Sarah M. Paul

Blood-based tumor mutational burden as a predictor of clinical benefit in non-small-cell lung cancer patients treated with atezolizumab

Although programmed death-ligand 1–programmed death 1 (PD-L1–PD-1) inhibitors are broadly efficacious, improved outcomes have been observed in patients with high PD-L1 expression or high tumor mutational burden (TMB). PD-L1 testing is required for checkpoint inhibitor monotherapy in front-line non-small-cell lung cancer (NSCLC). However, obtaining adequate tumor tissue for molecular testing in patients with advanced disease can be challenging. Thus, an unmet medical need exists for diagnostic approaches that do not require tissue to identify patients who may benefit from immunotherapy. Here, we describe a novel, technically robust, blood-based assay to measure TMB in plasma (bTMB) that is distinct from tissue-based approaches. Using a retrospective analysis of two large randomized trials as test and validation studies, we show that bTMB reproducibly identifies patients who derive clinically significant improvements in progression-free survival from atezolizumab (an anti-PD-L1) in second-line and higher NSCLC. Collectively, our data show that high bTMB is a clinically actionable biomarker for atezolizumab in NSCLC.A blood-based DNA sequencing assay to infer tumor mutational burden in the absence of tumor biopsy predicts response to PD-L1 blockade in patients with non-small-cell lung cancer.

Abstract B25: A comparison of resistance mechanisms to 1st, 2nd, and 3rd generation EGFR TKIs in NSCLC

Reversible EGFR tyrosine kinase inhibitors (TKIs) such as erlotinib and gefitinib offer significant clinical benefit to patients with EGFR mutation positive NSCLC compared to chemotherapy alone. In nearly all cases resistance emerges after approximately a year on therapy, often due to the EGFR T790M (50-60% of cases). Irreversible pan-HER TKIs, including dacomitnib and afatinib were developed in part to address the emergence of T790M mutations. Both molecules showed promise, but the T790M mutation is still a dominant resistance mechanism and the number and severity of adverse events in the clinic underscore the unmet need for better targeted treatments for NSCLC patients. Third generation TKIs—AZ9291 and CO-1686—were designed against activating EGFR mutations in order to spare the wild type pathway and developed for efficacy in the T790M setting. However, resistance to the new class of EGFR TKIs is inevitable, prompting us to investigate the impact of erlotinib resistance mechanisms on AZ9291 and ask what new mechanisms are involved. NSCLC cell lines harboring activating EGFR mutations with (H1975) or without T790M (HCC4006 and HCC827) were exposed to erlotinib, dacomitnib, or AZ9291 at IC 75 or greater for three months to generate resistance. The PTEN null line H1650 was found to be resistant to all three TKIs without long-term drug exposure, implicating constitutive PIK3 pathway signaling as one mechanism of resistance. In addition, AZ9291 resistance in H1975 resulted in an epithelial to mesenchymal transition (EMT) at the phenotypic level and a corresponding increase in vimentin staining, with at least a 1000-fold increase in IC 50. Preliminary results indicate both acquired and de novo mechanisms account for AZ9291 resistance in H1975. AZ9291 resistance in HCC827 resulted in resistance to erlotinib and dacomitnib, implicating either additional mutations in EGFR or in downstream genes. Expression of E-cadherin was markedly increased in the AZ9291 resistant HCC827 line. An analysis of EGFR mutation status, PTEN status, Met expression, and EMT gene expression and methylation signatures will be presented, and the possible implications for patients will be discussed. Citation Format: Sarah M. Paul, Xioafen Ye, Zora Modrusan, Somasekar Seshagiri, Robert Yauch, David S. Shames. A comparison of resistance mechanisms to 1st, 2nd, and 3rd generation EGFR TKIs in NSCLC. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Drug Sensitivity and Resistance: Improving Cancer Therapy; Jun 18-21, 2014; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(4 Suppl): Abstract nr B25.

Abstract 3602: Cross-resistance to 1st, 2nd, and 3rd generation EGFR tyrosine kinase inhibitors in vitro is characterized by MET amplification and PTEN loss

Reversible EGFR tyrosine kinase inhibitors (TKIs) such as erlotinib and gefitinib offer significant clinical benefit to patients with EGFR mutation positive non-small cell lung cancer (NSCLC) compared to chemotherapy alone, but high rates of resistance especially at EGFR T790M (50-60% of resistant cases) underscore the need for better targeted treatments for NSCLC patients. Third generation irreversible TKIs–AZD9291 and CO-1686–were developed for efficacy in the T790M setting. However, resistance to the new class of EGFR TKIs is inevitable, prompting us to investigate the relationship between erlotinib and AZD9291 resistance mechanisms and explore novel ones. NSCLC cell lines harboring activating EGFR mutations with (NCI-H1975) or without T790M (HCC4006, HCC827, and PC-9) were exposed to erlotinib, dacomitnib, or AZD9291 at IC 75 or greater for three months to generate resistance. We observed cross-resistance to other EGFR TKIs in nearly all resistant lines, suggesting that T790M was not the main driver of resistance in the erlotinib-treated lines and that a common mechanism might account for resistance in each cell line set. An epithelial to mesenchymal transition (EMT) was observed and a corresponding increase in vimentin staining was seen in resistant lines, consistent with cross-resistance. Previous studies showed that MET amplification caused erlotinib resistance in HCC827; our data suggest MET amplification also accounted for the AZD9291 and dacomitnib resistance in HCC827. In AZD9291-resistant HCC4006 and H1975 lines, PTEN levels were substantially reduced compared to the parental line, with a corresponding increase in PIK3 pathway markers. Whole exome sequencing data support homozygous deletion of PTEN loss as a mechanism of acquired resistance to AZD9291 in NCI-H1975. The PTEN null line NCI-H1650 was found to be resistant to all three TKIs without long-term drug exposure, implicating PTEN loss as a mechanism of de novo resistance. An analysis of EGFR mutation status, PTEN status, MET expression, and the kinetics of acquired resistance will be presented and clinical implications will be discussed. Citation Format: Sarah M. Paul, Dorothee Nickles, Xioafen Ye, Robert L. Yauch, David S. Shames. Cross-resistance to 1st, 2nd, and 3rd generation EGFR tyrosine kinase inhibitors in vitro is characterized by MET amplification and PTEN loss. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3602. doi:10.1158/1538-7445.AM2015-3602