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Background: Anaplastic lymphoma kinase (ALK) fusion, an important driven oncogene mutation, occurrs in 3%~7% non-small cell lung cancers (NSCLC), and EML4 is the most common partner gene. With the widespread application of next-generation sequencing (NGS), more gene breakpoint fusions have been discovered, and functional fusion transcripts can bring targeted clinical benefits. Clinical trials have shown that NSCLC patients with ALK fusion can obtain significant survival benefits through ALK tyrosine kinase inhibitor (ALK-TKI) treatment. In our case, a novel CLHC1/RNT4 intergenic region- ALK fusion was identified for the first time in a LUAD patient with BM, and the patient benefited from endostar and crizotinib sequential alectinib. Our case suggested the advantages of NGS for fusion detection and provided promising treatment options for NSCLC patients with BM harboring ALK fusions.

Clear cell meningioma (CCM), an unusual subtype of World Health Organization (WHO) grade II meningioma, represents only 0.2– 0.8% of meningiomas. Spinal CCMs are even rarer with unique clinical features: more common in younger patients; more likely to appear in lumbar spine; high recurrence rate. Although surgery and radiotherapy are the most common treatment for primary tumors and disease recurrence, there are lack of treatment options for recurrent or metastasis disease. It is urgent need to explore new effective treatment method. In our case, we firstly reported a rare spinal CCM patient with PD-L1 positive and multiple metastases benefiting from PD-1 inhibitor plus anti- angiogenesis therapy. This treatment program is effective, safe, and has a strong therapeutic reference value, which provides promising treatment options and the direction of future clinical trials for spinal CCMs.

Malignant gliomas are the most common brain tumors, with generally dismal prognosis, early clinical deterioration and high mortality. Recently, 2-aminoquinoline scaffold derivatives have shown pronounced activity in central nervous system disorders. We herein reported a series of 2-aminoquinoline-3-carboxamides as novel non-alkylator anti-glioblastoma agents. The synthesized compounds showed comparable activity to cisplatin against glioblastoma cell line U87 MG in vitro. Among them, we found that 6a displayed good inhibitory activity against A172 and U118 MG glioblastoma cell lines and induced cell cycle arrest in the G2/M phase and apoptosis in U87 MG by flow cytometry analysis. Additionally, 6a displayed low cytotoxicity to several normal human cell lines. In silico study showed 6a had promising physicochemical properties and was predicted to cross the blood-brain barrier. Moreover, preliminary structure-activity relationships are also investigated, shedding light on further modifications towards more potent agents on this series of compounds. Our results suggest this compound has a promising potential as an anti-glioblastoma agent with a differential effect between tumor and non-malignant cells.

Peritoneal metastasis of colorectal cancer is one of the most incident and fateful diseases among relapse cases. It shows a certain resistance to systemic chemotherapy. The perfusion system in clinic is complex and hard to be used in fundamental researches. This study aims at evaluating the effect of an improved hyperthermic intraperitoneal chemotherapy with Raltitrexed used in tumor-bearing mice with peritoneal metastatic colorectal carcinoma. The results showed that no severe adverse effect was observed. All control animals developed extensive peritoneal and mesenteric metastatic nodes. Tumor sites in the treatment groups were reduced significantly. The administration dose of Raltitrexed influenced concentration in systemic blood and peritoneal tissues. Temperature promoted the intracellular absorption of Raltitrexed significantly. Our findings reveal that hyperthermic intraperitoneal chemotherapy is an efficient therapy in treating peritoneal metastatic carcinoma in nude mice. It can effectively reduce the extension of carcinoma cells from macro and micro examination. The combination of hyperthermia and Raltitrexed resulted in an improved therapeutic effect on animal models.

Background: Human polypyrimidine tract binding protein 3 (PTBP3) was first discovered in 1999 and has been well characterised as a differentiation regulator. However, its role in human cancer has rarely been reported. Our previous study revealed increased PTBP3 protein level in gastric cancer tissues. Downregulation of PTBP3 suppressed the proliferation and differentiation of gastric cancer cells in vivo. Methods: PTBP3 mRNA levels in human gastric cancer and adjuvant non-tumour tissues were detected. Apoptosis and 5-FU effect were determined in PTBP3-silenced gastric cancer cells. Underlying molecular mechanisms were investigated. Results: MRNA expression of PTBP3 was upregulated in gastric cancer tissues, especially in those at an advanced stage. PTBP3 silencing led to apoptosis, under which modulation of PTB and thereby switch of Bcl-x pre-mRNA splicing pattern might be an important mechanism. Further research found that inhibition of PTBP3 expression enhanced the chemosensitivity of gastric cancer cells towards 5-FU treatment. This was mediated by reduced expression of histone deacetylase 6 (HDAC6), which further inhibited the phosphorylation of Akt and the expression of thymidylate synthase (TYMS), the critical determinant of 5-FU cytotoxicity. Conclusions: PTBP3 might serve as a biomarker of gastric cancer or potential target for anti-cancer therapy.

e21059 Background: Anaplastic lymphoma kinase ( ALK), c-ros oncogene 1 receptor tyrosine kinase ( ROS1), ret proto-oncogene ( RET) and neurotrophic receptor kinase ( NTRK) are important driver genes in lung cancer. Each of these four genes fusions as therapeutic targets have been recommended by the national comprehensive cancer network (NCCN) guidelines for non-small cell lung cancer (NSCLC). Malignant pleural effusion (MPE) is observed in multy types of malignancies, especially in lung cancer. Studies have indicated that next generation sequencing (NGS) detection of somatic mutations by MPE is reliable. Nevertheless, the distribution and comparison of fusions in lung cancer between malignant pleural effusion and tumor tissue specimen have not been well characterized yet. Methods: In this study, 9-gene, 69-gene and 539-gene panel NGS profiling were performed by MPE and tumor tissue in 640 and 10013 patients with lung cancer respectively. The variation analysis between patients with or without gene fusion in different sample groups were calculated by fisher exact test via SPSS v26.0. Results: In lung cancer patients using MPE detection, 59 of 640 (9.22%) were found harboring at least one of the four gene fusions, and the ratio of ALK, ROS1, RET and NTRK fusion was 6.25% (40/640), 1.41% (9/640), 0.63% (4/640) and 0.47% (3/640) respectively. Meanwhile, we found gene fusions in 6.25% (626/10013) lung cancer patients using tumor tissue detection, including 3.66% (366/10013) ALK fusion, 1.07% (107/10013) ROS1 fusion, 1.09% (108/10013) RET fusion and 0.22% (626/10013) NTRK fusion. Significant differences of gene fusion frequency between different sample groups were found via fisher exact test ( p = 0.005), especially ALK fusion ( p = 0.001). Conclusions: The frequency of fusion in MPE was significantly higher than in tumor tissue, especially ALK fusion. However, as a retrospective study, the conclusions and the specific mechanisms are needed to be verified.

10522 Background: Lung cancer is one of the most common types of cancer, ranking the first in the incidence and mortality of malignant tumors in the world and China. Although studies have been reported that genetic susceptibility to lung cancer is associated with certain germline mutations, the relationship between lung cancer risk and inherited genetic factors remains relatively elusive. However, the effect of germline mutation on TMB in lung cancer has not been explored. Herein, DNA genomic profiling was performed through NGS with a 539-gene panel to explore the germline mutations and the relationship with TMB in Chinese patients with lung cancer. Methods: We retrospectively analyzed the germline mutations through a comprehensive 539-gene profiling of 3541 Chinese patients with lung cancer. 539-gene panel contained germline mutations in 90 hereditary tumor-associated genes. We screened out the pathogenic and likely pathogenic germline mutations according to the standards and guidelines for the interpretation of sequence variants of The American College of Medical Genetics and Genomics (ACMG), and picked out there is no records in Clinvar database and no literature report. TMB of tissue or blood ctDNA in 3541 patients were further analyzed in with pathogenic mutations (P group), with likely pathogenic mutations (LP group), and no germline mutations group (Non-P group). The difference in TMB was analyzed via the Wilcoxon sign test. Results: In 3541 patients with lung cancer, 177 (4.999%) patients were identified harboring pathogenic or likely pathogenic germline mutations, in which 78 P group and 99 LP group, the rest 3364 were Non-P group. The highest prevalence of germline mutation was found in BRCA2 (0.565%), ATM (0.339%), MUTYH (0.282%), and BRCA1 (0.254%). In 177 patients with pathogenic or likely pathogenic germline mutations, 67 mutations were recorded as UNK (unknow) in Clinvar database and no literature report. The media TMB of tissue in P group, LP group and Non-P group were 5.149, 5.535 and 5.547 respectively. The media TMB of blood ctDNA in P group, LP group and Non-P group were 4.257, 3.945 and 4.483 respectively. There was no statistical difference in TMB between P and Non-P groups (tissue p = 0.98; ctDNA p = 0.5). Conclusions: In our study, we firstly identified 67 novel germline mutations and studied on the relationship between germline mutations and TMB in lung cancer, which expanded the understanding of germline mutations.

3119 Background: Li-Fraumeni syndrome (LFS, OMIM #151623) is an autosomal dominant cancer predisposition syndrome. Typical LFS tumors comprise adrenocortical carcinomas, sarcoma, breast cancer and central nervous system tumors. LFS is also associated with an increased risk of a multitude of other common types of cancer, such as prostate cancer, lung cancer, gastric cancer, colorectal cancer, ovarian cancer, melanoma, etc. TP53 germline mutations are the most common gene with LFS. Germline mutations of CHEK2 have been identified as another predisposing gene and associated with a range of cancer types. However, the pattern of TP53 and CHEK2 germline mutations in malignant tumors remains unknown. Methods: We identified 8535 malignant solid tumors patients without selecting age or family history in a retrospective cohort. Germline mutations were categorized based on ACMG (American College of Medical Genetics and Genomics) guidelines in pathogenicity. The patients were divided into three groups, P group (with pathogenic mutations), LP group (with likely-pathogenic mutations) and Non_P group (neither pathogenic nor likely-pathogenic mutation). Statistical significance was defined as P-value less than 0.05. Results: A total of 461 (461/8535) patients carried TP53 or CHEK2 germline mutations were identified, in which 15 patients with pathogenic mutations and 17 patients with likely-pathogenic mutations. One patient with lung cancer in LP group carried TP53 homozygous mutation ( p. Ser215Ile), and the remaining 31 patients all carried heterozygous mutations. Among these 31 carriers, 16 (51.6%) carried nonsense or missense mutations (10 for nonsense and 6 for missense mutations). 3 patients in the P group carried CHEK2 p. Y139* (one liver cancer patient and two lung cancer patients) were identified. The median age of group P, LP and Non_P was 55 (39 for TP53, 61 for CHEK2), 63 (52 for TP53, 66 for CHEK2) and 59, respectively. Somatic mutation analysis found no significant difference in tumor mutation burden (TMB) among three groups. The SNV/INDEL mutation frequency of LRP1B in the P or LP group was significantly lower than the Non_P group. Conclusions: Our data showed that 0.375% (32/8535) malignant solid tumor patients carried TP53 (16/8535) or CHEK2 (16/8535) germline pathogenic or likely-pathogenic mutations. The relationship between germline mutations and cancer susceptibility will be studied in the future.[Table: see text]

2638 Background: Immune checkpoint inhibitors (ICIs), such as programmed death(ligand)1 (PD-(L)1) and cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) inhibitors, have dramatic effects on treatment in patients with various malignancies. High tumor mutation burden (TMB) is predictive of clinical response to ICI in multiple cancer types. Although age-related immune dysfunction might induce difference on the efficacy of ICIs between younger and older patients, the potential effect of age on the efficacy of ICIs remains little known and controversial. Herein, we aimed to analysis the association between age and the efficacy of ICIs based on MSKCC cohort. Methods: We screened out 1661 patients having complete information with advanced cancer, whose tumors underwent next-generation sequencing (NGS) detection and who were treated with at least one dose of ICI in MSKCC cohort. All patients were divided into two groups according to age, the younger group (age ≤50-year old) and the older group (age > 50-year old). We further analyzed the differences in overall survival (OS) and TMB between the two groups. The pooled hazard ratios (HRs) and 95% confidence intervals (CIs) were calculated via Cox regression model for OS and P-values were calculated via the Wilcoxon sign test for TMB. We analyzed the effect of age on ICI in lung cancer using the same way. Results: In 1661 patients with cancer in our study, 312 (19%) younger and 1349 (81%) older patients were found. The pooled HRs for OS was 1.28 (95% CI: 1.09-1.52) in younger group compared with older group. In 1661 patients with cancer, there was 350 (21%) patients with lung cancer, including 30 (9%) younger and 320 (91%) older patients. The pooled HRs for OS was 1.45 (95% CI: 0.95-2.23) in younger group compared with older group in lung cancer. In addition, TMB in older group was higher than in younger group and significant difference of TMB was found via the Wilcoxon sign test (p = 2.6e-10) between the two groups, especially in lung cancer (p = 1e-4). Conclusions: Our study assessed the impact of age on the efficacy of ICIs using the threshold of 50 years old for the first time and we founded that patients in older group had higher TMB and longer OS than younger group.

e15001 Background: Overall survival (OS) is an important endpoint, with the advantage that there is minimal ambiguity in defining an OS event; the patient is either alive or dead. Some single gene mutations, such as TP53, were identified as important predictors of shorter OS in malignant tumors. However, the relationship between gene co-mutation and OS of malignant tumors is poorly defined. Methods: Genomic and OS data were derived from The Cancer Genome Atlas (TCGA) databases. We defined the double-mutant gene pair as “hit pair” in the specific cancer type if statistically significant OS difference was observed in at least one of three comparison (double-mutant samples vs gene A-only-mutant samples; double-mutant samples vs gene B-only-mutant samples; gene A-only-mutant samples vs gene B-only-mutant samples). Results: PIK3CA-TP53 co-mutation was evaluated as “hit pair” in seven cancer types: GBM (Glioblastoma multiforme), HNSC (Head and neck squamous cell carcinoma), LUSC (Lung squamous cell carcinoma), SARC (Sarcoma), SKCM (Skin cutaneous melanoma), UCEC (Uterine corpus endometrial carcinoma), and UCS (Uterine carcinosarcoma). In SARC and SKCM, the expected hazard ratio was higher in both-mutant group than TP53-only-mutant group, and neither genes were correlated with OS. Especially in SKCM, after we took “neither” group (both genes were wild-type) into comparison, hazard ratio of both-mutant group was significantly higher than neither group. Single-gene mutation didn’t make difference on hazard ratio, which indicated that the both-mutant interaction made the leading contribution. In GBM, LUSC, and UCS, the significant difference of hazard ratio between TP53-only-mutant group and PIK3CA-only-mutant group was neutralized in the both-mutant group, to some extent equivalent to “average effect (OS curve of double-mutant group was between that of only single gene mutant groups)". For PIK3CA-TP53 in HNSC, both-mutant group and TP53-only-mutant group carried with a higher hazard ratio than PIK3CA-only-mutant group. It suggested that the increasing hazard ratio in both-mutant group might be resulted from TP53 mutation. For the circumstance in UCEC, TP53 and PIK3CA were testified to be correlated with higher and lower hazard ratio, respectively and both-mutant group and TP53-only-mutant group both with significantly higher hazard ratio than PIK3CA-only-mutant group, which further verified the greater strength of TP53 than PIK3CA in both-mutant group. Conclusions: In summary, our study identified PIK3CA-TP53 co-mutation as a predictor of OS in seven cancer types: GBM, HNSC, LUSC, SARC, SKCM, UCEC, and UCS, especially in SARC and SKCM.

e15002 Background: Kinase fusions have been proved as driver factors and drug targets for lung cancer. DNA-based next-generation sequencing (NGS) has been widely used for gene fusion detection in patients with lung cancer and intergenic-breakpoint fusions can be detected, in which breakpoints localize to intergenic regions. Theoretically, intergenic-breakpoint fusions are unlikely to be functional, but recent studies have found that intergenic-breakpoint fusions are potentially activated. However, the proportion of intergene-breakpoint fusion occurrence in lung cancer is still unclear. Hereby, next generation sequencing was performed to describe lung intergenic-breakpoint fusions for further research. Methods: Tumor tissue, pleural effusion, or blood samples of 6798 patients with lung cancer from Simceredx were collected for targeted NGS panel sequencing from June 2020 to January 2021. Gene rearrangements of ALK, ROS1, RET, NTRK(1-3) were assessed. Results: We found gene fusions in 4.43% (301/6798) patients with lung cancer totally, of which 59.1% (178/301) ALK fusions, 18.9% (57/301) RET fusions, 15.9% (48/301) ROS1 fusions and 6.64% (20/301) NTRK(1-3) fusions. The intergenic-breakpoint fusions accounted for all fusions of ALK, RET and ROS1 genes were 24.2% (43/178), 22.8% (13 /57) and 18.8% (9/48), respectively. In patients with NTRK intergenic fusions (50.0%, 10/20), NTRK1, NTRK2 and NTRK3 was involved in 2, 1, 7, respectively. Co-occurrence of different fusions was rare. Conclusions: Our data firstly revealed the specific intergenic-breakpoint fusions ( ALK, RET, ROS1, NTRK1-3) in lung cancer. The functional consequences identifying by RNA or protein assay and clinical validation need to be carried out in the future.

e22500 Background: Breast cancer susceptibility genes BRCA1 and 2 are tumor suppressor genes and they play an important role in DNA damage response and repair during homologous recombination. Hereditary breast and ovarian cancer (HBOC) syndrome is an autosomal dominant disease due to BRCA1 and 2 germline mutation. Germline mutations of BRCA1 and 2 genes significantly increase the risk of developing breast cancer, ovarian cancer, prostate cancer and a broad range of cancers. PARP inhibitor (PARPi) monotherapy and combinations have shown promising efficacy against a variety of cancer types with BRCA mutations. Nevertheless, the knowledge of incidence of BRCA1 and 2 germline mutations in solid tumor remains poorly understood. Herein, next generation sequencing of 539-gene profiling was performed to explore the incidence of BRCA1 and 2 germline mutations in Chinese solid tumors. Methods: We retrospectively analyzed the BRCA1 and BRCA2 germline mutations from a comprehensive 539-gene profiling of 8535 Chinese patients with pan-cancer. 539-gene profiling contains the somatic mutations in tumor tissue or blood ctDNA and the germline mutations in blood leukocyte. We screened out the pathogenic and likely pathogenic mutations in BRCA germline mutations, and calculated the mutation frequency and the median age in every cancer type. Results: In 8535 patients with pan-cancer, 110 patients were found pathogenic or likely pathogenic germline mutations in BRCA gene and the mutation frequency was 1.29%, of which 40 BRCA1 mutations and 70 BRCA2 mutations were found in patients, respectively. The total median age was 58.15. Eliminated a few types of cancers that had a smaller number (less than 50), the higher frequency of mutations contained ovarian cancer (14.78%, media age 58), prostatic cancer (6%, media age 58.33), breast cancer (5.2% media age 57.33). The details of the top 8 cancer types with mutation frequency and media age were shown in Table. Conclusions: This was the first report of the incidence of BRCA1 and 2 germline mutations in Chinese solid tumors, which expanded the understanding of BRCA1/2 and provided a direction for clinical trial design of PARPi monotherapy and combinations.[Table: see text]

Disintegrin and metalloproteinase 28 (ADAM28) is a member of the disintegrin and metalloprotease domain (ADAM) family. It is associated with the growth and metastasis of various malignancies in vivo, but its role in gastric cancer remains unclear. The purpose of this study was to investigate the effect of ADAM28 derived from gastric cancer and endothelium on gastric cancer cells and its related mechanisms. In this study, Western blot analysis and q-PCR results showed that ADAM28 was up-regulated in gastric cancer cell lines. The TCGA database showed that patients with high ADAM28 expression had significantly shorter overall survival than those with low ADAM28 expression. By MTT analysis, wound healing assay, and flow cytometry, we found that overexpression/knockdown of ADAM28 expression in gastric cancer cells can regulate cell proliferation, apoptosis and migration in vitro. In addition, overexpression/knockdown of ADAM28 in human umbilical vein endothelial cells (HUVECs) in the upper ventricle can regulate the apoptosis of lower ventricular gastric cancer cells in the co-culture system. Furthermore, ELISA demonstrated that knockdown of ADAM28 from endothelial cells increased the expression of von Willebrand Factor (vWF) in the supernatant. We found that ADAM28 both from gastric cancer cells and HUVECs eliminated vWF-induced apoptosis of gastric cancer cells by cleaving vWF, and the addition of the vWF knockdown plasmid eliminated the increase of integrin β3, p-TP53 and c-Casp3 caused by ADAM28 knockdown. In conclusion, ADAM28 from endothelium and gastric cancer may cleave vWF to eliminate vWF-induced apoptosis of gastric cancer cells and play an pro-metastasis effect.

Background Platelet glycoprotein Ibα (GPIbα) extracellular domain, which is part of the receptor complex GPIb-IX-V, plays an important role in tumor metastasis. However, the mechanism through which GPIbα participates in the metastatic process remains unclear. In addition, potential bleeding complication remains an obstacle for the clinical use of anti-platelet agents in cancer therapy. Methods We established a series of screening models and obtained rat anti-mouse GPIbα monoclonal antibodies (mAb) 1D12 and 2B4 that demonstrated potential value in suppressing cancer metastasis. To validate our findings, we further obtained mouse anti-human GPIbα monoclonal antibody YQ3 through the same approach. Results 1D12 and 2B4 affected the von Willebrand factor (vWF)-GPIbα interaction via binding to GPIbα aa 41-50 and aa 277-290 respectively, which markedly inhibited the interaction among platelets, tumor cells, and endothelial cells in vitro, and reduced the mean number of surface nodules in the experimental and spontaneous metastasis models in vivo. As expected, YQ3 inhibited lung cancer adhesion and demonstrated similar value in metastasis. More importantly, for all three mAbs in our study, none of their Fabs induced thrombocytopenia. Conclusion Our results therefore supported the hypothesis that GPIbα contributes to tumor metastasis and suggested potential value of using anti-GPIbα mAb to suppress cancer metastasis.

Polypyrimidine tract-binding protein 3 (PTBP3) is an essential RNA-binding protein with roles in RNA splicing, 3′ end processing and translation. Although increasing evidence implicates PTBP3 in several cancers, its role in gastric cancer metastasis remains poorly explored. In this study, we found that PTBP3 was upregulated in the gastric cancer tissues of patients with lymph node metastasis. Patients with high PTBP3 expression levels had significantly shorter survival than those with low PTBP3 expression. Overexpression/knockdown of PTBP3 expression had no effect on proliferation, whereas it regulated migration and invasion in vitro. In addition, when a mouse xenotransplant model of MKN45 was established, knockdown of PTBP3 in MKN45 cells caused the formation of tumours that were smaller in size than their counterparts, with suppression of tumour lymphangiogenesis and metastasis to regional lymph nodes. Furthermore, we identified caveolin 1 (CAV1) as a downstream target of PTBP3. RNA immunoprecipitation (RIP) assays and dual-luciferase reporter gene assays indicated that PTBP3 interacted with the CU-rich region of the CAV1 gene to downregulate CAV1α expression. Knockdown of CAV1α abrogated the reduction of FAK and Src induced by PTBP3 knockdown. In summary, our findings provide experimental evidence that PTBP3 may function as a metastatic gene in gastric cancer by regulating CAV1 through alternative splicing.

The aerial parts of Salvia miltiorrhiza Bunge, as the non-medicinal parts, are always discarded during harvesting, resulting in a huge waste of resources and environmental pressure. Due to the high flavonoid content and their antioxidant activities characteristics, the aerial parts of S. miltiorrhiza can be developed into natural antioxidants and used in foods. A high-speed counter-current chromatography (HSCCC) method, using a two-phase solvent system composed of tert-butyl methyl ether/n-butanol/acetonitrile/water (3:1:1:20, v/v), was the first to successfully isolate five flavonoids from the aerial parts of S. miltiorrhiza in one attempt, and separately categorized as rutin (1), isoquercitrin (2), kaempferol-3-O-&alpha, l-rhamnopyranosyl-(1&rarr, 6)-&beta, d-glucopyranoside (3), kaempferol-3-O-&beta, d-glucopyranoside (4) and apigenin-7-O-&beta, d-glucopyranoside (5) after identification. The purities of these plant isolates were 97.3%, 99.5%, 92.8%, 98.1% and 98.7%, respectively. All the flavonoids were identified by HR-ESI-MS, 1D and 2D NMR. Compounds 3 and 5 were firstly isolated from the plant of S. miltiorrhiza. Results from antioxidant assays showed that rutin (1) and isoquercitrin (2) had higher antioxidant capacities compared to L-ascorbic acid as the positive control.