e14020 Background: Glioblastoma multiforme (GBM) is the most common primary brain tumor with complex genetic alterations and genomic profiles. EGFR amplification occurs in approximately 40% of primary GBM，EGFR mutation is also a prevalent genetic abnormality in GBM. A series of potential therapies targeting EGFR variants are currently in development, such as tyrosine kinase inhibitors (TKIs), monoclonal antibodies, vaccines, etc. However, to date, EGFR-targeted therapy continues to face significant challenges. A deeper understanding of EGFR variant profiles and pathobiology of GBM will be required. Methods: Next-generation sequencing of 131-gene profiling was performed to analyze EGFR activating mutations from 891 Chinese glioma patients in 2019-2020. Somatic mutations and copy number variations were detected following the standard operating procedure (SOP). We screened out the EGFR activating mutation and calculated the mutation frequency and the tumor location. Results: 51 GBM patients had activating mutations. The average age was 49 years (range,25-73 years). Most of the tumors occurred in the cerebral hemisphere (56.9%), followed by ventricles (3.9%) and thalamus (3.9%), etc. 43/51 (84.3%) were accompanied by high-fold EGFR amplification. 9/12 (75%) were EGFR intracellular mutation occurs with EGFR amplification, 31/35 (88.6%) were EGFR extracellular mutation occurs with EGFR amplification. Moreover, 4 patients carried EGFR intracellular and extracellular mutation occurs with EGFR amplification. There was no significant difference between the ratio of intracellular mutations and extracellular mutations with EGFR amplification (P=0.3496). Conclusions: In our GBM patients, EGFR activating mutations were accompanied by high-fold EGFR amplification. As we know that, GBM benefit from EGFR-TKIs may be limited. Therefore, multi-targeted combination therapy research could be considered in the future.
e14021 Background: The updated 2016 edition of the WHO Classification of CNS tumors indicates that IDH1 R132H, H3 K27M mutations, and co-deletion of 1p19q are strong stratification and prognostic markers glioma. FISH/IHC, as the commonly detected methods, present specific false-negative rates in the actual condition. Methods: In our study, IDH1 R132H status of 158 cases was assessed by IHC and NGS, and H3 K27M statuses of 83 patients were evaluated by IHC and NGS. 22 positive cases of 1p/19q co-deletion, all confirmed by FISH, were assessed by NGS. Results: For IDH1 R132H, 2 cases were IHC negative and were positive as confirmed by NGS. Another 10 patients with weakly IHC positive results were negative in NGS. Combined with histologic hallmarks, 6 cases of these samples could be diagnosed as glioblastoma, IDH wildtype; 1 case with POLE could be diagnosed as giant cell glioblastoma; 3 cases with BRAF V600E mutation, BRAF fusion, and ATRX mutation, respectively, could be diagnosed as pilocytic astrocytoma. Towards H3 K27M, 3 cases with IHC weakly positive were negative in NGS. Among these samples, 2 cases were diagnosed as glioblastoma, IDH wildtype by molecular and histologic hallmarks, and 1 case was medulloblastoma, SHH. Using NGS, IDH1 R132H /H3 K27M statues can be distinctly distinguished in which that is unknown by IHC. The results of NGS and FISH showed a 90.9%(20/22) consistent rate for the 1p19q co-deletion. 1 case was 1p deletion and intact 19q by FISH while 1p19q co-deletion by NGS because of the 19p deletion. 1 case was 1p19q co-deletion by FISH but 1p19q wildtype by NGS, diagnosed as glioblastoma, IDH wildtype with chr7+/10-. Conclusions: In our study, the agreement between NGS results and clinical pathology diagnosis was approximately 100%. NGS may act as the primary technology of molecular classification in glioma in the future.
e14012 Background: Diffuse midline gliomas (DMGs) with H3 K27 altered are extremely aggressive WHO grade IV tumors, with no significant therapeutic progress made in the past. DMGs with H3 K27 altered have been classified as a rare subtype of glial tumor according to 2021 WHO Classification of CNS Tumors. K27M mutation always occurs in the H3F3A gene or HISTIH3B/C gene, and the majority of DMGs harbor H3 K27M-mutation. Studies have been reported that systematic in vitro modification of the lysine 27 predicts K27I as the only substitution other than K27M to result in a repressive effect on H3K27me3. Herein, we reported two adult patients with DMGs harboring somatic H3-K27I mutation. It is important to identify H3 K27-mutation accurately for accurate diagnosis, prognostication, and also for treatment selection. Methods: Next-generation sequencing (NGS) 539-gene panel (Simceredx) profiling was performed using postoperative tissue. The molecular characteristics, K3 K27 mutation types, and other co-mutations were also evaluated. Results: Patient one was a 36-year-old female. Magnetic resonance images (MRI) of the brain were performed and showed the pons tumor in the brainstem. Postoperative pathology showed astrocytoma (WHO Grade II) with diffuse slightly dense cells. NGS results showed that the patient was carried H3F3B exon2 p.K27I (allele frequency, AF 57.64%), TP53 exon7 p.M237I (AF 80.27%). Patient two was a 44-year-old male and was diagnosed with spinal cord glioma, WHO grade III. NGS panel profiling was performed and H3F3B exon2 p.K27I (AF 30.46%), ATRX exon9 p.S1173* (AF 75.23%), KRAS exon2 p.G12A(AF 38.11%) and NF1 intron42 c.6580-1G > A(AF 36.44%) were identified. Conclusions: In our study, we identified the H3F3B K27I mutation in two adult DMG patients by NGS, which expanded the detection gene spectrum of DMG patients. The type of histone H3 mutated could also predict the outcome and accurate diagnosis and glioma grading of DMG patients, which was more efficient than clinical and radiological characteristics of the tumors. Accurate genetic testing should be given more attention to those patients.
e21062 Background: In previous studies, researchers have demonstrated that activation of the epidermal growth factor receptor (EGFR)-mutant pathway induced PD-L1 expression. In this study, we explored the correlation between PD-L1 expressions and EGFR variations. Methods: This study enrolled 2417 non-small cell lung cancer(NSCLC) patients harboring diverse EGFR mutations, including exon 19 deletions(19del)(n = 1045), L858R(n = 906), G719X(n = 176), S768I(n = 62), L861Q(n = 89) and exon 20 insertions(20ins)(n = 139). The stages range from I through IV, the majority were female(n = 1452, 60.07%) and adenocarcinoma(n = 1788, 73.98%), median age was 62(range 24-92). In this study, we retrospectively analyzed variants using next-generation sequencing(NGS). PD-L1 status was determined by VENTANA PD-L1 (SP263) Assay, TPS≥50% and TPS＜1% was the cut-off value for PD-L1 high-expression and negative separately. The 95% confidence interval (CI) was used to estimate the precision of the odds ratio (OR). Results: 1) Even though we observed subtle differences of PD-L1 high-expression ratio among various subtypes(19del：9.57%, L858R: 9.27%, G719X: 12.50%, S768I: 11.29%, L861Q: 7.87%, exon 20ins: 12.23%) in total patients, that PD-L1 high-expression was more likely to shown with G719X/S768I/exon 20ins than with 19del/L858R/L861Q, there was no statistically significant between 19del and other mutation：L858R(p = 0.8224), G719X (p = 0.2319), S768I(p = 0.6563), L861Q(p = 0.5982), exon 20ins(P = 0.3247). 2)We analyzed 742 treatment-naive EGFR-mutant NSCLC patients (PD-L1 negative, n = 624; high-expression, n = 118), PD-L1 high-expression group come with higher frequency of EGFR copy number variation(CNV)(OR = 2.2364, 95%CI, 1.4576-3.4312, p = 0.0002). Importantly, the ratio of high-level EGFR CNV(CN≥6) was higher in PD-L1 high-expression group than that in the negative group(OR = 3.0668, 95% CI, 1.3745-6.8427, p = 0.0062). Conclusions: In the newly diagnosed EGFR-mutant NSCLC patients, PD-L1 high-expression patients come with more frequent high-level EGFR CNV. There is no statistical significance between PD-L1 high-expression and EGFR mutation subtypes. As for how the EGFR signal affects PD-L1 expression, the mechanism needs to be further explored. The association between driver genes and immune checkpoints is a definite interest of future investigations.
10578 Background: DICER1 syndrome is a rare genetic condition predisposing to multiple cancer types and causes by germline DICER1 variants. Deleterious mutations identified were mostly located in the RNase III domain. VUSs found in other domains may be also crucial in the inheritance of high-risk neoplasms although there is insufficient evidence. Our study aimed at describing the spectrum of DICER1 variants detected in solid tumors to improve the identification of potentially high-risk DICER1 variants. Methods: Germline mutations including SNV, small INDEL in 448 patients with solid tumors were analyzed by next-generation sequencing (NGS) panel. The pathogenicity of germline mutations was categorized based on American College of Medical Genetics and Genomics (ACMG) guidelines. Results: In total, 3 (0.67%) patients (diagnosed as bladder cancer, schwannoma, and medulloblastoma) were identified harboring truncating pathogenic (P)/likely pathogenic (LP) germline mutations in the RNase III domain. The remaining 445 (99.33%) patients carried 447 uncertain significance (VUS) mutations, of which 416 (92%) were missense mutations lied in different domains and 52% (234/450) located in exon 20-23. The median age was 60 years old with an age range from 0 to 90. The higher frequency cancer type contained lung cancer (35.9%), glioma (10.0%), liver cancer (8.4%). In addition, the two highest frequencies of DICER1 missense variants were c.3334A > G (p. Asn1112Asp, n = 58) and c.3227G > A (p. Ser1076Asn, n = 53), which lied in unknown functional domain of the protein and had been reported in Clinvar. Their clinical significance and pathogenicity remain further study. Conclusions: In our study, DICER1 germline mutations mostly occurred in exon 20-23 and 92% were missense mutations. We reported 3 new cases of tumors associated with DICER1 syndrome, which expanded the DICER1-related tumor spectrum. Understanding the clinical significance of germline DICER1 VUS could improve the identification of potentially high-risk variants. Reclassifying these variants could make them useful for predictive, prognostic, and preventive purposes in clinical practice.
e14018 Background: Diffuse midline glioma H3 K27M–mutant is a specific entity added to the 2016 update of the WHO classification of CNS tumors. H3K27M-mutant gliomas are diagnosed primarily in children and adolescents with TP53 and/or ATRX mutations. However, the characteristics of H3 K27M-mutant gliomas in adults have not been explicitly described. Methods: We performed the 131-gene panel targeted sequencing on tumor samples from 33 adults H3 K27M-mutant gliomas( > 18 years) and 13 children and adolescents H3 K27M-mutant gliomas(≤18 years) in a CAP certified laboratory. Somatic mutations, copy number variations, and fusion genes were detected following the standard operating procedure (SOP). The MS-based assay measured MGMT promoter methylation. We calculated the tumor location and the median age in different cohorts. Results: In our adult cohort, 22/33（66.7%）had a midline location（spinal cord n = 2, thalamus n = 7, brainstem n = 6, cerebellum n = 3, pineal region n = 1, Basal ganglia region n = 3）, 6/33（18.1%）had a non-midline location (Lateral ventricle n = 2, Cerebral hemispheres n = 4). In the children and adolescents cohort, 11/13（84.6%）occurred in midline location, 1/13(7.7%) occurred in the Lateral ventricle. MGMT promoter methylation did not differ in adult and pediatric H3 K27M-mutant gliomas (12.1% vs. 0). H3 K27M-mutant adult gliomas significantly co-occurred with the NF1 mutation(P = 0.008937). The median age of H3 K27M-mutant adult gliomas with NF1 modification (13/33, 39.4%) is higher than the NF1 wild type (49 years vs. 38 years, P = 0.107), although the difference has no statistical significance. Conclusions: In Chinese adults, as in children, H3 K27M mutation gliomas are characterized by a constant midline location, low rate of MGMT promoter methylation. Inconsistently, H3 K27M mutant adult gliomas are featured by a higher rate of NF1 mutations. Our molecular profiling analysis revealed the H3 K27M mutation in adult gliomas. Our research suggests potential molecular pathogenesis of H3K27M mutant adult gliomas and identifies more therapeutic targets for precision medicine.
e14523 Background: ACVR2A (activin A receptor type 2A) encodes a receptor that mediates the functions of activins, and it contains two polyadenine (A8) microsatellite loci, which are located in exon 3 and exon 10. Recent studies have shown that ACVR2A mutant was associated with microsatellite instability-high (MSI-H) and high tumor mutational burden(TMB-H) in gastrointestinal cancers. However, this association remains unclear in other solid tumors. Methods: We retrospectively analyzed the ACVR2A mutations from comprehensive 539-gene profiling of 10434 Chinese patients with pan-cancer. Somatic mutations in tumor tissue were assessed. We screened out ACVR2A mutations, calculated the mutation frequency, TMB in different types of cancer. Results: ACVR2A mutants were detected in 340 (2.9%) samples. The top 5 frequently cancers were colorectal cancer (98, 28.8%), lung carcinoma (68, 20%), hepatocellular carcinoma (64, 18.8%), gastric cancer (48, 14.1%) and biliary tract cancers (19, 5.6%). And 12.7% were other cancers (e.g., pancreatic cancer, small bowel adenocarcinoma, brain cancer). ACVR2A variants included truncation (50%), splicing site variant (9.0%), and other mutant types(41.1%). Truncation was the most common type, recurring in a few hot spots (K437Rfs*5/ K437Rfs*19, D96Tfs*54/ D96Rfs*4, V433del, R438Efs*19). The TMB in ACVR2A mutation group was significantly higher than that in ACVR2A wild-type group (p < 0.001). In addition, TMB ≥10 muts/Mb was seen in 76.7% tumors with ACVR2A truncation or splicing site mutant(INACT) and 20.6% tumors with other mutant types(VUS)(p = 0.002). The median TMB of INACT group and VUS group was 35.46 muts/Mb (0.74-344.12) and 6.62 muts/Mb(0.5-598.53), respectively. Conclusions: We analyzed the distribution of ACVR2A mutants in Chinese patients with solid tumors. Our data shows that ACVR2A truncation or splice site mutant type is significantly associated with TMB-H, and this may be potential molecular marker of immunotherapy.
e15565 Background: As we all know that patients with advanced colorectal cancer (CRC) are often diagnosed with liver-only metastases (LM), pulmonary-only metastases (PM) and widespread metastasis (WM). Various methods have also been applied to molecular mutation detection in CRC, However, the differences in molecular mutation profiles between the different metastatic lesions are unclear. Methods: In the period of September 2019 to January 2022, 376 LM, 111 PM and 116 WM were included in this retrospective analysis. The average age of patients with LM, PM and WM were respectively 61, 63 and 63 (range, 21-86, 39-95, 29-83). The proportion of male patients accounted for about 60% of LM (246/376), PM (69/111) and WM (74/116) . In this study, we retrospectively analyzed the single nucleotide variants (SNV), copy number variation (CNV) and fusion variants, detected in patients with different metastatic lesions by using next-generation sequencing (NGS). Pearson’s chi-square test was used to test differences in mutation frequency. P-values < 0.05 were considered significant. SPSS 26.0 was used for the statistical analyses. Results: In LM, PM and WM analyzed cohorts, SNV was detected in 370, 108 and 114 patients. CNV was detected in 225, 58 and 60 patients, and fusions were detected in 25,7 and 9 patients. In LM, PM and WM analyzed cohorts, the genes with the highest mutation frequency were APC (11.60%, 8.73%, 6.40%), TP53 (8.90%, 7.70%, 5.29%), KRAS (5.90%, 6.67%, 5.03%) . In addition, the SMAD4 mutation frequency was 2.13%, 0.82% and 1.04%, respectively. APC and SMAD4 mutation frequency was higher in LM than PM ( P= 0.012, P= 0.007) and WM ( P < 0.0001, P= 0.008). 1026, 221 and 281 CNV were detected in LM, PM and WM, respectively. The genes with the highest mutation frequency were MYC (43%, 7.24%, 4.63%), MET (5.95%, 7.69%, 5.34%), EGFR (5.36%, 7.24%, 4.63%) and FLT3 (4.39%, 4.98%, 6.05%) in LM, PM and WM. The SMAD4 mutation frequency were 0.58% and 1.07% in LM and WM, which was not detected in PM. No significant difference was found in those CNVs. No characteristic fusions were found in the total analyzed cohorts. Conclusions: The SNV and CNV molecular mutation profiles were similar in LM, PM and WM. However, the genes with higher mutation probability show higher mutation frequency in LM. Compared with PM, SNV of SMAD4 displays a higher mutation frequency in LM. It has been previously reported that LM may display poorer overall survival (OS) compared with PM. Considering the effect of SMAD4 on the prognosis of CRC, this study may provide a possible analysis of the poor prognosis of LM patients. However, there is still a need for more clinical research to support these results.
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]
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