Structure-based classification predicts drug response in EGFR-mutant NSCLC.

Epidermal growth factor receptor (EGFR) mutations typically occur in exons 18-21 and are established driver mutations in non-small cell lung cancer (NSCLC)1-3. Targeted therapies are approved for patients with 'classical' mutations and a small number of other mutations4-6. However, effective therapies have not been identified for additional EGFR mutations. Furthermore, the frequency and effects of atypical EGFR mutations on drug sensitivity are unknown1,3,7-10. Here we characterize the mutational landscape in 16,715 patients with EGFR-mutant NSCLC, and establish the structure-function relationship of EGFR mutations on drug sensitivity. We found that EGFR mutations can be separated into four distinct subgroups on the basis of sensitivity and structural changes that retrospectively predict patient outcomes following treatment with EGFR inhibitors better than traditional exon-based groups. Together, these data delineate a structure-based approach for defining functional groups of EGFR mutations that can effectively guide treatment and clinical trial choices for patients with EGFR-mutant NSCLC and suggest that a structure-function-based approach may improve the prediction of drug sensitivity to targeted therapies in oncogenes with diverse mutations.

HER4 and EGFR Activate Cell Signaling in NRG1 Fusion-Driven Cancers: Implications for HER2-HER3-specific Versus Pan-HER Targeting Strategies.

INTRODUCTION: NRG1 gene fusions are clinically actionable alterations identified in NSCLC and other tumors. Previous studies have reported that NRG1 fusions signal through HER2 and HER3 but, thus far, strategies targeting HER3 specifically or HER2-HER3 signaling have exhibited modest activity in patients with NSCLC bearing NRG1 fusions. Although NRG1 fusion proteins can bind HER4 in addition to HER3, the contribution of HER4 and other HER family members in NRG1 fusion-positive cancers is not fully understood. METHODS: We investigated the role of HER4 and EGFR-HER3 signaling in NRG1 fusion-positive cancers using Ba/F3 models engineered to express various HER family members in combination with NRG1 fusions and in vitro and in vivo models of NRG1 fusion-positive cancer. RESULTS: We determined that NRG1 fusions can stimulate downstream signaling and tumor cell growth through HER4, independent of other HER family members. Moreover, EGFR-HER3 signaling is also activated in cells expressing NRG1 fusions, and inhibition of these receptors is also necessary to effectively inhibit tumor cell growth. We observed that cetuximab, an anti-EGFR antibody, in combination with anti-HER2 antibodies, trastuzumab and pertuzumab, yielded a synergistic effect. Furthermore, pan-HER tyrosine kinase inhibitors were more effective than tyrosine kinase inhibitors with greater specificity for EGFR, EGFR-HER2, or HER2-HER4, although the relative degree of dependence on EGFR or HER4 signaling varied between different NRG1 fusion-positive cancers. CONCLUSIONS: Our findings indicate that pan-HER inhibition including HER4 and EGFR blockade is more effective than selectively targeting HER3 or HER2-HER3 in NRG1 fusion-positive cancers.

CD70 is a therapeutic target upregulated in EMT-associated EGFR tyrosine kinase inhibitor resistance.

Effective therapeutic strategies are needed for non-small cell lung cancer (NSCLC) patients with epidermal growth factor receptor (EGFR) mutations that acquire resistance to EGFR tyrosine kinase inhibitors (TKIs) mediated by epithelial-to-mesenchymal transition (EMT). We investigate cell surface proteins that could be targeted by antibody-based or adoptive cell therapy approaches and identify CD70 as being highly upregulated in EMT-associated resistance. Moreover, CD70 upregulation is an early event in the evolution of resistance and occurs in drug-tolerant persister cells (DTPCs). CD70 promotes cell survival and invasiveness, and stimulation of CD70 triggers signal transduction pathways known to be re-activated with acquired TKI resistance. Anti-CD70 antibody drug conjugates (ADCs) and CD70-targeting chimeric antigen receptor (CAR) T cell and CAR NK cells show potent activity against EGFR TKI-resistant cells and DTPCs. These results identify CD70 as a therapeutic target for EGFR mutant tumors with acquired EGFR TKI resistance that merits clinical investigation.

Poziotinib for Patients With HER2 Exon 20 Mutant Non-Small-Cell Lung Cancer: Results From a Phase II Trial.

PURPOSE: Targeted therapies against non-small-cell lung cancer (NSCLC) harboring HER2 mutations remain an unmet need. In this study, we assessed the efficacy and safety of poziotinib in patients with HER2 exon 20 mutant advanced NSCLC in a single-arm, open-label, phase II study. PATIENTS AND METHODS: Patients with advanced HER2 exon 20 mutant NSCLC were enrolled to receive poziotinib at a dose of 16 mg/d for 28-day cycles. The primary end point was objective response rate per RECIST version 1.1. Confirmatory scans were performed at least 28 days from initial radiologic response. RESULTS: Thirty patients received poziotinib treatment. At baseline, 90% of patients received prior platinum-based chemotherapy and 53% had two lines or more prior systemic therapies. As of data cutoff on March 1, 2021, the confirmed objective response rate was 27% (95% CI, 12 to 46). Responses were observed across HER2 exon 20 mutation subtypes. The median duration of response was 5.0 months (95% CI, 4.0 to not estimable). The median progression-free survival was 5.5 months (95% CI, 4.0 to 7.0). The median overall survival was 15 months (95% CI, 9.0 to not estimable). The most common grade 3 treatment-related adverse events were skin rash (47%) and diarrhea (20%). There was one possible treatment-related death because of pneumonitis. CONCLUSION: Poziotinib showed promising antitumor activity in patients with HER2 exon 20 mutant NSCLC including patients who had previously received platinum-based chemotherapy.

Poziotinib for EGFR exon 20-mutant NSCLC: Clinical efficacy, resistance mechanisms, and impact of insertion location on drug sensitivity.

We report a phase II study of 50 advanced non-small cell lung cancer (NSCLC) patients with point mutations or insertions in EGFR exon 20 treated with poziotinib (NCT03066206). The study achieved its primary endpoint, with confirmed objective response rates (ORRs) of 32% and 31% by investigator and blinded independent review, respectively, with a median progression-free survival of 5.5 months. Using preclinical studies, in silico modeling, and molecular dynamics simulations, we found that poziotinib sensitivity was highly dependent on the insertion location, with near-loop insertions (amino acids A767 to P772) being more sensitive than far-loop insertions, an observation confirmed clinically with ORRs of 46% and 0% observed in near versus far-loop, respectively (p = 0.0015). Putative mechanisms of acquired resistance included EGFR T790M, MET amplifications, and epithelial-to-mesenchymal transition (EMT). Our data demonstrate that poziotinib is active in EGFR exon 20-mutant NSCLC, although this activity is influenced by insertion location.

ARTEMIS highlights VEGF inhibitors as effective partners for EGFR TKIs in EGFR mutant NSCLC.

The randomized ARTEMIS study demonstrates that adding the VEGF inhibitor bevacizumab to the EGFR inhibitor erlotinib improves progression-free survival in EGFR mutant non-small-cell lung cancer by more than 6 months, with even greater benefits seen in patients with brain metastases and EGFR L858R mutation. This provides further evidence for the tailored use of VEGF/EGFR combinations.

Oncogene-specific differences in tumor mutational burden, PD-L1 expression, and outcomes from immunotherapy in non-small cell lung cancer.

BACKGROUND: Non-small cell lung cancer (NSCLC) patients bearing targetable oncogene alterations typically derive limited benefit from immune checkpoint blockade (ICB), which has been attributed to low tumor mutation burden (TMB) and/or PD-L1 levels. We investigated oncogene-specific differences in these markers and clinical outcome. METHODS: Three cohorts of NSCLC patients with oncogene alterations (n=4189 total) were analyzed. Two clinical cohorts of advanced NSCLC patients treated with ICB monotherapy [MD Anderson (MDACC; n=172) and Flatiron Health-Foundation Medicine Clinico-Genomic Database (CGDB; n=894 patients)] were analyzed for clinical outcome. The FMI biomarker cohort (n=4017) was used to assess the association of oncogene alterations with TMB and PD-L1 expression. RESULTS: High PD-L1 expression (PD-L1 ≥50%) rate was 19%-20% in classic EGFR, EGFR exon 20 and HER2-mutant tumors, and 34%-55% in tumors with ALK, BRAF V600E, ROS1, RET, or MET alterations. Compared with KRAS-mutant tumors, BRAF non-V600E group had higher TMB (9.6 vs KRAS 7.8 mutations/Mb, p=0.003), while all other oncogene groups had lower TMB (p<0.001). In the two clinical cohorts treated with ICB, molecular groups with EGFR, HER2, ALK, ROS1, RET, or MET alterations had short progression-free survival (PFS; 1.8-3.7 months), while BRAF V600E group was associated with greater clinical benefit from ICB (CGDB cohort: PFS 9.8 months vs KRAS 3.7 months, HR 0.66, p=0.099; MDACC cohort: response rate 62% vs KRAS 24%; PFS 7.4 vs KRAS 2.8 months, HR 0.36, p=0.026). KRAS G12C and non-G12C subgroups had similar clinical benefit from ICB in both cohorts. In a multivariable analysis, BRAF V600E mutation (HR 0.58, p=0.041), PD-L1 expression (HR 0.57, p=0.022), and high TMB (HR 0.66, p<0.001) were associated with longer PFS. CONCLUSIONS: High TMB and PD-L1 expression are predictive for benefit from ICB treatment in oncogene-driven NSCLCs. NSCLC harboring BRAF mutations demonstrated superior benefit from ICB that may be attributed to higher TMB and higher PD-L1 expression in these tumors. Meanwhile EGFR and HER2 mutations and ALK, ROS1, RET, and MET fusions define NSCLC subsets with minimal benefit from ICB despite high PD-L1 expression in NSCLC harboring oncogene fusions. These findings indicate a TMB/PD-L1-independent impact on sensitivity to ICB for certain oncogene alterations.

Molecular Landscape of BRAF-Mutant NSCLC Reveals an Association Between Clonality and Driver Mutations and Identifies Targetable Non-V600 Driver Mutations.

INTRODUCTION: Approximately 4% of NSCLC harbor BRAF mutations, and approximately 50% of these are non-V600 mutations. Treatment of tumors harboring non-V600 mutations is challenging because of functional heterogeneity and lack of knowledge regarding their clinical significance and response to targeted agents. METHODS: We conducted an integrative analysis of BRAF non-V600 mutations using genomic profiles of BRAF-mutant NSCLC from the Guardant360 database. BRAF mutations were categorized by clonality and class (1 and 2: RAS-independent; 3: RAS-dependent). Cell viability assays were performed in Ba/F3 models. Drug screens were performed in NSCLC cell lines. RESULTS: A total of 305 unique BRAF mutations were identified. Missense mutations were most common (276, 90%), and 45% were variants of unknown significance. F468S and N581Y were identified as novel activating mutations. Class 1 to 3 mutations had higher clonality than mutations of unknown class (p < 0.01). Three patients were treated with MEK with or without BRAF inhibitors. Patients harboring G469V and D594G mutations did not respond, whereas a patient with the L597R mutation had a durable response. Trametinib with or without dabrafenib, LXH254, and lifirafenib had more potent inhibition of BRAF non-V600-mutant NSCLC cell lines than other MEK, BRAF, and ERK inhibitors, comparable with the inhibition of BRAF V600E cell line. CONCLUSIONS: In BRAF-mutant NSCLC, clonality is higher in known functional mutations and may allow identification of variants of unknown significance that are more likely to be oncogenic drivers. Our data indicate that certain non-V600 mutations are responsive to MEK and BRAF inhibitors. This integration of genomic profiling and drug sensitivity may guide the treatment for BRAF-mutant NSCLC.

Local Consolidation Therapy (LCT) After First Line Tyrosine Kinase Inhibitor (TKI) for Patients With EGFR Mutant Metastatic Non-small-cell Lung Cancer (NSCLC).

INTRODUCTION: Although most NSCLC patients with sensitizing epidermal growth factor receptor (EGFR) mutations have an impressive initial response, the vast majority has residual disease and develops acquired resistance after 9 to 14 months of EGFR tyrosine kinase (TKI) therapy. We recently reported a phase II trial showing that, for patients with molecularly unselected oligometastatic NSCLC who did not progress after first-line systemic therapy, local consolidation therapy (LCT) with surgery or radiation improved progression-free survival (PFS), compared with maintenance therapy alone. Herein, we report a retrospective analysis of LCT after TKI in patients with metastatic EGFR mutant NSCLC. PATIENTS AND METHODS: We identified patients with metastatic EGFR mutant NSCLC treated with TKI plus LCT or with TKI alone in the MD Anderson GEMINI (Genomic Marker-Guided Therapy Initiative) database and in our recently published LCT trial. PFS was compared between LCT plus TKI and TKI only treated patients using the log-rank test. RESULTS: We identified 129 patients with EGFR mutant NSCLC who were treated with first-line TKI and 12 that were treated with TKI followed by LCT. Among the 12 patients treated with TKI plus LCT, 8 patients had oligometastatic disease (defined as ≤ 3 metastases), and 4 patients had > 3 metastases. LCT regimens were hypofractionated radiotherapy or stereotactic ablative body radiotherapy for 11 patients and surgery for 1 patient. TKI followed by LCT resulted in a significantly longer PFS (36 months) compared with TKI alone (PFS, 14 months; log-rank P = .0024). CONCLUSIONS: Our data suggests that first-line TKI plus LCT is a promising therapeutic strategy for patients with EGFR mutant NSCLC that merits further investigation.

Pan-Cancer Landscape and Analysis of ERBB2 Mutations Identifies Poziotinib as a Clinically Active Inhibitor and Enhancer of T-DM1 Activity.

We characterized the landscape and drug sensitivity of ERBB2 (HER2) mutations in cancers. In 11 datasets (n = 211,726), ERBB2 mutational hotspots varied across 25 tumor types. Common HER2 mutants yielded differential sensitivities to eleven EGFR/HER2 tyrosine kinase inhibitors (TKIs) in vitro, and molecular dynamics simulations revealed that mutants with a reduced drug-binding pocket volume were associated with decreased affinity for larger TKIs. Overall, poziotinib was the most potent HER2 mutant-selective TKI tested. Phase II clinical testing in ERBB2 exon 20-mutant non-small cell lung cancer resulted in a confirmed objective response rate of 42% in the first 12 evaluable patients. In pre-clinical models, poziotinib upregulated HER2 cell-surface expression and potentiated the activity of T-DM1, resulting in complete tumor regression with combination treatment.

Mechanisms and clinical activity of an EGFR and HER2 exon 20-selective kinase inhibitor in non-small cell lung cancer.

Although most activating mutations of epidermal growth factor receptor (EGFR)-mutant non-small cell lung cancers (NSCLCs) are sensitive to available EGFR tyrosine kinase inhibitors (TKIs), a subset with alterations in exon 20 of EGFR and HER2 are intrinsically resistant and lack an effective therapy. We used in silico, in vitro, and in vivo testing to model structural alterations induced by exon 20 mutations and to identify effective inhibitors. 3D modeling indicated alterations restricted the size of the drug-binding pocket, limiting the binding of large, rigid inhibitors. We found that poziotinib, owing to its small size and flexibility, can circumvent these steric changes and is a potent inhibitor of the most common EGFR and HER2 exon 20 mutants. Poziotinib demonstrated greater activity than approved EGFR TKIs in vitro and in patient-derived xenograft models of EGFR or HER2 exon 20 mutant NSCLC and in genetically engineered mouse models of NSCLC. In a phase 2 trial, the first 11 patients with NSCLC with EGFR exon 20 mutations receiving poziotinib had a confirmed objective response rate of 64%. These data identify poziotinib as a potent, clinically active inhibitor of EGFR and HER2 exon 20 mutations and illuminate the molecular features of TKIs that may circumvent steric changes induced by these mutations.

Left-right analysis of mammary gland development in retinoid X receptor-α+/- mice.

Left-right (L-R) differences in mammographic parenchymal patterns are an early predictor of breast cancer risk; however, the basis for this asymmetry is unknown. Here, we use retinoid X receptor alpha heterozygous null (RXRα+/-) mice to propose a developmental origin: perturbation of coordinated anterior-posterior (A-P) and L-R axial body patterning. We hypothesized that by analogy to somitogenesis-in which retinoic acid (RA) attenuation causes anterior somite pairs to develop L-R asynchronously-that RA pathway perturbation would likewise result in asymmetric mammary development. To test this, mammary glands of RXRα+/- mice were quantitatively assessed to compare left- versus right-side ductal epithelial networks. Unlike wild-type controls, half of the RXRα+/- thoracic mammary gland (TMG) pairs exhibited significant L-R asymmetry, with left-side reduction in network size. In RXRα+/- TMGs in which symmetry was maintained, networks had bilaterally increased size, with left networks showing greater variability in area and pattern. Reminiscent of posterior somites, whose bilateral symmetry is refractory to RA attenuation, inguinal mammary glands (IMGs) also had bilaterally increased network size, but no loss of symmetry. Together, these results demonstrate that mammary glands exhibit differential A-P sensitivity to RXRα heterozygosity, with ductal network symmetry markedly compromised in anterior but not posterior glands. As TMGs more closely model human breast development than IMGs, these findings raise the possibility that for some women, breast cancer risk may initiate with subtle axial patterning defects that result in L-R asymmetric growth and pattern of the mammary ductal epithelium.This article is part of the themed issue 'Provocative questions in left-right asymmetry'.

Morphometric and fractal dimension analysis identifies early neoplastic changes in mammary epithelium of MMTV-cNeu mice.

Fractal dimension has emerged as a clinically useful tool in the diagnosis and management of breast cancer. The aim of the present study was to determine if fractal dimension can be applied for the analysis of a pre-clinical breast cancer mouse model, MMTV-cNeu. Using fractal dimension in conjunction with conventional morphometric measurements, the ductal epithelial networks of pubertal-stage MMTV-cNeu mice were quantitatively compared with those of wild-type mice. Significant alterations in ductal epithelial network growth and organization were detected during early neoplasia in MMTV-cNeu mice. Moreover, the left-side networks were significantly more affected relative to their wild-type counterparts than were the right-side networks, a finding that is consistent with elevated left-side tumor incidence reported for breast cancer patients. Taken together these results demonstrate that combined fractal dimension and morphometric analysis is an objective and sensitive approach to quantitatively identify ductal epithelial aberrancies that precede overt mammary carcinoma formation.