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Tumor metastasis is one of the important reasons for the poor prognosis of non-small cell lung cancer (NSCLC) patients, in which lymph node (LN) metastasis is the earliest and most frequent metastasis. With the development of adjuvant immunotherapy, increasing attention has been paid to the tumor-draining lymph nodes（TDLN）in early-stage NSCLC, especially tumor-metastatic lymph nodes, which provides poor prognostic information but has potential benefits in adjuvant treatment. Here, we showed the remodeled immune environment in TDLNs through using TCR-seq and multi-IHC to analyze primary lung cancer tissues and LNs from NSCLC patients with or without LN metastasis. Considering the complex communication between tumor and immunocytes, we subdivided TDLNs and found that metastasis-negative LNs from LN-metastatic patients (MNLN) exhibit more immune activation, exhaustion, and memory compared with both metastasis-positive LNs (MPLN) and TDLNs from non-LN-metastatic patients (NMLN). LN metastasis promotes tumor-specific antigen presentation in TDLNs and induces T cell priming, while existing tumor cells generate an immune-suppressive environment in MPLNs through multiple ways. The evidence implicates the comprehensive immunological mechanism by which LN metastasis influences tumor progression and plays a role in immunotherapy in NSCLC patients.

Aims: This trial explored the safety and efficacy of neoantigen-specific T cells (Nas-Ts) combined with anti-PD-1 (Nas-T + anti-PD-1). Patients & methods: This non-randomized trial recruited participants with solid tumors treated with at least two prior systemic treatment lines. For comparison, 1:1-matched controls who received anti-PD-1 alone were recruited. The primary end point was safety. Results: 15 participants were enrolled in the Nas-T + anti-PD-1 group, the objective response rate was 33.3%, and the disease control rate was 93.3%. The median progression-free survival was significantly different between the Nas-T + anti-PD-1 and control groups (13.8 vs 4.2 months; p = 0.024), but no difference in overall survival was found (p = 0.126). The most common adverse events were maculopapular skin reaction (53.3%), rash (53.3%), hepatotoxicity (53.3%) and fever (53.3%) in the Nas-T + anti-PD-1 group. No serious safety issues were experienced. Conclusion: Nas-Ts combined with anti-PD-1 could be more effective than anti-PD-1 alone in prolonging progression-free survival, with good safety.

Figure S4Comparison of somatic point mutation between paired baseline and relapse samples. (A) The changes of number of mutations from treatment-naïve to relapsed tissues are shown. The changes of transition, transversion and Indel rate between paired treatment-naïve and relapse samples are displayed in (B), (C) and (D), respectively.

Table S2Clinical characteristics for the 51 extensive-stage SCLCs receiving first-linechemotherapy (chemo-resistant, n=28 ; chemo-sensitive, n=23).

Purpose:Clonal architecture is fundamental for the understanding of cancer biology and therapy; however, multiregional sampling in advanced-stage cancers is not always applicable. This prospective clinical trial was to investigate whether paired tissue and circulating tumor DNA (ctDNA) could describe the clonal architecture of advanced non–small cell lung cancer (NSCLC) and its association with clinical outcome (NCT03059641).Patients and Methods:Paired tumor and plasma ctDNA samples were sequenced by target-capture deep sequencing of 1,021 genes. Clonal dominance analysis was performed on the basis of PyClone.Results:Overall, 300 treatment-naïve patients with stage IIIB–IV NSCLC were recruited from 14 centers. Of the 94 patients with available ctDNA data for EGFR clonal architecture analysis, 72 (76.6%) showed EGFR as the dominant clone. The median progression-free survival was longer for these patients than for the 22 patients whose EGFR was nondominant clone [11 vs. 10 months; HR, 0.46; 95% confidence interval (CI), 0.24–0.88; P = 0.02]. The difference was more significant if both tissue and ctDNA defined EGFR as dominant clone (n = 43) versus those not (n = 8; 11 vs. 6 months; HR, 0.13; 95% CI, 0.04–0.50; P = 0.003). Moreover, multivariate Cox proportional HR analysis demonstrated EGFR clonal architecture as an independent prognostic indicator of the efficacy of EGFR-tyrosine kinase inhibitors (TKIs).Conclusions:Paired tissue and ctDNA could be analyzed for clonal architecture in advanced cancer. EGFR mutations do not always make up a dominant clone in advanced NSCLC, which was associated with the efficacy of EGFR-TKIs in NSCLC.

Other driver mutations in 22 discordant cases.

Supplementary legendSupplementary legend

Kaplan-Meier curves for progression-free survival (A) EGFR detected in ctDNA/tissue of the 112 patients for EGFR-TKI efficacy analysis. (B) EGFR dominance analysis according to ctDNA or tissue of patients in (A). (C) Discordance of EGFR dominance analyzed by ctDNA or tissue of the 73 patients with EGFR detected in both ctDNA and tissue (D) Progression-free survival according to mutation clusters in ctDNA (n=85). (E) Progression-free survival according to mutation clusters in tissue (n=100). ctDNA=circulating free tumor-derived DNA. HR=hazard ratio.

Purpose This work assessed local and systemic alternations of the tumor immune microenvironment during concurrent chemoradiation therapy (CCRT) of local advanced cervical cancer to estimate the optimal timing for immune therapy in relation to CCRT. Methods and Materials In this single-center prospective clinical trial, 55 patients with stage IIA through IVA cervical cancer were enrolled between December 2016 and November 2017. The median follow-up was 32.1 months. All patients received cisplatin concurrently with external beam radiation therapy combined with high-dose-rate brachytherapy. Tumor tissues and peripheral blood mononuclear cells (PBMCs) were collected before, during and after CCRT. We analyzed the changes in lymphocyte subpopulations, programmed death-1 (PD-1) and programmed cell death ligand 1 (PD-L1) expression, and the T cell receptor (TCR) repertoire that occurred throughout CCRT. Results The frequencies of CD4+ and PD-1+ T cells in PBMCs decreased after the start of CCRT, whereas that of inhibitory regulatory T cells increased. In the tumor tissues, CCRT decreased the numbers of CD4+ and CD8+ T cells and reduced the median percentage of positive cells expressing PD-L1 from 78.1% to 49.8%. As indicated by the numbers of unique clones, the TCRs of PBMCs exhibited greater diversity before CCRT than after CCRT. Greater TCR diversity in PBMCs before CCRT was associated with superior 30-month progression-free survival (hazard ratio [HR], 0.12; 95% confidence interval [CI], 0.04-0.39; P = .001) and overall survival (HR, 0.17; 95% CI, 0.04-0.68; P = .004). Conclusions CCRT for cervical cancer altered the tumor immune microenvironment by reducing CD4+ and CD8+ T lymphocyte populations, PD-1/PD-L1 expression, and TCR diversity. Higher TCR diversity in PBMCs before CCRT resulted in better survival and prognosis, indicating that CCRT might inhibit immune activation. Our results suggest that it might be more effective to administer immune checkpoint inhibitors before CCRT of cervical cancer rather than during or after CCRT.

Purpose: Clonal architecture is fundamental for the understanding of cancer biology and therapy; however, multiregional sampling in advanced-stage cancers is not always applicable. This prospective clinical trial was to investigate whether paired tissue and circulating tumor DNA (ctDNA) could describe the clonal architecture of advanced non–small cell lung cancer (NSCLC) and its association with clinical outcome (NCT03059641). Patients and Methods: Paired tumor and plasma ctDNA samples were sequenced by target-capture deep sequencing of 1,021 genes. Clonal dominance analysis was performed on the basis of PyClone. Results: Overall, 300 treatment-naïve patients with stage IIIB–IV NSCLC were recruited from 14 centers. Of the 94 patients with available ctDNA data for EGFR clonal architecture analysis, 72 (76.6%) showed EGFR as the dominant clone. The median progression-free survival was longer for these patients than for the 22 patients whose EGFR was nondominant clone [11 vs. 10 months; HR, 0.46; 95% confidence interval (CI), 0.24–0.88; P = 0.02]. The difference was more significant if both tissue and ctDNA defined EGFR as dominant clone (n = 43) versus those not (n = 8; 11 vs. 6 months; HR, 0.13; 95% CI, 0.04–0.50; P = 0.003). Moreover, multivariate Cox proportional HR analysis demonstrated EGFR clonal architecture as an independent prognostic indicator of the efficacy of EGFR-tyrosine kinase inhibitors (TKIs). Conclusions: Paired tissue and ctDNA could be analyzed for clonal architecture in advanced cancer. EGFR mutations do not always make up a dominant clone in advanced NSCLC, which was associated with the efficacy of EGFR-TKIs in NSCLC.

Brain metastasis (BM) is a common malignant event in lung cancer. Here, we recruited 33 lung cancer patients with brain oligo-metastasis to explore the genomic features and tumor immune microenvironment (TIME) of the lung and BM independently. For genomic profiling, targeted sequencing was performed. We found that high-frequent ZFHX3 occurred in the lung (40%) and brain tumor (28%), which might relate to brain metastasis event; the vast majority of patients had lesions-shared mutations in primary tumor and BM, confirming the common clonal events; and EGFR was the most frequently clonal gene in both lung and BM, indicating its driver capability. To characterize TIME status, we also sequenced the T cell receptor (TCR) repertoires and performed immunohistochemistry (IHC) on CD8+ tumor-infiltrating lymphocytes (TILs) and PD-L1 expression in 28 patients who had paired samples. Through the comparison, the TCR clonality of BM was higher than lung tumor, indicating the distinct pattern of the stronger oligoclonal T cell expansion in BM; the primary tumor had a higher TMB than oligo-BM (13.9 vs 8.7 mutations, p = 0.019); CD8 + TILs of BM were significantly lower than lung tumor (10% vs 30%, p = 0.015), revealing the lower level of cytotoxic T cell infiltration; BM showed statistically equivalent level of PD-L1 compared with lung tumor (p = 0.722). We further investigated the potential biomarkers associated with overall survival (OS) after brain surgery. We found that higher TCR clonality was related to prolonged OS in EGFR-treated patients (HR 0.175, p p = 0.034). More CD8+ TILs were an independently positive indicator for OS, in EGFR-treated (HR 0.160, p = 0.001) and non-EGFR-treated patients (HR 0.308, p = 0.009). These findings provide a meaningful molecular and clinical understanding of lung carcinoma and brain oligo-metastasis.

Cancer-associated fibroblasts (CAFs) within the tumor microenvironment (TME) are critical for immune suppression by restricting immune cell infiltration in the tumor stromal zones from penetrating tumor islands and changing their function status, particularly for CD8The TME was characterized using immunohistochemical (IHC) analysis using a large-scale sample size of gene expression profiles. The CD8Compared with normal tissue, CAFs were enriched in cancer tissue, and the amount of CAFs was overwhelmingly higher than that in other immune cells. CAFs are positively correlated with the extent of immune infiltration. A higher CFR was strongly associated with improved survival in lung cancer, melanoma, and urothelial cancer immunotherapy cohorts. Within most cohorts, there was no clear evidence for an association between CFR and programmed death-ligand 1 (PD-L1) or tumor mutational burden (TMB). Compared with TMB and PD-L1, a higher correlation coefficient was observed between CFR and the ORR following pembrolizumab monotherapy in 20 solid tumor types (Spearman's r = 0.69CFR is a newly found and simple parameter that can be used for identifying patients unlikely to benefit from immunotherapy. Future studies are needed to confirm this finding.

Background Patient‐derived organoid (PDO) models are highly valuable and have potentially widespread clinical applications. However, limited information is available regarding organoid models of non‐small cell lung cancer (NSCLC). This study aimed to characterize the consistency between primary tumors in NSCLC and PDOs and to explore the applications of PDOs as preclinical models to understand and predict treatment response during lung cancer. Methods Fresh tumor samples were harvested for organoid culture. Primary tumor samples and PDOs were analyzed via whole‐exome sequencing. Paired samples were subjected to immunohistochemical analysis. There were 26 antineoplastic drugs tested in the PDOs. Cell viability was assessed using the Cell Titer Glo assay 7–10 days after drug treatment. A heatmap of log‐transformed values of the half‐maximal inhibitory concentrations was generated on the basis of drug responses of PDOs through nonlinear regression (curve fit). A total of 12 patients (stages I–III) were enrolled, and 7 paired surgical tumors and PDOs were analyzed. Results PDOs retained the histological and genetic characteristics of the primary tumors. The concordance between tumors and PDOs in mutations in the top 20 NSCLC‐related genes was >80% in five patients. Sample purity was significantly and positively associated with variant allele frequency (Pearson r = 0.82, P = 0.0005) and chromosome stability. The in vitro response to drug screening with PDOs revealed high correlation with the mutation profiles in the primary tumors. Conclusions PDOs are highly credible models for detecting NSCLC and for prospective prediction of the treatment response for personalized precision medicine. Key points Lung cancer organoid models could save precious time of drug testing on patients, and accurately select anticancer drugs according to the drug sensitivity results, so as to provide a powerful supplement and verification for the gene sequencing., Lung cancer organoid models could save the precious time of drug testing on patients, and accurately select anticancer drugs according to the drug sensitivity results, so as to provide a powerful supplement and verification for the gene sequencing.

Background: Pyroptosis has been recently identified as a hallmark of cancer biology; however, the potential of pyroptosis-related genes (PRGs) as prognostic markers has not been fully elucidated in hepatocellular carcinoma (HCC). Methods:35 PRGs were obtained from the published literature, and pyroptosis subtypes were identified by bioinformatics methods. The risk score model was established applying the LASSO Cox regression method in the TCGA cohort and validated in the ICGC datasets. Additionally, immune infiltration, enriched pathways and genomic alterations were analyzed between high and low risk-score subgroups. Finally, a nomogram containing the pyroptosis risk score and other prognostic related clinical factors was created for predicting the overall survival of HCC.Results:Based on the expression profile of PRGs, we determined two pyroptosis-related subtypes (cluster A and cluster B) of HCC with different immune characteristics and significantly different prognoses. The risk score model showed that up-regulation of GPX4, CASP8, NOD2, and GSDME was associated with poor prognosis, while NLRP6 was the opposite. Compared with patients with lower risk scores, the high-risk score group had a worse prognosis (P Conclusions:We developed and verified a prognostic risk model based on PRGs for HCC sufferers, which may provide a robust tool for forecasting prognosis in patients with HCC.

48 Background: Growing studies have suggested that personalized tumor-informed circulating tumor DNA (ctDNA) based minimal residual disease (MRD) detection can precisely predict recurrence in resectable patients (pts) with colorectal cancer (CRC). We developed an improved approach, a Tumor-Informed Patient-specific panel integrating with a tiny tumor-specific Core panel (TIPC) to monitor MRD by ultra-deep sequencing of plasma samples. The combination with a core panel confers TIPC overcoming the tumor temporal and spatial heterogeneity to some extent and providing more comprehensive information to guide individualized management. Here, we investigated the clinical utility of this assay and performed a head-to-head comparison with a Tumor-Informed Off-the-shelf panel approach (TIO). Methods: The retrospective study enrolled 34 I-IV CRC patients undergoing curative intent surgery from multiple centers and a median follow-up of 482 days. Tissue samples were collected at surgery. Peripheral blood samples collected postoperative and before relapse were analyzed. For all patients, at least one sample was available within 6 months before progression, defined as surveillance time point. TIPC is a whole exome sequencing (WES) based approach to identify somatic mutations from tissue samples, followed by customizing a patient-specific capture panel covering up to 50 top-ranked variants, and then combined with a CRC universal core panel containing 10 actionable or high prevalence cancer-related genes to conduct ultra-deep sequencing above 100,000X of follow-up blood samples. TIO assay was performed in serial plasma samples by using an off-the-shelf 338 gene panel at a sequencing depth of 30,000X. Results: As of August 31, 2022, 25 pts had confirmed recurrence. For the surveillance time point, 24/25 (96.0%) disease recurrence pts were detected MRD positive with TIPC assay, meanwhile, all disease-free pts were MRD negative (100%, 9/9). MRD detection preceded radiographic progression (median 5.6 months). TIO approach showed the same MRD detection specificity performance as TIPC, whereas the sensitivity was 88.0% (22/25). The ctDNA fraction of these two TIO undetectable pts was quantified using TIPC were 0.0168% and 0.0177%, while the positive mutations were beyond the 338 gene panel. The detectable postoperative ctDNA was associated with a poor prognosis for patients by either TIPC or TIO assay, with hazard ratios 20.8 (p＜0.0001) and 7.8 (p＜0.0001) respectively. To further verify TIPC may possess higher sensitivity, another cohort of 4 radiographic progressed CRC pts with TIO longitudinal undetectable MRD was analyzed, 3/4 (75.0%) was detectable by TIPC (ctDNA fraction range 0.0006%-0.0075%). Conclusions: WES-based patient-specific MRD detection assay demonstrated encouraging recurrence risk stratification value in CRC pts and may pave the way for the management of individual treatment.

Background Cervical cancer (CC) is the second leading cause of cancer death among women worldwide. Epigenetic regulation of gene expression through DNA methylation and hydroxymethylation plays a pivotal role during tumorigenesis. In this study, to analyze the epigenomic landscape and identify potential biomarkers for CCs, we selected a series of samples from normal to cervical intra‐epithelial neoplasia (CINs) to CCs and performed an integrative analysis of whole‐genome bisulfite sequencing (WGBS‐seq), oxidative WGBS, RNA‐seq, and external histone modifications profiling data. Results In the development and progression of CC, there were genome‐wide hypo‐methylation and hypo‐hydroxymethylation, accompanied by local hyper‐methylation and hyper‐hydroxymethylation. Hydroxymethylation prefers to distribute in the CpG islands and CpG shores, as displayed a trend of gradual decline from health to CIN2, while a trend of increase from CIN3 to CC. The differentially methylated and hydroxymethylated region‐associated genes both enriched in Hippo and other cancer‐related signaling pathways that drive cervical carcinogenesis. Furthermore, we identified eight novel differentially methylated/hydroxymethylated‐associated genes (DES, MAL, MTIF2, PIP5K1A, RPS6KA6, ANGEL2, MPP, and PAPSS2) significantly correlated with the overall survival of CC. In addition, no any correlation was observed between methylation or hydroxymethylation levels and somatic copy number variations in CINs and CCs. Conclusion Our current study systematically delineates the map of methylome and hydroxymethylome from CINs to CC, and some differentially methylated/hydroxymethylated‐associated genes can be used as the potential epigenetic biomarkers in CC prognosis., The differentially DMR/DhMR occurred earlier and much more than that of CNVs which would be more suitable for a marker to detect the disease progression.The differentially DMR/DhMR‐associated genes enrich in Hippo and other cancer‐related signaling pathways.Eight DMR/DhMR‐associated genes significantly correlate with the overall survival of CC.

Lung adenocarcinoma (LUAD) is most common pathological type of lung cancer. LUAD with brain metastases (BMs) usually have poor prognosis. To identify the potential genetic factors associated with BM, a genomic comparison for BM cerebrospinal fluid (CSF) and primary lung tumor samples obtained from 1082 early- and late-stage LUAD patients was performed. We found that single nucleotide variation (SNV) of EGFR was highly enriched in CSF (87% of samples). Compared with the other primary lung tissues, copy number gain of EGFR (27%), CDK4 (11%), PMS2 (11%), MET (10%), IL7R (8%), RICTOR (7%), FLT4 (5%), and FGFR4 (4%), and copy number loss of CDKN2A (28%) and CDKN2B (18%) were remarkably more frequent in CSF samples. CSF had significantly lower tumor mutation burden (TMB) level but more abundant copy number variant. It was also found that the relationships among co-occurrent and mutually exclusive genes were dynamically changing with LUAD development. Additionally, CSF (97% of samples) harbored more abundant targeted drugs related driver and fusion genes. The signature 15 associated with defective DNA mismatch repair (dMMR) was only identified in the CSF group. Cancer associated pathway analysis further revealed that ErbB (95%) and cell cycle (84%) were unique pathways in CSF samples. The tumor evolution analysis showed that CSF carried significantly fewer clusters, but subclonal proportion of EGFR was remarkably increased with tumor progression. Collectively, CSF sequencing showed unique genomic characteristics and the intense copy number instability associated with cell cycle disorder and dMMR might be the crucial genetic factors in BM of LUAD.

Background The genomic mutation and immune feature landscape of ERBB2 exon 20 insertion (ERBB2-ex20ins)-driven non-small cell lung cancer and the features associated with the response to chemoimmunotherapy are currently unknown. Methods The genomic landscape of ERBB2-ex20ins lung adenocarcinoma (LUAD) patients was characterized by next-generation sequencing (NGS) of 1021 cancer genes. The clinical outcomes of chemoimmunotherapy were evaluated among 13 patients with stage IV ERBB2-ex20ins LUAD, and potential biomarkers of the response to chemoimmunotherapy were explored using NGS and T cell receptor sequencing. Results Among 8247 LUAD patients, 207 (2.5%) had ERBB2-ex20ins, of whom 181 (87.4%) harbored more than one comutation. The most common comutations were in TP53. Patients with ERBB2-ex20ins had a low tumor mutational burden (TMB; median, 4.2 mutations/Mb), and most (66.7%) were PD-L1 negative. Thirteen of the 207 patients received chemoimmunotherapy, for whom the objective response rate, disease control rate, and median progression-free survival were 31%, 77%, and 8.0 months, respectively. Responders exhibited a higher TMB and a trend toward lower clonality in tumors compared with nonresponders (p = 0.0067 and p = 0.085, respectively). A high TMB combined with mutations in DNA damage repair pathways and SWI/SNF chromatin remodeling complexes was associated with a benefit from chemoimmunotherapy. Conclusions The efficacy and outcome of chemoimmunotherapy were encouraging among ERBB2-ex20ins LUAD patients, who were characterized by low TMB and negative PD-L1 expression. The combination of TMB and comutations is a potential biomarker to identify patients who will benefit from chemoimmunotherapy.

Esophageal squamous cell carcinoma (ESCC) is a highly malignant and deadly tumor. Radiation therapy is one of the primary treatments for locally advanced ESCC. However, the biomarkers for prognosis of definitive radiation remain undefined. Peripheral blood circulating tumor (ct)DNA provides information of tumor genetic alterations and has been confirmed as a potential non-invasive biomarker for several types of cancer. The present study investigated the clinical implications of ctDNA detection in patients with ESCC and receiving definitive radiation therapy. Patients with locally advanced ESCC were retrospectively recruited. Plasma samples were collected before, during and following radiation therapy. Next-generation sequencing was performed to identify somatic mutations in 180 genes. A total of 69 baseline and post-radiation plasma samples were collected from 25 patients. A total of 59 non-silent single nucleotide variants were present in 33 genes. All pre-radiation and 58.3% (14/24) of post-radiation samples had at least one mutation. Patients with lymph node metastases (LNM) exhibited a higher number of pre-radiation mutations compared with those without LNM. The variables, progression-free survival (PFS) and overall survival (OS) of the patients with one baseline mutation were not significantly different compared with that in patients with more than one baseline mutation. Patients with initial ctDNA-positive post-radiation samples exhibited significantly reduced PFS (P=0.047) and OS (P=0.005) compared with that in patients with ctDNA-negative samples. The post-radiation plasma ctDNA status was an independent prognostic factor from univariate and multivariate analyses. Dynamic monitoring of ctDNA during follow-up was examined. The results indicated that ctDNA was a predictive and prognostic marker in patients with ESCC and receiving definitive radiation therapy, which may guide subsequent treatment.

Highlights • The molecular characteristics of MSI and MSS CRC tumors are quite different. • MSI CRC postoperative plasma had distinct mutational characteristics with MSS CRC. • The prognostic value of ctDNA in postoperative plasma of MSI CRC should be independently evaluated in MSI., The mutation in postoperative plasma (molecular residues) was an independently prognostic factor in colorectal cancer (CRC). The status of postoperative plasma mutation of microsatellite instability (MSI) CRC has not been systematically examined. In this study, we enrolled 30 MSI and 46 microsatellite stability (MSS) CRCs, and performed next generation sequencing on surgical tissues, postoperative plasma, and plasma during follow-up. Compared with MSS, MSI tumors had dissimilar genomic profiles, higher tumor mutation burden (TMB), and more frameshift mutations. In the postoperative plasma, more MSI CRCs were detected with tumor-derived mutations (77% in MSI vs 33% in MSS, p < 0.001). The numbers of postoperative mutations were proportional to MSI tissues (Spearman r = 0.47, p = 0.023), while not for MSS. More proportion of postoperative plasma samples of MSI CRCs harbored frameshift mutations than MSS (p = 0.007). For the follow-up plasma, 93% (14 out of 15) MSI CRCs harbored tumor-derived mutations; 33% (4/12) MSS were mutation-positive, lower than MSI (p = 0.003). Thus, considering that MSI CRC had extremely distinct mutational characteristics in tumor and postoperative plasma compared with MSS CRC, we propose that the prognostic value of molecular residue identification in postoperative plasma needs to be independently evaluated in MSI and MSS CRCs.

Pulmonary sarcomatoid carcinoma (PSC) contains carcinomatous component (CaC) and sarcomatous component (SaC). Herein, we explored the genomic origin and intratumor heterogeneity (ITH) of PSC. We collected 31 resected PSC tumors and obtained CaC and SaC by laser capture microdissection for next-generation sequencing. The majority of PSCs (97%) had component-shared alterations. Driver mutations in EGFR, KRAS, MET, PIK3CA, and EML4-ALK fusion were mostly component-shared. Twenty-seven (87%) PSCs had component-private alterations. Compared with pure lung adenocarcinoma (LUAD), adenocarcinoma component of PSC showed lower EGFR incidence. Compared with other typical sarcomas, numerous genes of SaC exhibited significant differences. CaC and SaC had equivalent and proportional tumor mutation burden (TMB), as well as PD-L1 level. Compared with LUAD, SaC had significant higher TMB and more patients with high PD-L1 expression (tumor proportion score ≥50%). PSC with lower proportion of component-shared alterations (trunk-ratio) had a prolonged disease-free survival (DFS), regardless of the influence of clinical factors. We conclude that most PSCs originate from a monoclone accompanied by genomic ITH which is a potential independent prognostic factor, and more proportion of PSCs may be beneficial from immune checkpoint inhibitors.

BACKGROUND: Tracheal adenoid cystic carcinoma (TACC) is the second most common type of cancer in bronchial tumors with poor prognosis. Studies on the genomic profiles and tumor immune microenvironment (TIME) of TACC are still relatively rare. METHODS: Here, we performed whole-exome sequencing (WES), T cell repertoire (TCR) sequencing, and immunohistochemistry (IHC) on the resected tumors and matched peripheral blood leukocytes (PBLs) samples from 25 TACCs collected from April-2010 to Mar-2019. RESULTS: WES results revealed that LPAR3 and ALPI were recurrently mutated genes, with no classical lung cancer drivers in TACCs (n=8). The median tumor mutation burden (TMB) was 3.67, lower than other solid tumors. Unexpectedly, one patient showed high microsatellite instability (MSI). Recurrent copy number variations (CNVs) affected genes commonly involved in p53, cell cycle, and PI3K-Akt signaling pathways. For TCR estimators of 13 PBLs, the median clonality and Shannon index was 0.15 and 7.02, respectively. Shannon index showed marginally negative association with age (Pearson r =−0.53, P=0.062). Clonotype number and Shannon index of 7 TACC tissues were significantly lower than those of lung adenocarcinoma (LUAD) and lung squamous cell carcinoma (LUSC) (Mann-Whitney test, both P

Background: Although EGFR mutations in lung cancer are identified most often as trunk mutations in the early stage, cancer evolution often follows a branched trajectory. Whether EGFR would always be the dominant clone in the advanced stage is unknown. Methods: This prospective multicenter clinical trial recruited treatment naive patients with stage IIIB-IV non–small cell lung cancer from 14 centers in China. Paired tumor and plasma ctDNA samples were sequenced by target-capture deep sequencing of 1021 genes. Clonal dominance analysis was performed on the basis of PyClone. Findings: Overall, 300 patients were recruited and 132 patients treated with first line targeted therapy were followed. The median follow-up time was 10 months (IQR 6–13). Of the 94 patients with available ctDNA data for EGFR clonal architecture analysis, 72 (76.6%) showed EGFR as the dominant clone. The mPFS was longer for these patients than for the 22 patients who had EGFR non-dominant clone (11 months vs 10 months, hazard ratio [HR] 0.46, 95% CI 0.24–0.88, p = 0.02). The difference was more significant if patients who were defined as having EGFR dominant clone by both tumor tissue and plasma (n = 43) versus those not (n = 8) (11 vs 6 months, HR = 0.13, 95% CI 0.04–0.50, p = 0.003). Moreover, multivariate cox proportional hazard ratio analysis demonstrated EGFR clonal architecture as an independent prognostic indicator of the efficacy of EGFR-tyrosine kinase inhibitors. Interpretation: In advanced NSCLC,EGFR mutations do not always make up a dominant clone. Clonal architecture of EGFR in the pretreatment ctDNA is associated with the efficacy of EGFR-TKIs in NSCLC. Trial Registration: The trial has been registered with the number NCT03059641. Funding Statement: Geneplus-Beijing Co. Ltd. Declaration of Interests: RC, PL, LC and XY were employees of Geneplus-Beijing Co. Ltd. All other authors declare no conflict of interests. Ethics Approval Statement: This study was performed under a protocol (KS1707) approved by the Institutional Review Board of Shanghai Chest hospital (Shanghai, China).

Background: Although small-cell lung cancer (SCLC) is sensitive to chemotherapy and radiotherapy initially, nearly all patients recur often with treatment-resistant disease. Comparing genomic profiles of paired treatment-naïve and recurrent tumors to understand the clonal architecture and molecular evolution of SCLC under treatment may provide novel insights into mechanisms underlying recurrence and susceptibility to further treatment. Methods: Paired tumor samples procured at diagnosis and relapse were collected from 11 patients with limited-stage SCLC treated with concurrent chemoradiation (CCRT). All tissues underwent whole exome sequencing (WES). Genomic landscape including somatic mutations, somatic copy number alterations (SCNAs), and clonal architecture were compared between treatment-naïve and paired recurrent tumor samples. Baseline and paired recurrent plasma samples from another 9 patients with SCLC treated with CCRT were performed deep sequencing using a targeted panel containing 1021 cancer related genes. Results: In both pre- and post-treatment tumors, TP53 (73% vs 73%), FAM135B (55% vs 64%), RB1 (45% vs 55%), and CTNND2 (45% vs 55%) were the top four most frequently mutated genes. Tobacco exposure related mutational signature was predominant in all samples. Compared with primary tumors, relapsed tumors showed significantly increased number of mutations (220 in recurrent SCLC vs 210 in primary tumor, Wilcoxon paired test, p-value = 0.016). Six of the 11 patients had increased chromosomal instability (CIS) in recurrent tumors. Relapsed tumors had more genes with copy number amplification compared to pre-treatment primary tumors although the difference did not reach statistical significance (18 vs 10, p-value = 0.066). In all of the patients, an average of 94% mutations at baseline were present in relapsed tumors. The number of clones in recurrent samples was higher than that in pre-treatment samples (13 vs 12, p-value = 0.004), indicating that new clones emerged under the treatment and tumor heterogeneity increased. A total of 648 acquired mutations in 600 genes and 140 acquired SCNAs in 123 genes were identified, in which 126 mutations were clonal (CCF>0.6) in relapsed tumors. These genes were enriched in PI3K-ATK signaling pathway and covered 91% (10/11) patients, implying the potential mechanism of chemoradiation resistance. Based on the specific mutations and mutations with increased CCF in relapsed plasma samples from the other 9 SCLCs, acquired alterations were also enriched in PI3K-ATK signaling pathway. Conclusions: Acquired mutations and chromosomal copy number gains may play a role in relapse of limited stage SCLC post CCRT. PI3K-ATK pathway alterations are frequent in recurrent SCLCs, which may be a candidate resistant mechanism after chemoradiation. Citation Format: Ying Jin, Yamei Chen, Xiao Hu, Huarong Tang, Qian Li, Pansong Li, Xinze Lv, Xuefeng Xia, Jianjun Zhang, Ming Chen. Whole exome sequencing reveals PI3K-ATK pathway alterations are frequent in relapsed small cell-lung cancer after chemoradiation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1412.

Background: Immunotherapy can induce a long response in part patients of lung squamous cell carcinoma (LUSC). And accurate biomarkers are needed for suitable populations. As important tumor suppressor, tumor protein p53 (TP53) and low density lipoprotein receptor-related protein 1B (LRP1B) genes may be associated with immunogenicity. However, the interaction of TP53 and LRP1B and its potential association with response to immunotherapy are not fully understood in LUSC. Methods: Three cohorts were divided into four groups, respectively (A: TP53mut and LRP1Bmut, B: TP53mut and LRP1Bwild, C: TP53wild and LRP1Bmut, D: TP53wild and LRP1Bwild). 525 LUSCs form Geneplus database which performed next-generation sequencing were assessed the differences of genomic landscape, tumor mutation burden (TMB), and PD-L1 expression in four groups (cohort 1). TCGA-LUSC RNA data (n=466) from cBioPortal were used to evaluate immune cell infiltration thought single sample gene set enrichment analysis (cohort 2). The LUSCs (n=125) which received immunotherapy of POPLAR and OAK cohort were used to evaluate the efficacy of immunotherapy in four groups (cohort 3). Results: The proportion of patients in A, B, C and D groups of cohort 1 was 26%, 59%, 2%, 13%, respectively, suggesting that most patients with LRP1B mutation also carry TP53 mutation (98.6%) in LUSC. This similar ratio is 88.2% in cohort 2 and 74.3% in cohort 3. For group A, the most common mutated gene except TP53 and LRP1B was CDKN2A (31%). Group B was MLL2(28%). Group C was CDKN2A (33%). Group D was FAT1 (13%). The median TMB was 14.4 mutations/Mb (2.9-80.0) in group A, 9.8 (0.96-95.0) in group B, 11.5 (2-25) in group C, 3.8 (0.96-18.2) in group D. TP53 and LRP1B co-mutation had a highest TMB compared with the other three groups in all cohorts, while TP53wild and LRP1Bwild completely opposite. In cohort 1, PD-L1 expression had no significant difference in four groups. In cohort 3, progression-free survival (PFS) was longer in group D compared with the other three groups (p = 0.047, A: 1.5m, B: 1.6m, C: 2.1m, D: 4.2m). The same trend on overall survival (OS) were found but it was not significant. Group D also had a higher disease control rate (A: 50%, B: 46%, C: 44%, D: 69%). We than divide into two groups (D vs Other) to compare PFS and OS. It showed that group D had a prolonged PFS (p = 0.02) and OS (p = 0.03). Multivariate cox regression analysis showed that the results were independent of TMB, gender and age. Analysis of immune cell infiltration in cohort 2 showed that the better outcomes of group D might associated with elevated infiltrations of activated dendritic cell and activated CD8 T cell. Conclusion: TP53mut and LRP1Bmut, which accounted for a high proportion of LUSC, was associated with high TMB. And TP53wild and LRP1Bwild has better anti-PD-L1 outcome in LUSC. This might be related with a stronger activated dendritic cell and activated CD8 T cell infiltration despite lower TMB. Citation Format: Jiangyong Yu, Zhipeng Zhou, Ping Zhang, Pansong Li, Xu Li, Min Tang, Nannan Fan, Xiaonan Wu, Xin Nie, Xiaoyan Chen, Di Ma, Xi Chen, Yanfang Guan, Xuefeng Xia, Ling Yang, Xin Yi, Lin Li. Association of TP53 and LRP1B co-mutation status with response to immunotherapy in lung squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2492.

e20544 Background: The immunomodulatory effects of the tumor microenvironment (TME) significantly impact the T cell repertoire resulting in different anti-tumor immune responses. Previous work by our group and others has highlighted intratumor heterogeneity in T cell clonality, spatial distribution, and diversity, as well as infiltration of bystander T cells in early-stage non-small cell lung cancer (NSCLC). However, little is known about how the TME impacts the T cell repertoire in distant or surrounding adjacent uninvolved lung tissue. Methods: We investigated the interplay between tumor and surrounding lung tissues by investigating the T cell repertoire at the tumor site, tumor margin, and 1cm, 2cm, 5cm, and 10cm away from the tumor as well as in peripheral blood in 21 NSCLC patients with a total of 123 samples undergoing T cell receptor (TCR) CDR3b sequencing using the Geneplus technology. Differences in regional T cell repertoires were analyzed based on clonality, diversity, and Morisita Overlap Index (MOI). GLIPH was utilized to identify motifs associated with pathogen specificity in each region. Results: Clonality was lowest within the tumor and progressively increases from tumor margin to tumor adjacent lung toward distant lung with “hot spot” regions of T cell expansion located in adjacent lung tissue proximal to the tumor, namely 2cm away (p = 0.0025). Dominant T cell populations were better conserved between the tumor margins and “hot regions” of high clonality compared to the tumors (p = 0.0009) highlighting the differences between these environments and possible T cell exclusion in immunosuppressive TME. GLIPH analysis revealed an inverse correlation between clonality and the proportion of pathogen-associated TCR motifs (r = -0.83, p = 0.0195). Interestingly, tumor tissue contained the highest proportion of predicted pathogen TCRs whereas regions of high clonality contained the lowest (p < 0.0001), suggesting the antigen-independent nature of T cell trafficking. Conclusions: Combined, these results highlight isolated pockets of T cell expansion in the lungs outside the tumor and potential immune evasion mechanisms in NSCLC.

e20523 Background: Lung adenosquamous carcinoma (ASC) is a relatively uncommon malignancy that comprised of both adenocarcinoma component (ACC) and squamous cell carcinoma component (SCCC). We have reported its genomic profile, evolutionary origin, and clinical management. Information of tumor immune microenvironment (TIME) of ASC, including tumor mutation burden (TMB), tumor-infiltrating lymphocytes (TILs), T cell receptors (TCR) repertoire, and PD-L1 status remains scarce. Methods: 28 Surgical ASCs were collected and identified by immunohistochemistry (IHC). ACC and SCCC were obtained separately by microdissection. Targeted sequencing was performed for the two components using a 1021-gene-panel independently. TMB of ACC and SCCC were independently calculated. TMB of ASC calculation was based on aggregation (removing duplicates) of non-synonymous mutations from both ACC and SCCC. TILs and PD-L1were examined by multiplex immunohistochemistry (mIHC). The infiltration level of immune cells in tumor, stroma, and total region (named by both tumor and stroma regions) was investigated. T cell receptor (TCR) repertoires were sequenced and clonality and Shannon index were calculated. Results: SCCC (7.2 mutations/Mb) had a higher TMB level than ACC (6.5 muts/Mb), indicating the immunogenic heterogeneity between the different pathological components. The TMB value of ACC and SCCC were modestly proportional (Spearman r = 0.56, p = 0.001), which was related to the clonal origin. SCCC TMB was lower than the archival LUSC (10.1 muts/Mb), and ACC TMB was similar to LUAD (4.3 muts/Mb). The TMB of ASC was higher than LUAD and close to LUSC. The nonidentical TIL level in ACC and SCCC showed infiltration heterogeneity. For tumor region, CD3+ total T cells, CD4+ T cells, CD8+ T cells, and PD1+ cells accounted for the most proportion of immune cells, with no differences between ACC and SCCC; FOXP3+ CD4+ Treg cells were more abundant in SCCC than ACC ( p = 0.047), as well as CD3+ LAG3+ T cell ( p = 0.029). For stroma region, ACC and SCCC had similar level of CD4+, CD8+, and PD-1 immune cell infiltration; the amount of FOXP3+ CD4 + Treg cells was also higher in SCCC than ACC, as well as CD3+ LAG3+ T cells. For the total region, FOXP3+ CD4 + Treg cells, CD3+ LAG3+ T cells, and PD-1+ T cells were enriched in SCCC compared with ACC, while CD57+ natural killer T (NKT) cells were accumulated with a marginally higher level in ACC ( p = 0.049). TCR repertoires sequencing revealed a lower Shannon’s diversity ( p = 0.029) but higher clonality ( p = 0.047) of SCCC compared with ACC. PD-L1 expression identified by mIHC in SCCC was significantly higher than ACC, in the tumor ( p < 0.001), stroma ( p < 0.001), and total regions ( p < 0.001). Conclusions: SCCC of ASC had higher levels of TMB, Treg cells, TCR clonality, and PD-L1, and similar level of CD3+, CD4+, and CD8+ TILs, comprehensively reflecting the intratumor heterogeneity of TIME.

e20543 Background: CDKN2A and CDKN2B both acted as tumor suppressor genes by regulating the cell cycle, which in humans were located at chromosome 9, band p21.3. The frequencies of homozygous deletion (HomDel) in CDKN2A and CDKN2B in lung adenocarcinoma (LUAD) were 12.5% and 12.1%, respectively. However, the genomic, immunogenomic features and impact on the prognosis of LUAD patients with CDKN2A/B HomDel were still unclear. Methods: The cohort of this study was from The Cancer Genome Atlas (TCGA). A total of 508 LUAD patients, including 99 CDKN2A/B HomDel (homdel) and 509 CDKN2A/B wild (wild). This study explored the difference of genomic and immunogenomic landscape between homdel and wild by analysis of whole-exome sequencing (WES) and RNA sequencing data. Results: The most frequently mutated genes were TP53, TTN, MUC16, and CSMD3. Their frequencies in homdel and wild are 46% and 48%, 43% and 46%, 35% and 41%, 33% and 38%, respectively. There was no significant difference of tumor mutational burden (TMB) between homdel and wild (median TMB, 133 in homdel vs 177 in wild; Wilcoxon test, p = 0.11), and clinical characteristics including age, gender, smoking history, and tumor stage were not significantly different between homdel and wild. Homdel had a shorter overall survival (OS) than wild (Log-rank test, p = 0.04, Hazard Ratio: 0.7, 95% CI: 0.49-1.02), but there was no significant difference in progression-free survival (PFS) (Log-rank test, p = 0.05, Hazard Ratio: 0.73, 95% CI: 0.51-1.04). We used single sample gene set enrichment analysis (ssGSEA) to calculate the enrichment score (ES) of 25 immune-related pathways such as antigen presentation and T cell-mediated immunity, and then used the consensus clustering algorithm (ConsensusClusterPlus) to cluster homdel and wild respectively, and both clustered into low and high immune infiltration groups. For the high immune infiltration and low immune infiltration in homdel and wild, high immune infiltration had a longer OS (Log-rank test, p = 0.009, Hazard Ratio: 2.19, 95% CI: 1.22-3.94) and PFS (Log-rank test, p = 0.044, Hazard Ratio: 1.8, 95% CI: 1.01-3.2) than low immune infiltration in homdel. However, there was no significant heterogeneity between high and immune infiltration in terms of PFS (Log-rank test, p = 0.28, Hazard Ratio: 1.21, 95% CI: 0.87-1.68) and OS (Log-rank test, p = 0.96, Hazard Ratio: 1.01, 95% CI: 0.71-1.44) in the wild group, the wild group had longer OS than homdel group with low immune infiltration (Log-rank test, p = 0.003, Hazard Ratio: 0.5, 95% CI: 0.29-0.88), while had the same OS with homdel with high immune infiltration, irrespective of immune infiltration. And so was PFS (Log-rank test, p = 0.005, Hazard Ratio: 0.48, 95% CI: 0.27-0.82). Conclusions: CDKN2A/B homdel was an unfavorable prognostic factor for LUAD, but which with high immune infiltration might improve patient survival time.

e20573 Background: Although small-cell lung cancer (SCLC) is sensitive to chemotherapy and radiotherapy initially, nearly all patients recur often with treatment-resistant disease. Comparing genomic profiles and clonal architecture of paired treatment-naïve and recurrent tumors may provide novel insights into mechanisms underlying recurrence and susceptibility to further treatment. Methods: Paired tumor samples procured at diagnosis and relapse were collected from 11 patients (pts) with limited-stage SCLC treated with concurrent chemoradiation (CCRT). All tissues underwent whole exome sequencing (WES). Genomic landscape including somatic mutations, somatic copy number alterations (SCNAs), and clonal architecture were compared between treatment-naïve and paired recurrent tumor samples. Baseline and paired recurrent plasma samples from another 9 pts with SCLC treated with CCRT were performed deep sequencing using a targeted panel containing 1021 cancer related genes. Results: We observed TP53 and RB1 alterations including mutations and SCNAs in the majority of treatment-naïve ( TP53 in 10 of 11, RB1 in 7 of 11 pts) and relapse samples ( TP53 in 8 of 11, RB1 in 8 of 11 pts). Tobacco exposure related mutational signature was most or second predominant in 11 pre-treatment and 9 recurrent samples, respectively. Compared with primary tumors, relapsed tumors showed significantly increased number of mutations (220 vs 210, p = 0.016). A high proportion of shared mutations between baseline and PD samples ranging from 39% to 94% was observed. More than half (n = 6) of the 11 pts had increased chromosomal instability in recurrent tumors. A total of 687 relapse-specific also called acquired mutations in 633 genes were identified, in which 158 mutations with cancer cell fraction (CCF) > 0.6. These genes were enriched in PI3K-ATK signaling pathway and covered 73% (8/11) pts. Of the 9 paired plasma samples performed by targeted sequencing, genes with specific mutations and mutations with increased CCF in relapsed plasma samples were also enriched in PI3K-ATK pathway. In vitro proliferation assay indicated that PI3K inhibitor gave rise to a marked decrease in proliferation after combined with cisplatin, implying the potential mechanism of chemoradiation resistance. The number of clones in recurrent samples was higher than that in pre-treatment samples (13 vs 12, p = 0.004), indicating that new clones emerged under the treatment and tumor heterogeneity increased. Patients characterized by weakening of the major treatment-naïve clone tended to exhibit relatively longer PFS than those with sustaining major clone in both b baseline and relapsed tumors (p = 0.034). Conclusions: PI3K-ATK pathway alterations are frequent in recurrent SCLCs, which may be a candidate resistant mechanism after chemoradiation. PFS is probably associated with clonal evolutionary pattern.

Background: Tumor infiltrating stromal cells and immune cells are the main components of the tumor microenvironment. Cancer-associated fibroblasts (CAFs) make up the bulk of cancer stroma and play a key role in tumor progression. Evidence indicates that CAFs are highly related to treatment response and prognosis. However, the effect of CAFs on immunotherapy response still remains unknown. Methods: RNA-seq and clinical data were downloaded from TCGA and GEO. The sva package was used to remove batch effects. The ssGSEA algorithm was used to assess the level of infiltration of 24 immune cells and fibroblasts from each sample. ImmuneScore was calculated using the ESTIMATE method to characterize the degree of immune infiltration. OS and DFS were analyzed using the K-M method. GO enrichment analysis is used to identify the biological process of genes. The TIDE algorithm and subclass mapping were used to predict the clinical response to immune checkpoint blockade. Results: We evaluated the infiltration abundance of 24 types of immune cells and fibroblasts in 1768 NSCLC samples. Positive correlation between immune infiltration and CAFs infiltration through multiple methods (immune cells, Immunescore, and clustering of immune cells) (Kruskal-Wallis and Pearson test, p < 0.05) were found. There were only two types of immune cells in two cohorts are beneficial for prognosis, but the number of IMFRs (Immune cells / CAFs) that are positive correlated with prognosis is more than half of the total number of classes (log-rank test; P < 0.05). Univariate cox regression analysis showed that almost all IMFRs tended to be favorable for prognosis. This phenomenon is called “CAFs-mediated immune resistance pattern (CMIRP)”. At the best cut-off point, patients with high fibroblast content had worse prognosis (log-rank test; P Conclusion: The “CMIRP” we proposed shed light on a more accurate assessment of immune status. CFR is a potential marker for prognosis and predictive efficacy of immunotherapy in NSCLC. Citation Format: Xinlong Zheng, Dongqiang Zeng, Pansong Li, Wenying Pen, Xuan Gao, Zhipeng Zhou, Jing Bai, Junhui Li, Jianming Ding, Deqiang Wang, Suya Zheng, Gen Lin. Interaction between CAFs and CD8+ T in non-small cell lung cancer (NSCLC) affects prognosis and efficacy of immunotherapy [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 410.

e21103 Background: Small cell lung cancer (SCLC) is a type of aggressive malignancy with poor prognosis, accounting for 15% of lung cancers. Intratumoral heterogeneity (ITH) has been investigated in lung adenocarcinoma or squamous cell carcinoma, but its role in SCLC still remains unclear. Exploring ITH and tumor evolution of SCLC is essential for its treatment and prognosis. Methods: 102 multi-regional tumor tissues were collected from 34 operative SCLC patients (pts) before systemic therapy during Sep. 2009 to Sep.2018 from Sun Yat-sen University Cancer Center. All of the enrolled cases were confirmed as SCLC by immunohistochemistry. We performed whole-exome sequencing (WES) of all the tumor tissues and assessed the ITH using clonal and subclonal somatic mutations or copy number variants. ITH was defined as the proportion of subclonal genetic alterations of all. The relationship between ITH and overall survival (OS) was also explored. Results: Among the enrolled 34 SCLC pts, the median age was 64 year. 28 pts had the smoking history. 23 pts received adjuvant therapy after surgery. Stage I-II and III-IV pts accounted for 50% respectively. The most frequent mutated genes were TP53 (88%), RB1 (70%), TTN (68%), TCEB3CL (65%), MUC16 (56%), and all of them were clonal. The median overall ITH was 0.36 (ranging from 0 to 1), while the mutational ITH and the CNV ITH were 0.50 (0.22 to 1) and 0.49 (0.22 to 1). Univariate analysis revealed that postoperative treatment (HR = 0.27, 0.07-0.97, p= 0.006) and higher CNV ITH (HR = 0.28, 0.08-0.99, p= 0.009) were significant positive predictors of OS, while higher mutational heterogeneity was not associated with OS (HR = 3.34, 0.91-12.25, p= 0.21) significantly. Conclusions: TP53, RB1, TTN, TCEB3CL and MUC16 were probably clonal mutations as early events in SCLC evolution. CNV ITH might be a prognostic predictor in SCLC, which should be verified in a large-sample cohort.