Targeting HER2 genomic alterations in non-small cell lung cancer

Oncogenic mutations and amplifications in the erythroblastic oncogene B (ERBB2), or human epidermal growth factor receptor 2 (HER2), have emerged as distinct oncogenic drivers and drug targets in non-small cell lung cancer (NSCLC). Each genomic alteration occurs in 2–4% of NSCLC by next generation sequencing and is associated with constitutive HER2 activation. The most common HER2 mutations in NSCLC are exon 20 mutation A775_G776insYVMA mutation in the kinase domain and S310F mutation in the extracellular domain. Unlike in breast and gastric cancer, HER2 protein overexpression in NSCLC is not validated to be a biomarker predictive of clinical response to HER2-targeted agents. High HER2 protein overexpression by immunohistochemistry (3+) only occurs in 2–4% of NSCLC. Until now HER2-targeted agents (such as afatinib and ado-trastuzumab emtansine) only demonstrate modest clinical activity in patients with HER2-mutant NSCLC. Retrospective studies show concern for inferior clinical benefit of immune checkpoint inhibitors in HER2-mutated NSCLC. Therefore, platinum-based chemotherapy with or without an anti-angiogenesis inhibitor remains the first line standard treatment for this patient population. In May 2020 trastuzumab deruxtecan (T-DXd) received the U.S. Food and Drug Administration breakthrough therapy designation for HER2-mutant metastatic NSCLC, and was added as an option for HER2-mutant NSCLC to the NCCN guidelines V1.2021. A global phase III study of pyrotinib compared to docetaxel as a second line therapy for advanced NSCLC harboring HER2 exon 20 mutations was just opened for enrollment in September 2020. In this review, we highlight the current knowledge and perspectives on targeting-HER2 genomic alterations in NSCLC.


Introduction
Lung cancer is the leading cause of cancer related death for both men and women worldwide, with nearly 1.8 million deaths each year 1 .Non-small cell lung cancer (NSCLC) accounts for approximately 80-85% of all lung cancer cases.NSCLC is among the most genomically diverse and deranged of all cancers, which creates tremendous challenges for both prevention and treatment strategies.Increasingly over time, driver oncogenic mutations have been described by clinical use of multiplex next generation sequencing (NGS).Currently, both the National Comprehensive Cancer Network (NCCN) and American Society of Clinical Oncology (ASCO) guidelines recommend testing for mutations in EGFR, BRAF, MET, NTRK and gene fusions (or rearrangements) in ALK, ROS1, RET and NTRK for all NSCLC tumors that contain an adenocarcinoma component, regardless of histologic grade, dominant histologic subtype, clinical characteristics or demographic information 2 , 3 .Genomic alterations, mainly amplification and mutations, in the erythroblastic oncogene B ( ERBB2 ) or human epidermal growth factor receptor 2 ( HER2 ) have emerged as distinct oncogenic drivers in 2-4% of HER2 is normally expressed in the fetal period and at very low levels within some tissues in adults.HER2 plays a critical role in NSCLC development and progression by forming heterodimers with other HER family members (EGFR or HER1, HER2 and HER4).Additionally, HER2 may form homodimers when it is highly expressed 10 .HER2 can increase the activated HER proteins' affinity to ligands, affect the stability of the receptor and induce the amplification of the HER2 signal cascade.While the members of the HER family contain similar domains, each of the four proteins (EGFR, HER2, HER3 and HER4) have distinct properties.For example, HER2 has strong kinase activity but has no identified ligand; while HER3 is devoid of kinase activity due to substitutions in crucial tyrosine kinase (TK) domain residues 11 .The HER family members can form either homodimers or heterodimers upon ligand binding ( Fig. 2 ).This triggers the autophosphorylation of tyrosine residues within the cytoplasmic domain of receptors and initiates a variety of signaling pathways.These receptors work through a complex array of secondary messengers and play important roles in various cellular functions including adhesion, differentiation, growth, apoptosis and migration 12 .Overexpression of HER2 alone, or in co-overexpression with EGFR, could enhance down regulation of EGFR tyrosine kinase activity and induce aggressive tumor growth 13 .

Testing for HER2 alterations in NSCLC
HER2 alterations, which include mutations, gene amplification, and protein overexpression, have been detected in over 100 cancer types and are associated with poor prognosis [14][15][16] .There are significant variations in the type and frequency of HER2 alterations in different cancer types.These variations may be attributed to different tumor biology, tumor heterogeneity, sample size, race and ethnicity, detection methods, and definitions of overexpression.Table 1 summarizes the reported HER2 al-terations in human NSCLC specimens as compared to other major cancer types including breast, gastric, gastroesophageal junction (GEJ) adenocarcinomas, and colon cancer.
Currently, there is no guideline established for HER2 testing in lung cancer.This contrasts with breast, gastric and GEJ adenocarcinomas, which have amplification of wild -type HER2 with protein overexpression in up to 20% of cases.HER2 amplification, defined as a HER2/CEP17 ratio ≥ 2, was present in 10-20% of cases by fluorescence in situ hybridization (FISH), but only occurs in 2-4% of NSCLC cases by NGS 14 , 15 , 17 .Thus, the HER2 testing guidelines for gene amplification and protein overexpression in breast cancer, gastric cancer, and GEJ adenocarcinomas 18 are unlikely to be helpful for NSCLC.HER2 amplification is shown to increase the invasiveness of NSCLC cells in vitro and can elicit the constitutive activation of both HER2 and ligandindependent EGFR 19 .Higher HER2 amplification numbers are associated with increased clinical response to HER2-targeted agents in case reports and early phases of clinical trials when studied in different cancer types 20 , 21 .The optimal cutoff for predicting clinical benefit of HER2 amplification in NSCLC remains to be defined through clinical trials.
In addition to gene amplification, HER2 mutations were identified in 2-4% of NSCLC tumors by routine, clinical use of comprehensive molecular profiling via NGS 7 , 22 , 23 .Like EGFR mutations, the frequency of HER2 mutations is significantly increased in non-smokers, women, persons of Asian descent, and adenocarcinoma NSCLCs 8 .HER2 mutations are generally mutually exclusive with other oncogenes in NSCLC.The incidence of HER2 mutations can be as high as 6.7% in EGFR/ALK/ROS1 -negative NSCLC 24 , especially in those who were negative for EGFR .Pan-cancer analysis shows that the most common HER2 mutations are S310F/Y (11.0%),A775_G776insYVMA (5.7%), L755P/S (4.6%), V842I (4.4%), and V777L/M (4.0%).Although notably, the frequency and hotspots of HER2 mutation vary among different cancer types 15 .In breast cancer and colorectal cancer, the most common HER2 mutations are in exon 19 and exon 21, respectively.The most common HER2 mutations in lung cancer occur in exon 20.

Table 1
The prevalence of HER2 alterations in solid tumors.Abbreviations: FISH, fluorescent in situ hybridization; IHC, immunohistochemistry; NGS, next-generation sequencing; PCR, polymerase chain reaction.
Among the exon 20 mutations, the most common mutation is ERBB2 (HER2) A775_G776insYVMA.These HER2 mutations lead to constitutive activation of the receptor and the downstream activation of the phosphatidylinositol 3 -kinase (PI3K)/protein kinase B (AKT) and Ras/mitogen activated protein kinase (MAPK) signaling pathways 7 , 24 .HER2 exon 20 insertion mutations and exon 19 L755P mutation are resistant to most HER2 TK inhibitors (TKIs) due to an acquired sterically hindered drug binding pocket 25 .Mutations in the extracellular domain of the HER2 gene are oncogenic and associated with sensitivity to treatment with HER2 inhibitors 26 .There are many genomic alterations in the HER2 gene identified by NGS in NSCLC.Due to the rare frequency, most of the studies contained small sample sizes.Table 2 summarizes the types and frequencies of HER2 mutations in NSCLC reported from the two largest published or publicly available sequencing databases (Foundation Medicine, which contains both primary and metastatic tumors 14 , and cBioPortal [The Cancer Genome Atlas] dataset, which contains mainly primary tumors 27 .Furthermore, we summarized the known responses of each HER2 mutation to HER2-targeted therapy into a quick clinical reference for practicing physicians.The most common HER2 mutations in lung cancer occur in exon 20 (38.3%), followed by S310F mutations (14.9%) in the extracellular domain.The top "hotspot " HER2 mutations with frequencies are illustrated in Fig. 1 C.The molecular mechanisms that lead to these "hotspot " HER2 mutations are unknown.ERBB2 ( HER2 ) A775_G776insYVMA is also named Y772_A775dup, E770_A771insAYVM, or A771_M774dup, in the scientific literature depending on the hypothetical explanation for the molecular mechanism of the YVMA insertion (illustrated in Fig. 1 D).A recent large retrospective study shows that not only the HER2 mutation variants, but also co-mutations, affect the response to afatinib in HER2mutant NSCLC.TP53 is the most common co-mutation in HER2-mutant NSCLC.Co-mutations in TP53 and the PI3K/AKT/mTOR pathway confer additional resistance to anti-HER2 treatments in NSCLC.The underlying mechanisms and implication for treatment remain unknown 28 .Liquid biopsy for tumor genomonic profiling of plasma cell free DNA is being used more frequently to detect HER2 genomic alterations in many solid cancer types, including NSCLC 29 .Of note, HER2 mutations are rarely associated with HER2 amplification 30 .HER2 mutation or gene amplification usually does not demonstrate HER2 protein overexpression 30 .Each of these three HER2 genomic alterations is associated with clinical response to HER2-targeted therapy 18 , 21 .Thus, these HER2 genomic alterations define distinct tumor subtypes, and they are being evaluated as independent biomarkers for HER2-targeted therapy.

Table 2
Reported HER2 mutations in NSCLC.A total of 316 case were collected from Foundation Medicine report (N = 165) and cBioPortal (N = 151).Blue highlights the oncogenic mutations occurred in at least 6 (1.9%) cases and are illustrated in Fig. 1   Abbreviations: CR, complete response; mo, months; NA, not available; PFS, progression-free survival; PR, partial response; SD, stable disease.

Fig. 3.
Milestones for HER2-targeted therapy in solid tumors.Currently available HER2-targeted drugs were approved for breast cancer (pink) and gastric (brown) cancer.Trastuzumab deruxtecan (T-DXd, formerly DS-8201a) is the first HER2-targeted drug which received the U.S. FDA therapy designation for HER2 -mutant metastatic NSCLC (blue) in May 2020.On December 16, 2020, margetuximab-cmkb in combination with chemotherapy 97 , 98 joins trastuzumab deruxtecan, combination of tucatinib, capecitabine and trastuzumab treatment, and combination of neratinib, capecitabine and trastuzumab treatment as third-line treatment options for adult patients with HER2-positive metastatic breast cancer.

HER2-targeted therapies
All HER2-targeted agents (including antibodies, small molecular tyrosine kinase inhibitors and antibody drug conjugates) developed to date ( Fig. 2 ) were developed for amplification and protein overexpression of the HER2 wild-type genes in breast and gastric/ GEJ adenocarcinoma.Fig. 3 summarizes the milestones of the U.S. Food and Drug Administration (FDA) approval of these agents.For the rare subset of HER2mutant breast cancer, neratinib demonstrates a clinical benefit rate of 31-40% in two phase II clinical trials (MutHER and SUMMIT) 31 , 32 .Although ado-trastuzumab emtansine is recommended as a treatment op-tion for HER 2-mutant NSCLC by NCCN guidelines (category 2B) 3 , currently available HER2-targeted agents only demonstrate modest clinical activity in patients with HER2-positive NSCLC after platinum-containing chemotherapy ( Table 3 ).This is in sharp contrast to the efficacy of first line targeted therapy for NSCLC harboring other driver mutations, such as EGFR, ALK, ROS1 and BRAF , and NTRK 33 , 34 .Table 4 summarizes the FDA-approved HER2-targeted drugs for breast cancer.Further studies are needed to understand the molecular and cellular mechanisms driving HER2-positive NSCLC and to develop treatment strategies for these patients.The following section reviews current knowledge and ongoing studies for targeting HER2-positive NSCLC.Abbreviations: bid, twice daily; CI, confidence interval; DFS, disease-free survival; DOR, duration of response; HR, hazard ratio; IDFS, invasive disease-free survival; IV, intravenously; mo, month; NA, not available; NE, not estimable; NR, not reached; ORR, overall response rate; OS, overall survival; PFS, progression free survival; qd, once daily; q3w, every 3 weeks; wk, week.

Small molecule tyrosine kinase inhibitors (TKIs)
Small molecule TKIs enter the cell and bind to the cytoplastic ATP binding site at the tyrosine kinase domain to prevent phosphorylation and activation of the receptor.Monoclonal antibodies, in contrast, function extracellularly by inhibiting ligand binding and/or homo/heterodimerization of the receptor with activation of the signaling pathway ( Fig. 2 ).To date, no TKI that exclusively targets HER2 has been developed.On the other hand, selective EGFR TKIs such as osimertinib were successfully developed against EGFR.There are two classes of EGFR/HER2 TKIs: those that bind reversibly (such as lapatinib) and those that bind irreversibly (covalently) (such as afatinib, decomtinib) to the tyrosine kinase domain ATP binding site.Among these available drugs, afatinib is the most commonly used off-label TKI for HER2amplified or mutated NSCLC 35 , 36 .In the most comprehensive preclinical study to date, common HER2 mutants in exons 19-21 show various sensitivities to eleven EGFR/HER2 TKIs in vitro .Poziotinib was the most potent HER2 -mutant selected TKI tested, both alone or in combination with ado-trastuzumab emtansine 15 .However, the clinical development of poziotinib has been hampered by modest clinical activity with significant gastrointestinal and dermal toxicities as monotherapy 37 .Recently, a newer generation of pan-HER TKIs, such as pyrotinib, mobocertinib, tucatinib, BDTX-189, and tarloxotinib, show promising preclinical and clinical activity that is reviewed below.

Afatinib
Afatinib (BIBW2992) is a highly selective, potent, irreversible inhibitor for EGFR, HER2 and HER4 kinases 38 , 39 .The irreversible tyrosine kinase blockade may result in longer suppression of HER2 signaling compared to reversible inhibitors 40 , 41 .In a single arm, phase II study (NICHE), afatinib achieved disease control at 12 weeks in 53.8% of 13 patients with advanced NSCLC harboring HER2 exon 20 mutations.Median progression free survival (PFS) and overall survival (OS) were 15.9 weeks (95% CI: 6.0, 35.4) and 56 weeks (95% CI: 16.3, not reached), respectively 42 .In a retrospective study, 23 patients with stage IV or recurrent HER2 mutated lung adenocarcinomas were treated with afatinib 43 .Of the 23 patients, 13% of patients had a partial response (PR), 57% had stable disease (SD), and 30% showed disease progression.The median response time to afatinib was 6 months and median OS was 23 months.A preclinical study showed that afatinib in combination with mammalian TOR (mTOR) inhibitor rapamycin was the most effective way to inhibit the tumor growth in HER2 YVMA-mutant transgenic NSCLC mice when compared to trastuzumab and/or rapamycin treatment.Both upstream and downstream MAPK and Akt/mTOR axes were inhibited by afatinib and rapamycin combination 40 .A phase Ib study showed that afatinib combined with either intravenous vinorelbine or oral vinorelbine was safe in EGFR/HER2 overexpressed solid tumors 44 .Among the evaluated patients, 16 out of 28 (57.1%)patients in the intravenous group showed SD, and 3 out of 27 (11.1%)patients in the oral group achieved PR.The median PFS was 14.6 and 15.9 weeks in intravenous and oral group, respectively.Afatinib has different sensitivities in different HER2 mutation variants in NSCLC.In a large retrospective analysis containing 118 patients with HER2-mutated metastatic NSCLC, a total of 31 HER2 mutation variants and 35 concomitant genomic alterations were detected.This suggests HER2-mutant NSCLC is a group of very heterogenous disease.Certain variants, G778_P780dup and G776delinsVC, derive sustained clinical benefits from afatinib.Whereas the predominant variant, A775_G776insYVMA, is resistant to most available anti-HER2 treatments including afatinib 28 .Because it is commercially available for patients with EGFR-mutant NSCLC, afatinib is used in clinical settings with various responses.In some cases, this includes PR or SD for patients with HER2 mutations (including A775_G776insYVMA) 28 , 45 , 46 .

Dacomitinib
Dacomitinib (PF-00299804) is an irreversible pan-HER TKI that inhibits the kinase activity of wild-type EGFR, HER2, and HER4 .Further-more, it demonstrates an effect against gefitinib resistant NSCLC models harboring either HER2 amplifications or mutations in preclinical studies 47 .Dacomitinib is approved as an option for first line systemic therapy in patients with EGFR -mutant, advanced NSCLC cancer 48 .The median PFS was 14.7 months in the oral dacomitinib 45 mg/day group compared to 9.2 months in the oral gefitinib 250 mg/day group.The median OS was 34.1 months in dacomitinib group versus 26.8 months in gefitinib 49 .A phase II study of 26 patients with HER2-positive, advanced NSCLC, dacomitinib led to PR in 3 of 26 (11.5%) patients with HER2 mutations (25 insertion and 1 missense mutation) compared to no PR observed in 4 patients with HER2 amplification.The median OS was 9 months in the HER2 mutation group.OS ranged from 5 to 22 months in the HER2 amplification group 17 .Similar to afatinib, dacomitinib is used by clinicians for HER2-positive NSCLC patients when clinical trials are not available, or patients are not candidates or decline systemic chemotherapy.

Pyrotinib
Pyrotinib is an oral, irreversible pan-HER TKI with strong activity against EGFR and HER2 50 .Pyrotinib first received approval in China for metastatic breast cancer treatment 51 .Despite similar preclinical activity, pyrotinib has superior clinical activity compared to lapatinib in combination with capecitabine for patients with metastatic breast cancer (mBCA) 52 .Preclinical and phase 1 studies suggest that pyrotinib could effectively inhibit the proliferation of HER2-overexpressing cells in vitro and in vivo 53 , 54 .In HER2 exon 20 mutant NSCLC patient-derived organoid or xenograft models pyrotinib has shown potent tumor growth inhibition.This is achieved through inhibition of phosphorylated HER2 and downstream phosphorylated ERK and AKT pathways 50 .During early phase clinical trials, pyrotinib demonstrated promising clinical activity with good tolerability in both Chinese and US patients.In the Chinese cohort, among 28 enrolled HER2-positive metastatic breast cancer patients, 60.7% were trastuzumab-pretreated patients.The overall response rate (ORR) was 78.6% and median PFS of 22.1 months.The ORR was 90.9% and 70.6% in trastuzumab-naïve and trastuzumab-pretreated patients, respectively 55 .In the U.S. cohort, mean prior treatment was 3.Although 26% of the patients received more than 3 lines of prior therapy including HER2 targeted treatments (e.g.trastuzumab, ado-trastuzumab emtansine and other HER2 TKIs), the median PFS was 5.4 months and ORR was 13% 56 .Of 4 patients with confirmed PR and 5 patients with SD for more than 6 months, all had HER2 exon 20 mutations.Further analysis showed 6 cases with A775_G776insYVMA, 1 with E770A-A771insAYVM, 1 with G778-P780dup, and 1 with G778_779insCPG.The most common treatment emergent adverse effect was diarrhea with grade 3 and 4 toxicity present in 24.3% of patients.A phase II clinical trial (NCT02834936) enrolled 60 pretreated HER2-mutant NSCLC patients.Subjects received 400 mg of pyrotinib daily.The observed ORR was 31.7% (95% CI: 20.3%, 45.0%), median duration of response (DOR) and PFS were 7.0 months (95% CI: 5.5, 11.0) and 6.8 months (95% CI: 4.1, 8.3), respectively 57 .Prophylactic antidiarrheal treatment with loperamide was recommended.Based on these results, a phase III, global, randomized clinical trial (NCT04447118) comparing the efficacy of pyrotinib to docetaxel as second line systemic therapy in patients with advanced, non-squamous NSCLC harboring HER2 exon 20 mutations) who have failed platinum-based chemotherapy was recently opened to enroll patients in September 2020.

Mobocertinib
Mobocertinib (TAK-788) is an oral TKI with potent preclinical activity selectively against EGFR and HER2 mutations, including exon 20 insertions.A phase I/II clinical trial enrolled 34 patients with previously treated, advanced NSCLC with EGFR/HER2 exon 20 insertions or mutations.Patients received oral mobocertinib in daily doses of up to 180 mg during the dose-escalation phase 58 .Among patients with EGFR exon 20 insertions who received 80-to 160-mg doses of mobocertinib daily, 39% responded and 94% had radiographically demonstrated disease control.
Adverse effects were mostly mild and frequently included diarrhea, nausea, fatigue, and rash.These symptoms were similar to those with other TKIs.Investigators plan to study a 160-mg daily dose of mobocertinib in a phase 2 expansion cohort.Based on these results, the U.S. FDA granted mobocertinib Orphan Drug Designation for the treatment of lung cancer with HER2 mutations or EGFR mutations (including exon 20 insertion) in 2019.Additionally, mobocertinib received a breakthrough therapy designation for EGFR exon 20 insertion mutation NSCLC in 2020 59 .The efficacy of mobocertinib for patients with locally advanced or metastatic NSCLC whose tumors harbor HER2 exon 20 insertions has not been reported.

Tucatinib
Tucatinib is the newest oral HER2 TKI approved for metastatic HER2positive breast cancer.In vitro tucatinib inhibits phosphorylation of HER2 and HER3, which results in blockade of downstream PI3K/AKT and Ras/MAPK signaling pathways, thus inhibiting cell growth 60 , 61 .Although in vivo , tucatinib monotherapy demonstrates limited preclinical activity 62 .Tucatinib in combination with trastuzumab and capecitabine was approved by the US FDA for adult patients with advanced unresectable or metastatic HER2-positive breast cancer in April 2020.This includes patients with brain metastases who have received one or more prior anti-HER2-based regimens in the metastatic setting.In 6 HER2mutant patient derived xenograft models for colorectal, NSCLC, gallbladder and gastric cancers tucatinib and trastuzumab demonstrated promising synergistic antitumor effects in those harboring L755S, V77L or S310Y mutations 63 .Tucatinib and trastuzumab inhibited the growth of HER2 L755S mutations in NSCLC.In human lung cancer cell line NCI-H1781 it was observed that tucatinib can inhibit HER2 phosphorylation.A basket trial of tucatinib and trastuzumab (NCT04579380) is evaluating the clinical activity in solid tumors with HER2 alterations, including a cohort for NSCLC (accrual start date: January 11, 2021).

BDTX-189
A new EGFR/HER2 mutation selective TKI, BDTX-189, received the FDA Fast Track Designation in July 2020.BDTX-189 is an orally available, irreversible small molecule inhibitor that is designed to spare wildtype EGFR while blocking the function of EGFR and HER2 kinase domain exon 20 insertions.Additionally, it is designed to blockade other activating oncogenic drivers of HER2, including the S310F/Y mutation.Similar to the third generation EGFR TKI osimertinib, the sparing of wild-type EGFR by BDTX-189 has the potential to improve the toxicity profile of current HER2 kinase inhibitors and has high permeability into the brain 64 .The MasterKey-01 study (NCT04209465), a phase I/II trial of BDTX-189 is recruiting adult patients with solid tumors harboring an allosteric HER2 mutation, or an EGFR or HER2 Exon 20 insertion mutation, who have progressed following prior treatment and lack alternative therapy options.

Tarloxotinib
Tarloxotinib is the hypoxia-activated prodrug of a pan-ErbB kinase inhibitor that releases a potent irreversible active metabolite (tarloxotinib-E).This metabolite is preferentially delivered to the active moiety of tumor versus normal tissues.Tarloxotinib demonstrated preclinical efficacy in EGFR exon 20, HER2 -mutant NSCLC, HER2 -amplified NSCLC, and other oncogenic alterations in the ERBB gene family (such as NRG1 fusions) 65 .In July 2020, a phase II clinical trial (RAIN-701 trial, NCT03805841) evaluated 11 HER2 -mutant NSCLC patients who failed platinum-based chemotherapy to be treated with tarloxotinib.Four out of 9 evaluable patients demonstrated tumor shrink, 22% patients had PR, and 44% patients had SD 66 .Three patients remained on treatment after 6 months.Thus, tarloxotinib shows promising clinical efficacy in patients with HER2 -mutant NSCLC.

The role of chemotherapy in HER2-positive NSCLC
HER2-targeted therapies are well tolerated but lack clinical efficacy in HER2-positive NSCLC.Therefore, several clinical trials evaluated the efficacy of various chemotherapy in combination with HER2 targeting agents.A phase II trial evaluated the efficacy of carboplatin, paclitaxel and trastuzumab in patients with HER2-positive NSCLC.Of 139 patients screened, 53 were enrolled with HER2 positivity graded by IHC staining from 1 + to 3 + .The combination of chemotherapy and trastuzumab was well tolerated.For IHC 1 + and 2 + patients, the OS was observed to be consistent with historical data using carboplatin and paclitaxel alone.Patients with IHC 3 + HER2 had higher OS compared to IHC 1 + and 2 + patients, suggesting a potential benefit to trastuzumab therapy in this subset 67 .Similarly, a randomized phase II trial assessed the addition of trastuzumab to cisplatin/gemcitabine in previously untreated patients with HER2-positive NSCLC.Of the 619 patients screened, 103 patients were tested positive for HER2 and received gemcitabine and cisplatin with trastuzumab versus gemcitabine and cisplatin alone.Again, no clear clinical benefit was observed in the trastuzumab treated group versus chemotherapy alone.Although some benefit was observed in the HER2 IHC 3 + group, the subset was too small to provide definitive information 68 .Lastly, the addition of trastuzumab to weekly docetaxel had a PR rate of 8% in a small phase II trial with 13 patients who had IHC 2 + or 3 + HER2 positive tumors and progression after platinum-based chemotherapy 69 .Overall, the results support further development of HER2-targeted therapy and chemotherapy.

HER2-targeted antibody drug conjugates 4.3.1. Ado-tratuzumab emtansine (T-DM1)
Ado-tratuzumab emtansine (T-DM1) is a HER2 targeting antibodytoxin conjugate which is composed of trastuzumab, a stable thioether linker, and the potent cytotoxic agent DM1 70 .Preclinical studies demonstrated that ado-trastuzumab emtansine combines the distinct mechanisms of action of both trastuzumab and DM1.Furthermore, clinical experiments show a minimal amount of systemic exposure to free DM1, without evidence of DM1 accumulation after repeat doses.In a phase II basket trial 71 , 18 advanced HER2 -mutant lung adenocarcinomas received ado-trastuzumab emtansine and had a PR rate of 44% with median PFS of 5 months.Another phase II trial evaluated adotrastuzumab emtansine efficacy in previously treated HER2-overexpressing NSCLC 72 .Forty-nine patients were divided into HER2 immunohistochemistry (IHC) 2 + and IHC 3 + groups.None in the IHC 2 + group showed response to ado-trastuzumab emtansine, and 4 out of 20 patients with IHC 3 + expression showed PR.The ORR was 20%.Clinical benefit rates were significantly higher in the IHC 3 + group, 30% vs 7%.The response duration in those four responders range from 2.9 to 10.8 months.Of note, 3 out of 4 responders also had HER2 amplification.Currently, ado-trastuzumab emtansine is recommended by NCCN guidelines for advanced NSCLC with HER2 mutation (category 2A) 3 .
Trastuzumab deruxtecan also demonstrated promising clinical activity in patients with HER2 -positive metastatic NSCLC.In an ongoing phase 1 trial, trastuzumab deruxtecan (6.4 mg/kg) had a confirmed ORR of 58.8% (10/17) in HER2 -expressing or mutated NSCLC, and 72.7% (8/11) in HER2 -mutated NSCLC, with a manageable safety profile 77 .The first interim analysis from the ongoing phase II DESTINY-Lung01 trial (NCT03505710) was first presented at the ASCO 2020 Virtual Scientific Program 78 .The results showed that trastuzumab deruxtecan had a confirmed ORR of 61.9% (95% CI: 45.6%, 76.4%) in 42 patients with HER2 -mutated NSCLC.This included 1 patient (2.4%) with a complete remission and 59.5% of patients with a PR.Additionally, 28.6% of patients had SD.Thus, the disease control rate was 90.5% (95% CI: 77.4%, 97.3%).For most patients (90.5%), their HER2 mutations were located in the kinase domain.Only 1 patient had a mutation present in the extracellular domain, and 1 patient did not have this information reported.All patients reported treatment-emergent adverse events (TEAEs), and among them 64.3% were over grade 3. The overall safety and tolerability profile of trastuzumab deruxtecan was consistent with observations seen in the phase I trial.The most common adverse events to date (n = 42) were gastrointestinal and hematological, including nausea, alopecia, anemia, decreased appetite, and decreased neutrophil count.Drug-related interstitial lung disease was observed in 5 cases, and all of them were grade 2 78 .Based on these results, trastuzumab deruxtecan received the breakthrough therapy designation for HER2 -mutant metastatic NSCLC from the U.S. FDA in May 2020 79 .It was recently added to ado-trastuzumab emtansine for HER2 mutated NSCLC in the NCCN guidelines V1.2021.The activity of T-DXd patients with HER2 overexpression cohort was recently reported in WCLC 2020 80 .The ORR was 24.5%, the median PFS and median OS in HER2 overexpression by IHC cohort was reported as 5.4 months (95% CI: 2.8, 7.0 months) and 11.3 months (95% CI: 7.8, not evaluable) respectively.Compared to the HER2-mutant cohort, trastuzumab deruxtecan had lower ORR, PFS and OS in the HER2 overexpressed cohort by immunohistochemistry (IHC) 80 .Similar data were observed for afatinib 36 and ado-trastuzumab emtansine 71 , 72 , suggesting HER2 -mutant and HER2 -amplified NSCLC are distinct entities.Together with the variations in concurrent genomic alterations, further biomarker study is needed to select the appropriate patients with HER2-positive NSCLC.
Recently, HER2-targeted therapy has been expanded to include chimeric antigen receptor (CAR) T-cell therapy in lung cancer.Additionally, several new HER2-targeting drugs are in early phases of clinical development.Table 5 summarizes several ongoing studies for targeting HER2 alterations in NSCLC.Among these studies, ongoing phase II DESTINY-Lung01 trial (NCT03505710) and the newly activated phase III study of pyrotinib after first line platinum-based chemotherapy are the most advanced.

Systemic therapy with immune checkpoint inhibitor therapy
In a retrospective study of 122 patients with HER2 -mutant lung cancer, programmed cell death-ligand 1 (PD-L1) expression was lower but tumor mutation burden (TMB) was similar to those in unselected lung cancers 81 .There are only a few retrospective studies containing HER2mutant NSCLC patients treated with immune checkpoint inhibitors, each with about 20-30 cases [81][82][83][84][85] .Patients with HER2 -mutant NSCLC had an ORR of 6%-7.4% and a median PFS of 1.9-2.5 months with secondline immune checkpoint inhibitors 82 , 83 .This is inferior to an ORR of 10% and a median PFS of 4.3 months with second-line non-HER2 targeted chemotherapies 84 .None of the responders had a HER2 exon 20 YVMA mutation.In another retrospective study involving 23 patients with HER2 -mutant NSCLC, responses were seen in six patients (27.3%), but the effect is short lived with a median PFS of 2.2 months and an OS of 20.4 months 85 .Of note, in the unselected platinum-refractory NSCLC population, standard second-line chemotherapy, docetaxel, has an ORR of 7-13% and a median PFS of 2-4 months 3 .Therefore, platinum-based chemotherapy with or without an anti-angiogenesis inhibitor remains the first line standard treatment for this patient population.Further study is needed to define the role of immune checkpoint inhibitors in HER2 -mutant NSCLC.

Resistance mechanisms of HER2-targeted therapy
Unfortunately, many patients that received HER2-targeted therapy in NSCLC develop drug resistance in less than 6 months.There are unmet needs to both understand resistance mechanisms and to develop subsequent treatment strategies to overcome the resistance to HER2targeted therapy.Until now, most of the data have been from studies in breast cancer.Both HER2-dependent and HER2-independent resistance mechanisms were reported in NSCLC patients who developed resistance to HER2-targeted therapies.One of the independent resistance mechanisms is activation of compensatory pathways including reactivation of the PI3K/AKT and Ras/MAPK signaling pathways 24 .It was reported that PTEN-deficiency contributes to trastuzumab resistance in breast cancer patients and inhibition of PI3K could rescue PTEN-deficiency induced resistance 86 .Src activation, L755S mutation and T798I mutation were also reported in mediating drug resistance to HER2-targeted drugs including trastuzumab, lapatinib and neratinib [87][88][89] .Co-genomic alterations also contribute to HER2 drug resistance.For example, cyclin E overexpression was associated with trastuzumab resistance in breast cancer patients 90 , and a high level of HER2 somatic copy-number alterations (SCNAs) was correlated with innate trastuzumab resistance during disease progression 91 .In addition, HER2 mutations, such as L755S, V777L, D769Y, V842I, K753E, I655V, have been reported in resistance cases 92 .HER2 kinase domain mutation resulting in constitutive activation of HER2 and EGFR were described in HER2/EGFR drug resistance cases 93 .
Concurrent HER2 amplification has been reported in 3-5% of patients with EGFR -mutant NSCLC, which was an independent predictor of shorter time to progression (HR = 2.4, P = 0.015) on EGFR TKI treatment.This can be attributed the activation of HER2 share the same downstream network with EGFR, which is associated with very aggressive biology and poor clinical outcomes in NSCLC 94 .HER2 genomic alterations are identified as a primary or acquired resistance mechanism to first generation EGFR TKI (erlotinib) and second generation EGFR TKI (afatinib) in NSCLC 95 .Recently, the HER2 signaling pathway is also recognized as a driver of resistance to the third generation TKI osimertinib.For instance, the exon 16-skipping splice variant of HER2 (HER2D16) is a mediator of osimertinib resistance in EGFR L858R/T790M lung cancer.Combining afatinib and osimertinib could overcome HER2D16-mediated resistance 96 .Ongoing clinical trials (NCT04464967, NCT04144569) are evaluating the addition  Abbreviations: bid, twice daily; CAR-T cell, chimeric antigen receptor T cell; CNS, central nervous system; FISH, fluorescence in situ hybridization; IHC, immunohistochemistry; IV, intravenously; NGS, next-generation sequencing; od, every other day; qd, once daily; q2w, every 2 weeks; q3w, every 3 weeks.Blue highlights two most advanced studies for HER2-positive NSCLC.
of a HER2 inhibitor to an EGFR inhibitor, or an immune checkpoint inhibitor, to overcome the acquired resistance in patients with HER2mutated NSCLC.

Summary and perspective
Clinical application of multiplex biomarker testing by NGS identifies rare oncogenic mutations and amplification in the HER2 gene as two major genomic alterations in NSCLC.The most common HER2 mutation is in exon 20 A775_G776insYVMA.In patients with previously untreated or treated NSCLC, this mutation is associated with sensitivity to several HER2-targeting drugs including afatinib, dacomitinib, pyrotinib, BDTX-189, tarloxotinib, ado-trastuzumab emtansine, and trastuzumab deruxtecan.Unlike in breast and gastrointestinal cancer, currently available HER2-targeted agents only have modest clinical activity in HER2 -mutant or -amplified NSCLC.Trastuzumab deruxtecan received the U.S. FDA breakthrough therapy designation for HER2 -mutant metastatic NSCLC in May 2020.Two randomized studies are in late-stage clinical development.A global phase III study of pyrotinib compared to docetaxel as a second line therapy for advanced NSCLC harboring HER2 exon 20 mutations (PYRAMID-1, NCT04447118) was just opened for enrollment in September 2020.A randomized phase II study of tarloxotinib versus platinum-based chemotherapy as a first line systemic therapy is also actively recruiting patients with advanced NSCLC harboring HER2 exon 20 mutations (RAIN-701 trial, NCT03805841).Ongoing studies are also evaluating the feasibility and clinical benefit of HER2-targeted therapy in combination with other agents in advanced NSCLC patients.There are unmet needs to understand the biology of HER2 genomic alterations, to optimize biomarker testing, and to determine the resistance mechanisms of both primary and acquired resistance for patients with HER2 -altered NSCLC.

Declaration of Competing Interest
Dr. Li reports personal fees from Eisai, grants from Pfizer, grants from Merck, grants from Eureka, grants from OncoImmune, grants from Hengrui, grants from Tempus, outside the submitted work.

Fig. 1 .
Fig. 1.HER2 mutations in NSCLC.(A) Schema illustrates the location of HER2 ( ERRB2 ) gene in chromosome17.(B) and (C) Distribution and frequency of reported HER2 mutations collected from Foundation Medicine report and the cBioPortal database in the entire gene (B) and tyrosine kinase domain (C).(D) Depending on the proposed molecular mechanisms, the most common HER2 mutation A775_G776insYVMA (an insertion of the YVMA between A775 and G776) is also named Y772_A775dup (a duplication of Y772-A775 YVMA), E770_A771insAYVM (an insertion of the AYVM between E770 and A771), or A771_M774dup (a duplication of A771_M774 AYVM) in the scientific literature .

Table 3
Reported clinical activity of HER2-targeted therapy in phase II studies in patients with NSCLC.

Table 4
Summary of FDA-approved HER2-targeted drugs for breast cancer.

Table 5
Ongoing clinical trials of HER2-targeted agents for patients with metastatic NSCLC.
( continued on next page )