Mutation Subgroups Research Articles

HGG-46. Inter and Intra-tumor Heterogeneity of Pediatric-type Diffuse High-Grade Glioma Revealed by High-Dimensional Single-Cell Proteomics

Abstract Paediatric-type diffuse high-grade gliomas (PDHGG) are aggressive brain tumors, affecting children and young adults, with no effective treatments. A main constraint to the development of effective treatment is associated with their highly heterogeneous nature. In order to further dissect their intra and inter tumor heterogeneity, we exploited the mass cytometry technology, an advanced -OMIC approach that, by using metal-tagged antibodies, allows the simultaneous measurement of more than 40 markers, at single-cell level. Here we characterized 8 primary cell lines derived from diffuse pediatric-type high-grade glioma H3-wildtype (DHGG-WT), Diffuse hemispheric glioma H3G34-mutant (DHG-G34) and Diffuse midline glioma H3K27-altered (DMG-K27) patients. The adopted antibody panel was set to recognize antigens expressed by brain and tumor cells, including H3K27M and H3.3G34R variants, and it highlighted important intra- and inter- tumor heterogeneity in the expression of the 16 considered markers. Of these, CD56, CD44, CD29 and NESTIN were more expressed in the hemispheric cell lines, while CD90 was more expressed in the pontine. Even if there was not always a concordance between CyTOF and mRNA expression data from cell lines and tumor samples (e.g. CD90 and GFAP), CyTOF data were in line with the immunohistochemistry analysis for GFAP, whose expression was significantly higher in H3.1K27 compared to H3.3K27. The UMAP analysis allowed us to identify 10 cell clusters, with very minimal overlap between hemispheric and pontine location subgroups and with a peculiar antigenic profile, whose abundance strongly varied according to the mutational subgroups. For example, while the G34 subgroup was enriched for cluster 9 (CD29/CD63/CD56/PDGRFa), the H3.1K27 was enriched for cluster 3 (H3K27M/CD90/CD63/CD56) and cluster 4 (H3K27M/CD63/CD90/CD56/GFAP). In conclusion, single-cell mass cytometry reveals a significant inter and intra-tumoral heterogeneity at protein level, dependent on the molecular alterations. This approach could contribute to the identification of new clinically relevant biomarkers for PDHGG.

Uncommon EGFR mutations in lung carcinoma: features and treatment outcomes in a retrospective French cohort.

BackgroundThe best management for rare epidermal growth factor receptor (EGFR) mutations in advanced non-small cell lung carcinoma (NSCLC) remains uncertain. The literature indicates that response to usual treatment could differ in certain subgroups such as exon 20 insertion/duplication (E20ID), other single uncommon mutation (OSUM), and EGFR complex mutation (ECM).MethodsIn this observational, regional, multi-center, retrospective study, we gathered data on uncommon EGFR mutations in NSCLC from 2007 to 2021. We analyzed patient characteristics, prognostic factors and treatment outcomes [objective response rate (ORR), disease control rate (DCR), progression free survival (PFS) and overall survival (OS)].ResultsAmong 119 patients with an uncommon EGFR mutant, 34 harbored E20ID, 23 ECM, and 62 OSUM. There were significantly more non-smokers in E20ID. Female gender and performance status <2 were associated with a better prognosis. Among the 97 metastatic patients with available data for 1st line treatment, median estimated OS was 21 months (95% CI: 18–31 months), with better non-significant OS for ECM. Median estimated PFS was 7 months (95% CI: 4–9 months). We found significant differences in ORR, DCR and PFS favoring 1st line chemotherapy for E20ID, whereas the outcomes for OSUM and ECM were more favorable for tyrosine kinase inhibitor (TKI) (mainly 2nd and 3rd generation).ConclusionsThere were variations in treatment outcomes among subgroups in our cohort. Exon 20 insertions showed better ORR and PFS with 1st line chemotherapy compared to TKI. Conversely, other rare EGFR mutations including ECM had better ORR and PFS with TKI than chemotherapy. There was no significant difference in OS among treatment groups overall or within rare mutation subgroups.

139P KRAS-G12C NSCLC linked to female sex and high risk of CNS metastasis: Real-world data from the National Swedish Lung Cancer Registry 2016-2019

KRAS mutations represent common genetic driver aberrations in NSCLC. Real world data on demographics and outcome linked to different KRAS mutation subtypes is crucial as targeted drugs against the p.G12C variant become available in clinical practice. We used data from the National Swedish Lung Cancer Registry to evaluate 6183 patients diagnosed with NSCLC during 2016-2019 with reported NGS-based KRAS-status. Following exclusion of other targetable driver aberrations, three cohorts were studied further with regard to age, sex, smoking status, disease stage, performance status, histology, metastatic patterns and overall survival (OS, from diagnosis); KRAS-G12C (n=848), KRAS-other (n = 1161), driver negative (WT, n = 3349). Study sponsored by Amgen. The prevalence of KRAS driver mutations and KRAS-G12C respectively was 32%/14% in NSCLC, 38%/16% in adenocarcinoma, 28%/13% in NSCLC-NOS and 6%/2% in squamous cell carcinoma. Women were more common in the KRAS-G12C cohort (65%) compared to KRAS-other (59%) and WT (48%). Never smokers were rare in KRAS-G12C (2.5%) as opposed to KRAS-other and WT patients (7.2% and 12.3%). A high percentage of KRAS-G12C patients in stage IV (28%) had CNS metastases at diagnosis compared to KRAS-other or WT (19% and 18%). In stage I–IIIA disease we found no differences in OS between the three cohorts. In stage IV, both KRAS-G12C (5.8 months) and KRAS-other (5.2 months) had shorter median OS than the WT group (6.4 months). Women had better outcome in all stage IV mutational subgroups except in KRAS-G12C where OS was comparable between men and women (median 6.6 vs 6.1 months). Stage IV patients with KRAS-G12C, with and without CNS metastases had similar OS (median 6.1 months vs 6.2 months), as opposed to KRAS-other and WT where CNS metastasis was associated with poorer survival. In an unselected Swedish NSCLC population the KRAS-G12C mutation was associated with female sex, smoking, adenocarcinoma histology and linked to presence of CNS metastases. The relationship between OS, female sex and CNS metastases at diagnosis in the KRAS-G12C subgroup need to be further explored with regard to biological mechanisms and confounding clinical factors.

Spatially resolved transcriptomics deconvolutes histological prognostic subgroups in patients with colorectal cancer and synchronous liver metastases.

165 Background: Up to 50% of patients with Colorectal Cancer (CRC) will metastasise to the liver (CRLM). KRAS-mt liver metastases particularly when co-mutated with TP53 are associated with poor prognosis. We have used the Glasgow Microenvironment Score (GMS), a histological score performed on H+E slides utilising immune and stromal components of the microenvironment to robustly stratify outcome in primary CRC. The aim of the current study was firstly to determine the utility of GMS in metastatic CRC and secondly to employ the NanostringTM GeoMx Digital Spatial Profiler (DSP), a state-of-the art analysis platform enabling spatial transcriptomic characterisation while maintaining tumour microenvironment (TME) topographical features, to interrogate the functional biology underlying the GMS. Methods: FFPE specimens from primary and metastatic lesions from 44 patients undergoing synchronous resection of CRLM underwent GMS, IHC and panel genomic assessment. Primary endpoints were recurrence-free survival (RFS) and cancer-specific survival (CSS). In addition to bulk transcriptomic assessment, 4 matched pairs from the cohort were selected for GeoMx analysis: 2 samples were GMS0 (high-immune) and 2 were GMS1 (low-immune) with an equal distribution of KRASmt and wt. After multiplex IF staining (PanCK, CD45, DAPI, αSMA), 48 regions of interest were selected and Cancer Transcriptome Atlas Transcriptomic outputs (2000 genes) were analysed using Pathway enrichment analysis with immune deconvolution of the transcriptome performed. Results: GMS0 (high-immune) was associated with improved RFS (p=0.0048) and CSS (p=0.0012) remaining an independent predictor of survival on multivariate analysis (HR 2.90, 95% C.I 1.18-7.16 P=0.021). GMS0 lesions were enriched for adaptive immune (NES=2.20 p.adj&lt;0.0005) and IL-10 (NES=1.9 p.adj&lt;0.0005) pathways specifically at the invasive edge. In contrast, a poor prognostic KRAS/TP53 lesion demonstrated profound immunosuppression, upregulated NOTCH signalling (NES=2.13 p.adj&lt;0.0005) and neutrophil degranulation (NES=1.99 p.adj&lt;0.0005). Topographical Immune-cell deconvolution demonstrated significantly higher populations of CD4 (p=0.05) and CD8 (p=0.0003) cells in GMS0 leading edges. Conclusions: We have demonstrated that spatial transcriptomic analysis using the Nanostring GeoMx tool can reveal potential novel mechanisms underlying biologically relevant histological and mutational subgroups (KRAS-mt) of CRC, providing potential therapeutic targets requiring further investigation. Future studies will apply this technology to pre and post treatment biopsy samples.

Clinical efficacy of atezolizumab plus bevacizumab and chemotherapy in KRAS-mutated non-small cell lung cancer with STK11, KEAP1, or TP53 comutations: subgroup results from the phase III IMpower150 trial

BackgroundThe efficacy of atezolizumab (A) and/or bevacizumab (B) with carboplatin/paclitaxel (CP) chemotherapy was explored in the phase III, randomized IMpower150 study in patients with non-squamous non-small cell lung cancer (NSCLC)...

Broad spectrum of regorafenib activity on mutant KIT and absence of clonal selection in gastrointestinal stromal tumor (GIST): correlative analysis from the GRID trial

BackgroundIn the phase 3 GRID trial, regorafenib improved progression-free survival (PFS) independent of KIT mutations in exons 9 and 11. In this retrospective, exploratory analysis of the GRID trial, we investigated whether a more comprehensive KIT mutation analysis could identify mutations that impact treatment outcome with regorafenib and a regorafenib-induced mutation pattern.MethodsArchived tumor samples, collected at any time prior to enrollment in GRID, were analyzed by Sanger sequencing (n = 102) and next-generation sequencing (FoundationONE; n = 47). Plasma samples collected at baseline were analyzed by BEAMing (n = 163) and SafeSEQ (n = 96).ResultsIn archived tumor samples, 67% (68/102) had a KIT mutation; 61% (62/102) had primary KIT mutations (exons 9 and 11) and 12% (12/102) had secondary mutations (exons 13, 14, 17, and 18). At baseline, 81% of samples (78/96) had KIT mutations by SafeSEQ, including the M541L polymorphism (sole event in 6 patients). Coexisting mutations in other oncogenes were rare, as were mutations in PDGFR, KRAS, and BRAF. Regorafenib showed PFS benefit across all primary and secondary KIT mutational subgroups examined. Available patient-matched samples taken at baseline and end of treatment (n = 41; SafeSEQ), revealed heterogeneous KIT mutational changes with no specific mutation pattern emerging upon regorafenib treatment.ConclusionThese data support the results of the GRID trial, and suggest that patients may benefit from regorafenib in the presence of KIT mutations and without the selection of particular mutation patterns that confer resistance. The study was not powered to address biomarker-related questions, and the results are exploratory and hypothesis-generating.

Therapeutic effect of osimertinib plus cranial radiotherapy compared to osimertinib alone in NSCLC patients with EGFR-activating mutations and brain metastases: a retrospective study

BackgroundThe study aimed to compare the efficacy of osimertinib plus cranial radiotherapy (RT) with osimertinib alone in advanced non-small-cell lung cancer (NSCLC) patients harboring epidermal growth factor receptor (EGFR) mutations and brain metastases (BMs).MethodsThe clinical data of advanced NSCLC patients with BMs who received osimertinib were retrospectively collected. The patients were assigned to one of the two groups according to the therapeutic modality used: the osimertinib monotherapy group or the osimertinib plus RT group.ResultsThis was a retrospective study and 61 patients were included from December 2015 to August 2020. Forty patients received osimertinib monotherapy, and twenty-one patients received osimertinib plus RT. Radiotherapy included whole-brain radiation therapy (WBRT, n = 14), WBRT with simultaneous integrated boost (WBRT-SIB, n = 5) and stereotactic radiosurgery (SRS, n = 2). The median number of prior systemic therapies in the two groups was one. Intracranial and systemic ORR and DCR were not significantly different between the two groups. No difference in iPFS was observed between the two groups (median iPFS: 16.67 vs. 13.50 months, P = 0.836). The median OS was 29.20 months in the osimertinib plus RT group compared with 26.13 months in the osimertinib group (HR = 0.895, P = 0.826). In the L858R mutational subgroup of 31 patients, the osimertinib plus RT group had a longer OS (P = 0.046). In the exon 19 deletion mutational subgroup of 30 patients, OS in the osimertinib alone group was longer than that in the osimertinib plus RT group (P = 0.011). The incidence of any-grade adverse events was not significantly different between the osimertinib plus RT group and the osimertinib alone group (47.6% vs. 32.5%, P = 0.762). However, six patients (28.5%) experienced leukoencephalopathy in the osimertinib plus RT group, and 50% (3/6) of the leukoencephalopathy was greater than or equal to grade 3.ConclusionThe therapeutic effect of osimertinib with RT was similar to that of osimertinib alone in EGFR-positive NSCLC patients with BM. However, for patients with the L858R mutation, osimertinib plus RT could provide more benefit than osimertinib alone.

Prognostic Differences of RAS Mutations: Results from the South Australian Metastatic Colorectal Registry.

Effective targeting of RAS mutations has proven elusive until recently. Novel agents directly targeting KRAS G12C have shown promise in early-phase clinical trials that included patients with metastatic colorectal cancer. Prior reports have suggested that G12C mutation may be predictive of poor outcome. Assessment of the specific characteristics and prognostic implications of individual RAS mutation subtypes in patients with metastatic colorectal cancer. Retrospective review of individual RAS mutation types from the South Australian Metastatic Colorectal Registry between 2006 and 2020. Of the 5165 patients entered onto the registry, 2305 (45%) had RAS mutation results available. 772 (33%) had a RAS mutation. The nature of the RAS mutation was available in 668 (87% of those with RAS mutation). Rare mutations (outside codons 12 and 13) made up 12.6% of the total. There were numerical differences in survival between the specific RAS mutation subgroups, with the longest median overall survival (30 months) observed in those with G12S mutations. However, there was no statistical difference in survival when comparing the various RAS mutations, including the comparison of G12C to G12S (p=0.38). Patients with cancer harbouring rare RAS mutations had a median survival of 30 months. Whilst the G12S mutation was associated with the longest survival numerically, the observed survival for patients with the most common RAS mutations (G12C, G12V, G12A, G12D and G13D) did not significantly differ.

CSIG-27. PROTEOMIC ANALYSIS OF GENETICALLY STRATIFIED MENINGIOMA

Abstract WHO grade II and III as well as some WHO grade I meningioma are clinically aggressive. Approximately 60% sporadic meningiomas harbour mutations in the NF2 gene, others in genes including TRAF7, KLF4, AKT1, SMO and PIK3CA . However, the molecular mechanisms behind meningioma tumourigenesis are still unclear. We aim to identify novel biomarkers and therapeutic targets of meningioma by characterising the proteomic landscape. We performed (phosho)proteomic profiling of grade I, II and III meningiomas and three different mutational groups: AKT1 E17K /TRAF7, KLF4 K409Q /TRAF7 and NF2 -/-. We validated differential expression of proteins and phosphoproteins by Western blot on a meningioma validation set and by immunohistochemistry. Looking at all grades bioinformatics analysis revealed commonly upregulated proteins and phosphoproteins to be enriched in Gene Ontology terms associated with RNA metabolism. Validation studies confirmed significant overexpression of proteins such as EGFR and CKAP4 and upregulation and activation of the NIMA-related kinase, NEK9, involved in mitotic progression. Novel proteins described included the nuclear proto-oncogene SET, the splicing factor SF2/ASF and the higher-grade specific protein, Hexokinase 2. For the mutation subtypes we have quantified 4162 proteins across all mutational meningioma subgroups with proteomic profiles of mutational subgroups. Comparative analysis showed 10 proteins were commonly significantly upregulated among all mutational subtypes vs. normal meninges, indicating proteomic landscapes of mutational subtypes to be highly variable. 257 proteins were commonly significantly downregulated and enriched with molecular functions including aldehyde dehydrogenase and oxido-reductase. Mutational subtype-specific analysis identified 162 proteins significantly upregulated in AKT1 E17K /TRAF7 vs. remaining sample groups to be enriched in the oxidative phosphorylation pathway. Less proteins were commonly significantly upregulated in KLF4 K409Q /TRAF7 and NF2 -/- mutant meningioma subtypes respectively. Several of these up-regulated proteins including ANNEXIN-3, CRABP2, CLIC3 were verified. Analyses of 6600 phospho-sites predicted regulatory kinases. Further validation and functional verification of potential candidates is ongoing.

Phase II Trial of Ten-Day Decitabine with Venetoclax (DEC10-VEN) in Acute Myeloid Leukemia: Updated Outcomes in Genomic Subgroups

Phase II Trial of Ten-Day Decitabine with Venetoclax (DEC10-VEN) in Acute Myeloid Leukemia: Updated Outcomes in Genomic Subgroups

Proteomic analysis of genetically stratified low-grade meningioma

Abstract Aims Meningioma is the most common primary intracranial tumour. Although ~80% are benign WHO grade I and show high rates of recurrence. Surgery is the main therapeutic approach, yet location can hamper complete resection and chemotherapies are ineffective. Moreover, accurate biomarkers for clinical management are lacking. Approximately 60% sporadic meningiomas harbour mutations in the NF2gene, while mutations in genes including TRAF7, KLF4, AKT1, SMO and PIK3CAhave been identified majority in the NF2-positive low grade-tumours. Moreover, mutations in TRAF7 mostly co-occur with a KLF4K409Q or with AKT1E17K mutation. The mutations and their molecular manifestations consequently affect the signalling pathways at the protein level. The molecular mechanisms behind meningioma tumourigenesis are still obscure and the identification of specific biomarker is necessary to enable their implementation in routine diagnostics and therapeutics. Therefore, we aim to identify novel biomarkers and therapeutic targets of genetically stratified low-grade meningioma by characterising the proteomic landscape. Method Frozen tumour samples have already been analysed for NF2-/- by next generation sequencing and genotyped for common mutational hotspots in non-NF2 meningioma such as TRAF7, KLF4 and AKT1 and grouped in to three different mutational groups: AKT1E17K/TRAF7, KLF4K409Q/TRAF7and NF2-/- and all these mutations will be compared to normal healthy meninges. For global proteomics, proteins were separated by SDS-PAGE followed by in-gel tryptic digestion and sample preparation for LC-MS/MS analysis. Raw mass spectrometry data files were processed by MaxQuant (1.6.2.10) and Perseus software (1.6.1.3). Quantitative phospho-proteomics was performed using TMT 10plex labelling approach followed by motif analysis using motif-X algorithm. GO enrichment analyses were performed using (DAVID) v6.8 against all human proteins. Potential candidates from expression data analysis will be validated via Western Blot and immunohistochemistry. Results We have quantified 4162 proteins across all mutational meningioma subgroups and normal meninges (n=31). Hierarchical clustering analysis showed distinct proteomic profiles of mutational subgroups revealing clusters of differentially expressed proteins. Comparative analysis showed 10 proteins were commonly significantly upregulated (log2 fold-change≥1; p&amp;lt;0.05) among all mutational subtypes vs. normal meninges, indicating proteomic landscapes of mutational subtypes to be highly variable. In contrast, 257 proteins were commonly significantly downregulated (log2 fold-change≤-1; p&amp;lt;0.05) and enriched with molecular functions including aldehyde dehydrogenase and oxido-reductase. Mutational subtype-specific analysis identified 162 proteins significantly upregulated in AKT1E17K/TRAF7 vs. remaining sample groups to be enriched in the oxidative phosphorylation pathway. However, only 14 and 7 proteins were commonly significantly upregulated in KLF4K409Q/TRAF7 and NF2-/- mutant meningioma subtypes respectively. Several of these up-regulated proteins including ANNEXIN-3, CRABP2, CLIC3 and Endoglin were already verified via WB. Lastly, analyses of 6600 phospho-sites (n=8) predicted regulatory kinases including CHEK1, CHEK2 and LCK. Conclusion Global proteomic and phos-phoproteomics analysis has led to the identification of proteins differentially expressed in mutant subtypes. Results of this study to date suggest that a proteomic approach is an effective tool to identify distinct patterns in genetically distinct meningioma subgroups. Further validation and functional verification (with inhibitory or knockdown approaches) of potential candidates will allow us to identify potential drug targets/biomarkers for meningiomas.

Clinical Activity of Ripretinib in Patients with Advanced Gastrointestinal Stromal Tumor Harboring Heterogeneous KIT/PDGFRA Mutations in the Phase III INVICTUS Study.

Most patients with gastrointestinal stromal tumor (GIST) have activating mutations in KIT/PDGFRA and are initially responsive to tyrosine kinase inhibitors (TKI). The acquisition of secondary mutations leads to refractory/relapsed disease. This study reports the results of an analysis from the phase III INVICTUS study (NCT03353753) characterizing the genomic heterogeneity of tumors from patients with advanced GIST and evaluating ripretinib efficacy across KIT/PDGFRA mutation subgroups. Tumor tissue and liquid biopsy samples that captured circulating tumor DNA were collected prior to study enrollment and sequenced using next-generation sequencing. Subgroups were determined by KIT/PDGFRA mutations and correlation of clinical outcomes and KIT/PDGFRA mutational status was assessed. Overall, 129 patients enrolled (ripretinib 150 mg once daily, n = 85; placebo, n = 44). The most common primary mutation subgroup detected by combined tissue and liquid biopsies were in KIT exon 11 (ripretinib, 61.2%; placebo, 77.3%) and KIT exon 9 (ripretinib, 18.8%; placebo, 15.9%). Patients receiving ripretinib demonstrated progression-free survival (PFS) benefit versus placebo regardless of mutation status (HR 0.16) and in all assessed subgroups in Kaplan-Meier PFS analysis (exon 11, P < 0.0001; exon 9, P = 0.0023; exon 13, P < 0.0001; exon 17, P < 0.0001). Among patients with wild-type KIT/PDGFRA by tumor tissue, PFS ranged from 2 to 23 months for ripretinib versus 0.9 to 10.1 months for placebo. Ripretinib provided clinically meaningful activity across mutation subgroups in patients with advanced GIST, demonstrating that ripretinib inhibits a broad range of KIT/PDGFRA mutations in patients with advanced GIST who were previously treated with three or more TKIs.

OS08.5.A Proteomic analysis of meningioma

Abstract BACKGROUND Meningioma is the most common primary intracranial tumor. Although ~80% are benign some WHO grade I are clinically aggressive. Chemotherapies are ineffective and biomarkers for clinical management are lacking. Approximately 60% sporadic meningiomas harbor mutations in the NF2 gene andutations in TRAF7, KLF4, AKT1, SMO and PIK3CA have been identified in the majority NF2-positive tumors esp lower grade. However, the molecular mechanisms behind meningioma tumourigenesis is still unclear. We aim to identify novel biomarkers and therapeutic targets of meningioma by characterizing the proteomic landscape. MATERIAL AND METHODS We analysed grade I, II and III frozen meningioma specimens and three different mutational groups: AKT1/TRAF7, KLF4/TRAF7 and NF2 -/- using LC-MS/MS to analyse global proteins, enriched phosphoproteins and phosphopeptides. Differential expression and functional annotation of proteins was completed using Perseus, IPA® and DAVID. For mutational subtypes quantitative phosphoproteomics was performed using TMT 10plex labeling approach followed by motif analysis using motif-X algorithm. We validated differential expression of proteins and phosphoproteins by Western blot and immunohistochemistry. RESULTS We quantified 3888 proteins and 3074 phosphoproteins across all meningioma grades. Bioinformatics analysis revealed commonly upregulated (phospho)proteins to be enriched in Gene Ontology terms associated with RNA metabolism. Validation confirmed significant overexpression of proteins such as EGFR, CKAP4, the nuclear proto-oncogene SET, the splicing factor SF2/ASF as well as total and activated phosphorylated form of the NIMA-related kinase, NEK9, involved in mitotic progression. Hexokinase 2 was overexpressed in higher grades. For the mutation subtypes we have quantified 4162 proteins across all mutational meningioma subgroups. Analysis showed distinct proteomic profiles of mutational subgroups. Comparative analysis showed 10 proteins were commonly significantly upregulated among all mutational subtypes vs. normal meninges. 257 proteins were commonly significantly downregulated and enriched with molecular functions including aldehyde dehydrogenase and oxido-reductase. Mutational subtype-specific analysis identified 162 proteins significantly upregulated in AKT1/TRAF7 vs. remaining sample groups to be enriched in the oxidative phosphorylation pathway. 14 and 7 proteins were commonly significantly upregulated in KLF4/TRAF7 and NF2 -/- mutant meningioma subtypes respectively. Several of these up-regulated proteins including ANNEXIN-3, CRABP2, CLIC3 and Endoglin were verified via WB. Lastly, analyses of 6600 phosphosites predicted regulatory kinases CONCLUSION We show extensive proteomic and phospophoproteomics analysis of meningioma and suggest new therapeutic and biomarker candidates.

PL02.4.A Chromosome 1p19q codeletion frequency, but not survival, varies according to the IDH mutation subtypes: analysis of 1050 IDH-mutated diffuse gliomas

Abstract BACKGROUND recurring hotspot mutations in isocitrate dehydrogenase (IDH) 1 enzymes, and to a lesser extent IDH2, are the main oncogenic alteration in most of lower-grade diffuse gliomas and a subset of grade 4 gliomas. Although most commonly represented by the IDH1R132H mutation, non-canonical IDH1/2-nonR132H mutations are present in about 10% of cases. As the neomorphic enzymatic activity can vary depending on the type of the hotspot mutation, a different biological behaviour may be inferred. Nevertheless, the prognostic significance of the different IDH1/2 mutations remains a matter of debate. MATERIAL AND METHODS we queried the OncoNeuroTek tumor bank (Pitié-Salpêtrière Hospital, Paris) to identify all registered cases of IDH-mutated diffuse gliomas. Most relevant clinical and molecular data were collected. RESULTS We identified 1050 IDH mutated diffuse gliomas (481 grade 2 [46%], 459 grade 3 [44%], and 110 grade 4 [10%]), of which 1007 (96%) were IDH1 mutated (934 IDH1R132H [89%] and 73 IDH-nonR132H [7%]) and 43 (4%) were IDH2 mutated (24 IDH2R172K [2%] and 19 IDH2-nonR172K [2%]). The chromosomes 1p/19q codeletion was more frequent in IDH2-mutated tumors (25/42 [60%] versus 350/918 [38%] in IDH1-mutated cases, p=0.005). Nevertheless, only IDH2R172K mutation was associated with the codeletion (18/24 [75%], versus 7/18 [39%] in IDH2-nonR172K-mutated tumors, p=0.02). IDH1-nonR132H tumors showed the lowest rate of 1p/19q codeletion (9/69 [13%], versus 341/849 [40%] in IDH1R132H-mutated cases, p&amp;lt;0.001). At the time of observation, 536 patients were deceased (51%), with a median overall survival (OS) of 115 months (mo., 95% CI 108–125 mo.). Median follow up for censored patients was 77 mo. Codeleted patients had a significantly longer OS compared to non-codeleted ones (179 vs. 96 mo., p&amp;lt;0.001). No significant differences in survival were seen when stratifying according to IDH mutation type (115 mo. for IDH1R132H vs. 136 mo. for IDH1-nonR132H vs. 112 mo. for IDH2R172K vs. 150 mo. for IDH2-nonR172K, p=0.8). No differences were seen when restricting the analysis to codeleted or not-codeleted patients only, respectively. In a multivariate analysis including the main prognostic factors (age, sex, preoperative performance status, tumor grade, surgical resection, midline location, 1p/19q codeletion, and p16 homozygous deletion), no survival difference was associated with any of the IDH mutation subtype. CONCLUSION although significantly different rates of 1p/19q codeletion were seen according to the four main IDH mutation subgroups, these groups does not associate with different survival profiles in our cohort.

Clinical and molecular characteristics of epidermal growth factor receptor exon 20 insertion mutations in non-small-cell lung cancer

To evaluate the genomic and immune characteristics of non-small-cell lung cancer (NSCLC) patients with epidermal growth factor receptor (EGFR) exon 20 insertion (ex20ins) mutations from a retrospective dataset with molecular spectrum, tumor mutational burden (TMB), and programmed death-ligand 1 (PD-L1) expression, as well as to evaluate the efficacy of immune checkpoint inhibitors (ICIs). A total of 283 patients with EGFR ex20ins mutations who were diagnosed with NSCLC at our hospital from August 2013 to September 2020 were enrolled in this single-center retrospective study. Among the 283 patients with EGFR ex20ins mutations, 182 patients received next-generation sequencing (NGS) test, and 51 different subtypes of insertion variants were recorded. The most common mutations were A767_V769dup (21.4%), S768_D770dup (19.2%) and A763_Y764insFQEA (7.1%). The most common co-occurring mutations were EGFR amplification (37.9%), TP53 mutation (35.0%) and PIK3CA mutation (8.7%). PD-L1 status was available for 141 patients, and 75.9% (107/141) of these samples showed negative PD-L1 expression. In the 36 cases with TMB tested by NGS, the median TMB was 4.6 mutations/Mb. Then 12 patients received ICIs monotherapy or combination therapy. No severe adverse events were observed. Low PD-L1 expression and TMB were observed in NSCLC patients harboring EGFR ex20ins mutations. Further investigations are needed to confirm the therapeutic sensitivity of ICIs in this subgroup of EGFR mutations.

Retrospective Case Series Analysis of RAF Family Alterations in Pancreatic Cancer: Real-World Outcomes From Targeted and Standard Therapies.

PURPOSEIn pancreatic cancer (PC), the RAF family alterations define a rare subset of patients that may predict response to inhibition of the BRAF/MEK/ERK signaling pathway. A comprehensive understanding of the molecular and clinical characteristics of RAF-mutated PC may support future development of RAF-directed strategies.METHODSClinical outcomes were assessed across a multi-institutional case series of 81 patients with RAF family-mutated PC. Mutational subgroups were defined on the basis of RAF alteration hotspots and therapeutic implications.RESULTSThe frequency of RAF alterations in PC was 2.2% (84 of 3,781) within a prevalence cohort derived from large molecular databases where BRAF V600E (Exon 15), BRAF ΔNVTAP (Exon 11), and SND1-BRAF fusions were the most common variants. In our retrospective case series, we identified 17 of 81 (21.0%) molecular profiles with a BRAF V600/Exon 15 mutation without any confounding drivers, 25 of 81 (30.9%) with BRAF or RAF1 fusions, and 18 of 81 (22.2%) with Exon 11 mutations. The remaining 21 of 81 (25.9%) profiles had atypical RAF variants and/or multiple oncogenic drivers. Clinical benefit from BRAF/MEK/ERK inhibitors was observed in 3 of 3 subjects within the V600 subgroup (two partial responses), 4 of 6 with fusions (two partial responses), 2 of 6 with Exon 11 mutations (one partial response), and 0 of 3 with confounding drivers. Outcomes analyses also suggested a trend favoring fluorouracil-based regimens over gemcitabine/nab-paclitaxel within the fusion subgroup (P = .027).CONCLUSIONProspective evaluation of RAF-directed therapies is warranted in RAF-mutated PC; however, differential responses to targeted agents or standard regimens for each mutational subgroup should be a consideration when designing clinical trials.

Uncommon single and compound EGFR mutations: clinical outcomes of a heterogeneous subgroup of NSCLC

Molecular characterization of non-small-cell lung cancer (NSCLC) is essential to define the correct therapeutic algorithm in metastatic disease. Approximately 90% of epidermal growth factor receptor (EGFR) mutations are usually associated with sensitivity to EGFR tyrosine kinase inhibitors (TKIs). The remaining 10% defines a small, extremely heterogeneous subgroup of mutations, with a varied profile of sensitivity and response to target therapies.This retrospective observational study includes 47 patients affected by metastatic NSCLC harboring uncommon EGFR mutations (single or compound mutation). Patients were treated with EGFR-targeting TKIs or platinum-based chemotherapy as first-line treatment.Median OS resulted longer in the compound mutation group when compared to single rare mutations (33.6 vs 12 months; P = 0.473); a similar result was observed for PFS (16 vs 7.6 months; P = 0.281), although statistical significance was not reached. ORR, PFS and OS resulted similar for patients treated with first-line EGFR TKIs or chemotherapy. No difference in terms of PFS and OS was found according to the TKI administered.Compound mutations seem to be a good prognostic indicator for OS; they are also predictive of response to 1st and 2nd generation EGFR TKIs, as well as exon 19 insertions and mutations in codon 719 of exon 18. For mutations in exon 18 (not in codon 719) and exon 20 insertions, chemotherapy seems the most effective available option. The addition of immunotherapy to chemotherapy could change this approach in the next future.

Variability in the type and layer distribution of cortical Aβ pathology in familial Alzheimer's disease.

Familial Alzheimer's disease (FAD) is caused by autosomal dominant mutations in the PSEN1, PSEN2 or APP genes, giving rise to considerable clinical and pathological heterogeneity in FAD. Here we investigate variability in clinical data and the type and distribution of Aβ pathologies throughout the cortical layers of different FAD mutation cases. Brain tissue from 20 FAD cases [PSEN1 pre‐codon 200 (n = 10), PSEN1 post‐codon 200 (n = 6), APP (n = 4)] were investigated. Frontal cortex sections were stained immunohistochemically for Aβ, and Nissl to define the cortical layers. The frequency of different amyloid‐beta plaque types was graded for each cortical layer and the severity of cerebral amyloid angiopathy (CAA) was determined in cortical and leptomeningeal blood vessels. Comparisons were made between FAD mutations and APOE4 status, with associations between pathology, clinical and genetic data investigated. In this cohort, possession of an APOE4 allele was associated with increased disease duration but not with age at onset, after adjusting for mutation sub‐group and sex. We found Aβ pathology to be heterogeneous between cases although Aβ load was highest in cortical layer 3 for all mutation groups and a higher Aβ load was associated with APOE4. The PSEN1 post‐codon 200 group had a higher Aβ load in lower cortical layers, with a small number of this group having increased cotton wool plaque pathology in lower layers. Cotton wool plaque frequency was positively associated with the severity of CAA in the whole cohort and in the PSEN1 post‐codon 200 group. Carriers of the same PSEN1 mutation can have differing patterns of Aβ deposition, potentially because of differences in risk factors. Our results highlight possible influences of APOE4 genotype, and PSEN1 mutation type on Aβ deposition, which may have effects on the clinical heterogeneity of FAD.

Longitudinal genotype-phenotype analysis in 86 patients with PAX6-related aniridia.

Aniridia is most commonly caused by haploinsufficiency of the PAX6 gene, characterized by variable iris and foveal hypoplasia, nystagmus, cataracts, glaucoma, and aniridia-related keratopathy (ARK). Genotype-phenotype correlations have previously been described; however, detailed longitudinal studies of aniridia are less commonly reported. We identified 86 patients from 62 unrelated families with molecularly confirmed heterozygous PAX6 variants from a UK-based single-center ocular genetics service. They were categorized into mutation groups, and a retrospective review of clinical characteristics (ocular and systemic) from baseline to most recent was recorded. One hundred and seventy-two eyes were evaluated, with a mean follow-up period of 16.3 ± 12.7 years. Nystagmus was recorded in 87.2% of the eyes, and foveal hypoplasia was found in 75%. Cataracts were diagnosed in 70.3%, glaucoma in 20.6%, and ARK in 68.6% of eyes. Prevalence, age of diagnosis and surgical intervention, and need for surgical intervention varied among mutation groups. Overall, the missense mutation subgroup had the mildest phenotype, and surgically naive eyes maintained better visual acuity. Systemic evaluation identified type 2 diabetes in 12.8% of the study group, which is twice the UK prevalence. This is the largest longitudinal study of aniridia in the UK, and as such, it can provide insights into prognostic indicators for patients and guiding clinical management of both ocular and systemic features.

IDH Mutation Subgroup Status Associates with Intratumor Heterogeneity and the Tumor Microenvironment in Intrahepatic Cholangiocarcinoma.

Intrahepatic cholangiocarcinoma (ICC) is highly heterogeneous. Here, the authors perform exome sequencing and bulk RNA sequencing on 73 tumor regions from 14 ICC patients to portray the multi‐faceted intratumor heterogeneity (ITH) landscape of ICC. The authors show that ITH is highly concordant across genomic, transcriptomic, and immune levels. Comparison of these data to 8 published datasets reveals significantly higher degrees of ITH in ICC than hepatocellular carcinoma. Remarkably, the authors find that high‐ITH tumors highly overlap with the IDH (isocitrate dehydrogenase)‐mutant subgroup (IDH‐SG), comprising of IDH‐mutated tumors and IDH‐like tumors, that is, those IDH‐wildtype tumors that exhibit similar molecular profiles to the IDH‐mutated ones. Furthermore, IDH‐SG exhibits less T cell infiltration and lower T cell cytotoxicity, indicating a colder tumor microenvironment (TME). The higher ITH and colder TME of IDH‐SG are successfully validated by single‐cell RNA sequencing on 17 503 cells from 4 patients. Collectively, the study shows that IDH mutant subgroup status, rather than IDH mutation alone, is associated with ITH and the TME of ICC tumors. The results highlight that IDH‐like patients may also benefit from IDH targeted therapies and provide important implications for the diagnosis and treatment of ICC.