BRAF Hotspot Mutations Research Articles

Abstract 3700: Genomic alterations on first-line panitumumab plus FOLFIRI in metastatic colorectal cancer

Abstract Background: IMPROVE is a randomized, non-comparative multicenter, phase 2 study evaluating continuous or intermittent FOLFIRI/PANI, in first-line in patients (pts) with unresectable RAS/BRAF wild-type metastatic colorectal cancer (mCRC). In this trial intermittent FOLFIRI/PANI strategy produced a longer progression-free survival (PFS) on treatment, 17.6 months (mts) vs 11.3 mts, a reduced skin severe toxicity, 18% vs 30%, without any detrimental effect on overall survival (OS), 36.2 mts vs 36.2 mts. The trial also included a prospective genomic alterations (GAs) analysis on circulating tumor DNA with the aim to explore emergence of GAs under the selective pressure of FOLFIRI/PANI. Methods: We analyzed paired plasma samples (basal and PD) by a commercially available NGS panel enabled to simultaneously analyze KRAS, NRAS, BRAF and PIK3CA hotspot mutations from blood samples. Among them, inspecting default calling parameters on proprietary analysis software (Ion Reporter software 5.2.0), only variants with ⩾5X allele coverage and a quality score ⩾20, within an amplicon that covered at least 1000X alleles was considered. As clonality, the threshold of 0.5% of variant allele frequency (VAF) was established to classify mutations as clonal (VAF⩾0.5%) or subclonal (VAF<0.5%). Results: Of the 137 pts enrolled in the trial, GAs basal analyses were available for 84 pts. Among these, in 55 (65.5%) no GAs were detected (ND). In the remaining cases, the analysis relieved the presence of mutations for PIK3CA (N=14), KRAS (N=12), NRAS (N=2) and BRAF (N=1). According to the clonality threshold, these GAs were classified as subclonal (SC) in 12 cases (14.3%) and clonal (CL) in 17 (20.2%) cases. Survival analysis according to basal GAs clonality showed a median PFS of 16.6 mts (95% IC 13.0-20.2), 12 mts (95% IC 9.1-14.9) and 25.8 mts (95% IC 17.2-34.5) for ND, CL and SC group, respectively. Median OS was 47.7 mts for ND group (95% IC 34.9-60.5), 26.8 mts for CL (95% IC 25.8-27.8) and not reached for SC group. Moreover, we performed GAs analysis on 61 cases of which basal and PD samples were available. Forty-two cases (68.9%) resulted ND, 7 (11.5%) with SC and 12 (19.7%) with CL GAs.At PD, among 42 ND basal cases, 29 remained ND (69%), 8 (19%) and 5 cases (11.9%) developed SC and CL GAs, respectively. In the SC group, 1 kept SC (14.3%), 4 (57.1%) and 2 (28.6%) cases became ND and CL GAs, respectively. In the CL group, 10 (83.3%) kept CL GAs, 1 (8.3%) case became ND and 1 (8.3%) CL. Conclusions: These findings suggest that classical GAs associated with anti-EGFR resistance are infrequent with up-front use of anti-EGFR/chemotherapy. Moreover, our data show that basal SC GAs rarely evolve to CL GAs at progression disease and do not affect survival of pts with anti-EGFR/chemotherapy treatment. An update will be provided at the congress. NCT04425239 Citation Format: Antonio Avallone, Francesco Giuliani, Guglielmo Nasti, Vincenzo Montesarchio, Giuseppe Santabarbara, Silvana Leo, Umberto Malapelle, Gerardo Rosati, Claudio Lotesoriere, Emiliano Tamburini, Alfredo Colombo, Francesco Pepe, Claudia Cardone, Alessandra Leone, Carlo Vitagliano, Gianluca Russo, Giancarlo Troncone, Diana Giannarelli, Alfonso De Stefano, Alfredo Budillon. Genomic alterations on first-line panitumumab plus FOLFIRI in metastatic colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3700.

Mutations in the Serine/Threonine Kinase BRAF: Oncogenic Drivers in Solid Tumors.

Since their discovery in 2002, BRAF mutations have been identified as clear drivers of oncogenesis in several cancer types. Currently, their incidence rate is nearly 7% of all solid tumors with BRAF V600E constituting approximately 90% of these diagnoses. In melanoma, thyroid cancer, and histiocytic neoplasms, BRAF hotspot mutations are found at a rate of about 50%, while in lung and colorectal cancers they range from 3% to 10% of reported cases. Though present in other malignancies such as breast and ovarian cancers, they constitute a small portion of diagnoses (<1%). Given their frequency along with advancements in screening technologies, various methods are used for the detection of BRAF-mutant cancers. Among these are targeted next-generation sequencing (NGS) on tumor tissue or circulating tumor DNA (ctDNA) and immunohistochemistry (IHC)-based assays. With advancements in detection technologies, several approaches to the treatment of BRAF-mutant cancers have been taken. In this review, we retrace the milestones that led to the clinical development of targeted therapies currently available for these tumors.

Genomic profiling of primary and metastatic thyroid cancers.

The genetic repertoire of primary thyroid cancers (TCs) is well documented, but there is a considerable lack of molecular profiling in metastatic TCs. Here, we retrieved and analyzed the molecular and clinical features of 475 primary and metastatic TCs subjected to targeted DNA sequencing, from the cBioPortal database. The cohort included primary and metastatic samples from 276 papillary thyroid carcinomas (PTCs), 5 follicular thyroid carcinomas, 22 Hürthle cell carcinomas (HCCs), 127 poorly differentiated thyroid carcinomas (PDTCs), 30 anaplastic thyroid carcinomas (ATCs) and 15 medullary thyroid carcinomas. The ATCs had the highest tumor mutational burden and the HCCs the highest fraction of the genome altered. Compared to primary PTCs, the metastases had a significantly higher frequency of genetic alterations affecting TERT (51% vs 77%, P < 0.001), CDKN2A (2% vs 10%, P < 0.01), RET (2% vs 7%, P < 0.05), CDKN2B (1% vs 6%, P < 0.05) and BCOR (0% vs 4%, P < 0.05). The distant metastases had a significantly lower frequency of BRAF (64% vs 85%, P < 0.01) and a significantly higher frequency of NRAS (13% vs 3%, P < 0.05) hotspot mutations than the lymph node metastases. Metastases from HCCs and PDTCs were found to be enriched for NF1 (29%) and TP53 (18%) biallelic alterations, respectively. The frequency of subclonal mutations in ATCs was significantly higher than in PTCs (43% vs 25%, P < 0.01) and PDTCs (43% vs 22%, P < 0.01). Metastatic TCs are enriched in clinically informative genetic alterations such as RET translocations, BRAF hotspot mutations and NF1 biallelic losses that may be explored therapeutically.

Revolutionizing Non-Small Cell Lung Cancer Diagnosis: Ultra-High-Sensitive ctDNA Analysis for Detecting Hotspot Mutations with Long-term Stored Plasma.

Circulating cell-free DNA (cfDNA) has great potential in clinical oncology. The prognostic and predictive values of cfDNA in non-small cell lung cancer (NSCLC) have been reported, with epidermal growth factor receptor (EGFR), KRAS, and BRAF mutations in tumor-derived cfDNAs acting as biomarkers during the early stages of tumor progression and recurrence. However, extremely low tumor-derived DNA rates hinder cfDNA application. We developed an ultra-high-sensitivity lung version 1 (ULV1) panel targeting BRAF, KRAS, and EGFR hotspot mutations using small amounts of cfDNA, allowing for semi-quantitative analysis with excellent limit-of-detection (0.05%). Mutation analysis was performed on cfDNAs extracted from the plasma of 104 patients with NSCLC by using the ULV1 panel and targeted next-generation sequencing (CT-ULTRA), followed by comparison analysis of mutation patterns previously screened using matched tumor tissue DNA. The ULV1 panel demonstrated robust selective amplification of mutant alleles, enabling the detection of mutations with a high degree of analytical sensitivity (limit-of-detection, 0.025%-0.1%) and specificity (87.9%-100%). Applying ULV1 to NSCLC cfDNA revealed 51.1% (23/45) samples with EGFR mutations, increasing with tumor stage: 8.33% (stage I) to 78.26% (stage IV). Semi-quantitative analysis proved effective for low-mutation-fraction clinical samples. Comparative analysis with PANAMutyper EGFR exhibited substantial concordance (κ=0.84). Good detection sensitivity (~80%) was observed despite the limited volume (1 mL) and long-term storage (12-50 months) of plasma used and is expected to increase with high cfDNA inputs. Thus, the ULV1 panel is a fast and cost-effective method for early diagnosis, treatment selection, and clinical follow-up of patients with NSCLC.

Next-generation sequencing in dermatology.

Over the past decade, Next-Generation Sequencing (NGS) has advanced our understanding, diagnosis, and management of several areas within dermatology. NGS has emerged as a powerful tool for diagnosing genetic diseases of the skin, improving upon traditional PCR-based techniques limited by significant genetic heterogeneity associated with these disorders. Epidermolysis bullosa and ichthyosis are two of the most extensively studied genetic diseases of the skin, with a well-characterized spectrum of genetic changes occurring in these conditions. NGS has also played a critical role in expanding the mutational landscape of cutaneous squamous cell carcinoma, enhancing our understanding of its molecular pathogenesis. Similarly, genetic testing has greatly benefited melanoma diagnosis and treatment, primarily due to the high prevalence of BRAF hot spot mutations and other well-characterized genetic alterations. Additionally, NGS provides a valuable tool for measuring tumor mutational burden, which can aid in management of melanoma. Lastly, NGS demonstrates promise in improving the sensitivity of diagnosing cutaneous T-cell lymphoma. This article provides a comprehensive summary of NGS applications in the diagnosis and management of genodermatoses, cutaneous squamous cell carcinoma, melanoma, and cutaneous T-cell lymphoma, highlighting the impact of NGS on the field of dermatology.

Detection and Monitoring of Tumor-Derived Mutations in Circulating Tumor DNA Using the UltraSEEK Lung Panel on the MassARRAY System in Metastatic Non-Small Cell Lung Cancer Patients.

Analysis of circulating tumor DNA (ctDNA) is a potential minimally invasive molecular tool to guide treatment decision-making and disease monitoring. A suitable diagnostic-grade platform is required for the detection of tumor-specific mutations with high sensitivity in the circulating cell-free DNA (ccfDNA) of cancer patients. In this multicenter study, the ccfDNA of 72 patients treated for advanced-stage non-small cell lung cancer (NSCLC) was evaluated using the UltraSEEK® Lung Panel on the MassARRAY® System, covering 73 hotspot mutations in EGFR, KRAS, BRAF, ERBB2, and PIK3CA against mutation-specific droplet digital PCR (ddPCR) and routine tumor tissue NGS. Variant detection accuracy at primary diagnosis and during disease progression, and ctDNA dynamics as a marker of treatment efficacy, were analyzed. A multicenter evaluation using reference material demonstrated an overall detection rate of over 90% for variant allele frequencies (VAFs) > 0.5%, irrespective of ccfDNA input. A comparison of UltraSEEK® and ddPCR analyses revealed a 90% concordance. An 80% concordance between therapeutically targetable mutations detected in tumor tissue NGS and ccfDNA UltraSEEK® analysis at baseline was observed. Nine of 84 (11%) tumor tissue mutations were not covered by UltraSEEK®. A decrease in ctDNA levels at 4-6 weeks after treatment initiation detected with UltraSEEK® correlated with prolonged median PFS (46 vs. 6 weeks; p < 0.05) and OS (145 vs. 30 weeks; p < 0.01). Using plasma-derived ccfDNA, the UltraSEEK® Lung Panel with a mid-density set of the most common predictive markers for NSCLC is an alternative tool to detect mutations both at diagnosis and during disease progression and to monitor treatment response.

Small Gastric Stromal Tumors: An Underestimated Risk

Small gastrointestinal stromal tumors (GISTs) are defined as tumors less than 2 cm in diameter, which are often found incidentally during gastroscopy. There is controversy regarding the management of small GISTs, and a certain percentage of small GISTs become malignant during follow-up. Previous studies which used Sanger targeted sequencing have shown that the mutation rate of small GISTs is significantly lower than that of large tumors. The aim of this study was to investigate the overall mutational profile of small GISTs, including those of wild-type tumors, using whole-exome sequencing (WES) and Sanger sequencing. Thirty-six paired small GIST specimens, which were resected by endoscopy, were analyzed by WES. Somatic mutations identified by WES were confirmed by Sanger sequencing. Sanger sequencing was performed in an additional 38 small gastric stromal tumor samples for examining hotspot mutations in KIT, PDGFRA, and BRAF. Somatic C-KIT/PDGFRA mutations accounted for 81% of the mutations, including three novel mutation sites in C-KIT at exon 11, across the entire small gastric stromal tumor cohort (n = 74). In addition, 15% of small GISTs harbored previously undescribed BRAF-V600E hotspot mutations. No significant correlation was observed among the genotype, pathological features, and clinical classification. Our data revealed a high overall mutation rate (~96%) in small GISTs, indicating that genetic alterations are common events in early GIST generation. We also identified a high frequency of oncogenic BRAF-V600E mutations (15%) in small GISTs, which has not been previously reported.

Analytical and clinical validation of a custom 15-gene next-generation sequencing panel for the evaluation of circulating tumor DNA mutations in patients with advanced non-small-cell lung cancer.

BackgroundThis is a pilot proof-of-concept study to evaluate the utility of a custom 15-gene circulating tumor DNA (ctDNA) panel as a potential companion molecular next-generation sequencing (NGS) assay for identifying somatic single nucleotide variants and indels in non-small-cell lung cancer (NSCLC) patients. The custom panel covers the hotspot mutations in EGFR, KRAS, NRAS, BRAF, PIK3CA, ERBB2, MET, KIT, PDGFRA, ALK, ROS1, RET, NTRK1, NTRK2 and NTRK3 genes which serve as biomarkers for guiding treatment decisions in NSCLC patients.MethodThe custom 15-gene ctDNA NGS panel was designed using ArcherDX Assay Designer. A total of 20 ng or 50 ng input ctDNA was used to construct the libraries. The analytical performance was evaluated using reference standards at different allellic frequencies (0.1%, 1%, 5% and parental). The clinical performance was evaluated using plasma samples collected from 10 treatment naïve advanced stage III or IV NSCLC patients who were tested for tissue EGFR mutations. The bioinformatics analysis was performed using the proprietary Archer Analysis Software.ResultsFor the analytical validation, we achieved 100% sensitivity and specificity for the detection of known mutations in the reference standards. The limit of detection was 1% allelic frequency. Clinical validation showed that the clinical sensitivity and specificity of the assay for detecting EGFR mutation were 83.3% and 100% respectively. In addition, the NGS panel also detected other mutations of uncertain significance in 6 out of 10 patients.ConclusionThis preliminary analysis showed that the custom 15-gene ctDNA NGS panel demonstrated good analytical and clinical performances for the EGFR mutation. Further studies incorporating the validation of other candidate gene mutations are warranted.

Liquid biopsy for KRAS, NRAS and BRAF mutation testing in advanced colorectal cancer patients: the Argentinean experience.

Objective: To analyze the frequency of KRAS, NRAS and BRAF hotspot mutations in circulating tumor DNA (ctDNA) from patients with metastatic colorectal cancer (mCRC). Methods: Observational, descriptive and retrospective study in mCRC patients with available ctDNA-based genotype of KRAS, NRAS and BRAF. Results: The frequencies of plasma mutations for KRAS, NRAS and BRAF were 34% (±7), 4% (±3) and 4% (±3), respectively. Median overall survival of plasma-tested RAS/BRAF-mutated patients was 26.6months (95% CI: 14.4-not estimable [NE]), while RAS/BRAF wild-type patients did not reach the median survival during follow-up. Median progression-free survival for RAS/BRAF wild-type and RAS/BRAF-mutated patients was 12 (95% CI: 7-NE) and 4months (95% CI: 4-NE), respectively. Conclusion: Our work supports the utility of KRAS, NRAS and BRAF analysis in liquid biopsy from mCRC patients.

Abstract 3411: Detection and monitoring of tumor-derived mutations in ctDNA using the UltraSEEK Lung Panel on the MassARRAY System in metastatic NSCLC patients

Abstract Background: The analysis of circulating tumor DNA (ctDNA) is a potential minimally invasive molecular tool to guide treatment decision making and disease monitoring. A suitable diagnostic-grade platform is required for detection of tumor-specific mutations with high sensitivity in circulating cell-free DNA (ccfDNA) of cancer patients. Methods: In an international multicenter study conducted by three laboratories participating in the CANCER-ID consortium, a cohort containing 177 cell-free plasma samples at baseline, follow-up or disease progression derived from 72 patients treated for advanced-stage non-small cell lung cancer (NSCLC), relevant mutations were tested using the UltraSEEK® Lung Panel on the MassARRAY® System covering 73 hotspot mutations in EGFR, KRAS, BRAF, ERBB2 and PIK3CA, and compared against mutation-specific droplet digital PCR (ddPCR) and tumor tissue next-generation sequencing (NGS) performed in routine diagnostics. Variant detection accuracy at primary diagnosis and during disease progression, and ctDNA dynamics as a marker of treatment efficacy were analyzed. Results: The UltraSEEK® Lung Panel revealed an overall detection rate of 87.2% irrespective of ccfDNA input, and a specificity of &amp;gt;99.5% using the reference material. When comparing the 131 variants identified in the patient-derived cell-free plasma using UltraSEEK® with mutation-specific ddPCR analyses, a concordance of 90% was found. The 77 diagnostically or clinically relevant variants identified in 66 pretreatment tumor tissue showed an overall concordance with UltraSEEK® of 73% at baseline with a false-negative rate of 3%. When restricting to therapeutically targetable mutations, the concordance elevated to 85%. A decrease in ctDNA levels at 4-6 weeks after start of treatment detected with UltraSEEK® correlated with a prolonged median PFS (46 vs 11 weeks; P&amp;lt;0.05) and OS (145 vs 39 weeks; P&amp;lt;0.001). In four out of thirteen NSCLC patients treated with targeted therapy, resistance mutations were identified using UltraSEEK® at disease progression, implicating the requirement of a therapy switch. Conclusion: Using plasma-derived ccfDNA, the UltraSEEK® Lung Panel with a mid-density set of the most common predictive markers for NSCLC is an alternative tool to detect both mutations at diagnosis and disease progression, and to monitor treatment response. Citation Format: Paul van der Leest, Melanie Janning, Naomi Rifaela, Maria L. Aguirre Azpurua, Jolanthe Kropidlowski, Sonja Loges, Nicholas Lozano, Alexander Sartori, Darryl Irwin, Pierre-Jean Lamy, T. Jeroen N. Hiltermann, Harry J. Groen, Klaus Pantel, Léon C. van Kempen, Harriet Wikman, Ed Schuuring. Detection and monitoring of tumor-derived mutations in ctDNA using the UltraSEEK Lung Panel on the MassARRAY System in metastatic NSCLC patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3411.

Characterization and clustering of kinase isoform expression in metastatic melanoma.

Mutations to the human kinome are known to play causal roles in cancer. The kinome regulates numerous cell processes including growth, proliferation, differentiation, and apoptosis. In addition to aberrant expression, aberrant alternative splicing of cancer-driver genes is receiving increased attention as it could lead to loss or gain of functional domains, altering a kinase's downstream impact. The present study quantifies changes in gene expression and isoform ratios in the kinome of metastatic melanoma cells relative to primary tumors. We contrast 538 total kinases and 3,040 known kinase isoforms between 103 primary tumor and 367 metastatic samples from The Cancer Genome Atlas (TCGA). We find strong evidence of differential expression (DE) at the gene level in 123 kinases (23%). Additionally, of the 468 kinases with alternative isoforms, 60 (13%) had significant difference in isoform ratios (DIR). Notably, DE and DIR have little correlation; for instance, although DE highlights enrichment in receptor tyrosine kinases (RTKs), DIR identifies altered splicing in non-receptor tyrosine kinases (nRTKs). Using exon junction mapping, we identify five examples of splicing events favored in metastatic samples. We demonstrate differential apoptosis and protein localization between SLK isoforms in metastatic melanoma. We cluster isoform expression data and identify subgroups that correlate with genomic subtypes and anatomic tumor locations. Notably, distinct DE and DIR patterns separate samples with BRAF hotspot mutations and (N/K/H)RAS hotspot mutations, the latter of which lacks effective kinase inhibitor treatments. DE in RAS mutants concentrates in CMGC kinases (a group including cell cycle and splicing regulators) rather than RTKs as in BRAF mutants. Furthermore, isoforms in the RAS kinase subgroup show enrichment for cancer-related processes such as angiogenesis and cell migration. Our results reveal a new approach to therapeutic target identification and demonstrate how different mutational subtypes may respond differently to treatments highlighting possible new driver events in cancer.

Utility of Select Gene Mutation Detection in Tumors by the Idylla Rapid Multiplex PCR Platform in Comparison to Next-Generation Sequencing.

Testing of tumors by next generation sequencing (NGS) is impacted by relatively long turnaround times and a need for highly trained personnel. Recently, Idylla oncology assays were introduced to test for BRAF, EGFR, KRAS, and NRAS common hotspot mutations that do not require specialized trained personnel. Moreover, the interpretation of results is fully automated, with rapid turnaround time. Though Idylla testing and NGS have been shown to have high concordance in identifying EGFR, BRAF, KRAS, and NRAS hotspot mutations, there is limited experience on optimal ways the Idylla system can be used in routine practice. We retrospectively evaluated all cases with EGFR, BRAF, KRAS, or NRAS mutations identified in clinical specimens sequenced on two different NGS panels at the University of Rochester Medical Center (URMC) molecular diagnostics laboratory between July 2020 and July 2021 and assessed if these mutations would be detected by the Idylla cartridges if used. We found that the Idylla system could accurately identify Tier 1 or 2 actionable genomic alterations in select associated disease pathologies if used. Yet, in a minority of cases, we would have been unable to detect NGS-identified pathogenic mutations due to their absence on the Idylla panels. We derived algorithmic practice guidelines for the use of the Idylla cartridges. Overall, Idylla molecular testing could be implemented either as a first-line standalone diagnostic tool in select indications or for orthogonal confirmation of uncertain results.

The potential of PIK3CA, KRAS, BRAF, and APC hotspot mutations as a non-invasive detection method for colorectal cancer

BackgroundEarly diagnosis of colorectal cancer (CRC) can lead to prompt treatment modalities. Circulating cell-free DNA (cfDNA) analysis provides an alternative non-invasive procedure for the study of the molecular profiles of the corresponding tumor tissue. In this study, we aimed to investigate PIK3CA, KRAS, BRAF, and APC hotspot mutations in CRC tumor tissue, besides evaluating the diagnostic performance of KRAS, BRAF, and PIK3CA mutations in the plasma cfDNA. MethodPrimary CRC tissue samples and paired plasma samples were collected from 70 patients. After DNA extraction, PCR-direct sequencing was used to screen for mutations in PIK3CA exon 9 and APC exon 15 in tumor tissues. Amplification Refractory Mutation System (ARMS)-quantitative PCR (qPCR) was used to evaluate KRAS codon 12 and 13, BRAF V600E, and PIK3CA exon 9 hotspot mutations. ResultsPIK3CA exon 9 hotspot mutations were detected in 47.1% of tumor tissues and 20% of paired plasma cfDNA samples by ARMS-qPCR method, while Sanger sequencing did not identify any mutation in PIK3CA exon 9. The KRAS exon 2 mutations were detected in 71.4% and 34.3% of tumor tissue samples and paired plasma cfDNA respectively. BRAF V600E mutation was observed in 17.1% and 4.3% of tissue DNA and plasma cfDNA respectively. A panel of PIK3CA, KRAS, and BRAF showed a sensitivity of 61% and a specificity of 100% (AUC = 0.803). APC hotspot mutations were observed in 76.8% of CRC tissue samples. APC mutations were not analyzed in the plasma samples. The co-existence of KRAS/PIK3CA/APC gene mutations encompassed the highest frequency among all combinations of mutations. BRAF and PIK3CA mutations were significantly more frequent in older patients. ConclusionWe demonstrated that a panel consisting of PIK3CA, KRAS, and BRAF mutations showed good diagnostic performance for detecting CRC in the plasma cfDNA.

Innovating Strategies and Tailored Approaches in Neuro-Oncology.

Simple SummaryDiffuse gliomas, including the most aggressive subtype glioblastoma, represent the most frequent primary central nervous system tumors. Despite intense chemoradiation protocols that represent the current standard of care, these cancers inevitably recur, and median overall survival does not exceed 18 months. New therapeutic options are compellingly needed for these tumors, particularly those lacking the favorable prognostic marker IDH mutation. Nonetheless, potentially druggable alterations are increasingly identified in distinct subsets of patients harboring gliomas. Targeted treatments, along with improved immunotherapeutic schedules, gene therapy, cell therapy, and physical strategies to improve drug delivery to the nervous system, are currently under extensive investigation. They bring hope for more effective therapies in these diseases with currently often a dismal outcome.Diffuse gliomas, the most frequent and aggressive primary central nervous system neoplasms, currently lack effective curative treatments, particularly for cases lacking the favorable prognostic marker IDH mutation. Nonetheless, advances in molecular biology allowed to identify several druggable alterations in a subset of IDH wild-type gliomas, such as NTRK and FGFR-TACC fusions, and BRAF hotspot mutations. Multi-tyrosine kinase inhibitors, such as regorafenib, also showed efficacy in the setting of recurrent glioblastoma. IDH inhibitors are currently in the advanced phase of clinical evaluation for patients with IDH-mutant gliomas. Several immunotherapeutic approaches, such as tumor vaccines or checkpoint inhibitors, failed to improve patients’ outcomes. Even so, they may be still beneficial in a subset of them. New methods, such as using pulsed ultrasound to disrupt the blood–brain barrier, gene therapy, and oncolytic virotherapy, are well tolerated and may be included in the therapeutic armamentarium soon.

Appendiceal sessile serrated lesions are distinct from their right-sided colonic counterparts and may be precursors for appendiceal mucinous neoplasms

Low-grade appendiceal mucinous neoplasms (LAMNs) can occur concurrently with appendiceal sessile serrated lesions (SSLs). To interrogate relatedness, we performed multigene and immunohistochemical characterizations of paired and unpaired SSLs and LAMNs. We evaluated 62 serrated lesions from 50 appendectomy specimens for hotspot mutations in BRAF, KRAS, and GNAS genes. Cases were subdivided into 3 groups: 20 unpaired SSLs, 18 unpaired LAMNs, and 12 with an SSL and concurrent LAMN. β-catenin and Annexin A10 immunostaining were performed on the SSL and LAMN components in the 12 paired cases, and 14 colonic SSLs served as controls. There was no significant difference in KRAS hotspot mutation rates in appendiceal SSLs (17/26; 65.4%) and LAMNs (16/30; 53.3%) (p=0.42). BRAF V600E was identified in a single case (1/50; 2.0%) of SSL and concurrent LAMN (p=1.0). Mutations in GNAS were more common in LAMNs (6/30; 20.0%) than in SSLs (1/31; 3.2%) (p=0.05). The molecular genotypes between paired SSLs and LAMNs were concordant in most cases (10/12; 83.3%). Annexin A10 immunostaining was significantly greater in colonic SSLs (14/14; 100%) than in appendiceal SSLs (1/12; 8.3%) (p<0.0001). β-catenin immunostaining was significantly increased in LAMNs (10/12; 83.3%) compared with their paired appendiceal SSLs (2/12; 16.7%) (p=0.003). Overall, appendiceal SSLs are predominantly driven by KRAS mutations and are not characterized by Annexin A10 immunostaining. Our data suggestthat at least a subset of LAMNs may arise from a precursor SSL in which GNAS mutations and/or upregulation of the WNT signaling pathway are likely key events modulating this progression.

GEN-8 Real-time PCR based intraoperative genetic analysis for gliomas

An individual therapeutic strategy based on the genetic characterization is important in gliomas. However, it has been difficult to obtain genetic features during surgery. In this study, we present an overview of intraoperative genetic analysis using modified real-time PCR method. The tumor specimen was crushed with liquid nitrogen, then extract DNA within 60 minutes. Reagents of real-time PCR for detecting IDH, TERT, and BRAF hot spot mutations were stocked and real-time PCR was performed after mixing the extracted DNA. We used PNA and LNA to detect single nucleotide variant (SNV). The average time from tumor extraction to intraoperative tentative judgement was approximately 100 minutes. Using this system. we preliminary performed intraoperative genomic analysis in10 glioma patients. We confirmed that 8 of 10 cases (80%) of intraoperative genomic diagnosis were consistent with post-operative diagnosis by Sanger sequencing. However, we experienced 2 (20%) unmatched cases due to low allele of SNV, which indicates that more advanced system is required for clinical application.

Diverse alterations associated with resistance to KRAS(G12C) inhibition.

Inactive state-selective KRAS(G12C) inhibitors1-8 demonstrate a 30-40% response rate and result inapproximately 6-month median progression-free survival in patients with lung cancer9. The genetic basis for resistance to these first-in-class mutant GTPase inhibitors remains under investigation. Here we evaluated matched pre-treatment and post-treatment specimens from 43 patients treated with the KRAS(G12C) inhibitor sotorasib. Multiple treatment-emergent alterations were observed across 27 patients, including alterations in KRAS, NRAS, BRAF, EGFR, FGFR2, MYC and other genes. In preclinical patient-derived xenograft and cell line models, resistance to KRAS(G12C) inhibition was associated with low allele frequency hotspot mutations in KRAS(G12V or G13D), NRAS(Q61K or G13R), MRAS(Q71R) and/or BRAF(G596R), mirroring observations in patients. Single-cell sequencing in an isogenic lineage identified secondary RAS and/or BRAF mutations in the same cells as KRAS(G12C), where they bypassed inhibition without affecting target inactivation. Genetic or pharmacological targeting of ERK signalling intermediates enhanced the antiproliferative effect of G12C inhibitor treatment in models with acquired RAS or BRAF mutations. Our study thus suggests a heterogenous pattern of resistance with multiple subclonal events emerging during G12C inhibitor treatment. A subset of patients in our cohort acquired oncogenic KRAS, NRAS or BRAF mutations, and resistance in this setting may be delayed by co-targeting of ERK signalling intermediates. These findings merit broader evaluation in prospective clinical trials.

Definition of Biologically Distinct Groups of Conjunctival Melanomas According to Etiological Factors and Implications for Precision Medicine.

Simple SummaryConjunctival melanoma (ConjMel) is a rare but potentially deadly eye tumor developing on the ocular mucosal surface, which is partially exposed to sunlight. The relationships between potential etiological factors such as ultraviolet exposure and ConjMel mutational landscape have not been precisely described in large cohorts. Here, we report the sequencing of 400 cancer genes in 47 primary ConjMel and show several associations between mutations and etiological factors. BRAF- and CDKN2A-mutated ConjMel affect younger patients while NF1-mutated tumors tend to develop in older ones. CTNNB1 mutations are more frequent in nevi-derived ConjMel, suggesting that the Wnt pathway is pivotal in their tumorigenesis. We further identified concomitant KIT/SF3B1 mutations in BRAF/RAS-wild type, sun-protected tumors, suggesting a similar profile as previously observed in genital and anorectal melanomas, thus unveiling a distinct, mucosal-specific, tumorigenic pathway. Finally, we report for the first time new targetable oncogenic mutations, opening new therapeutic options for these patients.Conjunctival melanoma (ConjMel) is a potentially deadly ocular melanoma, originating from partially sunlight-exposed mucosa. We explored the mutational landscape of ConjMel and studied the correlation with etiological factors. We collected 47 primary ConjMel samples and performed next-generation sequencing of 400 genes. Hotspot mutations in BRAF, NRAS, HRAS, and KIT were observed in 16 (34%), 5 (11%), 2, and 2 cases, respectively. Patients with BRAF and CDKN2A-mutated ConjMel tended to be younger while the NF1-mutated one tended to be older. The eight tumors arising from nevi were enriched in CTNNB1 mutations (63% vs. 8%; Fisher’s exact p-test = 0.001) compared to non-nevi ConjMel and five were devoid of BRAF, RAS, NF1, or KIT mutations, suggesting a specific oncogenic process in these tumors. The two KIT-mutated cases carried SF3B1 mutations and were located on sun-protected mucosa, a genotype shared with genital and anorectal mucosal melanomas. Targetable mutations were observed in ERBB2, IDH1, MET, and MAP2K1 (one occurrence each). Mutational landscape of ConjMel characterizes distinct molecular subtypes with oncogenic drivers common with mucosal and skin melanomas. CTNNB1 mutations were associated with nevus-derived ConjMel. Concomitant KIT/SF3B1 mutations in sun-protected cases suggest a common tumorigenic process with genital and anorectal mucosal melanomas.

Mesonephric and mesonephric-like carcinomas of the female genital tract: molecular characterization including cases with mixed histology and matched metastases.

Mesonephric carcinoma of the cervix is a rare tumor derived from Wolffian remnants. Mesonephric-like carcinomas of the ovary and endometrium, while morphologically similar, do not have obvious Wolffian derivation. Here, we sought to characterize the repertoire of genetic alterations in primary mesonephric and mesonephric-like carcinomas, in the distinct histologic components of mixed cases, as well as in matched primary tumors and metastases. DNA from microdissected tumor and normal tissue from mesonephric carcinomas (cervix, n = 8) and mesonephric-like carcinomas (ovarian n = 15, endometrial n = 13) were subjected to sequencing targeting 468 cancer-related genes. The histologically distinct components of four cases with mixed histology and four primary tumors and their matched metastases were microdissected and analyzed separately. Mesonephric-like carcinomas were underpinned by somatic KRAS mutations (25/28, 89%) akin to mesonephric carcinomas (8/8, 100%), but also harbored genetic alterations more frequently reported in Müllerian tumors. Mesonephric-like carcinomas that lacked KRAS mutations harbored NRAS (n = 2, ovary) or BRAF (n = 1, endometrium) hotspot mutations. PIK3CA mutations were identified in both mesonephric-like (8/28, 28%) and mesonephric carcinomas (2/8, 25%). Only mesonephric-like tumors harbored CTNNB1 hotspot (4/28, 14%) and PTEN (3/13, 23%) mutations. Copy number analysis revealed frequent gains of chromosomes 1q and 10 in both mesonephric (87% 1q; 50% chromosome 10) and mesonephric-like tumors (89% 1q; 43% chromosome 10). Chromosome 12 gains were more frequent in ovarian mesonephric-like carcinomas, and losses of chromosome 9 were more frequent in mesonephric than in mesonephric-like carcinomas (both p = 0.01, Fisher's exact test). The histologically distinct components of four mixed cases were molecularly related and shared similar patterns of genetic alterations. The progression from primary to metastatic lesions involved the acquisition of additional mutations, and/or shifts from subclonal to clonal mutations. Our findings suggest that mesonephric-like carcinomas are derived from a Müllerian substrate with differentiation along Wolffian/mesonephric lines.

Craniopharyngiomas, including Recurrent Cases, Lack TERT Promoter Hotspot Mutations.

Adamantinomatous craniopharyngiomas (ACP) are characterized by alterations in the CTNNB1 gene while almost all papillary craniopharyngiomas (PCP) harbor a canonical V600E mutation in the BRAF gene. Although other recurrent driver genes have not been described to date in craniopharyngiomas, the heterogeneous clinical course of these tumors might be associated with the acquisition of further genomic alterations. It is well known that telomerase reverse transcriptase (TERT) promoter (TERTp) alterations, including mutations or methylation, upregulate the expression of TERT and increase telomerase activity, promoting tumorigenesis. We investigated whether TERTp mutations or methylation are associated with tumor relapse in a subset of craniopharyngiomas. Samples from 42 patients with histologically confirmed craniopharyngioma were retrieved. We determined TERTp, BRAF, and CTNNB1 hotspot mutations in all samples using targeted sequencing and the TERTp methylation status by methylation-specific polymerase chain reaction (PCR) in 30 samples. While BRAF V600E mutations and CTNNB1 mutations were detected in 12 (28.6%) and 21 patients (50%) in the initial tumors and subsequent recurrences, respectively, none of the patients in our cohort, including those with multiple relapses, harbored a TERTp mutation. Furthermore, TERTp methylation was detected in 14 out of 24 cases (58.3%) with available primary samples; however, no correlation between TERTp methylation with the pathological subtype, genotype, or tumor aggressiveness was detected. These data suggest that elevated telomerase activity via acquisition of TERTp mutations is an infrequent pathway in the tumorigenesis of craniopharyngiomas, regardless of their clinical course.