New Targets for Therapy in Lung Cancer

Human Epidermal Growth Factor Receptor Type 2

HER2 (also known as ERBB2) belongs to the same family of HER RTKs as EGFR. In contrast to EGFR, HER2 does not interact with any ligand directly but serves as the preferred dimerization partner of EGFR and other ErbB family members, such as HER3 and HER4, to trigger autophosphorylation and downstream signaling through both the MAPK and PI3K pathways described earlier. HER2 gene amplification (defined as HER2/CEP17 ratio per cell of 2 or greater, and absolute HER2 signals in more than 4; or more than 15 copies in more than 10% of cells using fluorescence in situ hybridization [FISH] assay) is found in approximately 1% to 3% of lung cancers. HER2 exon 20 insertions are found in approximately 3% of adenocarcinomas. HER2 amplification is correlated with histologic subtype and tumor grade, such that high-level amplification appears to be concentrated in the subgroup of high-grade adenocarcinomas. Intratumor heterogeneity in the level of HER2 amplification also appears frequently. This latter feature may partly account for the negative results of clinical trials conducted a decade ago using trastuzumab in combination with chemotherapy for patients with NSCLC. In addition to patients with tumor heterogeneity, these studies included patients with potentially low-to-absent HER2 amplification (e.g., inclusion of patients with 2+ HER2 protein expression as determined by IHC).

Afatinib, an oral pan-HER inhibitor, has been used as monotherapy in genotypically selected solid tumors (lung cancers excluded) with either EGFR or HER2 amplification but showed limited activity, with an objective response rate of 5%. Lapatinib, an oral dual EGFR/HER2 inhibitor, demonstrated limited activity as monotherapy in a molecularly unselected population of NSCLC tumors. Of interest is that one of two patients with HER2 amplification (determined retrospectively) had a partial response, although this result was not confirmed. Dacomitinib is another oral pan-HER inhibitor with demonstrated in vitro activity in selected HER2 -amplified cell lines resistant to trastuzumab and lapatinib, which resulted in a 12% response rate in patients with HER2 exon 20 insertions, but no responses in patients with HER2 amplification.

HER2 amplification is also implicated in acquired resistance to therapy with EGFR tyrosine kinase inhibitors (TKIs) in laboratory models and in the clinical setting. HER2 amplification is found in 12% to 13% of cases of acquired resistance to EGFR-TKIs wherein it is mutually exclusive with the EGFR T790M mutation among tumors with acquired EGFR-TKI resistance. In contrast, no HER2 exon 20 mutations were identified in patients who developed resistance to the irreversible EGFR-TKI afatinib. HER2 amplification is also a putative mechanism of acquired resistance to ALK inhibitors in EML4–ALK-translocated lung cancer cells in vitro, although clinical studies have not confirmed this possibility to date.

In comparison, mutations in HER2 occur in 2% to 4% of NSCLC tumors, largely in high-grade and moderately to poorly differentiated adenocarcinomas. More than 95% of the mutations described to date are small insertions in exon 20, mostly represented by an in-frame insertion of 12 base pairs that causes duplication of the amino acids YVMA. Functional studies of this insertion mutation show that it confers greater transforming and antiapoptotic potential, in addition to its stronger catalytic activity, compared with wild-type HER2 . It can also trigger EGFR activation in the absence of cognate ligands and EGFR kinase activity. HER2 mutations appear to occur in greater proportions among women and never-smokers and are generally mutually exclusive with EGFR and Kirsten rat sarcoma ( KRAS ) mutations as well as HER2 amplification, with rare exceptions. An activating HER2 V659E mutation sensitive to lapatinib was described in a specimen of lung adenocarcinoma from a patient with Li-Fraumeni syndrome. It is anticipated that secondary HER2 mutations, such as L755S, T862A, and the gatekeeper T798M mutation, can arise as a mechanism of acquired drug resistance similar to what is seen in HER2 -amplified breast cancers after chronic therapy with lapatinib.

Afatinib has been reported to induce tumor response or disease stabilization when used as monotherapy in HER2 -mutant lung adenocarcinomas. Similarly, trastuzumab-based combinations have induced partial responses. Although lapatinib demonstrated preclinical activity against cells expressing the HER2 insertion mutation, tumor response has not been documented with monotherapy in the very limited number of patients reported on thus far. Clinical activity has been documented, however, when lapatinib is used in combination with either chemotherapy or trastuzumab-based regimens in patients with NSCLC who have either a HER2 exon 20 insertion or the HER2 V659E mutation. As per above, dacomitinib resulted in a 12% response rate in patients with HER2 exon 20 insertions. HER2 exon 20 mutations, especially HER2YVMA, appear to be a promising target in lung cancer but needs to be validated.

Human Epidermal Growth Factor Receptor Type 3

HER3 (also known as ERBB3) is another representative of the four-member group in the HER RTK family. HER3 is generally considered to have functionally weaker kinase activity compared with EGFR. Heterodimerization with other HER members, such as with HER2 upon binding of the ligand neuregulin, triggers autophosphorylation and recruitment of downstream signaling molecules. HER3 mutations have been reported in approximately 1% of lung adenocarcinomas and 1% of squamous cell lung cancers. Most of the HER3 mutations identified to date are clustered in the extracellular domain (ECD), although some are mapped to the kinase domain. The functional characteristics of most specific mutants described in NSCLC are yet to be verified; however, several HER3 mutations in either the ECD or kinase domain have been shown to promote oncogenesis in a ligand-independent manner, although this effect required the presence of kinase-active HER2. Recently, an activating HER3 V855A mutation that is homologous to EGFR L858R was identified in a patient with chemotherapy resistant NSCLC. This mutation was transforming in murine and human cell line studies in the presence of wild-type HER2 . Various small-molecule inhibitors and monoclonal antibodies against HER2 and HER3, as well as PI3K inhibitors, have demonstrated variable effectiveness depending on the specific HER3 mutation.

The recurrent fusion gene CD74-NRG1 found in mucinous lung adenocarcinomas of never-smokers appears to correlate with increased HER3 phosphorylation in tumor tissue. This chimeric transcript results in the expression of the EGF-like domain in tumor tissue that is otherwise negative for neuregulin. Functional characterization of this fusion protein in vitro showed activation of the PI3K-AKT pathway. Indeed, HER3 plays an integral role in activation of the PI3K survival pathway upon its heterodimerization with EGFR or HER2 in malignant cell lines. EGFR-TKI-sensitive NSCLC cancer cell lines rely on HER3 signaling to activate the PI3K/AKT pathway.

HER3 signaling is also implicated in acquired resistance to EGFR-TKIs. Persistent HER3-activated PI3K signaling is uncoupled from EGFR and is mediated instead through its interaction with MET, which is amplified in this setting. Another mechanism that can sustain HER3-activated PI3K signaling is disruption of negative feedback networks. ERK signaling leads to feedback phosphorylation of the conserved T669 residue within the juxtamembrane domain of EGFR, HER2, and HER4, and this prevents transphosphorylation of HER3 ( Fig. 48.1 ). The loss of this dominant negative feedback suppression of HER3 by intact MEK/ERK signaling is thought to account for the increased AKT phosphorylation found with MEK inhibitors in EGFR- and HER2-driven malignant tumors.

Blockade of HER3 signaling, through monoclonal antibody therapies or antisense oligonucleotides, improves the antitumor activity of EGFR and HER2 TKIs in preclinical models, including cell lines with acquired resistance to EGFR-TKIs. Another proposed approach for reducing HER3-mediated activation to enhance EGFR-TKI activity involves modulation of circulating neuregulin ligands via inhibition of ADAM17, a membrane-associated metalloprotease that cleaves and releases HER ligands from cells to enable receptor binding. Afatinib and dacomitinib are both oral irreversible pan-HER TKIs that have produced marked tumor regression in xenograft models that contained EGFR mutations, including the T790M mutation, which is associated with acquired resistance to EGFR-TKIs. However, subsequent modeling showed that cytotoxicity against T790M can be accomplished only at clinically unachievable concentrations, thus accounting for the limited efficacy seen for this patient subset in the clinic. Nonetheless, these agents can block HER2 heterodimerization with EGFR or HER3, thus explaining the potential to overcome acquired resistance mediated by HER3. In a randomized phase II study comparing dacomitinib and erlotinib, dacomitinib demonstrated significantly improved progression-free survival in KRAS wild-type NSCLC with or without EGFR mutation. A phase III study comparing first-line dacomitinib with gefitinib for patients who have NSCLC with EGFR-activating mutations was ongoing at the time of publication. Another phase III study comparing second- or third-line dacomitinib and erlotinib in patients with KRAS wild-type NSCLC has completed enrollment at the time of the publication of this chapter ( ClinicalTrials.gov identifier: NCT01360554 ). A phase III clinical trial recently demonstrated statistically significant but modest improvements in progression-free and overall survival with afatinib compared with erlotinib in patients receiving second-line therapy for squamous cell NSCLC; however, enthusiasm for these results has been muted by the advances seen with immunotherapy for this patient population. Various monoclonal antibodies against HER3 are being investigated in clinical trials, combining them with other inhibitors of the EGFR or HER2 pathway.

Hepatocyte Growth Factor Receptor

Binding of the ligand hepatocyte growth factor (HGF, also known as scatter factor), a paracrine factor secreted by stromal cells, to its cognate receptor MET facilitates receptor phosphorylation, leading to the activation of downstream signaling through the MAPK and PI3K pathways, which promote epithelial-to-mesenchymal transition (EMT), invasion, and metastasis. Ubiquitin-mediated receptor degradation is regulated at the Cbl E3-ligase RTK binding domain, similar to what has been described for EGFR and HER2. Mechanisms of aberrant activation of MET described in NSCLC include receptor overexpression (with or without HGF), c-MET gene amplification, or exon 14 skipping abnormalities. Levels of MET expression as high as 60% have been reported in various studies of NSCLC. Both the EGFR and c-MET genes are located on chromosome 7, and an increased copy number of the c-MET gene as determined by FISH is associated with an increased copy number of the EGFR gene and confers a worse prognosis. Coamplification of c-MET and EGFR is also described in up to 8.5% of NSCLCs not previously treated with EGFR-TKIs. Amplification of c-MET also occurs infrequently with an incidence of 3% to 7% among patients not treated with EGFR-TKIs, but this incidence increases to 10% to 22% among patients with acquired resistance to EGFR-TKIs. Exon 14 skipping mutations occur in about 3% of patients with NSCLC. Patients with sarcomatoid pulmonary carcinomas are enriched for MET exon 14 skipping mutations, and one study identified that 22% of patients with this relatively rare form of lung cancer have this mutation. MET can be transactivated through a variety of protein interactions and can heterodimerize with other RTKs, such as EGFR, HER2, HER3, and ret proto-oncogene (RET), in cells with c-MET amplification, representing an escape or bypass mechanism mediating resistance to inhibitors of these RTK-activated signaling pathways. In a preclinical model, MET activation through paracrine secretion of HGF also served as a mechanism for resistance to second-generation selective ALK inhibitors such as ceritinib (LDK378), but not to the MET/ALK inhibitor crizotinib.

Somatic intronic mutations of c-MET that lead to an alternatively spliced transcript, encoding a deletion of the exon-14 juxtamembrane domain spanning the amino acids 964 through 1010, result in loss of the Cbl binding site at Y1003. This skipping mutation in exon 14 yields a functional MET protein with decreased ubiquitination and consequently sustained activation of the MAPK pathway through altered receptor downregulation. This mutation variant appears to be mutually exclusive with mutations of other genes involved in the MAPK pathway (e.g., EGFR , RAS , and RAF ). Additional mutations have been described in both the ECD and juxtamembrane domains, some of which have transforming potential. No reports have indicated nonsynonymous mutations in the kinase domain to date. Of note is that most of these other mutations reported are in fact germline. The germline mutation N375S, which occurred at the highest frequency, appears to be associated with smoking and squamous histology. The MET -N375S mutation seems to confer resistance to the small-molecule MET kinase inhibitor SU11274.

Various approaches for inhibiting the MET pathway have been tested or are in clinical development. These strategies include anti-HGF and anti-MET monoclonal antibodies, or small-molecule MET kinase inhibitors. The MET/ALK/ROS1 inhibitor crizotinib was reported anecdotally to induce a rapid and durable response in a patient with de novo c-MET amplification without ALK rearrangement. Models of resistance to MET inhibitors predict the emergence of either secondary mutations or activation of EGFR signaling through increased expression of transforming growth factor-α. A randomized phase III study in previously treated patients with NSCLC of erlotinib with or without tivantinib, initially thought to be a selective nonadenosine triphosphate (non-ATP) competitive MET inhibitor, was halted because of an increased incidence of interstitial lung disease; regardless, there was no improvement in overall survival. Similarly, a separate phase III clinical trial conducted throughout Europe and the United States failed to show an overall survival benefit of erlotinib and tivantinib compared with erlotinib and placebo. Nonetheless, preclinical data generated from two different groups suggest that although tivantinib can mitigate HGF-dependent MET activation, this is not its major mechanism of action. Tivantinib (in contrast to crizotinib) did not inhibit MET autophosphorylation at doses that induced apoptosis. Instead, it exhibited cytotoxicity regardless of activation status of the MET pathway or the presence or absence of a functional MET kinase. In fact, growth inhibition and cytotoxicity reported with tivantinib may be mainly due to its effect on microtubule dynamics, which is not found with other MET inhibitors.

Onartuzumab (MetMAb), a monoclonal antibody that binds to the ECD of MET to prevent ligand binding, was evaluated in a randomized phase III study in combination with erlotinib as compared with placebo plus erlotinib in patients with NSCLC and MET-positive status as determined by IHC. The rationale for the study was based on promising results for progression-free survival (2.9 vs. 1.5 months; hazard ratio [HR], 0.53; p = 0.04) and overall survival (12.6 vs. 3.8 months; HR, 0.37; p = 0.002) noted in patients with MET-positive NSCLC who received the onartuzumab plus erlotinib combination compared with the placebo plus erlotinib treatment in the preceding randomized phase II study. In contrast, patients with MET-negative NSCLC who received the combination had worse progression-free survival (1.4 vs. 2.7 months; HR, 1.82; p = 0.05) and overall survival (8.1 vs. 15.3 months; HR, 1.78; p = 0.16) compared with patients who received placebo plus erlotinib. Unfortunately, the phase III clinical trial was stopped early due to futility as the experimental arm did not improve overall survival (OS), progression free survival (PFS), or overall response rate (ORR) compared with erlotinib alone.

Whereas targeting MET based on protein expression has had challenges, MET amplification and MET exon 14 skipping mutations are emerging as promising targets. The interim analysis of a small study demonstrated that 4 of 12 patients with intermediate (2.2–5) or high (>5) ratios of MET to CEP7 responded to crizotinib. An even more impressive response rate was observed in an interim analysis of a trial of the safety and efficacy of crizotinib for patients with MET exon 14 skipping mutations where 10 of 15 patients had confirmed or unconfirmed partial responses. One case report showed that a patient with a MET exon 14 skipping mutation who demonstrated a response to crizotonib acquired a mutation in the MET kinase domain, D1228N, at the time of progression. Responses have also been observed in patients with MET exon 14 skipping mutations treated with cabozantinib. Other MET inhibitors in development include AMG337 and capmatinib (INC280). These ongoing trials will help clarify which MET abnormalities are potentially targetable in NSCLC, but MET exon 14 skipping mutations and amplification seem to be better predictors of response than MET expression by IHC at this time.