Exon Skipping Mutations Research Articles

Capmatinib in MET exon 14-mutated non-small-cell lung cancer: final results from the open-label, phase 2 GEOMETRY mono-1 trial

Capmatinib has previously shown activity in treatment-naive and previously treated patients with non-small-cell lung cancer (NSCLC) and a MET exon 14-skipping mutation (METex14). Here, we report the final outcomes from the phase 2 GEOMETRY mono-1 study with an aim to provide further evidence for the activity of capmatinib. In this non-randomised, multi-cohort, open-label, phase 2 trial conducted in 152 centres and hospitals in 25 countries, with patients treated in 95 centres in 20 countries, eligible patients (aged ≥18 years) with MET-dysregulated, EGFR wild-type, and ALK rearrangement-negative advanced NSCLC (stage IIIB/IV) and an Eastern Cooperative Oncology Group performance status of 0 or 1 were assigned to cohorts (1a, 1b, 2, 3, 4, 5a, 5b, 6 and 7) based on their MET status (METex14 or MET amplification) and previous therapy lines. Patients received capmatinib (400 mg orally twice daily) in 21-day treatment cycles. The primary endpoint was overall response rate by blinded independent central review per Response Evaluation Criteria in Solid Tumours version 1.1 and was performed on the full analysis set (all patients who received at least one dose of capmatinib). Previous reports of this study had published interim or primary data for cohorts 1-7. Here, we report the final clinical outcomes from all METex14 cohorts (4, 5b, 6, and 7) and safety from all study cohorts (1-7). The trial is registered with ClinicalTrials.gov, NCT02414139, and has been completed. Of 373 treated patients enrolled from June 11, 2015, to March 12, 2020, 160 (97 [61%] female) patients had METex14 NSCLC and were enrolled in four cohorts: 60 treatment-naive (cohorts 5b and 7) and 100 previously treated (cohorts 4 and 6). The overall median study follow-up was 46·4 months (IQR 41·8-65·4) for the treatment-naïve patients and 66·9 months (56·7-73·9) for previously treated patients, respectively. Overall responses were recorded in 41 (68%; 95% CI 55·0-79·7) of 60 treatment-naive patients and 44 (44%; 95% CI 34·1-54·3) of 100 previously treated patients. In all 373 treated patients, the most common treatment-related adverse events were peripheral oedema (n=174; 47%), nausea (n=130; 35%), increased blood creatinine (n=78; 21%), and vomiting (n=74; 20%). Grade 3-4 serious adverse events occurred in 164 (44%) patients, dyspnoea being the most common (18 patients [5%]). Treatment-related deaths occurred in four (1%) patients (one each of cardiac arrest, hepatitis, organising pneumonia, and pneumonitis). No new safety signals were reported. These long-term results support METex14 as a targetable oncogenic driver in NSCLC and add to the evidence supporting capmatinib as a targeted treatment option for treatment-naive and previously treated patients with METex14 NSCLC. Novartis Pharmaceuticals.

Landscape of gene fusions and METex14 skipping in non-small cell lung carcinoma among the Chinese population: Insights from targeted RNA sequencing.

e20041 Background: Targeted RNA Sequencing is crucial for detecting gene fusions and exon-skipping mutations in non-small cell lung carcinoma (NSCLC). This study delineates the landscape of RNA-based fusion and METex14 skipping in the Chinese NSCLC population, compares the consistency with DNA-based fusion detection, and further explores the relationship between fusion-positive cases and immune markers. Methods: We profiled 294 NSCLC tumor tissues using targeted RNA sequencing, focusing on 29 fusion genes and 6 reference genes. Of these, 229 samples were concurrently assessed for fusions using a DNA panel. Patients with gene fusions or METex14 skipping were categorized as 'FUSION-POS'. Additionally, the expression of PD-L1 was evaluated. Results: In 294 NSCLC patients, 33 types of fusions and METex14 skipping were detected from 45 patients based on RNA detection. Actionable gene fusions/METex14 skipping were present in 11.22% (33/294) of patients, with the most prevalent being ALK (4.4%), METex14 skipping (2.7%), ROS1 (1.4%), and RET (1.0%). The partners of ALK, RET and ROS1 were EML4 (n= 12)/ LCLAT1(n=1), KIF5B (n=3) /PARD3(n=1) and CD74(n=2)/LRIG3(n=1)/EZR (n=1), respectively. Notably, we also identified two patients harboring FGFR2-ATE1 fusion and CD74-NRG1 fusion, respectively. Except for MET 14skipping, ST7-MET fusion was detected in 2 patients. Comparing the consistency of actionable fusions/METex14 skipping between RNA and DNA assessments in 229 NSCLC patients, RNA detection yielded a higher number of cases (24 patients vs 15 patients, 10.5% vs 6.5%), with all 15 fusions/ METex14 skipping detected by DNA also identified by RNA. Furthermore, we observed that PD-L1 expression was significantly elevated in the FUSION-POS group within lung adenocarcinoma patients (TPS: P=0.015). Conclusions: Our findings indicate that 11.22% of Chinese LUAD patients possess actionable fusions and METex14 skipping. The detection rate of fusions/ METex14 skipping via RNA is higher than that via DNA, with RNA detection fully encompassing all fusions/ METex14 skipping identified by DNA. Meanwhile, the association between fusions and PD-L1 expression suggests potential synergies for combining targeted therapy and immunotherapy in NSCLC.

Clinical characteristics and prognosis analysis of pulmonary sarcomatoid carcinoma

Objective: To summarize the clinical characteristics, treatment, and outcomes of patients with pulmonary sarcomatoid carcinoma (PSC) in order to improve clinicians' understanding of this disease. Methods: The clinical data of patients diagnosed with PSC in our hospital from January 1, 2015 to November 30, 2023 were retrospectively analyzed. According to whether radical resection was performed, the patients were divided into resectable group and unresectable group. The characteristics and treatments of PSC in different groups were compared. The survival curves were drawn by Kaplan-Meier method to compare the prognosis of different groups of patients. Results: A total of 43 PSC patients were included, including 32 males, with an average age of (62.79±9.59) years, and 31 smokers. Peripheral-type tumors were more common, with imaging showing predominantly solid soft tissue masses, and the maximum diameter of the tumor was more than 5 cm in 14 patients. Among the 23 patients who underwent NGS gene testing, the KRAS mutation rate was 43.5%, the TP53 mutation rate was 30.4%, and the MET mutation rate was 8.7%, all of which were MET-14 exon skipping mutations. PD-L1 expression was detected in 13 patients, 10 of whom showed high expression. The median overall survival (mOS) of the 43 patients with PSC was 24.6 months (13.0-52.7 months). Among them, 22 patients underwent radical lobectomy plus mediastinal lymph node dissection, 13 patients had postoperative recurrence, and 7 patients died during follow-up. The median disease-free survival (mDFS) was 12.3 months, the mOS was not achieved and the 1-year OS rate was 77.3 %. Twenty-one patients had unresectable locally advanced or advanced stage, and 15 patients died. The mDFS was 2.5 months, the mOS was 6.2 months, and the 1-year OS rate was 42.9 %. Seventeen patients received immunotherapy, and 1 patient received targeted therapy with the MET inhibitor glumetinib. Conclusions: PSC has a higher incidence in the elderly, smokers, and males, is highly malignant and has a poor prognosis. Based on its molecular biological characteristics, PD-L1 expression and tumor molecular detection can be performed to guide treatment options.

Abstract 2116: Telisotuzumab vedotin (Teliso-V)-mediated c-Met internalization, endolysosomal trafficking, and potent cytotoxic activity are unaffected by MET exon-14 skipping mutation

Abstract Background: Telisotuzumab vedotin (Teliso-V) is an antibody-drug conjugate (ADC) composed of a c-Met (MET protein) antibody coupled to the microtubule inhibitor monomethyl auristatin E (MMAE). Teliso-V was granted FDA Breakthrough Therapy Designation for advanced/metastatic EGFR WT, NSQ NSCLC with high c-MET overexpression. For MMAE drug delivery, Teliso-V must first undergo receptor internalization and endolysosomal (EL) trafficking. Hepatocyte growth factor (HGF)-dependent MET EL trafficking and degradation depend on the E3 ubiquitin ligase Casitas B-lineage lymphoma (CBL). METex14 skipping and CBL-binding site mutations, which delete the binding site for CBL, occur in ~ 3% of NSCLC. The role of CBL in Teliso-V-MMAE delivery and activity was assessed in this work. Methods: WT-human c-Met, c-Met-Y1003F/N (CBL binding site mutations in MET), and c-Met-ex14∆ (47 aa deletion to mimic ex14 skipping) or CBL-L620f, G807*, and W802* (CBL inactivating mutants) were expressed in NIH3T3, Ba/F3 cells, or A549 tumor cells using retroviral transduction. Genetically modified cells were then used to define the role of CBL in c-Met surface expression, intracellular signaling, EL trafficking (using pHrodo Red-labeled Teliso-V), and Teliso-V sensitivity in 2D/3D cell cultures. Results: c-Met expression was required for Teliso-V cytotoxicity and intracellular payload delivery in all cell lines. While minimal changes in cell surface c-Met levels were observed, CBL binding mutants had increased phosphorylated c-Met in the absence of HGF, and ex14∆-MET and CBL binding site mutants showed increased sensitivity to small molecule c-Met inhibitors. Compared to WT c-Met, ex14∆ and CBL-binding mutants showed similar EL trafficking and no change in sensitivity to Teliso-V. Co-treatment with a pan-CBL inhibitor also failed to alter Teliso-V EL trafficking and anti-tumor activity. Like most ADCs, EL trafficking was dependent on a dynamin-mediated process with reduced EL trafficking by co-treatment with Dyngo4a. To further understand the role of CBL in Teliso-V sensitivity, we expressed CBL inactivating mutations in A549 tumor cells. While the L602F and W802* mutants increased spheroid size, as well as activated p-MET, there was no change in sensitivity to Teliso-V. Conclusions: Loss of CBL-binding sites on c-Met via ex14 deletion or Y1003F/N leads to increased MET-targeted TKI sensitivity and increased phospho-c-Met levels but does not impact Teliso-V sensitivity or EL trafficking. Our results indicate that Teliso-V traffics to the EL via a dynamin-mediated process. Therefore, Teliso-V delivers MMAE through a robust ADC-mediated EL-dependent mechanism that is independent of CBL, suggesting that high c-Met expression in Ex14skipping or CBL binding site mutant NSCLC patients should be further investigated for response to Teliso-V. Citation Format: Izhar Batth, Ronald Pandoy, Shamim Khaja, Dolonchampa Maji, Tamar Uziel, Athan Vasilopoulos, Peter Ansell, PK Epling-Burnette. Telisotuzumab vedotin (Teliso-V)-mediated c-Met internalization, endolysosomal trafficking, and potent cytotoxic activity are unaffected by MET exon-14 skipping mutation [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 2116.

Non-Small Cell Lung Cancer Testing on Reference Specimens: An Italian Multicenter Experience.

Biomarker testing is mandatory for the clinical management of patients with advanced non-small cell lung cancer (NSCLC). Myriads of technical platforms are now available for biomarker analysis with differences in terms of multiplexing capability, analytical sensitivity, and turnaround time (TAT). We evaluated the technical performance of the diagnostic workflows of 24 representative Italian institutions performing molecular tests on a series of artificial reference specimens built to mimic routine diagnostic samples. Sample sets of eight slides from cell blocks of artificial reference specimens harboring exon19 EGFR (epidermal growth factor receptor) p.E746_AT50del, exon2 KRAS (Kirsten rat sarcoma viral oncogene homologue) p.G12C, ROS1 (c-ros oncogene1)-unknown gene fusion, and MET (MET proto-oncogene, receptor tyrosine kinase) Δ exon14 skipping were distributed to each participating institution. Two independent cell block specimens were validated by the University of Naples FedericoII before shipment. Methodological and molecular data from reference specimens were annotated. Overall, a median DNA concentration of 3.3ng/µL (range 0.1-10.0ng/µL) and 13.4ng/µL (range 2.0-45.8ng/µL) were obtained with automated and manual technical procedures, respectively. RNA concentrations of 5.7ng/µL (range 0.2-11.9ng/µL) and 9.3ng/µL (range 0.5-18.0ng/µL) were also detected. KRAS exon2 p.G12C, EGFR exon19 p.E736_A750del hotspot mutations, and ROS1 aberrant transcripts were identified in all tested cases, whereas 15 out of 16 (93.7%) centers detected MET exon14 skipping mutation. Optimized technical workflows are crucial in the decision-making strategy of patients with NSCLC. Artificial reference specimens enable optimization of diagnostic workflows for predictive molecular analysis in routine clinical practice.

A phosphorylation-deficient mutant of Sik3, a homolog of Sleepy, alters circadian sleep regulation by PDF neurons in Drosophila.

Sleep behavior has been observed from non-vertebrates to humans. Sleepy mutation in mice resulted in a notable increase in sleep and was identified as an exon-skipping mutation of the salt-inducible kinase 3 (Sik3) gene, conserved among animals. The skipped exon includes a serine residue that is phosphorylated by protein kinase A. Overexpression of a mutant gene with the conversion of this serine into alanine (Sik3-SA) increased sleep in both mice and the fruit fly Drosophila melanogaster. However, the mechanism by which Sik3-SA increases sleep remains unclear. Here, we found that Sik3-SA overexpression in all neurons increased sleep under both light-dark (LD) conditions and constant dark (DD) conditions in Drosophila. Additionally, overexpression of Sik3-SA only in PDF neurons, which are a cluster of clock neurons regulating the circadian rhythm, increased sleep during subjective daytime while decreasing the amplitude of circadian rhythm. Furthermore, suppressing Sik3-SA overexpression specifically in PDF neurons in flies overexpressing Sik3-SA in all neurons reversed the sleep increase during subjective daytime. These results indicate that Sik3-SA alters the circadian function of PDF neurons and leads to an increase in sleep during subjective daytime under constant dark conditions.

MET exon 14 skipping mutation is a hepatocyte growth factor (HGF)-dependent oncogenic driver in vitro and in humanized HGF knock-in mice.

Exon skipping mutations of the MET receptor tyrosine kinase (METex14), increasingly reported in cancers, occur in 3-4% of non-small cell lung cancer (NSCLC). Only 50% of patients have a beneficial response to treatment with MET-tyrosine kinase inhibitors (TKIs), underlying the need to understand the mechanism of METex14 oncogenicity and sensitivity to TKIs. Whether METex14 is a driver mutation and whether it requires hepatocyte growth factor (HGF) for its oncogenicity in a range of in vitro functions and in vivo has not been fully elucidated from previous preclinical models. Using CRISPR/Cas9, we developed a METex14/WT isogenic model in non-transformed human lung cells, and report that the METex14 single alteration was sufficient to drive MET-dependent in vitro anchorage-independent survival and motility and in vivo tumorigenesis, sensitising tumours to MET-TKIs. However, we also show that human HGF (hHGF) is required, as demonstrated in vivo using a humanized HGF knock-in strain of mice and further detected in tumor cells of METex14 NSCLC patient samples. Our results also suggest that METex14 oncogenicity is not a consequence of an escape from degradation in our cell model. Thus, we developed a valuable model for preclinical studies and present results that have potential clinical implication.

Detection of multiple types of cancer driver mutations using targeted RNA sequencing in non-small cell lung cancer.

DNA-based next-generation sequencing has been widely used in the selection of target therapies for patients with nonsmall cell lung cancer (NSCLC). RNA-based next-generation sequencing has been proven to be valuable in detecting fusion and exon-skipping mutations and is recommended by National Comprehensive Cancer Network guidelines for these mutation types. The authors developed an RNA-based hybridization panel targeting actionable driver oncogenes in solid tumors. Experimental and bioinformatics pipelines were optimized for the detection of fusions, single-nucleotide variants (SNVs), and insertion/deletion (indels). In total, 1253 formalin-fixed, paraffin-embedded samples from patients with NSCLC were analyzed by DNA and RNA panel sequencing in parallel to assess the performance of the RNA panel in detecting multiple types of mutations. In analytical validation, the RNA panel achieved a limit of detection of 1.45-3.15 copies per nanogram for SNVs and 0.21-6.48 copies per nanogram for fusions. In 1253 formalin-fixed, paraffin-embedded NSCLC samples, the RNA panel identified a total of 124 fusion events and 26 MET exon 14-skipping events, in which 14 fusions and six MET exon 14-skipping mutations were missed by DNA panel sequencing. By using the DNA panel as the reference, the positive percent agreement and the positive predictive value of the RNA panel were 98.08% and 98.62%, respectively, for detecting targetable SNVs and 98.15% and 99.38%, respectively, for detecting targetable indels. Parallel DNA and RNA sequencing analyses demonstrated the accuracy and robustness of the RNA sequencing panel in detecting multiple types of clinically actionable mutations. The simplified experimental workflow and low sample consumption will make RNA panel sequencing a potentially effective method in clinical testing.

Chinese multidisciplinary expert consensus on the management of adverse drug reactions associated with savolitinib

MET gene is a proto-oncogene, which encodes MET protein with tyrosine kinase activity. After binding to its ligand, hepatocyte growth factor, MET protein can induce MET dimerization and activate downstream signaling pathways, which plays a crucial role in tumor formation and metastasis. Savolitinib, as a specific tyrosine kinase inhibitor (TKI) targeting MET, selectively inhibits the phosphorylation of MET kinase with a significant inhibitory effect on tumors with MET abnormalities. Based on its significant efficacy shown in the registration studies, savolitinib was approved for marketing in China on June 22, 2021 for the treatment of advanced non-small cell lung cancer with MET 14 exon skipping mutations. In addition, many studies have shown that MET TKIs are equally effective in patients with advanced solid tumors with MET gene amplification or MET protein overexpression, and relevant registration clinical studies are ongoing. The most common adverse reactions during treatment with savolitinib include nausea, vomiting, peripheral edema, pyrexia, and hepatotoxicity. Based on two rounds of extensive nationwide investigations to guide clinicians, the consensus is compiled to use savolitinib rationally, prevent and treat various adverse reactions scientifically, and improve the clinical benefits and quality of life of patients. This consensus was prepared under the guidance of multidisciplinary experts, especially including the whole-process participation and valuable suggestions of experts in Traditional Chinese Medicine, thus reflecting the clinical treatment concept of integrated Chinese and western medicines.

Chinese multidisciplinary expert consensus on the management of adverse drug reactions associated with savolitinib.

Savolitinib is a highly selective mesenchymal-epithelial transition (MET) tyrosine kinase inhibitor (TKI). Based on its significant efficacy shown in clinical studies, savolitinib was conditionally approved for marketing in China on 22 June 2021, for the treatment of advanced non-small cell lung cancer (NSCLC) with MET 14 exon skipping mutation. Additionally, many studies showed that MET TKIs were equally effective in patients with advanced solid tumors with MET gene amplification or MET protein overexpression. Several relevant registered clinical studies are in progress. The most common adverse reactions (ARs) due to savolitinib administration are nausea, vomiting, peripheral edema, pyrexia, and hepatotoxicity. This consensus was developed through two rounds of extensive national surveys involving multidisciplinary experts in China, aiming to guide clinicians to prevent and treat various ARs scientifically, and improve the efficacy of the drug and the quality of life of patients.

A lung adenocarcinoma patient with co-mutations of MET and EGFR exon20 insertion responded to crizotinib

BackgroundTargeted therapy has revolutionized the treatment of patients with malignancies harboring mutations in driver genes and has brought a favorable survival benefit to the population with actionable oncogenic mutations. In recent years, the MET exon14 skipping mutation has been recognized as a potentially promising therapeutic target in non-small cell lung cancer (NSCLC). These changes are mutually exclusive with molecular drivers such as EGFR, KRAS, HER-2, BRAF, ALK and ROS1. The prevalence rate of coexisting MET exon 14 mutations and EGFR sensitive mutations (L858R, exon 19 deletions) in Chinese population was reported to be 0.2% (3/1590). However, the coexistence of MET exon 14 mutations with EGFR exon 20 insertion mutations has never been reported and the management of this subtype is not identified.Case presentationA 69-year-old male with a right lung adenocarcinoma (T4N2M0, IIIB) was confirmed to be positive for MET exon 14 skipping (c.3028_3028+1delGGinsTT, 44.4%), MET amplification (copy number 4.4), and EGFR exon 20 insertion (p. N771_H773dup, 22.1%) mutations. After the progression of one cycle of chemotherapy (Pemetrexed 0.8 g d1), the patient was subsequently accepted treatment with Crizotinib (250 mg twice a day) and achieved an important clinical remission for six months until the development of brain metastases. Then, he was submitted to a cycle of anti-programmed cell death-1 (PD-1) therapy after failure of Crizotinib and eventually acquired resistance despite of the high expression of programmed death ligand-1 (PD-L1) and tumor mutational burden (TMB) status.ConclusionThis case report provides treatment strategies for epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs)-untreated lung adenocarcinoma patients simultaneously carrying MET alterations and EGFR exon 20 insertion mutations. In addition, the signatures of PD-L1 or TMB expression were not the candidate for predicting the efficacy of immunotherapy in this context.

Detection of multiple types of cancer driver mutations using targeted RNA sequencing in NSCLC.

e15043 Background: DNA NGS sequencing has been widely used as a companion diagnostic method for matching NSCLC patients with appropriate personalized target therapy. RNA-based NGS has been proved to be valuable in detecting fusion and exon skipping mutations and therefore recommended by NCCN guidelines for these mutation types. It would be helpful to have a diagnostic method that suits different types of clinically actionable mutations in a single tube. Methods: We developed an RNA-based hybridization panel targeting actionable driver oncogenes in solid tumors. Experimental and bioinformatics pipelines were optimized for detection of fusion, SNV and indel mutations, as well as gene expression quantification. Reference materials were used for analytical validation of the panel, and 1253 FFPE samples from NSCLC patients were analyzed by DNA panel sequencing and RNA panel sequencing in parallel to assess the performance of the RNA panel in detection of multiple types of clinically actionable mutations. Results: Analytical validation showed a detection specificity of 100.0% for SNVs and fusions. The targeted RNA panel achieved a 1.45-3.15 copies/ng limit of detection (LOD) for SNVs and a 0.21-6.48 copies/ng LOD for fusions. Gene expression analysis revealed a correlation greater than 0.9 between the RNA-seq and targeted RNA panel results. Among 1253 NSCLC FFPE samples, multiple mutation types were called at both DNA and RNA levels and compared between the two assays. Twenty-one fusion and six METex14 skipping events were only detected by the RNA panel. The positive percent agreement (PPA) and positive prediction value (PPV) were 98.08% and 98.62% for targetable SNVs and 98.15% and 99.38% for targetable Indels, respectively. We also explored the relationship of RNA expression with gene copy number and protein expression. The RPKM (reads per kilobase per million mapped reads) of EGFR transcripts exhibited a linear relationship with copy number, whereas MET gene expression seemed to be regulated in a more complexed manner. In IHC analysis, all MET IHC 3+ samples had significantly higher RPKM levels than samples with MET IHC level of 2+/1+. Conclusions: Parallel DNA and RNA systematic analysis demonstrated the accuracy and robustness of the RNA sequencing panel for detection of multiple types of clinically actionable mutations. The simplified experimental workflow, lowered cost and sample consumption, along with the superior performance in simultaneous detection of multiple types of mutations will make RNA panel sequencing an effective method in clinical testing for NSCLC.

MET Exon 14 Splice-Site Mutations Preferentially Activate KRAS Signaling to Drive Tumourigenesis.

Simple SummaryMET exon 14 splice-site mutations occur in ~3–4% of lung adenocarcinoma cases, defining a cohort of patients which might benefit from anti-MET targeted therapy. Such therapies have yielded mixed results, however, pointing to the need for better treatment design. Our study sought to aid this by characterizing key changes in mutant MET signaling behaviour. We first compared the transcriptional profiles of lung tumours with either METΔex14 or wild-type MET-amplification. METΔex14-mutant tumours exhibited increased activation of the Ras-MAPK pathway, consistent with our observations in an isogenic model system. Furthermore, sustained activity of this pathway is necessary for proliferation and maintenance of METΔex14 tumours, while forced reactivation of this pathway is sufficient to restore growth in the absence of MET activity. Our findings suggest that the MAPK pathway represents a main effector of METΔex14-driven cancer, lending credence to the possibility of combined MET-MAPK inhibition to improve therapeutic outcomes.Targeted therapies for MET exon 14-skipping (METΔex14)-driven lung cancers have generated some promising results but response rates remain below that seen for other kinase-driven cancers. One strategy for improving treatment outcomes is to employ rational combination therapies to enhance the suppression of tumour growth and delay or prevent the emergence of resistance. To this end, we profiled the transcriptomes of MET-addicted lung tumours and cell lines and identified the RAS-mitogen-activated protein kinase (MAPK) pathway as a critical effector required for METΔex14-dependent growth. Ectopic expression of MET in an isogenic cell line model showed that overexpression of the mutant MET receptor led to higher levels of MAPK phosphorylation and nuclear import, resulting in increased expression and phosphorylation of nuclear MAPK targets. In comparison, other known MET effectors were unaffected. Inhibition of this pathway by KRAS knockdown in MET-addicted cells in vitro led to decreased viability in only the METΔex14-mutant cells. Conversely, decoupling RAS-MAPK axis, but not other effector pathways, from MET activity via the introduction of constitutively active mutants conferred resistance to MET inhibitors in vitro. Our results suggest that aberrant hyperactivity of the MET receptor caused by the exon 14-skipping mutation does not uniformly upregulate all known downstream effectors, rather gaining a predilection for aberrantly activating and subsequently relying on the RAS-MAPK pathway. These findings provide a rationale for the co-targeting of the RAS-MAPK pathway alongside MET to prolong therapeutic response and circumvent resistance to improve patient survival.

The parathyroid glands and parathyroid hormone: Insights from PTH gene mutations.

Nine mutations have been discovered in the parathyroid hormone (PTH) gene since it was initially sequenced in 1983. An autosomal dominant C18R mutation in the signal peptide was first reported in 1990, followed by an exon skipping mutation, leading to loss of exon 2 in 1992; the latter mutation prevents PTH biosynthesis, as exon 2 contains the initiation codon. The S23P and S23X mutations affecting the same residue were reported in 1999 and 2012, respectively, while in 2008, the somatic mutation, R83X, was detected in a parathyroid adenoma tissue sample from a patient with overt hyperparathyroidism. In 2013, the heterozygous p.Met1_Asp6del mutation was discovered incidentally in a case-control study, while another heterozygous mutation, M14K, was detected in the signal peptide 4 years later. In 2015, a homozygous R56C mutation was reported, and was the first hypoparathyroidism-causing mutation identified that affects the mature bioactive portion of PTH; this mutation has significantly contributed to the understanding of the molecular mechanisms involved in signal transduction through the PTH receptor. Recently, a novel homozygous S32P mutation was identified, which is also situated in the bioactive portion of PTH. The discovery of these nine mutations in the PTH gene and determination of the molecular mechanisms underlying their effects has provided deep insights into the synthesis, processing, and secretion of PTH. Future attempts to discover other such mutations will help elucidate as yet unknown functions of PTH, with potential clinical implications.

4-Phenylbutyric acid enhances the mineralization of osteogenesis imperfecta iPSC-derived osteoblasts

Osteogenesis imperfecta (OI) is a heritable brittle bone disease mainly caused by mutations in the two type I collagen genes. Collagen synthesis is a complex process including trimer formation, glycosylation, secretion, extracellular matrix (ECM) formation, and mineralization. Using OI patient-derived fibroblasts and induced pluripotent stem cells (iPSCs), we investigated the effect of 4-phenylbutyric acid (4-PBA) on collagen synthesis to test its potential as a new treatment for OI. Endoplasmic reticulum (ER) retention of type I collagen was observed by immunofluorescence staining in OI patient-derived fibroblasts with glycine substitution and exon skipping mutations. Liquid chromatography–mass spectrometry analysis revealed excessive glycosylation of secreted type I collagen at the specific sites in OI cells. The misfolding of the type I collagen triple helix in the ECM was demonstrated by the incorporation of heat-dissociated collagen hybridizing peptide in OI cells. Type I collagen was produced excessively by OI fibroblasts with a glycine mutation, but this excessive production was normalized when OI fibroblasts were cultured on control fibroblast-derived ECM. We also found that mineralization was impaired in osteoblasts differentiated from OI iPSCs. In summary, treatment with 4-PBA normalizes the excessive production of type I collagen, reduces ER retention, partially improves misfolding of the type I collagen helix in ECM, and improves osteoblast mineralization. Thus, 4-PBA may improve not only ER retention, but also type I collagen synthesis and mineralization in human cells from OI patients.

CRISPR/Cas9 induces exon skipping that facilitates development of fragrant rice.

Splicing of precursor-mRNAs (pre-mRNAs) is a critical biological process of gene expression. Normally, pre-mRNAs are processed by spliceosomes to produce mature mRNAs by removing introns at 5'-(donor) and 3'-(acceptor) splice sites based on the canonical GU-AG rule (Reddy et al., 2013). Mutation at either the intron donor or acceptor sites should cause mRNA mis-splicing. Recently, an exon skipping method has been developed to generate loss of gene function in mammalian cells using base editors to mutate nucleotides at the acceptor sites (Gapinske et al., 2018).

Dnmt3b regulates DUX4 expression in a tissue-dependent manner in transgenic D4Z4 mice

BackgroundFacioscapulohumeral muscular dystrophy (FSHD) is a skeletal muscle disorder that is caused by derepression of the transcription factor DUX4 in skeletal muscle cells. Apart from SMCHD1, DNMT3B was recently identified as a disease gene and disease modifier in FSHD. However, the exact role of DNMT3B at the D4Z4 repeat array remains unknown.MethodsTo determine the role of Dnmt3b on DUX4 repression, hemizygous mice with a FSHD-sized D4Z4 repeat array (D4Z4-2.5 mice) were cross-bred with mice carrying an in-frame exon skipping mutation in Dnmt3b (Dnmt3bMommeD14 mice). Additionally, siRNA knockdowns of Dnmt3b were performed in mouse embryonic stem cells (mESCs) derived from the D4Z4-2.5 mouse model.ResultsIn mESCs derived from D4Z4-2.5 mice, Dnmt3b was enriched at the D4Z4 repeat array and DUX4 transcript levels were upregulated after a knockdown of Dnmt3b. In D4Z4-2.5/Dnmt3bMommeD14 mice, Dnmt3b protein levels were reduced; however, DUX4 RNA levels in skeletal muscles were not enhanced and no pathology was observed. Interestingly, D4Z4-2.5/Dnmt3bMommeD14 mice showed a loss of DNA methylation at the D4Z4 repeat array and significantly higher DUX4 transcript levels in secondary lymphoid organs. As these lymphoid organs seem to be more sensitive to epigenetic modifiers of the D4Z4 repeat array, different immune cell populations were quantified in the spleen and inguinal lymph nodes of D4Z4-2.5 mice crossed with Dnmt3bMommeD14 mice or Smchd1MommeD1 mice. Only in D4Z4-2.5/Smchd1MommeD1 mice the immune cell populations were disturbed.ConclusionsOur data demonstrates that loss of Dnmt3b results in derepression of DUX4 in lymphoid tissues and mESCs but not in myogenic cells of D4Z4-2.5/Dnmt3bMommeD14 mice. In addition, the Smchd1MommeD1 variant seems to have a more potent role in DUX4 derepression. Our studies suggest that the immune system is particularly but differentially sensitive to D4Z4 chromatin modifiers which may provide a molecular basis for the yet underexplored immune involvement in FSHD.

Abstract 5454: A data driven pipeline to detect somatic mutations in oncology

Abstract The analysis of Next Generation Sequencing (NGS) data relies on workflows that integrate different bioinformatics tools. Designing workflows that are assay agnostic and have a robust computational framework for result generation in a timely manner is particularly important for clinical applications of NGS. These technologies play an important role in classifying a patient's tumor at a molecular level however, there is still a need to develop workflows that generate consistent mutation profiles for oncology. We developed a bioinformatics tool to analyze DNA and RNA sequencing data in a single pipeline and to specifically detect somatic mutations in oncology clinical workflows. Our automated pipeline report contains extensive quality metrics for each sample and generates a robust mutation annotation and filtration module vital for clinical use. We use data driven filters and algorithms that predict the effectiveness of mutations to filter out mutations of poor quality or mutations without clinical significance. We applied our pipeline to standard cell lines and various cancer FFPE samples acquired from the Intermountain Healthcare Biorepository. Our pipeline successfully processes different mutation classes including Single Nucleotide Variants (SNVs), small (<20 bp) and large (>21 bp) insertions and deletions (InDels), gene fusions, and exon skipping mutations. In addition, we established a reference set that allows us to call Copy Number Variants (CNVs) in a gene panel designed to detect clinically relevant mutations in lung cancers. Comparison of this pipeline to a validated external workflow yield high concordance of clinically significant mutations. We conclude that our pipeline offers a sensitive and specific methodology to analyze DNA and RNA sequencing data to clinically relevant results from low input amplicon-based assays for various solid tumor cancer types. Citation Format: Katherine Shortt, Christopher Johnson, Archana Ramesh, Tom Neuwerth, Chris Giauque, Sharanya Raghunath. A data driven pipeline to detect somatic mutations in oncology [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5454.

Progress on Mechanism of MET Gene Mutation and Targeted Drugs in Non-small Cell Lung Cancer

间质-上皮细胞转化因子（mesenchymal-epithelial transition factor, MET）基因是非小细胞肺癌（non-small cell lung cancer, NSCLC）的一种重要肿瘤驱动基因，针对MET 14外显子的跳跃突变的靶向治疗药物给患者带来新的希望。目前已经上市或者即将上市的MET抑制剂包括：克唑替尼、卡博替尼、沃利替尼和Tepotinib等。MET抑制剂的客观缓解率较高，并且安全性良好。但是，MET抑制剂的耐药不可避免，因此需要重视对于耐药机制的研究。肝细胞生长因子（hepatocyte growth factor, HGF）/MET信号通路抑制剂与其他药物的联合应用，对于抑制和逆转耐药可能发挥重要作用。

Analysis of multigene detection in patients with advanced lung adenocarcinoma using cytological specimens

ObjectiveTo investigate the mutation status and clinical characteristics of multigene detection in advanced lung adenocarcinoma using cytological specimens. Materials and Methods137 advanced lung adenocarcinoma patients with 10 driver genes detection in the Fourth Hospital Hebei Medical University from January 2019 to November 2019 was analysized. 137 cytological specimens including fine-needle aspiration specimens and maligant serous cavity effusion (pleural effusion, peritoneal and pericardial effusion). Ten driver mutations of EGFR, ALK, ROS1, BRAF, KRAS, NRAS, HER2, RET, PIK3CA and MET were detected by the amplification refractory mutation system (ARMS). Meanwhile, 90 of 137 patients were detected with biopsies for parallel gene detection. Results78.10 % (107/137) of patients with advanced lung adenocarcinoma harbored at least one of 10 driver mutations. The three main mutations were EGFR (69.16 %, 74/137), ALK (6.57 %, 9/137)and ROS1 (3.65 %, 5/137) mutations. Besides, we found 6 cases including two concomitant mutations: EGFR Exon19 del/HER2 (1/137), EGFR Exon21 L858R/PIK3CA (2/137), EGFR Exon21 L858R/RET (1/137), and ALK/KRAS (2/137). Among 137 patients, women aged 64 or older were more likely to have the mutations (P < 0.05). Female patients (P = 0.003) older or equal to 64 years (P = 0.015) with non-smoking habbit (P = 0.027) were more detected with EGFR mutations, while ALK was more detectable in patients yonger than 64 years. Parallel analysis showed that rates of single EGFR, ALK, ROS1, RET, KRAS, NRAS, HER2, MET mutations and concomitant different mutations were not significantly different between cytological specimens and matched histological specimens. ConclusionsIn the study, cytological specimens and biopsy samples have a very high coincidence rate of gene detection. EGFR, ALK and ROS1 mutations were the main driver mutations in patients with advanced lung adenocarcinoma.We speculate that EGFR and ALK are more prone to concomitant mutations respectively and the treatment of advanced lung adenocarcinoma patients with concomitant mutations deserves further study. The rate of KRAS, NRAS, BRAF, PIK3CA, RET and MET exon14 skipping mutation were low but may had a significant impact on the targeted therapy of patients with advanced lung adenocarcinoma.