Prognostic and Predictive Value of KRAS Mutations in Advanced Non-Small Cell Lung Cancer

<div><p>Clinical implications of KRAS mutations in advanced non-small cell lung cancer remain unclear. We retrospectively evaluated the prognostic and predictive value of KRAS mutations in patients with advanced NSCLC. Among 484 patients with available results for both KRAS and EGFR mutations, 39 (8%) had KRAS and 182 (38%) EGFR mutations, with two cases having both mutations. The median overall survivals for patients with KRAS mutations, EGFR mutations, or both wild types were 7.7, 38.0, and 15.0 months, respectively (P<0.001). The KRAS mutation was an independent poor prognostic factor in the multivariate analysis (hazard ratio = 2.6, 95% CI: 1.8–3.7). Response rates and progression-free survival (PFS) for the pemetrexed-based regimen in the KRAS mutation group were 14% and 2.1 months, inferior to those (28% and 3.9 months) in the KRAS wild type group. KRAS mutation tended to be associated with inferior treatment outcomes after gemcitabine-based chemotherapy, while there was no difference regarding taxane-based regimen. Although the clinical outcomes to EGFR tyrosine kinase inhibitors (TKIs) seemed to be better in patients with KRAS wild type than those with KRAS mutations, there was no statistical difference in response rates and PFS according to KRAS mutation status when EGFR mutation status was considered. Two patients with both KRAS and EGFR mutations showed partial response to EGFR TKIs. Although G12D mutation appeared more frequently in never smokers, there was no difference in clinical outcomes according to KRAS genotypes. These results suggested KRAS mutations have an independent prognostic value but a limited predictive role for EGFR TKIs or cytotoxic chemotherapy in advanced NSCLC.</p></div

The combination of AZD9291 with a MEK inhibitor effectively inhibits the growth of AZD9291-resistant cells.

<p>(A) PC9 and (B) PC9/AZDR cells were treated with various concentrations of AZD9291 alone, AZD6244 alone, or their combinations for 72 h. The data are mean ± SE of six replicates. (C) Cells were treated with 100 nM AZD9291 alone, or 100 nM AZD6244 alone, and the combination of AZD9291 with AZD6244 for 24 h. Western blotting was carried out to determine the levels of p-ERK. β-actin was used as a loading control. (D) TUNEL staining was performed after 48 h treatment of AZD9291 (100 nM), AZD6244 (100 nM), and the combination of AZD9291 with AZD6244. Nuclei were counterstained with DAPI. Scale bar, 50 μm. (E) PC9/AZDR xenografts were treated with vehicle, AZD9291 (5 mg/kg/d), AZD6244 (10 mg/kg/d), or AZD9291 plus AZD6244 by oral gavage for 5 days each week for a total of 4 weeks. Tumor sizes were measured as indicated. Each measurement is mean ± SE of 9–10 replicates. (F) Xenograft tumor sections were stained with H&E and Ki-67. Nuclei were counterstained with hematoxylin in Ki-67 stained slides. Scale bar, 100 μm.</p

Acquired resistance to AZD9291 as an upfront treatment is dependent on ERK signaling in a preclinical model

<div><p>AZD9291 (osimertinib) is approved for standard care in patients with EGFR T790M-positive non-small cell lung cancer (NSCLC) after prior EGFR TKI progression. Furthermore, AZD9291 is now being evaluated as a first-line treatment for NSCLC patients with activation EGFR mutations. Based on previous experiments, resistance to AZD9291 as a first-line treatment may also emerge. Thus, identification and understanding of resistance mechanisms to AZD9291 as a first-line treatment can help direct development of future therapies. AZD9291-resistant cells (PC9/AZDR) were established using EGFR inhibitor-naïve PC9 cells. Resistance mechanisms were analyzed using next-generation sequencing (NGS) and a proteome profiler array. Resistance to AZD9291 developed through aberrant activation of ERK signaling by an EGFR-independent mechanism. The combination of a MEK inhibitor with AZD9291 restored the sensitivity of PC9/AZDR cells <i>in vitro</i> and <i>in vivo</i>. PC9/AZDR cells also showed increased MET expression and an HRAS G13R mutation. In addition, maspin expression was higher after AZD9291 treatment in PC9/AZDR cells. Sustained ERK activation confers resistance to AZD9291 as a first-line therapy. Thus, co-targeting EGFR and MEK may be an effective strategy to overcome resistance to AZD9291.</p></div

AZD9291-resistant PC9 cells contain an HRAS mutation.

<p>(A) Summary of genetic alterations in PC9 and PC9/AZDR cells. (B) Cell viability was measured by CCK-8 assay of cells transfected with HRAS siRNA for 48 h. The data are mean ± SE of six replicates. **, <i>P</i> < 0.01 for comparison of the indicated pairs. C. Cells were transfected with either nontargeting control siRNA (siCTL) or HRAS siRNA (siHRAS) for 48 h. HRAS knockdown and phosphorylation of AKT and ERK were detected by Western blotting. β-actin was used as a loading control.</p

Final diagnosis of subjects with positive and negative PET/CT results for indicating the primary cancer.

<p>Final diagnosis of subjects with positive and negative PET/CT results for indicating the primary cancer.</p

Acquired resistance to AZD9291 as first-line treatment is mediated by EGFR-independent mechanisms.

<p>(A) PC9 and PC9/AZDR cells were treated with the indicated concentrations of AZD1775 for 72 h. Cell viability was determined using CCK-8 assay. The data are mean ± SE of six replicates. (B) Cell proliferation was measured by BrdU cell incorporation at 48 h after treatment. The data are mean ± SE of six replicates. (C) Sanger sequencing of EGFR cDNA clones derived from PC9/AZDR. (D) PC9 and PC9/AZDR cells were treated with the indicated concentrations of CO-1686, afatinib, gefitinib, erlotinib for 72 h. Cell viability was determined using the CCK-8 assay. The data are mean ± SE of six replicates.</p

Final diagnosis of patients that were PET/CT negative for primary cancer.

<p>Final diagnosis of patients that were PET/CT negative for primary cancer.</p

A 55-year-old male with gastric cancer identified by FDG PET/CT.

<p>(a) The maximum intensity projection image shows multiple hypermetabolic metastatic lesions in the skeleton, liver and hepatoduodenal lymph nodes. (b) A FDG lesion was detected in the stomach that was interpreted as primary gastric cancer (arrow). Endoscopy revealed a 3-cm-sized ulcerative gastric mass and biopsy confirmed adenocarcinoma of the stomach.</p

PET/CT features indicating multiple myeloma as cause for bone lesions.

<p>PET/CT features indicating multiple myeloma as cause for bone lesions.</p

AZD9291-resistant PC9 cells show increased SerpinB5 levels after AZD9291 treatment.

<p>(A) A Human XL Oncology Array was used to determine candidates for resistance-related proteins by comparison of PC9 and PC9/AZDR cell lysate after 24 h treatment with 100 nM AZD9291. (B) Cells were transfected with either nontargeting control siRNA (siCTL) or maspin siRNA (siMaspin) for 24 h and treated with 100 nM AZD9291 for 24 h. Maspin knockdown was confirmed by Western blotting. β-actin was used as a loading control. (C) After 24 h after siRNA transfection, cells were treated with 100 nM AZD9291 for 48 h. Cell viability was measured by CCK-8 assay. The data are mean ± SE of six replicates. *, <i>P</i> < 0.05 for comparison of the indicated pairs.</p