EGFR 19del Research Articles

Abstract 5891: Plasma-based BDA-NGS test holds potential in circulating nucleic acid based oncogenic aberration detection

Abstract Introduction: Liquid biopsy has been increasingly acknowledged as a substitute of invasive tissue biopsies in the era of targeted therapies, with the booming of plasma-based test platforms. However, the optimal platform for liquid biopsy has not come to a consensus yet and no approved platforms have been approved for oncogene fusion detection. This study aimed to investigate the feasibility of BDA-NGS in plasma-based molecular detection for patients with advanced NSCLC. Methods: This was a preliminary study. 20 paired tissue and blood samples from untreated advanced NSCLC patients underwent molecular detection, using ARMS-PCR (Amoy Diagnostics Co, Ltd) for tissue samples and BDA-NGS (Carrier Gene Biotech Co, Ltd) for blood samples. Specificity, sensitivity and consistency were calculated and compared. Statistical analysis was performed with SAS (version 16.0). Results: Among 20 paired samples, tissue-based ARMS-PCR identified 13 oncogene aberrations, including 11 EGFR mutations, 1 ALK fusion and 1 KRAS mutation. While, plasma-based BDA-NGS also identified 13 oncogene aberrations, including 11 EGFR mutations, 1 ALK fusion and 2 KRAS mutations. The specificity of plasma-based BDA-NGS was 92.3%, sensitivity was 85.7% and consistency was 90.0%, when compared with tissue-based AMRS-PCR. Plasma-based BDA-NGS also identified one EGFR 19del from wild type EGFR and one KRAS co-mutation from ALK fusion by tissue-based ARMS-PCR. However, one patient confirmed as EGFR G719X by tissue-based ARMS-PCR was identified as EGFR wild type by plasma-based BDA-NGS. Since plasma-based BDA-NGS successfully identified ALK fusion, we then mainly focus on the feasibility of BDA-NGS in oncogene fusion detection, results shall be reported lately. Conclusion: Plasma-based BDA-NGS demonstrated high concordant detection rate compared with tissue-based tests, also further increased the detection of oncogene aberrations. The feasibility of plasma-based BDA-NGS in oncogene fusion detection are under evaluation, results shall be reported in the near future. Molecular detection according to different testsNo.AgeGenderHistologytissue-based ARMS-PCRplasma-based BDA-NGS#132FadenocarcinomaWTWT#264FadenocarcinomaL858R + S768IL858R + S768I#355FadenocarcinomaWT19DEL#466Fadenocarcinoma19DEL19DEL#567FNSCLC-NOSWTWT#653Madenocarcinoma19DEL19DEL#757MadenocarcinomaKRASKRAS#874MadenocarcinomaWTWT#962MNSCLC-NOSWTWT#1081FadenocarcinomaL858RL858R#1171MNSCLC-NOSWTWT#1267MadenocarcinomaL858RL858R#1385Msquamous carcinomaWTWT#1449FadenocarcimonaG719XWT#1567MadenocarcinomaL858RL858R#1659FadenocarcinomaL858RL858R#1738MNSCLC-NOSALK fusionKRAS + ALK fusion#1866Msquamous carcinoma19DEL19DEL#1954MadenocarcinomaL858RL858R#2070MadenocarcinomaL858RL858R Citation Format: Shengxiang Ren, Shiqi Mao, Shuo Yang, Yiwei Liu, Xuefei Li, Chao Zhao, Guanghui Gao, Wei Li, Anwen Xiong, Yayi He, Fengying Wu, Xiaoxia Chen, Chunxia Su, Caicun Zhou, David Yu Zhang, Fred R. Hirsch. Plasma-based BDA-NGS test holds potential in circulating nucleic acid based oncogenic aberration detection [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5891.

EGFR mutation genotypes affect efficacy and resistance mechanisms of osimertinib in T790M-positive NSCLC patients.

BackgroundOsimertinib is a potent third-generation EGFR tyrosine kinase inhibitor (TKI) with robust activity in advanced EGFR-mutant non-small cell lung cancer (NSCLC), including those with T790M resistance mutation. However, a broad interpatient variability was observed. This study aimed to evaluate whether EGFR-mutant genotypes affect the clinical outcomes and resistance mechanisms in T790M-positive NSCLC patients receiving osimertinib therapy.MethodsAll NSCLC patients treated with osimertinib in our institute were screened. We included those with known EGFR-mutant genotypes and T790M positivity. Clinical outcomes including objective response rate (ORR), clinical benefit rate (CBR), progression-free survival (PFS), and overall survival (OS), were evaluated and compared between different EGFR genotypes. Patients with next-generation sequencing testing or tumor rebiopsy after osimertinib treatment were analyzed for resistance mechanisms.ResultsORR, CBR, PFS, and OS were all non-significantly different among patients harboring EGFR exon 19 deletion (19Del, n=136), L858R (n=93), and uncommon mutations (n=6). However, a subset of tumors with deletion starting at E746 (ΔE746, n=98), but not non-ΔE746 tumors (n=38), had better clinical outcomes than L858R tumors (n=93). Frequencies of T790M loss and C797S acquisition after osimertinib treatment were similar between 19Del (n=56) and L858R tumors (n=33). However, compared with L858R tumors (n=33), those with 19Del ΔE746 subtype (n=40) had a higher whereas non-ΔE746 subtype (n=16) had a similar frequency of acquired C797S mutation. Combined analysis of our cohort and public cohort confirmed these findings.ConclusionsOur findings indicate that the EGFR 19Del subtypes affect the clinical outcomes and resistance mechanisms to osimertinib in T790M-positive patients. Identifying patients with relatively worse treatment outcomes may be informative for establishing new therapies for these patients.

A rare family case study of multiple siblings with multiple synchronous primary lung cancers.

e21028 Background: Familial lung cancer is rare, let alone inherited lung cancer with multiple synchronous primary lung cancer lesions. Study of inherited lung cancer may facilitate the understanding of the molecular mechanism of the tumorigenesis. Methods: We identified an extremely rare family of five siblings, four with multiple primary lung adenocarcinomas by pathological examination. DNA samples were extracted from the patient tumor tissues and libraries were prepared for next-generation sequencing (NGS) analysis using a custom 500-gene cancer panel. Results: EGFR L858R or 19DEL mutations were identified in at least one lesion in all of the four lung cancer patients. Interestingly, lung cancer patient L1 had EGFR 19DEL in one lesion and L858R in the other, lung cancer patient L2 had EGFR L858R and KRAS G12C mutation in lesion 1 and lesion 2, respectively. The mutation statuses of EGFR and KRAS were confirmed by ARMS-PCR. The mutational spectrum of the two lesions from the same patient distinguished significantly based on the 500-gene NGS panel analysis, further demonstrating the heterogeneity of cancer mutations. Taken together, these results indicate that patients L1 and L2 both had synchronous primary lung cancers. Due to the small lesions, the two lesions for patient L3 were mixed together; the concurrent EGFR L858R and 19DEL mutations in the tissue sample implicated different drivers in the two different lesions since co-occurring of these two mutations was very rare. For patient L4, EGFR L858R was identified in one lesion and uncommon mutations with low frequencies were observed in the other lesion, consistent with different driving mechanisms for the two lesions. Conclusions: To our knowledge, this is the first study identifying a family of multiple siblings with multiple synchronous primary lung cancers and using NGS to reveal the genetics. Together with germline mutation analysis, this study may shed light on the tumorigenesis of familial lung cancer.

Technical validation of a high-sensitivity target capture NGS assay using unique molecular identifier approach.

e13657 Background: Identification of a broad spectrum of somatic mutations is crucial to guide targeted therapy such as for non-small cell lung cancer (NSCLC) patients. In the clinical environment, it requires well validated NGS workflow both for the wet-lab and dry-lab procedures. Here we describe a high sensitivity target NGS assay to accurately capture single nucleotide variants (SNVs), short insertions and deletions (indels), copy number alterations and gene rearrangements for formalin-fixed paraffin-embedded (FFPE) NSCLC patient samples. Extensive analytical validation was performed following the checklists of College of American Pathologists. Methods: Next generation sequencing (NGS) libraries were prepared using extracted DNA from FFPE tissue NSCLC patient samples. The protocol for library generation was optimized in several steps and incorporated 10bp unique molecular identifiers (UMIs). The libraries were sequenced on Illumina HiSeq X-Ten platform. The sequence data was analyzed by an in-house bioinformatics pipeline to call somatic mutations at an average depth of 4000X. Results: We tested the accuracy of 68 clinical tumor samples that were also validated by conventional or alternative methods in the third party CAP accredited labs. We observed 100% sensitivity and 100% specificity compared with the other lab¡¯s validation results. To define the limit of detection (LOD) for different mutation types, clinical DNA samples containing different variants were diluted with normal DNA. The LODs for SNV (as in EGFR L858R) and indel (as in EGFR 19del) were 0.5% and 1%, respectively. Addressing the LOD of fusion and copy number alteration is usually challenging. Our NGS assay was able to achieve 2% LOD for gene rearrangement (fusion) and 3.5 copies for copy number amplification. The high reproducibility was also achieved by inter- and intra- replicate experiments. Our NGS assay showed better performance than other widely used commercial NGS assay panels. Conclusions: We have validated an NGS based approach with UMI technology that is able to achieve high accuracy and sensitivity as low as 0.5% for detection of somatic mutations, which will improve the clinical testing performance for NSCLC FFPE samples with low allele frequencies of driver mutations.

A novel method for enrichment and detection of the six major subtypes of EGFR exon 19 deletions simultaneously by capturing mutant-alleles.

e15539 Background: EGFR exon 19 deletions (19DELs) are important precision therapy targets for NSCLC patients. Due to over twenty subtypes of 19DEL mutations, it is usually challenging for detecting different deletion forms in one assay using limited DNA preparation from plasma sample. Methods: Previously we had developed a high sensitivity detection method, namely PEAC, to detect single somatic mutation in one assay. The method used locked¨Cnucleic acid modified short DNA probe to capture mutant fragment under an optimized temperature. In the present study, we have established a multiplexing PEAC approach for simultaneous enrichment and high sensitivity detection of six major subtypes of EGFR 19DELs.The six subtypes overall account for >86% EGFR exon 19 deletions in the patient population. Results: Genomic DNA of EGFR wild-type was mixed with DNA samples of known EGFR 19DEL mutations. The mutation allele fractions of 5%, 1%, and 0.1% for the six 19del subtypes were constructed, which included c.2235-2249del15 p.E746_A750del, c.2236-2250del15 p.E746_A750del, c.2237-2255delinsT p.E746_A752delinsV, c.2235-2249delinsC p.L747_A750delinsP, c.2240-2254del15 p.L747_T751del, and c.2240-2257del18 p.L747_P753delinsS. The mixed DNA samples were sonicated to mimic the fragment distribution of circulating-free DNA (cfDNA) and PCR amplified for exon 19 regions. Multiplexing enrichment using the six specific probes targeted to the 19DEL subtypes were performed to the amplified DNA fragments under the optimal binding conditions. Sanger sequencing all showed detectable variants in the enriched samples starting with 5%, 1%, or 0.1% mutants, which indicated the capacity and high sensitivity of multiplexing PEAC technology. To explore the potential of multiplexing with other EGFR mutants, the 0.1% mixed mutants of 19DEL were PCR amplified for EGFR exon 20 and exon 21 in addition to exon 19, and followed with enrichment and detection of the six 19DEL variants. Sanger sequencing can detect all the six subtypes of 19DELs. Our method is superior to ddPCR in some aspects such as high throughput and low starting DNA amounts. Conclusions: Multiplexing PEAC technology can simultaneously enrich and detect the six major subtypes of EGFR 19 deletions with high sensitivity. It also has potential to multiplex with other EGFR driver mutations such as in exons 18-21. The technology may serve as an attractive method for detecting mutations in liquid biopsies in clinical practice.

Effective Treatment of Lung Adenocarcinoma Harboring EGFR-Activating Mutation, T790M, and cis-C797S Triple Mutations by Brigatinib and Cetuximab Combination Therapy.

Acquired resistance to osimertinib mediated by EGFR cis-C797S is now a growing challenge. No effective treatment strategy is currently available to overcome cis-C797S-mediated resistance. In this retrospective cohort study, 15 patients with advanced lung adenocarcinoma and EGFR-activating mutation, T790M, and cis-C797S after osimertinib progression were identified by targeted next-generation sequencing. Five of these patients received a combined therapy of brigatinib and cetuximab, and 10 patients received cisplatin-based doublet chemotherapy. Among the five patients who were positive for EGFR 19del-T790M-cis-C797S mutations, and who received brigatinib and cetuximab combination therapy, three patients achieved partial response, and two had stable disease, resulting in an overall objective response rate of 60% and disease control rate of 100%. Among the 10 patients who were positive for EGFR 19del or L858R-T790M-cis-C797S mutations and received chemotherapy, only one patient achieved partial response, five had stable disease, and the other four did not benefit from chemotherapy, resulting in an overall objective response rate and disease control rate of 10% and 60%, respectively. The median progression-free survival of patients who received combined targeted therapy was 14 months, and 3 months for those treated with chemotherapy. No grade III to IV adverse events were observed in any patient. Our retrospective study provides clinical evidence that a combined targeted therapy of brigatinib and cetuximab could be of benefit and may potentially be an effective treatment strategy to improve survival outcomes in patients who acquire EGFR T790M-cis-C797S-mediated resistance to osimertinib.

Investigation of efficacy and acquired resistance for EGFR-TKI plus bevacizumab as first-line treatment in patients with EGFR sensitive mutant non-small cell lung cancer in a Real world population

ObjectivesWe aimed to investigate the clinical efficacy of EGFR tyrosine kinase inhibitor (TKI, T) plus bevacizumab (an antiangiogenic therapy, A) in a real-world population and to provide insights into their mechanism of resistance. MethodsThis study included 256 NSCLC patients harboring EGFR sensitizing mutations (EGFR 19del and L858R) who underwent nextgeneration sequencing (NGS) with 168-gene panel prior to treatment between Jan 2015 to Aug 2018. Cohort A included 60 patients treated with A + T; while cohort B consisted of 120 patients treated with EGFR-TKI monotherapy with the patients identified using Propensity Score Matching (Ratio of 1:2). Clinical outcomes and potential resistance mechanism were evaluated. ResultsBaseline clinical characteristics were not significantly different between Cohort A and B. Compared with cohort B, cohort A had significantly better overall response rate (95% vs 74.2%, p = 0.001) and longer median progression-free survival (PFS, 16.5m vs.12.0 m, HR = 0.7, p = 0.001). Until Jan 2019, 31 and 103 patients in cohort A and B, respectively, were evaluated with progressive disease and underwent tissue re-biopsy and NGS profiling with 168-gene panel. In cohort B, T790M was the predominant acquired resistance mechanism, detected in 51.5% (53/103) of progressive tumors, followed by amplifications in EGFR (15.5%, 16/103) and MET (6.8%, 7/103). Contrastingly, cohort A had a significantly lower rate of T790 M mutation (35.5%, 11/31, p = 0.0003), followed by mutations in TP53 (29.0%, 9/31), RB1 (9.7%, 3/31), SMAD4 (3.2%, 1/31) and EGFR V834 L (3.2%, 1/31) and amplifications in EGFR (9.7%, 3/31), and MET(6.5%, 2/31). ConclusionTreatment with first-line A + T significantly extends the time to progression and increases the response rate with acceptable safety profile. T790 M was the most common acquired resistance mechanism but it was less common in patients who received A + T.

Association Of Initial Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitors Treatment And EGFR Exon 19 Deletion With Frequency Of The T790M Mutation In Non-Small Cell Lung Cancer Patients After Resistance To First-Line Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitors.

BackgroundThe present study analyzed the relationship between clinical features and the T790M mutation in non-small cell lung cancer (NSCLC) patients resistant to epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) treatment.MethodsNSCLC patients with resistance to first-generation EGFR-TKIs in which the disease control time was more than 6 months after initial TKI treatment were enrolled. T790M mutation analysis was performed using one of the following methods according to each manufacturer’s protocols: Cobas EGFR mutation test (41/105, 39.0%), digital PCR (42/105, 40.0%) or Scorpion amplification refractory mutation system (ARMS) (22/105, 21.0%). Sample type of T790M was from tissue only (53/105, 50.5%), plasma only (46/105, 43.8%), tissue and plasma (6/105, 5.7%).ResultsOf 105 patients, 57 were T790M-positive and 48 were T790M-negative. T790M-positive patients had longer progression-free survival (PFS) after initial EGFR-TKI treatment (p = 0.019). T790M positivity was more frequent in patients treated with gefitinib than in those treated with icotinib (65% vs 40.54%, p = 0.018). The rate of T790M positivity was lower in patients with EGFR L858R (44.44%, 12/27) before TKI treatment than in those with EGFR 19del (72.0%, 36/50, p = 0.036). Patients who achieved PR after initial EGFR-TKI treatment had a higher rate of T790M positivity than those with SD (75.76% vs 50%, p = 0.023). There was no relationship between T790M status and age, gender, primary site, metastasis site, or treatment before TKI.ConclusionProgression-free survival (PFS), drug type, response to initial EGFR-TKI treatment, and EGFR status before initial EGFR treatment were associated with the frequency of T790M mutation.

EP1.01-52 Preferable Intracranial Chemotherapy for Lung Cancer Patients with Meningeal Metastasis Harboring CIN in CSF

Brain metastases occur in about half of the NSCLC patients throughout the course of disease, including brain parenchymal metastasis and meningeal metastasis. It has been reported that compared with plasma detection, next-generation sequencing (NGS) of circulating tumor DNA (ctDNA) from cerebrospinal fluid (CSF) is capable of more comprehensively characterizing the genomic alterations of brain tumor, so as to identify actionable variants. We conducted a small sample study consisting of 7 patients with brain metastasis, including 3 lung cancer patients with meningeal metastasis (P314, P316, P318), a lung cancer patient with brain parenchymal metastasis (P323), 2 breast cancer patients with meningeal metastasis (P324 and P328) as well as a gastric cancer patient with meningeal metastasis (P326). Profiling of the specimens from CSF, brain parenchyma and plasma of these 7 patients was performed using NGS along with bioinformatics analysis. Among the three patients P314, P316 and P318, high-frequency driver mutations including ERBB2 p.V659E, EGFR p.L858R & MET amplification, EGFR 19del were detected in the CSF respectively, but all of these variants were not observed in the plasma. Notably, these three patients’ brain lesions continued to progress after receiving corresponding targeted agents, while their conditions remained stable in the presence of switching to intracranial chemotherapy. Following bioinformatic analysis revealed the occurrence of chromosome instability (CIN). We wonder if it is a special case or a common phenomenon. To further explore whether there is similar phenomenon in brain parenchymal metastasis, we examined the patient P323. The detection results of his brain parenchyma, CSF and plasma showed that there was no variation of CIN in brain parenchyma and CSF. In view of this, we hypothesized that CIN variants might exist only in lung cancer patients with meningeal metastasis. In addition, our results displayed that no chromosome instability was identified in the CSF of patients P324, P328 and P326. In summary, CIN is likely an important genomic feature of lung cancer with meningeal metastasis and is apt to be detected in the CSF. According to our observation, NGS of CSF specimen rather than plasma sample may be more favorable to the selection of appropriate treatment options for lung cancer patients with meningeal metastasis. For such patients with CIN variation, intracranial chemotherapy possibly offers more significant clinical benefit than other treatments such as targeted therapy. It still requires more investigations to verify above hypothesis.

EP1.14-37 Anlotinib-Induced Broncho-Pericardial/Pleural Fistula in Patients Suffering from Lung Cancer (First Report)

Anlotinib is an small molecule inhibitor of multiple receptor tyrosine kinases, with inhibitory effects on tumor angiogenesis, and approved in China for treatment of patients with non-small cell lung cancer (NSCLC) who have undergone progression after≥2 lines of chemotherapy. The occurrence of bronchial fistula related to anlotinib has not been reported yet. We presented 2 cases of NSCLC patients with bronchial fistula after treatment with anlotinib. Case 1: A 69-year-old male diagnosed central squamous cell lung carcinoma(CSCLC) of the left (stage IV) on July 2018, who refused chemotherapy but treated with anlotinib for 3 months, aggravated with short breath, with CT indicating broncho-pericardial fistula. After catheter was set into pericardium and drained for 5 days, CT showed significant reduction of gas and pericardium partially conglutinated. Case 2: 63-year-old male diagnosed CSCLC of the right (EGFR 19del) in 2016, with a history of diabetes, who successively received 2 lines of chemotherapy, target therapy, radiotherapy, shifted to anlotinib in August 2018 for 4 months, then aggravated with coughing pyohemosputum and fever, with CT indicating broncho-pleural fistula. After performed a closed thoracic drainage and anti-bacterium therapy, the patient improved and drainage decreased. Two cases developed bronchial fistula during the treatment of anlotinib alone, suggesting that the adverse effects mainly related to the antiangiogenics effect of it and causing ischemic necrosis. Other possible factors include: ①central lung cancer,②squamous cell carcinoma,③cachexia,④multi-line treatment,⑤long diameter of tumor≥5cm) and cavity formation,⑥radiotherapy,⑦accompanied with underlying diseases easy to complicated with infection, such as diabetes. Although the incidence of bronchial fistula caused by anlotinib in NSCLC is extremely rare, it seriously affects the quality of life and overall survival of patients. Therefore, we need to use it selectively and make a close observation of the high-risk patients.

P2.17-36 Molecular Markers, Therapeutic and Prognostic Analysis of Lung Sarcomatoid Carcinomas

Several molecular markers have been established for therapeutic intervention and prognostic prediction of lung cancer. Less is known about their therapeutic potential and prognostic significance in less common lung cancer subtypes. The present study was therefore designed to assess a set of well-defined molecular markers in patients with lung sarcomatoid carcinomas and their therapeutic and prognostic significance. This is a single-center retrospective study of lung cancer patients with histologic types of the sarcomatoid carcinomas who underwent surgical resection at our center during August 2008 to August 2018. The molecular markers analyzed were driver mutations in EGFR, ERBB2, PIK3CA, C-MET, RAS, BRAF, EML4-ALK, RET and ROS1 by ARMS-PCR or NGS, protein expressions of PD-L1 (clone SP-142) in tumors and/or associated lymphocytes by immunohistochemistry. Log-rank test was used to compare the overall survival of patients. A total of 74 (1.18%) patients with sarcomatoid carcinomas were identified from 6,285 patients underwent surgical resection. Of the 35 patients underwent mutation testing (including 14 test EGFR and RAS by ARMS), 17 (48.57%) harbored driver mutations (12 RAS, 4 EGFR L859R and 4 EGFR 19del). Proteins expressions of PD-L1 were found in 67.86% of patients. No prognostic significance (DFS and OS) was noted regarding to any driver mutations and PD-L1 expression. Half of the patients (37/74, 50.00%) received adjuvant therapy, 27 of whom used platinum-based chemotherapy (72.97%). Platinum-based adjuvant chemotherapy showed improved DFS (P=0.011) but similar OS (P=0.079). A part of lung sarcomatoid carcinomas harbor driver mutation or PD-L1 expression, although they are not prognostic. Platinum-based chemotherapy was preferable in these patients. The role of targeted or immune agents as adjuvant therapy needs further study.

P1.03-04 Use Supernatant of Malignant Pleural Effusion to Identify Driver Mutants and Monitor Response to Targeted Therapy

Malignant pleural effusion (MPE) from patients with non-small cell lung cancer (NSCLC) is useful for genetic testing due to advantages including availability, less-invasiveness, and less-heterogeneity. Generally, cell pellets of MPE are used. This study is to further address whether supernatant of MPE is a suitable source to identify key oncogenic mutants in NSCLC patients and provide evidence for clinical molecular testing. MPE samples from 12 NSCLC patients were centrifuged to obtain supernatants and cell pellets, and DNA were extracted. The DNA samples were analyzed by next generation sequencing (NGS) panels using Illumina HiSeq platform. First, MPE samples with the corresponding cancer tissues were collected from 3 NSCLC patients and analyzed with a 500-gene comprehensive cancer panel. Nine mutants were identified in both the paired MPE and cancer tissue samples. We then analyzed nine more NSCLC patient samples using an 18-gene panel to detect key oncogenic mutants; in total, 8 mutants including EGFR L858R, 19DEL, or T790M were identified in the MPE samples. For all of the 17 mutants from the 12 MPE samples, 10 mutants were observed in both the supernatants and pellets of the matched sample sets, of which more pairs (6 out of 10) had supernatants with higher abundance of mutants than the corresponding cell pellets. Importantly, 7 of the 17 mutants were detected only in the supernatants but not the pellets of the paired MPE samples. These results suggest that supernatant of MPE is a better source to detect key oncogenic mutants of NSCLC. Interestingly, 2 patients had both sensitive and resistant mutants to EGFR tyrosine kinase inhibitor (TKI) detected in supernatants of MPE; both patients had treated with EGFR TKI previously, suggesting the development of TKI resistant mutant and supporting the usage of MPE supernatants in monitoring TKI resistance. This study demonstrates that supernatant of MPE is a suitable source for identifying key oncogenic driver mutants for NSCLC and can also be used to monitor response to targeted therapy. The study provides evidence of using supernatant of MPE as an alternative for molecular testing and thus direct precise targeted therapy and surveillance of the therapy effect.

1530P - Different stories for different EGFR exon 19 deletion variants

BackgroundEpidermal growth factor receptor (EGFR) exon 19 deletion (19del) and L858R mutation respond differently to first generation tyrosine kinase inhibitors (TKIs), however, the clinical outcomes of different 19del variants to TKIs and the resistance patterns are not well studied. MethodsFrom April 2016 to August 2017, 194 treatment-naive NSCLC patients detected as EGFR exon 19 deletions using amplification-refractory mutation system were included. DNA sequencing was used to detect the subtypes. Clinicopathological features as well as patients’ outcomes to first-line EGFR TKIs were analyzed. ResultsTwelve EGFR 19del variants were confirmed in 169 of the 194 samples. Among these, p.E746_A750del was the most frequent variant (147/169, 87.0%). p.E746_A750del was more frequently found in females (p=0.008) and never smokers (p=0.004) than other deletions. And deletions starting with E746 was also more frequently found in these patients than L747 (p=0.014 and p=0.018, respectively). Interestingly, T790M occurred more frequently in p.E746_A750del than other deletions (64.3% vs 18.2%, p=0.017) or deletions started with E746 than L747 (63.6% vs 11.1%, p=0.012). Patients harboringdeletions starting with L747 plus insertions had significantly shorter progression-free survival (PFS) than no insertions (8.3m vs 15.0m, p=0.008), as well as other subtypes (8.3m vs 12.8m, p=0.016). Multivariate analysis showed that only L747 deletion plus insertions and age were independent factors influencing PFS (p=0.015 and 0.046, respectively). Conclusionsp.E746_A750del was the most frequent variant of EGFR exon 19del in NSCLC patients. It was more frequently found in female and never smoking patients. T790M mutation had a higher ratio in these patients when they were resistant to first-line TKI treatment. Deletions starting with L747 plus insertions had shorter PFS than other variants. Legal entity responsible for the studyThe authors. FundingHas not received any funding. DisclosureAll authors have declared no conflicts of interest.

Abstract 1277: Honokiol, a natural product, overcomes acquired resistance of EGFR mutant NSCLC cells to the third generation EGFR inhibitor, osimertinib

Abstract Osimertinib (AZD9291) is a FDA-approved EGFR inhibitor for treating non-small cell lung cancer (NSCLC) patients with activating EGFR mutations (first-line) or those who have become resistant to 1st generation EGFR inhibitors through the T790M mutation (second-line). The development of acquired resistance to osimertinib limits the long-term benefits of patients, and effective treatment options are urgently needed. We have identified that honokiol, a natural product with potential antitumor activity, may be used to overcome osimertinib resistance. In several osimertinib-resistant cell lines including HCC827/AR, PC-9/AR, PC-9/GR/AR and PC-9/3M (with EGFR 19del, T790M and C797S mutations), the combination of honokiol and osimertinib had synergistic effects on decreasing the survival of these cell lines. The combination inhibited cell colony formation and growth, and augmented induction of apoptosis of resistant cells. This combination enhanced reduction of Mcl-1, a critical mechanism by which osimertinib induces apoptosis in sensitive EGFR mutant NSCLC cells; this effect is likely due to enhanced Mcl-1 degradation since Mcl-1 was degraded much faster in cells exposed to the combination of honokiol and osimertinib than in DMSO-treated control cells. Moreover enforced overexpression of ectopic Mcl-1 abolished the enhanced effect of honokiol and osimertinib combination on induction of apoptosis, supporting a critical role of Mcl-1 reduction in inducing apoptosis of the osimertinib-resistant cells by this combination. Our findings warrant further study of this combination for overcoming osimertinib-resistance in vivo and potentially in the clinic. (This work was supported by the NIH/NCI R01 CA223220 to SYS and Winship lung cancer pilot award to SYS). Citation Format: Hongjing ZANG, Songqing Fan, Jack Arbiser, Taofeek K. Owonikoko, Suresh S. Ramalingam, Shi-Yong Sun. Honokiol, a natural product, overcomes acquired resistance of EGFR mutant NSCLC cells to the third generation EGFR inhibitor, osimertinib [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1277.

Association between certain non-small cell lung cancer driver mutations and predictive markers for chemotherapy or programmed death-ligand 1 inhibition.

This study aimed to analyze the association between driver mutations and predictive markers for some anti–tumor agents in non–small cell lung cancer (NSCLC). A cohort of 785 Chinese patients with NSCLC who underwent resection from March 2016 to November 2017 in the First Affiliated Hospital of Guangzhou Medical University was investigated. The specimens were subjected to hybridization capture and sequence of 8 important NSCLC‐related driver genes. In addition, the slides were tested for PD‐L1, excision repair cross‐complementation group 1 (ERCC1), ribonucleotide reductase subunit M1 (RRM1), thymidylate synthase (TS) and β‐tubulin III by immunohistochemical staining. A total of 498 (63.4%) patients had at least 1 driver gene alteration. Wild‐type, EGFR rare mutation (mut), ALK fusion (fus), RAS mut, RET fus and MET mut had relatively higher proportions of lower ERCC1 expression. EGFR 19del, EGFR L858R, EGFR rare mut, ALK fus, HER2 mut, ROS1 fus and MET mut were more likely to have TS low expression. Wild‐type, EGFR L858R, EGFR rare mut and BRAF mut were associated with lower β‐tubulin III expression. In addition, wild‐type, RAS mut, ROS1 fus, BRAF and MET mut had higher proportion of PD‐L1 high expression. As a pilot validation, 21 wild‐type patients with advanced NSCLC showed better depth of response and response rate to taxanes compared with pemetrexed/gemcitabine (31.2%/60.0% vs 26.6%/45.5%). Our study may aid in selecting the optimal salvage regimen after targeted therapy failure, or the chemo‐regimen where targeted therapy has not been a routine option. Further validation is warranted.

The clinical prognostic factors in advanced lung adenocarcinoma patients with different EGFR mutation subtype.

e20680 Background: The prognostic factors of advanced NSCLC patients with EGFR mutation subtypes remains controversial. This study aimed to investgate the association of clinical prognostic factors with EGFR-TKI efficacy in advanced lung adenocacinoma patients with different EGFR mutation subtypes. Methods: The clinical characteristics, treatment and survival data of advanced lung adenocarcinoma patients with EGFR sensitive mutations who were treated with Icotinib (Betta Pharmaceuticals, China) from January 2011 to December 2017 in our medical center were reviewed. The prognositic factors of patients between with EGFR exon 19 deletion (19-Del) and exon 21 Leu858Arg mutation (L858R) were retrospectively analyzed by using the Kaplan-Meier method, the chi-square test, and multivariate Cox hazards model. Results: A total of 187 patients were enrolled in the study, including 104 patients with EGFR19-Del and 83 patients with EGFR21-L858R. The results showed that no significant difference in mPFS between the two groups (19-Del vs. L858R: 15.0 months, 95% CI 17.2-32.8months vs. 13.0 months 95% CI 11.7-14.3 months, p = 0.347), while the mOS of patients with 19-Del (35months, 95% CI 28.6-41.4 months) was significantly better than that of patients with EGFR21-L858R (25 months, 95%CI 17.2-32.8 months, p = 0.020). For PFS, liver metastasis was an independent prognostic factor in EGFR19-DEL patients (HR 5.009, 95%CI 1.296-19.354, p = 0.019), while gender (HR 5.881, 95%CI 2.105-16.431, p = 0.001), smoking status (HR 6.972, 95%CI 2.851-17.049, p = 0.001), lymph node metastasis (HR 2.676, 95%CI 1.002-7.148, p = 0.050) were independent prognostic factors in patients with L858R. As for OS, age (HR 1.080, 95%CI 1.017-1.147, p = 0.012), brain metastases (HR 23.166, 95%CI 3.946-136.009, p = 0.001), liver metastases (HR 14.088, 95%CI 1.782-111.385, p = 0.012), renal or adrenal metastasis (HR 10.558, 95%CI 1.480-75.326, p = 0.019), TKI initial efficacy (HR 6.638, 95%CI 2.743-16.067, p = 0.001) were independent prognostic factors in patients with 19-del, however, the initial efficacy of TKI (HR 6.089, 95% CI 2.669-13.893, p = 0.001) was an independent prognostic factor in patients with L858R. Conclusions: The mOS of patients with EGFR 19-Del treated with Icotinib was significantly better than that of patients with L858R, while the mPFS of both were similar. Moreover, there are significant differences in prognostic factors of advanced lung adenocarcinoma patients between with 19-Del and L858R subtypes, which may be helpful for the clinical treatment strategy of EGFR-TKI.

The relationship between preliminary efficacy and prognosis after first-line EGFR tyrosine kinase inhibitor (EGFR-TKI) treatment of advanced non-small cell lung cancer.

Nowadays, patients with EGFR tyrosine kinase inhibitor (EGFR-TKI)-sensitive advanced non-small cell lung cancer (NSCLC) receive EGFR-TKIs as first-line treatment. We aimed to analyze the relationship between preliminary efficacy (tumor shrinkage within 1 month) and progression-free survival (PFS) after first-line EGFR-TKI treatment. A total of 82 patients with EGFR-TKI-sensitive advanced NSCLC confirmed by histopathology from January 2013 to January 2017 were retrospectively analyzed. All patients received first-line EGFR-TKI treatment and follow-up at Shanghai Chest Hospital. Of a total of 82 patients, 42 (51.2%) patients achieved partial response (PR) within 1 month, and 40 (48.8%) patients achieved stable disease (SD: -30% to 0) within 1 month. The median PFS among all patients was 10 months. The median PFS in patients achieving PR within 1 month was 10.0 months. The median PFS in patients achieving SD (-30% to 0) within 1 month was 9.3 months. There was no statistically significant difference between PR within 1 month and SD (-30% to 0) within 1 month (P=0.620). In the EGFR-sensitive mutation subgroup, there was also no statistically significant difference between PR within 1 month and SD (-30% to 0) within 1 month. Univariate and multivariate analysis of first-line EGFR-TKI treatment showed that age, EGFR mutation type, and T staging had effects on PFS. Patients who were more than 65 years old, had EGFR 19del mutation, along with a T staging less than 4, had a longer PFS; these differences were statistically significant. Liver metastasis, bone metastasis, and brain metastasis were not shown to be related to PFS. For patients with EGFR-TKI-sensitive advanced NSCLC, there is no correlation between preliminary efficacy (tumor shrinkage within 1 month) and PFS after first-line EGFR-TKI treatment.

The in cis compound EGFR mutations in Chinese advanced non-small cell lung cancer patients

ABSTRACT Literatures regarding the prevalence and clinical significance of compound EGFR mutations are limited. Until now, none of retrospective or prospective research has focused on in cis compound EGFR mutations except case reports. In this study, we screened a cohort of 3,000 treatment-naïve Chinese advanced NSCLC patients using capture-based ultra-deep targeted sequencing to evaluate the prevalence of EGFR in cis compound mutations and the efficacy of EGFR-TKI in this population. Of the 3,000 patients screened, 1,266 (42.2%) had EGFR mutation; among them, 15 patients (1.2%) harboring in cis compound EGFR mutations, with 10 patients carrying EGFR L858R in combination with a rare mutation and five patients carrying two rare EGFR mutations. No patient with EGFR 19del was observed. Interestingly, no in trans configuration was identified in this cohort. All of the patients harboring in cis compound EGFR mutations were non-smokers, histologically diagnosed with adenocarcinoma and received first-generation EGFR-TKI. Furthermore, our data also revealed that patients with in cis compound EGFR mutations exhibit comparable PFS to first generation EGFR-TKI comparing to patients with single activating EGFR mutation. This observation was further supported by in silico molecular modeling analyses which demonstrated in cis compound mutations do not alter the ATP-binding pocket of EGFR, thus having no effect on the interaction between gefitinib and EGFR.

Next-Generation Sequencing for Genotyping of Endobronchial Ultrasound-Guided Transbronchial Needle Aspiration Samples in Lung Cancer

Endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) can obtain a small amount of specimen. This study aims to evaluate the feasibility and robustness of using EBUS-EBNA samples to perform capture-based targeted next-generation sequencing (NGS). Tissue samples from patients with advanced non-small cell lung cancer were collected byEBUS-TBNA and were formalin-fixed paraffin-embedded. Three representative genes, EGFR, ALK, and ROS1, were examined by amplification refractory mutation system polymerase chain reaction, immunohistochemistry, and quantitative reverse transcription polymerase chain reaction. The remaining samples were processed with NGS assay with a 56-gene panel. Classic driver mutations detected by NGS were verified by conventional methods. Of the 85 samples from patients with advanced non-small cell lung cancer, 77 were performed successfully with all assays. Forty-one mutations in EGFR, ALK, and ROS1 were detected in both conventional methods and NGS, representing a 100% concordance. In contrast, four EGFR mutations detected by NGS were not covered in the targeted regions of amplification refractory mutation system polymerase chain reaction, leading to a negative call in these patients. Altogether, NGS detected 12 additional variants, including six KRAS mutations, one BRAF mutation, one RET fusion, one MET amplification concurrent with EGFR L858R, one KRAS amplification together with EGFR 19del, and one ERBB2 amplification. The mean number of needle passes per lymph node was 5.2 in samples successfully applied in all assays. NGS assay can be successfully conducted with limited tissue samples obtained from EBUS-TBNA. Compared with conventional methods, NGS assay provides more comprehensive information on genetic alterations in tumors, which greatly assists therapeutic decision making for advanced lung cancer.

Short-Term Responders of Non–Small Cell Lung Cancer Patients to EGFR Tyrosine Kinase Inhibitors Display High Prevalence of TP53 Mutations and Primary Resistance Mechanisms

Non–small cell lung cancer (NSCLC) with activating EGFR mutations in exon 19 and 21 typically responds to EGFR tyrosine kinase inhibitors (TKI); however, for some patients, responses last only a few months. The underlying mechanisms of such short responses have not been fully elucidated. Here, we sequenced the genomes of 16 short-term responders (SR) that had progression-free survival (PFS) of less than 6 months on the first-generation EGFR TKI and compared them to 12 long-term responders (LR) that had more than 24 months of PFS. All patients were diagnosed with advanced lung adenocarcinoma and harbored EGFR 19del or L858R mutations before treatment. Paired tumor samples collected before treatment and after relapse (or at the last follow-up) were subjected to targeted next-generation sequencing of 416 cancer-related genes. SR patients were significantly younger than LR patients (P < .001). Collectively, 88% of SR patients had TP53 variations compared to 13% of LR patients (P < .001). Additionally, 37.5% of SR patients carried EGFR amplifications compared to 8% of LR patients. Other potential primary resistance factors were also identified in the pretreatment samples of 12 SR patients (75%), including PTEN loss; BIM deletion polymorphism; and amplifications of EGFR, ERBB2, MET, HRAS, and AKT2. Comparatively, only three LR patients (25%) were detected with EGFR or AKT1 amplifications that could possibly exert resistance. The diverse preexisting resistance mechanisms in SR patients revealed the complexity of defining treatment strategies even for EGFR-sensitive mutations.