Patterns of resistance to first-line osimertinib are not well-established and have primarily been evaluated using plasma assays which cannot detect histologic transformation and have differential sensitivity for copy number changes and chromosomal rearrangements. Experimental Design:To characterize mechanisms of resistance to osimertinib, patients with metastatic EGFR-mutant lung cancers who received osimertinib at Memorial Sloan Kettering and had next-generation sequencing performed on tumor tissue before osimertinib initiation and after progression were identified.Results: Among 62 patients who met eligibility critieria, histologic transformation, primarily squamous transformation, was identified in 15% of first-line osimertinib cases and 14% of laterline cases. Nineteen percent (5/27) of patients treated with first-line osimertinib had off-target genetic resistance (2 MET amplification, 1 KRAS mutation, 1 RET fusion, and 1 BRAF fusion) whereas 4% (1/27) had an acquired EGFR mutation (EGFR G724S). Patients with squamous transformation exhibited considerable genomic complexity; acquired PIK3CA mutation, chromosome 3q amplification and FGF amplification were all seen. Patients with transformation had shorter time on osimertinib and shorter survival compared to patients with on-target resistance. Initial EGFR sensitizing mutation, time on osimertinib treatment and line of therapy also influenced resistance mechanism that emerged. The compound mutation EGFR S768 + V769L and the mutation MET H1094Y were identified and validated as resistance mechanisms with potential treatment options. Conclusion:Histologic transformation and other off-target molecular alterations are frequent early emerging resistance mechanisms to osimertinib and are associated with poor clinical outcomes.Research.

Salivary duct carcinoma (SDC) is an uncommon, aggressive malignant neoplasm histologically resembling high-grade mammary ductal carcinoma. SDC can arise de novo or ex pleomorphic adenoma. To clarify the correlation of biomarker immunoprofile with clinicopathological findings and clinical outcome of SDC, we conducted immunohistochemistry for EGFR, HER2, HER3, AR, CK5/6, p53, and Ki-67, along with HER2 fluorescence in situ hybridization in 151 SDCs. SDCs ex pleomorphic adenoma more commonly overexpressed EGFR, HER2, HER3, and Ki-67 than de novo SDCs (P = 0.015, < 0.001, 0.045, and 0.02, respectively). In multivariate analysis, AR− and CK5/6+ were associated with shorter progression-free survival (P = 0.027 and 0.004, respectively). Moreover, patients with p53-extreme negative/positive demonstrated poorer overall survival (P = 0.007). On assessing the revised classification by the combination of biomarker expression, the percentages of each subtype were as follows: ‘apocrine A’ (AR+/HER2−/Ki-67-low) (24%), ‘apocrine B’ (AR+/HER2−/Ki-67-high) (18%), ‘apocrine HER2’ (AR+/HER2+) (35%), ‘HER2-enriched’ (AR−/HER2+) (12%), and ‘double negative’ (AR−/HER2−) (11%). ‘Double negative’ was further subclassified into ‘basal-like’ (EGFR and/or CK5/6+) (7%) and ‘unclassified’ (3%). Consequently, patients with ‘apocrine A’ showed a better progression-free survival than those with any other subtypes. Our revised immunoprofiling classification was valuable for predicting the survival and might be useful in personalized therapy for patients with SDC.

Background:Glioma stem-like cell (GSC) properties are responsible for gliomagenesis and recurrence. GSCs are invasive but its mechanism remains to be elucidated. Here, we attempted to identify the molecules that promote invasion in GSCs.Methods:Neurospheres and CD133+ cells were collected from glioblastoma (GBM) specimens and glioma cell lines by sphere-formation method and magnetic affinity cell sorting, respectively. Differential expression of gene candidates, its role in invasion and its signaling pathway were evaluated in glioma cell lines.Results:Neurospheres from surgical specimens attached to fibronectin and laminin, the receptors of which belong to the integrin family. Integrin α3 was overexpressed in CD133+ cells compared with CD133− cells in all the glioma cell lines (4 out of 4). Immunohistochemistry demonstrated the localisation of integrin α3 in GBM cells, including invading cells, and in the tumour cells around the vessels, which is believed to be a stem cell niche. The expression of integrin α3 was correlated with migration and invasion. The invasion activity of glioma cells was linked to the phosphorylation of extracellular signal–regulated kinase (ERK) 1/2.Conclusion:Our results suggest that integrin α3 contributes to the invasive nature of GSCs via ERK1/2, which renders integrin α3 a prime candidate for anti-invasion therapy for GBM.

Osimertinib (AZD9291) has an efficacy superior to that of standard EGFR-tyrosine kinase inhibitors for the first-line treatment of patients with EGFR-mutant advanced non-small cell lung cancer (NSCLC). However, patients treated with osimertinib eventually acquire drug resistance, and novel therapeutic strategies to overcome acquired resistance are needed. In clinical or preclinical models, several mechanisms of acquired resistance to osimertinib have been elucidated. However, the acquired resistance mechanisms when osimertinib is initially used for EGFR-mutant NSCLC remain unclear. In this study, we experimentally established acquired osimertinib-resistant cell lines from EGFR-mutant NSCLC cell lines and investigated the molecular profiles of resistant cells to uncover the mechanisms of acquired resistance. Various resistance mechanisms were identified, including the acquisition of MET amplification, EMT induction, and the upregulation of AXL. Using targeted next-generation sequencing with a multigene panel, no secondary mutations were detected in our resistant cell lines. Among three MET-amplified cell lines, one cell line was sensitive to a combination of osimertinib and crizotinib. Acquired resistance cell lines derived from H1975 harboring the T790M mutation showed AXL upregulation, and the cell growth of these cell lines was suppressed by a combination of osimertinib and cabozantinib, an inhibitor of multiple tyrosine kinases including AXL, both in vitro and in vivo. Our results suggest that AXL might be a therapeutic target for overcoming acquired resistance to osimertinib.Implications: Upregulation of AXL is one of the mechanisms of acquired resistance to osimertinib, and combination of osimertinib and cabozantinib might be a key treatment for overcoming osimertinib resistance.

BackgroundPeri-implantitis (PI) is an inflammatory reaction associated with functional deterioration of supporting bones around the dental implant. Recent studies suggested Epstein–Barr virus (EBV) is involved in the pathogenesis of periodontitis. We investigated the association between EBV and Porphyromonas gingivalis in Japanese PI patients.MethodsFifteen periodontally healthy individuals, 15 healthy implant patients and 15 PI patients were recruited. Forty five subgingival plaque samples were collected from the deepest probing pocket depth (PPD) site from each patient. Real-time PCR was used to detect EBV DNA and P. gingivalis.ResultsEBV and P. gingivalis were detected in 7 and 3 PPD sites of the healthy controls, in 9 and 4 PPD sites of the healthy implants, and in 13 and 14 PPD sites of the PI patients. P. gingivalis and coexistence of EBV and P. gingivalis were detected significantly higher in the PI patients than healthy controls and healthy implant patients. EBV was detected significantly higher in the PI patients than healthy controls.ConclusionsHigher levels of EBV and P. gingivalis were detected in PPD sites of PI patients. These results suggest that coexistence of EBV and P. gingivalis may serve pathogenic factors cause for PI in Japanese dental patients.