Reduced sensitivity for EGFR T790M mutations using the Idylla EGFR Mutation Test.

AIMS: Osimertinib is a third-generation EGFR (epidermal growth factor receptor) tyrosine kinase inhibitor that is effective in non-small cell lung cancer (NSCLC) harbouring the EGFR T790M mutation. The Idylla EGFR Mutation Test is a rapid cartridge-based method for detecting T790M and other EGFR mutations. However, false negative T790M results have been reported, and the sensitivity of the assay for this mutation is uncertain. METHODS: Eighty NSCLC samples were tested by both Idylla and a next-generation sequencing (NGS) assay; 46 were from patients at disease progression, and 24 of these had known T790M mutations. Droplet digital PCR (ddPCR) was used to confirm NGS findings in samples with the T790M mutation. RESULTS: Of 19 samples with T790M variant allele frequencies (VAF) higher than the stated 5% limit of detection, 14 were detected by Idylla (sensitivity 74%, 95% CI 49% to 90%). Where sufficient sample remained, ddPCR was consistent with NGS findings in all samples. False negative T790M results were associated with higher EGFR control Cq values (median 22.8 vs 19.8), presence of the EGFR Q787Q polymorphism in cis (80% vs 44%) and presence of an invalid T790M amplification curve. An EGFR exon 19 indel with VAF >5% was also not detected by the Idylla assay in two samples. CONCLUSIONS: The Idylla EGFR Mutation Test has reduced sensitivity for the T790M mutation compared with NGS and ddPCR methods. The presence of an invalid T790M amplification curve may indicate a possible false negative result that warrants further testing by an orthogonal method.

Prevalence of the EGFR T790M and other resistance mutations in the Australian population and histopathological correlation in a small subset of cases.

We sought to review the prevalence of EGFR T790M and other EGFR mutations associated with either proven or probable tyrosine kinase inhibitor (TKI) resistance in the Australasian lung cancer population and to perform histopathological correlation in a subset of cases. Retrospective statistical analysis was performed on a set of targeted lung cancer gene mutation tests (FIND IT gene panel) performed at Sonic Healthcare during 2018 and early 2019. A total of 1833 lung adenocarcinoma tumour samples underwent somatic mutation testing. EGFR mutations were found in 28% (n=514) of patients, in whom 9.3% (n=48) T790M mutations were present (always combined with other EGFR mutations) and 4.8% (n=25) exon 20 insertions were found. We also compared the prevalence of EGFR mutations identified in our population with that of the four largest publicly available lung cancer cohorts (total n=576 samples). Finally, a subset of 38 samples of primary/and or metastatic lung adenocarcinomas from 23 patients, including five with serial biopsies, underwent detailed morphological analysis. No reproducible morphological correlates were found to be associated with T790M, exon 20 resistance mutations or rarer co-occurring EGFR mutations. Although this may be subject to referral bias towards patients with resistant disease, the incidence of EGFR and T790M mutations is higher in this series from an Australasian population than in other similar publicly available lung adenocarcinoma cohorts. We conclude that histopathological features cannot be used to predict the acquisition of EGFR resistance.

Auranofin improves overall survival when combined with standard of care in a pilot study involving dogs with osteosarcoma.

Osteosarcoma is the most common paediatric primary bone malignancy. The major cause of death in osteosarcoma is drug-resistant pulmonary metastasis. Previous studies have shown that thioredoxin reductase 2 is a driver of metastasis in osteosarcoma and can be inhibited by auranofin (AF). Moreover, studies have shown that AF significantly reduces pulmonary metastases in xenotransplant models. Here, we describe a phase I/II study of AF in canine osteosarcoma, a well-recognized spontaneous model of human osteosarcoma. We performed a single-arm multicentre pilot study of AF in combination with standard of care (SOC) (amputation + carboplatin). We recruited 40 dogs to the trial and used a historical SOC-only control group (n = 26). Dogs >15 kg received 9 mg AF q3d PO and dogs <15 kg received 6 mg q3d. Follow-up occurred over at least a 3-year period. Auranofin plus SOC improved overall survival (OS) (P = .036) in all dogs treated. The improved outcome was attributable entirely to improved OS in male dogs (P = .009). At the time of writing, 10 dogs (25%) survive without measurable disease in the treatment group with survival times ranging between 806 and 1525 days. Our study shows that AF improves OS in male dogs when combined with SOC. Our findings have translational relevance for the management of canine and human osteosarcoma. Our data justify a larger multicentre phase 2 trial in dogs and a phase I/II trial in human patients with refractory disease at the time of initial surgery.

Extracellular vesicles secreted by highly metastatic clonal variants of osteosarcoma preferentially localize to the lungs and induce metastatic behaviour in poorly metastatic clones.

Osteosarcoma (OS) is the most common pediatric bone tumor and is associated with the emergence of pulmonary metastasis. Unfortunately, the mechanistic basis for metastasis remains unclear. Tumor-derived extracellular vesicles (EVs) have been shown to play critical roles in cell-to-cell communication and metastatic progression in other cancers, but their role in OS has not been explored. We show that EVs secreted by cells derived from a highly metastatic clonal variant of the KHOS cell line can be internalized by a poorly metastatic clonal variant of the same cell line and induce a migratory and invasive phenotype. This horizontal phenotypic transfer is unidirectional and provides evidence that metastatic potential may arise via interclonal co-operation. Proteomic analysis of the EVs secreted by highly metastatic OS clonal variants results in the identification of a number of proteins and G-protein coupled receptor signaling events as potential drivers of OS metastasis and novel therapeutic targets. Finally, multiphoton microscopy with fluorescence lifetime imaging in vivo, demonstrated a preferential seeding of lung tissue by EVs derived from highly metastatic OS clonal variants. Thus, we show that EVs derived from highly metastatic clonal variants of OS may drive metastatic behaviour via interclonal co-operation and preferential colonization of the lungs.

Auranofin is a potent suppressor of osteosarcoma metastasis.

Osteosarcoma (OS) accounts for 56% of malignant bone cancers in children and adolescents. Patients with localized disease rarely develop metastasis; however, pulmonary metastasis occurs in approximately 50% of patients and leads to a 5-year survival rate of only 10-20%. Therefore, identifying the genes and pathways involved in metastasis, as new therapeutic targets, is crucial to improve long-term survival of OS patients. Novel markers that define metastatic OS were identified using comparative transcriptomic analyses of two highly metastatic (C1 and C6) and two poorly metastatic clonal variants (C4 and C5) isolated from the metastatic OS cell line, KHOS. Using this approach, we determined that the metastatic phenotype correlated with overexpression of thioredoxin reductase 2 (TXNRD2) or vascular endothelial growth factor (VEGF). Validation in patient biopsies confirmed TXNRD2 and VEGF targets were highly expressed in 29-42% of metastatic OS patient biopsies, with no detectable expression in non-malignant bone or samples from OS patients with localised disease. Auranofin (AF) was used to selectively target and inhibit thioredoxin reductase (TrxR). At low doses, AF was able to inhibit TrxR activity without a significant effect on cell viability whereas at higher doses, AF could induce ROS-dependent apoptosis. AF treatment, in vivo, significantly reduced the development of pulmonary metastasis and we provide evidence that this effect may be due to an AF-dependent increase in cellular ROS. Thus, TXNRD2 may represent a novel druggable target that could be deployed to reduce the development of fatal pulmonary metastases in patients with OS.

Progression of Osteosarcoma from a Non-Metastatic to a Metastatic Phenotype Is Causally Associated with Activation of an Autocrine and Paracrine uPA Axis.

Pulmonary metastasis is the major untreatable complication of osteosarcoma (OS) resulting in 10-20% long-term survival. The factors and pathways regulating these processes remain unclear, yet their identification is crucial in order to find new therapeutic targets. In this study we used a multi-omics approach to identify molecules in metastatic and non-metastatic OS cells that may contribute to OS metastasis, followed by validation in vitro and in vivo. We found elevated levels of the urokinase plasminogen activator (uPA) and of the uPA receptor (uPAR) exclusively in metastatic OS cells. uPA was secreted in soluble form and as part of the protein cargo of OS-secreted extracellular vesicles, including exosomes. In addition, in the tumour microenvironment, uPA was expressed and secreted by bone marrow cells (BMC), and OS- and BMC-derived uPA significantly and specifically stimulated migration of metastatic OS cells via uPA-dependent signaling pathways. Silencing of uPAR in metastatic OS cells abrogated the migratory response to uPA in vitro and decreased metastasis in vivo. Finally, a novel small-molecule inhibitor of uPA significantly (P = 0.0004) inhibited metastasis in an orthotopic mouse model of OS. Thus, we show for the first time that malignant conversion of OS cells to a metastatic phenotype is defined by activation of the uPA/uPAR axis in both an autocrine and paracrine fashion. Furthermore, metastasis is driven by changes in OS cells as well as in the microenvironment. Finally, our data show that pharmacological inhibition of the uPA/uPAR axis with a novel small-molecule inhibitor can prevent the emergence of metastatic foci.

Confluence-Induced Squamous Differentiation Is Not Accompanied by Changes in H3K27me3 Repressive Epigenetic Mark.

Recent studies have reported that epigenetic mechanisms may regulate the initiation and progress of squamous differentiation in normal and transformed keratinocytes. In particular, the role of the repressive H3K27me3 mark in the regulation of squamous differentiation has been prominent. However, there is conflicting literature showing that squamous differentiation may be dependent upon or independent of changes in H3K27me3 status. In this study we have examined the binding of trimethylated H3K27 to the promoters of proliferation or differentiation genes in keratinocytes undergoing squamous differentiation in vitro and in vivo. Initially, we examined the expression levels for EZH1, EZH2, and H3K27me3 in differentiating keratinocytes in vitro and in vivo. We extended this to include H3K27me3 chromatin immunoprecipitation sequencing (ChIP-seq). Based on these studies, we could find no evidence for an association between widespread gain or loss of H3K27me3 on the promoters of proliferation-specific or differentiation-specific target genes, respectively, during squamous differentiation in adult human keratinocytes. These data suggest that squamous differentiation may occur independent of regulation by H3K27me3 on proliferation and differentiation genes of normal adult human keratinocytes.

A novel E2F/sphingosine kinase 1 axis regulates anthracycline response in squamous cell carcinoma.

PURPOSE: Head and neck squamous cell carcinomas (HNSCC) are frequently drug resistant and have a mortality rate of 45%. We have previously shown that E2F7 may contribute to drug resistance in SCC cells. However, the mechanism and pathways involved remain unknown. EXPERIMENTAL DESIGN: We used transcriptomic profiling to identify candidate pathways that may contribute to E2F7-dependent resistance to anthracyclines. We then manipulated the activity/expression of the candidate pathway using overexpression, knockdown, and pharmacological inhibitors in in vitro and in vivo models of SCC to demonstrate causality. In addition, we examined the expression of E2F7 and a downstream effector in a tissue microarray (TMA) generated from HNSCC patient samples. RESULTS: E2F7-deficient keratinocytes were selectively sensitive to doxorubicin and this was reversed by overexpressing E2F7. Transcriptomic profiling identified Sphingosine kinase 1 (Sphk1) as a potential mediator of E2F7-dependent drug resistance. Knockdown and overexpression studies revealed that Sphk1 was a downstream target of E2F7. TMA studies showed that E2F7 overexpression correlated with Sphk1 overexpression in human HNSCC. Moreover, inhibition of Sphk1 by shRNA or the Sphk1-specific inhibitor, SK1-I (BML-EI411), enhanced the sensitivity of SCC cells to doxorubicin in vitro and in vivo. Furthermore, E2F7-induced doxorubicin resistance was mediated via Sphk1-dependent activation of AKT in vitro and in vivo. CONCLUSION: We identify a novel drugable pathway in which E2F7 directly increases the transcription and activity of the Sphk1/S1P axis resulting in activation of AKT and subsequent drug resistance. Collectively, this novel combinatorial therapy can potentially be trialed in humans using existing agents.

Focal overexpression of CEACAM6 contributes to enhanced tumourigenesis in head and neck cancer via suppression of apoptosis.

BACKGROUND: Overexpression of CEACAM6 has been reported for a number of malignancies. However, the mechanism of how CEACAM6 contributes to cancer formation and its role in head and neck squamous cell carcinoma (HNSCC) remains unclear. Therefore, we examined the role of CEACAM6 in head and neck squamous cell carcinoma (HNSCC). METHODS: CEACAM6 expression was examined in normal squamous epithelia as well as a number of patient HNSCC samples and tumours derived from HNSCC cell lines injected into NOD/SCID mice. CEACAM6 expression was manipulated in HNSCC cell lines by shRNA-mediated CEACAM6 knockdown or virally-delivered overexpression of CEACAM6. The role of CEACAM6 in tumour growth and chemotherapeutic sensitivity was then assessed in vivo and in vitro respectively. RESULTS: CEACAM6 expression was significantly increased in highly tumourigenic HNSCC cell lines when compared to poorly tumourigenic HNSCC cell lines. Moreover, HNSCC patient tumours demonstrated focal expression of CEACAM6. Functional investigation of CEACAM6, involving over-expression and knock down studies, demonstrated that CEACAM6 over-expression could enhance tumour initiating activity and tumour growth via activation of AKT and suppression of caspase-3 mediated cell death. CONCLUSION: We report that CEACAM6 is focally overexpressed in a large fraction of human HNSCCs in situ. We also show that over-expression of CEACAM6 increases tumour growth and tumour initiating activity by suppressing PI3K/AKT-dependent apoptosis of HNSCC in a xenotransplant model of HNSCC. Finally, our studies indicate that foci of CEACAM6 expressing cells are selectively ablated by treatment of xenotransplant tumours with pharmacological inhibitors of PI3K/AKT in vivo.

Evaluation of saliva collection devices for the analysis of proteins.

BACKGROUND: Human saliva mirrors the body's health and can be collected non-invasively, does not require specialized skills and is suitable for large population based screening programs. The aims were twofold: to evaluate the suitability of commercially available saliva collection devices for quantifying proteins present in saliva and to provide levels for C-reactive protein (CRP), myoglobin, and immunoglobin E (IgE) in saliva of healthy individuals as a baseline for future studies. METHODS: Saliva was collected from healthy volunteers (n=17, ages 18-33years). The following collection methods were evaluated: drool; Salimetrics® Oral Swab (SOS); Salivette® Cotton and Synthetic (Sarstedt) and Greiner Bio-One Saliva Collection System (GBO SCS®). We used AlphaLISA® assays to measure CRP, IgE and myoglobin levels in human saliva. RESULTS: Significant (p<0.05) differences in the salivary flow rates were observed based on the method of collection, i.e. salivary flow rates were significantly lower (p<0.05) in unstimulated saliva (i.e. drool and SOS), when compared with mechanically stimulated methods (p<0.05) (Salivette® Cotton and Synthetic) and acid stimulated method (p<0.05) (SCS®). Saliva collected using SOS yielded significantly (p<0.05) lower concentrations of myoglobin and CRP, whilst, saliva collected using the Salivette® Cotton and Synthetic swab yielded significantly (p<0.05) lower myoglobin and IgE concentrations respectively. CONCLUSIONS: The results demonstrated significantly relevant differences in analyte levels based on the collection method. Significant differences in the salivary flow rates were also observed depending on the saliva collection method. The data provide preliminary baseline values for salivary CRP, myoglobin, and IgE levels in healthy participants and based on the collection method.