PIK3CA Mutations in Diffuse Gliomas: An Update on Molecular Stratification, Prognosis, Recurrence, and Aggressiveness

Introduction: PIK3CA is one of the most mutated oncogenes in solid tumors. In breast cancer (ER-positive, HER2-negative), these events represent a predictive biomarker of response to alpelisib. In glioblastomas (GBM), PIK3CA mutations were described as early constitutive events. Here, we investigated PIK3CA mutational profile across glioma molecular subgroups and its relevance during glioma recurrence. Furthermore, PIK3CA mutations’ effect in PI3K pathway, prognosis, and response to therapy was also explored. Material and Methods: Exons 10 and 21 of PIK3CA mutations were evaluated in 394 gliomas and 19 glioma recurrences from Instituto Português de Oncologia Lisboa Francisco Gentil (IPOLFG) and compared with The Cancer Genome Atlas (TCGA) data. TIMER2.0 and NetMHCpan4.1 were used to assess the immune-microenvironment contribution. Results: PIK3CA mutations were identified among all glioma subgroups, although with no impact on their stratification or prognosis. In both cohorts (IPOLFG and TCGA), PIK3CA mutation frequencies in IDH-mutant and IDH-wild-type GBM were similar (IPOLFG: 9% and 3%; TCGA: 8% and 2%). These mutations were not mutually exclusive with PTEN deletion and EGFR amplification. Despite their reduced frequency, we discovered PIK3CA mutations were maintained during glioma recurrence regardless of administered therapies. The immune microenvironment might not contribute to this phenotype as PIK3CA mutations did not influence immune cell infiltration. Conclusions: Despite the absence of a predominant effect in glioma stratification, PIK3CA mutations were maintained during glioma recurrence, possibly contributing to glioma cell survival, representing promising therapeutic targets in recurrent glioma. Nevertheless, understanding the potential synergistic effects between PIK3CA mutations, PTEN deletion, and EGFR amplification is pivotal to targeted therapies’ efficiency.

Cinobufagin Is a Selective Anti-Cancer Agent against Tumors with EGFR Amplification and PTEN Deletion

Glioblastoma multiforme (GBM) is the most common and malignant brain tumor, and almost half of the patients carrying EGFR-driven tumor with PTEN deficiency are resistant to EGFR-targeted therapy. EGFR amplification and/or mutation is reported in various epithelial tumors. This series of studies aimed to identify a potent compound against EGFR-driven tumor. We screened a chemical library containing over 600 individual compounds purified from Traditional Chinese Medicine against GBM cells with EGFR amplification and found that cinobufagin, the major active ingredient of Chansu, inhibited the proliferation of EGFR amplified GBM cells and PTEN deficiency enhanced its anti-proliferation effects. Cinobufagin also strongly inhibited the proliferation of carcinoma cell lines with wild-type or mutant EGFR expression. In contrast, the compound only weakly inhibited the proliferation of cancer cells with low or without EGFR expression. Cinobufagin blocked EGFR phosphorylation and its downstream signaling, which additionally induced apoptosis and cytotoxicity in EGFR amplified cancer cells. In vivo, cinobufagin blocked EGFR signaling, inhibited cell proliferation, and elicited apoptosis, thereby suppressing tumor growth in both subcutaneous and intracranial U87MG-EGFR xenograft mouse models and increasing the median survival of nude mice bearing intracranial U87MG-EGFR tumors. Cinobufagin is a potential therapeutic agent for treating malignant glioma and other human cancers expressing EGFR.

AI and High-Grade Glioma for Diagnosis and Outcome Prediction: Do All Machine Learning Models Perform Equally Well?

Radiomic models outperform clinical data for outcome prediction in high-grade gliomas (HGG). However, lack of parameter standardization limits clinical applications. Many machine learning (ML) radiomic models employ single classifiers rather than ensemble learning, which is known to boost performance, and comparative analyses are lacking in the literature. We aimed to compare ML classifiers to predict clinically relevant tasks for HGG: overall survival (OS), isocitrate dehydrogenase (IDH) mutation, O-6-methylguanine-DNA-methyltransferase (MGMT) promoter methylation, epidermal growth factor receptor vIII (EGFR) amplification, and Ki-67 expression, based on radiomic features from conventional and advanced magnetic resonance imaging (MRI). Our objective was to identify the best algorithm for each task. One hundred fifty-six adult patients with pathologic diagnosis of HGG were included. Three tumoral regions were manually segmented: contrast-enhancing tumor, necrosis, and non-enhancing tumor. Radiomic features were extracted with a custom version of Pyradiomics and selected through Boruta algorithm. A Grid Search algorithm was applied when computing ten times K-fold cross-validation (K=10) to get the highest mean and lowest spread of accuracy. Model performance was assessed as AUC-ROC curve mean values with 95% confidence intervals (CI). Extreme Gradient Boosting (xGB) obtained highest accuracy for OS (74,5%), Adaboost (AB) for IDH mutation (87.5%), MGMT methylation (70,8%), Ki-67 expression (86%), and EGFR amplification (81%). Ensemble classifiers showed the best performance across tasks. High-scoring radiomic features shed light on possible correlations between MRI and tumor histology.

EXTH-67. THERAPEUTIC EFFICACY OF GC1118, A NOVEL ANTI-EGFR ANTIBODY, AGAINST GLIOBLASTOMA WITH HIGH EGFR AMPLIFICATION IN PATIENT-DERIVED XENOGRAFTS

We aimed to evaluate the preclinical efficacy of GC1118, a novel anti-epidermal growth factor receptor (EGFR) monoclonal antibody (mAb), against glioblastoma (GBM) tumors using patient-derived xenograft (PDX) models. A total of 15 distinct GBM PDX models were used to evaluate the therapeutic efficacy of GC1118. Genomic data derived from PDX models were analyzed to identify potential biomarkers associated with the anti-tumor efficacy of GC1118. A patient-derived cell-based high-throughput drug screening assay was performed to further validate the efficacy of GC1118. Compared to cetuximab, GC1118 exerted comparable growth inhibitory effects on the GBM tumors in the PDX models. We confirmed that GC1118 accumulated within the tumor by crossing the blood–brain barrier in in vivo specimens and observed the survival benefit in GC1118-treated intracranial models. Genomic analysis revealed high EGFR amplification as a potent biomarker for predicting the therapeutic efficacy of GC1118 in GBM tumors. In summary, GC1118 exerted a potent anti-tumor effect on GBM tumors in PDX models, and its therapeutic efficacy was especially pronounced in the tumors with high EGFR amplification. Our study supports the importance of patient stratification based on EGFR copy number variation in clinical trials for GBM. The superiority of GC1118 over other EGFR mAbs in GBM tumors should be assessed in future studies.

The EGFRvIII transcriptome in glioblastoma, a meta-omics analysis

Background EGFR is among the genes most frequently altered in glioblastoma, with exons 2-7 deletions (EGFRvIII) being amongst its most common genomic mutations. There are conflicting reports about its prognostic role and it remains unclear whether and how it differs in signalling compared with wildtype EGFR. Methods To better understand the oncogenic role of EGFRvIII, we leveraged four large datasets into one large glioblastoma transcriptome dataset (n=741) alongside 81 whole-genome samples from two datasets. Results The EGFRvIII/EGFR expression ratios differ strongly between tumours and ranges from 1% to 95%. Interestingly, the slope of relative EGFRvIII expression is near-linear, which argues against a more positive selection pressure than EGFR wildtype. An absence of selection pressure is also suggested by the similar survival between EGFRvIII positive and negative glioblastoma patients. EGFRvIII levels are inversely correlated with pan-EGFR (all wildtype and mutant variants) expression, which indicates that EGFRvIII has a higher potency in downstream pathway activation. EGFRvIII-positive glioblastomas have a lower CDK4 or MDM2 amplification incidence than EGFRvIII-negative (p=0.007), which may point towards crosstalk between these pathways. EGFRvIII-expressing tumours have an upregulation of ‘classical’ subtype genes compared to those with EGFR-amplification only (p=3.873e-6). Genomic breakpoints of the EGFRvIII deletions have a preference towards the 3’ end of the large intron-1. These preferred breakpoints preserve a cryptic exon resulting in a novel EGFRvIII variant and preserve an intronic enhancer. Conclusions These data provide deeper insights into the complex EGFRvIII biology and provide new insights for targeting EGFRvIII mutated tumours.

Efficacy of Osimertinib Plus Bevacizumab In Glioblastoma Patients With Simultaneous EGFR Amplification And EGFRvIII Mutation

Background: Amplification of EGFR and its active mutant EGFRvIII are common in glioblastoma (GB). While EGFR and EGFRvIII play critical roles in pathogenesis, targeted therapy with EGFR-tyrosine kinase inhibitors (TKIs) or antibodies has shown limited efficacy. To improve the likelihood of effectiveness, we targeted adult patients with recurrent GB enriched for simultaneous EGFR amplification and EGFRvIII mutation, with osimertinib/bevacizumab at doses described for non-small cell lung cancer (NSCLC). Methods: We retrospectively explored whether previously described EGFRvIII mutation in association with EGFR gene amplification could predict response to osimertinib/bevacizumab combination in a subset of 15 patients treated at recurrence. The resistance pattern in a subgroup of subjects is described using a commercial NGS panel in liquid biopsy.Results: There were ten males (66.7%), and the median patient’s age was 56 years (range 38-70 years). After their initial diagnosis, 12 patients underwent partial (26.7%) or total resection (53.3%). Subsequently, all cases received IMRT and concurrent and adjuvant temozolomide (TMZ; the median number of cycles 9, range 6-12). The median follow-up after recurrence was 17.1 months (95% CI 12.3-22.6). All patients received osimertinib/bevacizumab as a second-line intervention with a median progression-free survival (PFS) of 5.1 months (95% CI 2.8-7.3) and overall survival (OS) of 9.0 months (95% CI 3.9-14.0). The PFS6 was 46.7%, and the overall response rate (ORR) was 13.3%. After exposure to the osimertinib/bevacizumab combination, the main secondary alterations were MET amplification, STAT3, IGF1R, PTEN, and PDGFR.Conclusions: While the osimertinib/bevacizumab combination was marginally effective in most GB patients with simultaneous EGFR amplification plus EGFRvIII mutation, a subgroup experienced a long-lasting meaningful benefit. The findings of this brief cohort justify the continuation of the research in a clinical trial. The pattern of resistance after exposure to osimertinib/bevacizumab includes known mechanisms in the regulation of EGFR, findings that contribute to the understanding and targeting in a stepwise rational this pathway.

Primary and secondary resistance mechanisms in first, second and third generation tyrosine kinase inhibitors in EGFR mutant non-small cell lung cancer patients.

e21142 Background: Different molecular mechanisms of on target and off target primary and secondary resistance have been observed in EGFR mutant NSCLC patients after first(1st), second(2nd) and third (3rd) generation of tyrosine kinase inhibitors(TKIs). Next generation sequencing(NGS) offers a comprehensive method of detecting these mechanisms to decide next line of treatment. Methods: We retrospectively analyzed 430 samples of NSCLC for primary and secondary resistance to 1st, 2nd and 3rd TKIs. NGS was performed using thermofischer Ion Torrent Oncomine Focus 52 gene Assay. These cases were divided into 4 groups.1)Primary resistance to first and second generation TKIs 2)Primary resistance to 3rd generation TKI 3)Secondary resistance to 1st and 2nd generation TKI 4) Secondary resistance to 3rd generation TKI.Last group was further subgrouped into A when 3rd generation TKI was offered as second line after 1st or 2nd generation TKIs on detection of T790M and subgroup B when it was given as first line. Results: Group1 had 13 cases. There were 2 cases of complex EGFR exon 19 mutation p.Glu746_Leu747delinsValPro, 4 cases of EGFR exon 20 insertion, 1 case of dual EGFR L833V & H835L mutation, 2 cases with EGFR amplification with EGFR exon 19 del and PIK3CA C420_P421del along with EGFR exon 19 del. Four cases had no additional abnormality. Group 2 had 5 cases:1 case had L858R and E709A dual mutation, 2 cases had KRAS G13C and KRAS G12V along with EGFR exon 19 del. One case had EGFR amplification and one case had MET amplification along with EGFR exon 19 del respectively.Group 3 had 34 cases including 10 cases of EGFR L858R and 24 cases of exon 19 deletion.T790M mutation was detected in 8 patients, MET amplification in 7 cases,one case had both T790M and MET amplification. One case lost the primary EGFR exon 19 del. Others mutations detected were KRAS G13C, PIK3CA H1047R, TP53 R213Q and TP53 C242fs. Group3 had 15 cases with 7 cases in subgroup A and 9 cases in subgroup B. In subgroup A T790M mutation was lost in 6 out of 7 cases.One case which lost T790M developed ALK translocation.One case of EGFR exon 19 del retained EGFR T790M with EGFR C797S in cis allele. Other mutations detected were PIK3CA E542K and KRAS G12C. In subgroup B one case showed EGFR C797S(both cis and trans) besides the primary EGFR exon 19 del. One case showed BRAF G469A along with EGFR exon 19 del. Other mutations detected were CTNNB1 D32N, KRAS G12V, and PIK3CA E542K. Conclusions: Primary and secondary acquired resistance is unavoidable in EGFR mutant advanced NSCLC on any generation of TKIs. NGS offers an advantage in diagnosing mechanism of resistance for further choice of therapy.

Novel biomarkers for head and neck squamous cell carcinoma: A retrospective study.

e18010 Background: PDL-1 and tumor mutation burden are only useful to select a small portion of patients with head and neck squamous cell carcinoma for immunotherapy, more biomarkers are in an urgent need for the majority. Methods: Ninety-nine recurrent/metastatic patients were analyzed. PD-1 cohort including 78 patients; Non-PD-1(NPD-1) cohort including 28 patients received anti-EGFR antibody and harbored at least one of EGFR amplification/mutation, CCND1 amplification, FGF3/4/19 amplification, or CDKN2A/B mutations (including 7 patients received with both treatment). Patients were evaluated as no clinical benefit (NCB) if experiencing progressive disease or stable disease lasting < 6 months and were discontinued from immunotherapy within 3 months; otherwise, patients were evaluated as clinical benefit (CB). Tumor genomic DNA was isolated from formalin-fixed paraffin-embedded tissue for the targeted sequencing by a 769-gene panel. R package was used for fisher test to evaluate the variants. p < 0.05 was set as significant. Results: With median age of 57 years old, this study included 75 (75.8%) oral squamous cell carcinoma patients,17 (17.2%) oropharyngeal carcinoma patients and 7 others. Sixty-nine (69.7%) patients have PD-L1 CPS≥1, 27 (27.3%) patients have CPS＜1 and 3 (3.0%) have an unknown CPS. The estimated 10-month progression-free survival of the NPD-1 cohort and PD-1cohort were 60.0%and 47.6% (p = 0.06) respectively. In NPD-1 cohort, 23 patients were evaluated as CB (78.3%), and in PD-1 cohort, 41 were evaluated as CB (52.6%) (p = 0.00682), indicating EGFR amplification/mutation, CCND1 amplification, FGF3/4/19 amplification, or CDKN2A/B mutations may be negatively correlated with immunotherapies. There were 14 patients who harbored either EGFR amplification or SNV mutation. Of the 8 patients who received NPD-1 treatment, 7 were CB (87.5%); Of the 8 patients who received PD-1 treatment, 2 were CB (25%) Statistically, the difference between NPD-1 treatment group and PD-1 treatment group was significant with a p value of 0.04056. There were 16 patients who harbored CCND1 amplification, or FGF3/4/19 amplification. Of the 12 patients who received NPD-1 treatment, 10 were evaluated as CB (83.3%); Of the 7 patients who received PD-1 treatment, 1 was evaluated as CB (14.3%). The difference was significant with a p value of 0.00627. There were 44 patients who obtained CDKN2A/B mutations. Of the 12 patients who received NPD-1 treatment, 11 acquired CB (81.7%); Of the 33 patients who received PD-1 treatment, 18 acquired CB (54.5%). The difference was significant with a p value of 0.03325. Conclusions: We for the first time showed that genetic-altered EGFR, FGF3/4/19, CCND1 and CDKN2A/B were negatively correlated with anti-PD-1 therapy in clinical cohorts retrospectively and these genetic aberrations may serve as novel immune-negative biomarkers for immunotherapies.