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e21051 Background: Differentiating multiple pulmonary lesions as multiple primary lung cancer (MLC) or intra-pulmonary metastasis (IPM) is critical for clinics. Radiological or histological (dis)concordance may be informative, while, genetic information may aid tracing lineage information on multiple lung lesions. This study aims to apply comprehensive genomic profiling deciphering intrinsic genetics of multiple lung lesions. Methods: 32 patients, 69 FFPE samples were applied to perform high-through put sequencing. 636 cancer-related genes were captured and sequenced by MGI-seq 2000. Raw data was processed via in-house developed pipeline. Unsupervised clustering was applied to cluster samples based on genetic characters after variant calling and annotation. Patients were categorized into “Genomic related” and “Genomic unrelated” groups. Another cohort consisting of 402 patients with mono-lesion was used as control cohort. Results: No difference of age of onset was found between 32 patients and 403 patients with mono lesion (60.5 versus 60 years). Male had relatively lower incidence of multiple lung lesions (RR:0.46,95%CI: 0.20-0.93, P = 0.04). Eight patients were classified as “Genomic related” by presenting same mutation in driver or tumor suppressor genes. Six of eight patients were characterized to carry EGFR L858R, in addition, two patients presented co-mutation of RB1(c.1814+1G > T, p.E48Kfs*18) and TP53 mutations (p.H193R, p.R249S). Another two patients carried EGFR 19 Deletion. Two lesions from one patient with predefined diagnosis of intra-pulmonary metastasis showed biallelic loss of function mutations in same loci of NF1 gene (p.S2311*/ p.Q1360*) together with same KRAS mutation (p.G13C). Radiologically, no same lobe as well as left-right preference were revealed between “Genomic related” and “Genomic unrelated” groups. Histologically, there was no enrichment of patients with same histology subtype in “genomic related” group, verse visa. While, patients in “genomic related” group had higher ages at initial diagnosis (median age 69.5 vs 56.5 years, P = 0.04). Conclusions: Comprehensive genomic profiling should be applied to clinics distinguishing the nature of multiple lung lesion irrespective of radiological and histological diagnosis. [Table: see text]

e21024 Background: Alterations of TP53, EGFR, KRAS genes are of importance in LUAD etiology, vital prognostic markers as well as therapeutic targets as recently reported. In addition, in advanced or metastatic NSCLC, concomitant mutations of TP53 with EGFR or KRAS closely associated with prognosis and TKIs’ efficacy. While, distribution of concomitant mutations of TP53, EGFR and KRAS in early stage or resectable LUAD remained to be elucidated. Methods: 434 patients with defined pathological diagnosis of LUAD were recruited from 1st.Jan.2019-31st.Dec.2019, which made 468 FFPE blocks for the study. Histology and composition were given by authorized by pathologist. Genomic DNA from FFPE samples and peripheral blood was extracted. 636 cancer-related genes were specifically captured and sequenced by MGI-seq 2000. Association of concomitant status with clinical and genomic characteristics was further analyzed. Results: Generally, TP53, EGFR and KRAS mutation rate is 26.7%, 60.9% and 10% respectively. Among these samples, Subtypes of EGFR mutations significantly differed in between tumors rising in left and right lung (P = 0.048). Tumors with KRAS mutations occurred more easily in right lung than those with TP53 or EGFR mutations (P = 0.012, P = 0.043 respectively). In addition, histology subtype and clinical stage were closely associated with TP53, EGFR and KRAS mutation status (P < 0.001). 90 samples (19.2%) carried concomitant TP53 and EGFR mutation, 14 samples (3%) were TP53/KRAS co-mutated. LUAD samples with EGFR amplification had higher rate of concomitant TP53/EGFR mutations (RR:1.1,95%CI: 1.032 to 1.159,P = 0.0009). Moreover, KRAS G13 mutated samples more easily co-mutated with TP53 mutations compared with those with mutations in G12 loci (RR:1.33,95%CI: 1.24 to 1.37,P = 0.024). Further, samples with non-concomitant TP53 mutations had highest level of tumor mutation burden (muts/Mb) (median TMB = 9.74 vs 2.05 for TP53/EGFR/KRAS wild type, P < 0.0001). Samples with concomitant TP53/EGFR mutations and TP53/KRAS mutations displayed higher TMB level than their non-concomitant compartment (P < 0.001 and P = 0.05 respectively). Conclusions: TP53,EGFR and KRAS mutations and their concomitant mutation status displayed diverse correlation with spatial, clinical and genomic characteristics in resectable LUAD patients. This correlation may indicate complex biological heterogeneity of LUAD which may need further exploration.

e21023 Background: TMB is associated with mono-immunotherapy efficacy for patients with advanced or metastatic non-small-cell lung cancer (NSCLC). For early-stage NSCLC or EGFR mutated non-resectable NSCLC, TMB-H predicted better prognosis or poor response to TKIs. This research might develop potential predictive parameters for distinguishing NSCLC with high or low TMB. Methods: Samples of surgically resected, cancer tissues were collected from 499 patients with NSCLC: 29 (5.8%) lung squamous cell carcinoma (LUSC) and 468 (94.2%) lung adenocarcinoma (LUAD). The mutations and TMB were confirmed by target region capture sequencing (Oseq™-508). Results: According to the median value (3.08), TMB is divided into high (N = 244) and low (N = 255). Interactions between TMB and sex, age, location, histology, pathological subtype, lymph node, parabronchial lymph node, LVI, neuro-invasive, stage and mutation status were evaluated. The distribution of TMB-H was significantly (p < 0.001) correlated with sex, age, histology, pathological subtype, stage (TN stage) and partial mutations. Among all mutations, 6 genes (TP53 / FAT3 / KMT2D / TSHZ3 / NAV3 / EPHA3) were confirmed to be significantly related to TMB-H in NSCLC. We also found that TMB-H was significantly affected by mutation status of KRAS gene (P. G12X, p < 0.001) and missense mutations in FAT3 (p < 0.001). The Kruskal–Wallis H test results showed that the mutation types of FAT3, KRAS, TP53 and PIK3CA were closely related to TMB-H. Lasso linear regression analysis was applied and resulted the better predictors of TMB status including TSHZ3, KMT2D, TP53, gender and T stage. Conclusions: Instead of comprehensive genomic profiling to evaluate TMB, clinical characteristics and special mutation types may help to effectively screen and predicate patients with TMB-H status. [Table: see text]

e13542 Background: Environment factors are associated with lung cancer occurrence. Genetic susceptibility to lung cancer remains unclear. This study assessed germline mutations in Chinese non-small-cell lung cancer (NSCLC) patients. Methods: 506 FFPE samples were collected from 469 patients pathologically confirmed as lung adenocarcinoma. A 508-gene panel including 63 hereditary cancer genes was applied to detect mutations on MGI-seq 2000. Raw data was processed and analyzed. Variation pathogenicity was categorized following ACMG 2015 guideline. Results: 21 patients (4%) carried (likely) pathogenic (P or LP) mutations in 15 cancer predisposition genes. 11 germline variations included MUTYH (1/21), BLM (1/21), BRIP1(2/21), RAD50(1/21), PMS1(1/21), TP53(1/21), BRCA2(1/2), PALB2(1/21), NF1(1/21), CDH1(1/21) and MRE11A (1/21) genes. Nine likely pathogenic mutations were identified in MRE11A, RAD51C, RAD50, ATR, BRCA2, BLM, PMS1 and VHL genes. These mutations are involved in homologous recombination repair, mismatch repair, Fanconi anemia, and Li-Fraumeni syndrome. Germline mutations were commonly occurred in females (female vs Male: 13 vs 7) without statistical significance. No significant correlations of age of onset and TMB were observed between NSCLC patients with germline wild type and mutation. Other clinical characters like histology and clinical stage presented similar results. Somatic mutations with high frequency like EGFR, KRAS were distributed equally in patients with both germline mutation positive and negative. Conclusions: In this study, there is no significant correlation of germline susceptibility gene mutations with clinical and genetic characters of NSCLC patients. Further investigation on germline genetic aberrations of NSCLC is definitely needed to clarify germline impact on the etiology of NSCLC. [Table: see text]

e21049 Background: Inhibition of PI3K/AKT/mTOR pathway has emerged as a promising anticancer strategy. Measuring genetic alterations in PI3K/AKT/mTOR pathway are critical for clinical decision making for patients with lung cancer. This study aims to analyze PI3K/AKT/mTOR pathway-related gene mutations in non-small cell lung cancer (NSCLC) in a consecutive cohort. Methods: 536 surgically resected tumor tissues were collected. Target region capture sequencing for 508 cancer-related genes was conducted on MGI-seq 2000 platform. Results: PI3K/AKT/mTOR pathway genes were mutated in 120 samples (22.4%). Female or older patients displayed higher mutation incidence of PI3K/AKT/mTOR pathway (P = 0.06 for female, P = 0.05 for patients ≥60 y). In NSCLC samples, mutations mainly occurred in NF1/2(18/499), PTEN (7/499), MTOR (4/499), TSC1/2(18/499), PIK3CA (26/499), PDK1 (2/499), AKT1/2/3 (5/499) and KRAS (49/499) genes. Genetic alterations of AKT2 (p = 0.003), mTOR (P = 0.03), PTEN (P = 0.005) and PIK3CA (P < 0.001) are preferred to occur in squamous NSCLC. PIK3CA variants (p.E542K, p.E545K, p.Q546K) were more enriched in non-squamous NSCLC, while amplification of PIK3CA was more prevalent in squamous NSCLC (P = 0.038). Histologically, TSC1/2 mutations were more correlate with micro-invasion subtype in non-squamous NSCLC (P = 0.002). Meanwhile, PIK3CA alterations, especially, amplification occurred more often in keratinizing squamous NSCLC (P < 0.001). In addition, four patients with biallelic NF1 loss of function mutations were exclusively found in non-squamous NSCLC without co-occurrence of mutation in known driver genes like EGFR or TP53, which indicated biallelic loss of function of NF1 might be sufficient to drive oncogenesis of non-squamous NSCLC. Conclusions: PI3K/AKT/mTOR pathway alterations present a heterogenous distribution across various types of lung malignancies especially in NSCLC. As more small molecule inhibitors developed to target PI3K/AKT/mTOR pathway, this study might shed light on further clinical investigation of these lately developed therapeutic approaches. [Table: see text]