Discovery of Pyridopyrimidinones that Selectively Inhibit the H1047R PI3Kα Mutant Protein.

The H1047R mutation of PIK3CA is highly prevalent in breast cancers and other solid tumors. Selectively targeting PI3KαH1047R over PI3KαWT is crucial due to the role that PI3KαWT plays in normal cellular processes, including glucose homeostasis. Currently, only one PI3KαH1047R-selective inhibitor has progressed into clinical trials, while three pan mutant (H1047R, H1047L, H1047Y, E542K, and E545K) selective PI3Kα inhibitors have also reached the clinical stage. Herein, we report the design and discovery of a series of pyridopyrimidinones that inhibit PI3KαH1047R with high selectivity over PI3KαWT, resulting in the discovery of compound 17. When dosed in the HCC1954 tumor model in mice, 17 provided tumor regressions and a clear pharmacodynamic response. X-ray cocrystal structures from several PI3Kα inhibitors were obtained, revealing three distinct binding modes within PI3KαH1047R including a previously reported cryptic pocket in the C-terminus of the kinase domain wherein we observe a ligand-induced interaction with Arg1047.

PIK3CA testing in hormone receptor-positive/HER2-negative metastatic breast cancer: real-world data from Italian molecular pathology laboratories.

Introduction: PIK3CA gene mutations occur in approximately 40% of hormone receptor-positive/HER2-negative (HR+/HER2-) metastatic breast cancers (MBCs), electing them to targeted therapy. Testing PIK3CA status is complex due to selection of biological specimen and testing method. Materials & methods: This work investigates real-life experience on PIK3CA testing in HR+/HER2- MBC. Clinical, technical and molecular data on PIK3CA testing were collected from two referral laboratories. Additionally, the results of a nationwide PIK3CA survey involving 116 institutions were assessed. Results: Overall, n = 35 MBCs were PIK3CA-mutated, with mutations mostly occurring in exons 9 (n = 19; 51.4%) and 20 (n = 15; 40.5%). The nationwide survey revealed significant variability across laboratories in terms of sampling methodology, technical assessment and clinical report signing healthcare figures for PIK3CA molecular testing in diagnostic routine practice. Conclusion: This study provides insights into the real-world routine of PIK3CA testing in HR+/HER2- MBC and highlights the need for standardization and networking in predictive pathology.

Network pharmacology, molecular docking and experimental study of CEP in nasopharyngeal carcinoma.

ETHNOPHARMACOLOGICAL RELEVANCE: The Stephania cephalantha Hayata is an important traditional medicinal plant widely used in traditional medicine to treat cancer. Cepharanthine (CEP) was extracted from the roots of Stephania cephalantha Hayata. It has been found to exhibit anticancer activity in different types of cancer cells. Nevertheless, the activity of CEP against nasopharyngeal carcinoma (NPC) and its underlying mechanism warrant further investigation. AIMS OF THE STUDY: NPC is an invasive and highly metastatic malignancy that affects the head and neck region. This research aimed to investigate the pharmacological properties and underlying mechanism of CEP against NPC, aiming to offer novel perspectives on treating NPC using CEP. MATERIALS AND METHODS: In vitro, the pharmacological activity of CEP against NPC was evaluated using the CCK-8 assay. To predict and elucidate the anticancer mechanism of CEP against NPC, we employed network pharmacology, conducted molecular docking analysis, and performed Western blot experiments. In vivo validation was performed through a nude mice xenograft model of human NPC, Western blot and immunohistochemical (IHC) assays to confirm pharmacological activity and the mechanism. RESULTS: In a dose-dependent manner, the proliferation and clonogenic capacity of NPC cells were significantly inhibited by CEP. Additionally, NPC cell migration was suppressed by CEP. The results obtained from network pharmacology experiments revealed that anti-NPC effect of CEP was associated with 8 core targets, including EGFR, AKT1, PIK3CA, and mTOR. By performing molecular docking, the binding capacity of CEP to the candidate core proteins (EGFR, AKT1, PIK3CA, and mTOR) was predicted, resulting in docking energies of -10.0 kcal/mol for EGFR, -12.4 kcal/mol for PIK3CA, -10.8 kcal/mol for AKT1, and -8.6 kcal/mol for mTOR. The Western blot analysis showed that CEP effectively suppressed the expression of EGFR and the phosphorylation levels of downstream signaling proteins, including PI3K, AKT, mTOR, and ERK. After CEP intervention, a noteworthy decrease in tumor size, without inducing any toxicity, was observed in NPC xenograft nude mice undergoing in vivo treatment. Additionally, IHC analysis demonstrated a significant reduction in the expression levels of EGFR and Ki-67 following CEP treatment. CONCLUSION: CEP exhibits significant pharmacological effects on NPC, and its mechanistic action involves restraining the activation of the EGFR/PI3K/AKT pathway. CEP represents a promising pharmaceutical agent for addressing and mitigating NPC.

Identification of genomic alterations with clinical impact in canine splenic hemangiosarcoma.

Canine hemangiosarcoma (HSA) is an aggressive cancer of endothelial cells with short survival times. Understanding the genomic landscape of HSA may aid in developing therapeutic strategies for dogs and may also inform therapies for the rare and aggressive human cancer angiosarcoma. The objectives of this study were to build a framework for leveraging real-world genomic and clinical data that could provide the foundation for precision medicine in veterinary oncology, and to determine the relationships between genomic and clinical features in canine splenic HSA. One hundred and nine dogs with primary splenic HSA treated by splenectomy that had tumour sequencing via the FidoCure® Precision Medicine Platform targeted sequencing panel were enrolled. Patient signalment, weight, metastasis at diagnosis and overall survival time were retrospectively evaluated. The incidence of genomic alterations in individual genes and their relationship to patient variables including outcome were assessed. Somatic mutations in TP53 (n = 44), NRAS (n = 20) and PIK3CA (n = 19) were most common. Survival was associated with presence of metastases at diagnosis and germline variants in SETD2 and NOTCH1. Age at diagnosis was associated with somatic NRAS mutations and breed. TP53 and PIK3CA somatic mutations were found in larger dogs, while germline SETD2 variants were found in smaller dogs. We identified both somatic mutations and germline variants associated with clinical variables including age, breed and overall survival. These genetic changes may be useful prognostic factors and provide insight into the genomic landscape of hemangiosarcoma.

Application of precision medicine based on next-generation sequencing and immunohistochemistry in ovarian cancer: a real-world experience.

OBJECTIVE: To evaluate the landscape of gene alterations and immunohistochemistry (IHC) profiles of patients with ovarian cancer for targeted therapy and investigate the real-world experience of applying precision medicine. METHODS: Patients diagnosed with ovarian cancer between January 2015 and May 2021 at Severance Hospital and who underwent tumor next-generation sequencing (NGS) were reviewed. Data on germline mutation, IHC markers for mismatch repair deficiency (MMRd), programmed death ligand 1 (PD-L1) expression, and human epidermal growth factor receptor 2 (HER2) expression were acquired. The use of matched therapy and its clinical outcomes were evaluated. RESULTS: Of the 512 patients who underwent tumor NGS, 403 underwent panel-based germline testing. In patients who underwent both tests, tumor NGS identified 39 patients (9.7%) with BRCA mutations and 16 patients (4.0%) with other homologous recombination repair (HRR)-associated gene mutations, which were not found in germline testing. The most common single nucleotide variants were TP53 (82.2%), ARID1A (10.4%), PIK3CA (9.7%), and KRAS (8.4%). Copy number aberrations were found in 122 patients. MMRd was found in 3.2% of patients, high PD-L1 expression in 10.1%, and HER2 overexpression in 6.5%. Subsequently, 75 patients (14.6%) received a poly (ADP-ribose) polymerase inhibitor based on BRCA mutation and 11 patients (2.1%) based on other HRR-associated gene mutations. Six patients (1.2%) with MMRd underwent immunotherapy. Twenty-eight patients (5.5%) received other matched therapies targeting HER2, fibroblast growth factor receptor, folate receptor alpha, RAS, and PIK3CA. CONCLUSION: A comprehensive review of germline mutation, IHC, and tumor NGS helped identify candidates for precision therapy in patients with ovarian cancer, a proportion of whom received matched therapy.

Clinical practice guideline for activated phosphatidyl inositol 3-kinase-delta syndrome in Japan.

Activated phosphatidyl inositol 3-kinase-delta syndrome (APDS) due to gain-of-function variant in the class IA PI3K catalytic subunit p110δ (responsible gene: PIK3CD) was described in 2013. The disease is characterized by recurrent airway infections and bronchiectasis. It is associated with hyper-IgM syndrome due to the defect of immunoglobulin class switch recombination and decreased CD27-positive memory B cells. Patients also suffered from immune dysregulations, such as lymphadenopathy, autoimmune cytopenia or enteropathy. T-cell dysfunction due to increased senescence is associated with a decrease in CD4-positive T lymphocytes and CD45RA-positive naive T lymphocytes, along with increased susceptibility to Epstein-Barr virus/cytomegalovirus infections. In 2014, loss-of-function (LOF) mutation of p85α (responsible gene: PIK3R1), a regulatory subunit of p110δ, was identified as a causative gene, followed in 2016 by the identification of the LOF mutation of PTEN, which dephosphorylates PIP3, leading to the differentiation of APDS1 (PIK3CD-GOF), APDS2 (PIK3R1-LOF) and APDS-L (PTEN-LOF). Since the pathophysiology of patients with APDS varies with a wide range of severity, it is crucial that patients receive appropriate treatment and management. Our research group created a disease outline and a diagnostic flow chart and summarized clinical information such as the severity classification of APDS and treatment options.

Beyond Skin Rash: Alpelisib-Induced Anaphylactic Reactions.

Alpelisib is a specific oral PI3K inhibitor used combined with fulvestrant for the treatment of patients with HR+/HER2-/PIK3CA-mutated metastatic breast cancer. Adverse drug reactions with alpelisib are common, including hyperglycemia and rash. Here we describe extraordinary and life-threatening reactions beyond skin rash in two patients with progressive PIK3CA-mutated metastatic cancer in whom alpelisib was initiated. Case-A (vaginal cancer): After 10 days on treatment, she developed dry eyes, generalized rash and itching. Alpelisib was interrupted and symptomatic treatment initiated. Because of an initial tumor response, a rechallenge was done. Ninety minutes after a reduced dose of alpelisib, she developed an anaphylactic reaction with angioedema, hypotension, and skin rash. Case-B (breast cancer): After 11 days on treatment, she developed skin rash and alpelisib was interrupted. At re-initiation, she felt tingles in her face and ears and some skin erythema. Given the mild rash, a second rechallenge with premedication was performed. Ninety minutes after a reduced dose of alpelisib, she developed a type-1 allergic reaction with angioedema, tingles, and skin rash. In both cases, a type-1 allergic reaction was diagnosed and symptomatic treatment was initiated, alpelisib was permanently discontinued and the patients fully recovered the next week(s). This report underlines the critical importance to consider type-I allergic reactions in the differential diagnosis in cases of rash associated with alpelisib. Even if a reaction develops after days on treatment, a type-I allergic reaction cannot be excluded. A rechallenge can be dangerous and should always be well contemplated or even avoided.

PTEN mutation predicts unfavorable fertility preserving treatment outcome in the young patients with endometrioid endometrial cancer and atypical hyperplasia.

OBJECTIVE: This study aimed to investigate the impact of molecular classification and PTEN, KRAS and PIK3CA gene mutation on the outcome of fertility-preserving treatment in the patients with endometrioid endometrial cancer (EEC) and endometrial atypical hyperplasia (EAH). METHODS: This is a single-center retrospective study. A total of 135 patients with EEC and EAH receiving fertility-preserving treatment and molecular classification were reviewed. The distribution of the four types of molecular classification was described. The impact of non-specific molecular profile (NSMP), mismatch repair-deficiency (MMRd), and PTEN, KRAS and PIK3CA gene mutation on the outcome of fertility-preserving treatment was analyzed. RESULTS: Of the patients analyzed, 86.7% (117/136) were classified as having NSMP; 14 (10.4%), MMRd; 1 (0.7%), POLEmut EAH; and 3 (2.2%), p53abn EEC. The patients having NSMP and MMRd achieved similar 16-, 32-, and 48-week complete response rates. The patients harboring tier I and tier II PTEN mutations (PTENmut-Clin) achieved lower cumulative 32-week CR rates than those with PTEN-others (without PTENmut-Clin) (22/47, 46.8% vs. 50/74, 67.6%; p=0.023; odds ratio=0.422; 95% confidence interval [CI]=0.199-0.896). Insulin-resistance (hazard ratio [HR]=0.435; 95% CI=0.269-0.702; p=0.001) and PTENmut-Clin (HR=0.535; 95% CI=0.324-0.885; p=0.015) were independent negative predictors for lower 32-week CR rates. CONCLUSION: PTENmut-Clin is an independent risk factor for unfavorable fertility-preserving treatment outcomes in the patients with EEC and EAH. The patients with MMRd receiving fertility-preserving treatment achieved outcomes similar to those of the patients with NSMP. The molecular profiles might guide fertility-preserving treatment in the prognosis and clinical decisions.

Ipatasertib, an oral AKT inhibitor, in combination with carboplatin exhibits anti-proliferative effects in uterine serous carcinoma.

PURPOSE: Uterine serous carcinoma (USC) exhibits worse survival rates compared to the endometrioid subtype, and there is currently no effective treatment options for recurrence of this disease after platinum-based chemotherapy. Activation of PIK3CA/AKT/mTOR signaling pathway is a common biological feature in USC. MATERIALS AND METHODS: Ipatasertib (IPAT) is an investigational, orally administered, ATP-competitive, highly selective inhibitor of pan AKT that has demonstrated anti-proliferative activity in a variety of tumor cells and tumor models. In this study, we used IPAT, carboplatin and their combination to investigate the anti-tumor activity in SPEC-2 and ARK-1 cells. RESULTS: Our results indicate that IPAT combined with carboplatin at low doses was more effective at reducing proliferation, inducing apoptosis and causing cellular stress than IPAT or carboplatin alone. In particular, inhibition of the PIK3CA/AKT/mTOR pathway and induction of DNA damage were involved in the synergistic inhibition by combination treatment of cell viability in USC cells treated with the combination. Furthermore, IPAT in combination with carboplatin significantly reduced cell adhesion and inhibited cell invasion. CONCLUSIONS: These findings suggest that the combination of IPAT and carboplatin has potential clinical implications for developing new USC treatment strategies.

Circulating tumor DNA profile and its clinical significance in patients with hormone receptor-positive and HER2-negative mBC.

Background: After early-line (first- and second-line) endocrine therapy, hormone-receptor (HR)-positive and human epidermal growth factor receptor 2 (HER2)-negative metastatic breast cancers (mBCs) become resistant to endocrine therapy. Genetic alterations may underlie resistance to endocrine therapies. This study aims to investigate the circulating tumor DNA (ctDNA) alterations and the clinical implication in hormone-receptor-positive, HER2-negative metastatic breast cancer patients with multiline endocrine therapy failure. Methods: This registered study (NCT05079074, ClinicalTrials.gov) enrolled 104 patients with hormone-receptor-positive, HER2-negative metastatic breast cancer who progressed after the early-line endocrine therapy. ctDNA alterations were analyzed by next generation sequencing (NGS). ctDNA alterations were ranked and clustered by using R 'ComplexHeatmap' and 'hclust' function. ctDNA-guided therapy was administrated. Progression-free survival (PFS) was assessed COX regression analysis, and Kaplan-Meier curves were plotted. Findings: The top ctDNA altered genes were TP53 (39%), PIK3CA (38%), BRCA1/2 (13%), ESR1 (12%), FGFR (11%), ERBB2 (11%), and GATA3 (9%). Among these genes, TP53, PIK3CA helix domain mutation (PIK3CA-HD), FGFR, ESR1 and GATA3 were related to endocrine therapy resistance. The genetic landscapes changed and tumor mutation burden increased in both TP53-altered and PIK3CA-altered patients. Both BRCA1/2 and ERBB2 alterations correlated with TP53 alterations (P=0.02 and P=0.04, respectively). However, while 93% BRCA1/2 alterations concentrated in PIK3CA-wildtype patients, 82% ERBB2 alterations concentrated in PIK3CA-altered patients. Kaplan-Meier curves showed that patients who received druggable ctDNA alteration-guided treatment (DDAT) had significantly longer PFS than those who received physician-chosen therapy, with median PFS of 6.1 months versus 4.6 months (hazard ratio = 0.53, 95% CI: 0.34-0.85, Logrank P = 0.006). Conclusion: Multiple genetic alterations were important reasons for the failure of endocrine therapy for HR-positive and HER2-negative mBC. Targeting these genes might restore the treatment sensitivity and benefit survival.

Discovery of GDC-0077 (Inavolisib), a Highly Selective Inhibitor and Degrader of Mutant PI3Kα.

Small molecule inhibitors that target the phosphatidylinositol 3-kinase (PI3K) signaling pathway have received significant interest for the treatment of cancers. The class I isoform PI3Kα is most commonly associated with solid tumors via gene amplification or activating mutations. However, inhibitors demonstrating both PI3K isoform and mutant specificity have remained elusive. Herein, we describe the optimization and characterization of a series of benzoxazepin-oxazolidinone ATP-competitive inhibitors of PI3Kα which also induce the selective degradation of the mutant p110α protein, the catalytic subunit of PI3Kα. Structure-based design informed isoform-specific interactions within the binding site, leading to potent inhibitors with greater than 300-fold selectivity over the other Class I PI3K isoforms. Further optimization of pharmacokinetic properties led to excellent in vivo exposure and efficacy and the identification of clinical candidate GDC-0077 (inavolisib, 32), which is now under evaluation in a Phase III clinical trial as a treatment for patients with PIK3CA-mutant breast cancer.

Quantifying the Value of the Molecular Tumor Board: Discordance Recommendation Rate and Drug Cost Avoidance.

PURPOSE: Molecular tumor boards (MTBs) provide interventions that assist the patient's primary oncologist's interpretation and application of precision oncology and avoid clinical and financial toxicities of prescribing inappropriate targeted therapy. In this article, we describe a novel method for illustrating MTBs value and recommendation discordance rate and report associated drug cost avoidance data. METHODS: From January 1, 2021, to December 31, 2021, patients assessed by our program's MTB were retrospectively evaluated. Recommendation discordance was defined as any disagreement between MTB therapeutic recommendations and those provided in the next-generation sequencing vendor's report. RESULTS: In 2021, our program processed 1,119 next-generation sequencing orders via external vendors for 1,029 unique patients with a variety of solid tumor and hematologic malignancies. During this period, 962 patients were reviewed through our MTB process. MTB recommendation discordance rate was high (229 of 502; 45.6%) and varied across test vendors. Rationales for discordance included the following: low level of evidence (88% of patients), alternative standard of care available (60%), and tolerability concerns (42%), among others. Discordance was highest for Vendor C (30%), followed by Vendor A (24%) and Vendor B (8%). The most common drug classes not supported were mTOR, PARP, MEK, and PIK3CA inhibitors when recommended by vendors in off-label settings. MTB interventions accounted for $3,209,070 in US dollars in potential drug cost avoidance. CONCLUSION: Therapeutic recommendation discordance rates can provide quantitative insight into the benefit of MTB. Discordance-associated drug cost avoidance further demonstrates MTB's financial value. These measures may be used as part of the justification for this service line within a cancer care program.

Integrated Metabolomics and Network Pharmacology to Reveal the Mechanisms of Guizhi-Fuling Treatment for Myocardial Ischemia.

Myocardial ischemia is a cardio-physiological condition due to a decrease in blood perfusion to the heart, leading to reduced oxygen supply and abnormal myocardial energy metabolism. Guizhi-Fuling (GZFL) is effective in treating Myocardial ischemia. However, its mechanism of action is unclear and requires further exploration. We attempt to decipher the mechanisms behind GZFL treating Myocardial ischemia by integrating metabolomics and network pharmacology. In this study, myocardial metabolomic analysis was performed using GC/MS to identify the potential mechanism of action of GZFL during myocardial ischemia. Then, network pharmacology was utilized to analyze key pathways and construct a pathway-core target network. Molecular docking was incorporated to validate core targets within network pharmacological signaling pathways. Finally, western blots were utilized to verify core targets of metabolomics, network pharmacology integrated pathways, and key signaling targets. Thus, 22 critical biomarkers of GZFL for treating myocardial ischemia were identified. Most of these metabolites were restored using modulation after GZFL treatment. Based on the network pharmacology, 297 targets of GZFL in treating myocardial ischemia were identified. The further comprehensive analysis focused on three key targets, such as Tyrosine hydroxylase (TH), myeloperoxidase (MPO), and phosphatidylinositol 3-kinases (PIK3CA), and their related metabolites and pathways. Compared with the model group, the protein expression levels of TH, MPO and PIK3CA were reduced in GZFL. Therefore, the mechanism of GZFL for treating myocardial ischemia could inhibit myocardial inflammatory factors, reduce myocardial inflammation, and restore endothelial function while controlling norepinephrine release and uric acid concentration.

Paeoniflorin binds to VEGFR2 to restore autophagy and inhibit apoptosis for podocyte protection in diabetic kidney disease through PI3K-AKT signaling pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Paeoniflorin (PF) was found to exhibit renal protection from diabetic kidney disease (DKD) in previous trials, but its specific mechanism remains to be elucidated. AIM OF THE STUDY: This study furtherly explored the specific mechanism of PF in protect podocyte injury in DKD. MATERIALS AND METHODS: We observed the effects of PF on renal tissue and podocytes in DKD by constructing the vitro and vivo models after measuring the pharmacokinetic characteristics of PF. Target proteins of PF were found through target prediction, and verified by molecular docking, CESTA, and SPR, and then furtherly explored the downstream regulation mechanism related to podocyte autophagy and apoptosis by network prediction and co-immunoprecipitation. Finally, by using the target protein inhibitor in vivo and knocking down the target protein gene in vitro, it was verified that PF played a role in regulating autophagy and apoptosis through the target protein in diabetic nephropathy. RESULTS: This study found that in STZ-induced mice model, PF could improve the renal biochemical and pathological damage and podocyte injure (p < 0.05), upregulate autophagy activity (p < 0.05), but inhibit apoptosis (p < 0.01). Vascular endothelial growth factor receptor 2 (VEGFR2), predicted as the target of PF, directly bind with PF reflected by molecular docking and surface plasmon resonance detection. Animal studies demonstrated that VEGFR2 inhibitors have a protective effect similar to that of PF on DKD. Network prediction and co-immunoprecipitation further confirmed that VEGFR2 was able to bind PIK3CA to regulate PI3K-AKT signaling pathway. Furthermore, PF downregulated the phosphorylation of PI3K and AKT (p < 0.05). In vitro, similarly to autophagy inhibitors, PF was also found to improve podocyte markers (p < 0.05) and autophagy activity (p < 0.05), decrease caspase 3 protein (p < 0.05) and further inhibited VEGFR2-PI3K-AKT activity (p < 0.05). Finally, the results of VEGFR2 knockdown were similar to the effect of PF in HG-stimulated podocytes. CONCLUSION: In conclusion, PF restores autophagy and inhibits apoptosis by targeting the VEGFR2-mediated PI3K-AKT pathway to improve renal injury in DKD, that provided a theoretical basis for PF treatment in DKD.

Audit of Molecular Mechanisms of Primary and Secondary Resistance to Various Generations of Tyrosine Kinase Inhibitors in Known Epidermal Growth Factor Receptor-Mutant Non-small Cell Lung Cancer Patients in a Tertiary Centre.

AIMS: Presently, three generations of epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) are approved against oncogene addicted EGFR-mutant non-small cell lung cancer (NSCLC). Patients with actionable EGFR mutations invariably develop resistance. This resistance can be intrinsic (primary) or acquired (secondary). MATERIALS AND METHODS: This was a retrospective study carried out between January 2016 and April 2021 analysing 486 samples of NSCLC for primary and secondary resistance to first- (erlotinib, gefitinb), second- (afatinib) and/or third-generation (osimertinib) TKIs in EGFR-mutant NSCLCs by next generation sequencing (NGS). Tissue NGS was carried out using the Thermofischer Ion Torrent™ Oncomine™ Focus 52 gene assay; liquid biopsy NGS was carried out using the Oncomine Lung Cell-Free Total Nucleic Acid assay. All cases were previously tested for a single EGFR gene with the Therascreen® EGFR RGQ PCR kit. RESULTS: The results were divided into four groups: (i) group 1: primary resistance to first- and/or second-generation TKIs. This group, with 21 cases, showed EGFR exon 20 insertions, dual, complex mutations and variant of unknown significance, de novo MET gene amplification besides other mutations. (ii) Group 2: primary resistance to third-generation TKIs. This group showed two cases, with one showing dual EGFR mutation (L858R and E709A) and EGFR gene amplification. (iii) Group 3: secondary resistance to first- and second-generation TKIs. This group had 27 cases, which were previously reported negative for EGFR T790M by single gene testing. Significant findings were MET gene amplification in four cases, with one also showing MET exon 14 skipping mutation. Three cases showed small cell change and one showed loss of primary mutation. (iv) Group 4: secondary resistance to third-generation TKIs. The latter group was further subgrouped into group 4A: secondary resistance to osimertinib (third-generation TKI) when offered as second-line therapy after first- and second-generation TKIs on detection of T790M mutation. This group had 15 cases. EGFR T790M mutation was lost in 10 (10/15; 67%) cases and was retained in five cases. Patients with T790M loss experienced early resistance (6.9 months versus 12.6 months mean, P = 0.0024) compared with cases that retained T790M. Two cases gained MET amplification as the resistance mechanisms. Other mutations that were found when EGFR T790M was lost were in FGFR3, KRAS, PIK3CA, CTNNB1, BRAF genes. One case had EML4-ALK translocation. Two cases showed driver EGFR deletion 19, retained T790M and C797S mutation in Cis form. Group 4B: secondary resistance to osimertinib (when given as first-line therapy) in EGFR-mutant NSCLC. This group had three cases. The duration of osimertinib treatment ranged from 11 to 17 months. Two patients showed additional C797S mutation along with primary EGFR mutation. CONCLUSION: This study shows the wide spectrum of primary and secondary EGFR resistance mechanisms to first, second and third generation of TKIs and helps us to identify newer therapeutic targets that could carry forward the initial advantage offered by EGFR TKIs.

New Ameloblastoma Cell Lines Enable Preclinical Study of Targeted Therapies.

Ameloblastoma (AB) is an odontogenic tumor that arises from ameloblast-lineage cells. Although relatively uncommon and rarely metastatic, AB tumors are locally invasive and destructive to the jawbone and surrounding structures. Standard-of-care surgical resection often leads to disfigurement, and many tumors will locally recur, necessitating increasingly challenging surgeries. Recent genomic studies of AB have uncovered oncogenic driver mutations, including in the mitogen-activated protein kinase (MAPK) and Hedgehog signaling pathways. Medical therapies targeting those drivers would be a highly desirable alternative or addition to surgery; however, a paucity of existing AB cell lines has stymied clinical translation. To bridge this gap, here we report the establishment of 6 new AB cell lines-generated by "conditional reprogramming"-and their genomic characterization that reveals driver mutations in FGFR2, KRAS, NRAS, BRAF, PIK3CA, and SMO. Furthermore, in proof-of-principle studies, we use the new cell lines to investigate AB oncogene dependency and drug sensitivity. Among our findings, AB cells with KRAS or NRAS mutation (MAPK pathway) are exquisitely sensitive to MEK inhibition, which propels ameloblast differentiation. AB cells with activating SMO-L412F mutation (Hedgehog pathway) are insensitive to vismodegib; however, a distinct small-molecule SMO inhibitor, BMS-833923, significantly reduces both downstream Hedgehog signaling and tumor cell viability. The novel cell line resource enables preclinical studies and promises to speed the translation of new molecularly targeted therapies for the management of ameloblastoma and related odontogenic neoplasms.

Medical management of vascular anomalies of the head and neck.

Depending on impairment, treatment of vascular anomalies is decided on a case-by-case basis in pluridisciplinary consultations. Interventional treatments, especially surgery and sclerotherapy, are usually partially efficient and management of patients with vascular anomalies increasingly involves the use of medical drugs. The most common vascular tumor is infantile hemangioma where first-line medical treatment, when necessary, is propranolol. Kasabach-Merritt phenomenon is now largely treated with sirolimus whereas first-line treatment of coagulation disorders associated with venous malformations is based on low-molecular-weight heparins or direct anticoagulants. Sirolimus is the standard treatment for painful inflammatory manifestations of low-flow vascular malformations such capillary, venous, and lymphatic malformations that can occur singly or in combination but PIK3CA inhibitors, originally developed in oncology, have shown promising results in patients with PIK3CA-related overgrowth spectrum. Currently, medical treatments are poorly developed for high-flow malformations such as arteriovenous malformations. However, new research aimed at delineating the different arteriovenous malformations based on molecular findings has given new hope for future development of targeted therapies.

Effects of Musk Volatile Compounds on Attenuated Nerve Injury and Improving Post-cerebral Ischemic Exercise Functions.

BACKGROUND: Reperfusion Injury Acute ischemic stroke is increasing in people recently and Musk, as a commonly used Traditional Chinese Medicine (TCM), has been suggested as a potential agent against acute ischemic stroke, but the efficacies and underlying mechanisms of it remain unknown. OBJECTIVE: This study was aimed to test the hypotheses that volatile compounds of musk could attenuate nerve injury and identify the bioactive compounds and potential mechanisms of Musk. METHODS: Transient middle cerebral artery occlusion (MCAO) model in vivo in Sprague-Dawley rats (SD rats) was used to test this hypothesis. Collecting ingredients of Musk and their related targets were discerned from the Gas chromatography-olfactory mass spectrometry (GC-O-MS) experiment. Then the potential mechanisms and targets of the compounds were searched by network pharmacology techniques. Finally, the pathway was verified by Western Bolt (WB). RESULTS: First, Musk treatment significantly up-regulated the relative levels of AKT1, PI3KA, and VEGFA in the hippocampus, and improved the sport functions in the post-MCAO ischemic rats in vivo. Next, twenty potential flavor active compounds were recognized by GC-O-MS. A total of 89 key targets including HIF-1, PIK3CA, TNF signaling pathway, and VEGF were identified. AKT1, HIF1A, PIK3CA, and VEGFA were viewed as the most important genes, which were validated by molecular docking simulation. CONCLUSION: The Volatile compounds of musk can attenuate nerve injury and improving post-cerebral ischemic exercise functions by HIF1A pathways, and the combined data provide novel insight for Musk volatile compounds developed as new drug for improving reperfusion injury in acute ischemic stroke.