS OS patients Mazieres et Retrospectiv Trastuzumab 58 75.5% 50.9% 4.8 13.3

al (14)

e EURHER study Phase II trial

and chemotherapy, T-DM1 Trastuzumab 14 and pertuzumab

Hotta et al (84)

Phase II trial

T-DM1

15

53.4%

Li et al (85)

Phase II basket trial

T-DM1

18

83%

Planchard et al (87)

Phase I trial

Trastuzumab deruxtecan

11

-

Hainsworth et al (83)

21% after 120 days

21% (95%CI: 5-51) after 6 weeks 6.7% (95%CI: 0.327.9) 44% (95%CI: 22-69) 72.7%

months (95%CI: 3.4-6.5) -

months (95%CI: 8.1-15) -

2.0 months (95%CI: 1.4-4.0) 5.0 months (95%CI: 3.0-9.0) -

10.9 months (95%CI: 4.4-12.0) -

-

Table 3. Clinical trials targeting HER2 amplification as a primary driver with antibody and antibody drug conjugates. Author (ref) Trial Drug No DCR ORR PFS OS patients Hainsworth Phase II trial Trastuzumab 16 13% 13% et al (83) and after (95%CI: pertuzumab 120 2-38) days after 6 weeks Li et al (85) Phase II T-DM1 18 11% 5.5% basket trial Table 4. Clinical trials targeting HER2 overexpression as a primary driver with antibody and antibody conjugated drugs. Author (ref) Trial Drug No DCR ORR PFS OS patients Clamon et al Phase II Trastuzumab 24 4.2% (89) trial Herbst et al Pertuzumab 38 41.9% 6.1 (91) after 6 weeks weeks, (95%: 20.9% 5.3-11.3 after weeks) 12 weeks Johnson et al Phase II Trastuzumab 33 0% (90) trial and pertuzumab Hotta et al Phase II T-DM1 8 37.5% 0% (84) trial Peters et al Phase II T-DM1 49 IHC2+: IHC2+: IHC2+: (92) trial (IHC2+ 0% 2.6 12.2

29, IHC3+ 20)

IHC3+: 20% (95% CI: 5.743.7%)

months (95%CI: 1.4-2.8) IHC3+: 2.7 months (95% CI, 1.4–8.3) 6.7 months (IQR 4.410.2) -

Doi et al (86)

Phase I trial

Trastuzumab deruxtecan

24

91% (95%CI 72.098.9)

43% (95%CI: 23.265.5)

Planchard et al (87)

Phase I trial

Trastuzumab deruxtecan

17

-

58.8%

months (95% CI, 3.8– 23.3) IHC3+: 15.3 months (95% CI, 4.1–NE) -

-

Figure 1 Prevalence of HER2 (ERBB2) cancer hotspots in NSCLC. Schematic diagram of the HER2 (ERBB2) gene showing the frequency and location of HER2 hotspot mutations in 46 NSCLCs (source: cBioPortal). Bar lengths represents the frequency of reported amino acid change. For exon 20, codons 772 to 780 are highlighted to show insertions and mutations in this region. TM, trans membrane domain.

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Highlights Three principal mechanisms of HER2 alterations can be identified: protein overexpression, gene amplification and gene mutations. No consensus about HER2 subgroup definitions has been obtained, complicating the interpretation of the results of the many trials studying possible treatment strategies. Future research should focus on the definition of HER2 alteration subgroups and test therapeutic agents in these different subgroups.