Multidimensional biomarker predicts disease control in response to immunotherapy in recurrent or metastatic head and neck squamous-cell carcinoma.

PURPOSE: Anti-PD-1 therapy provides clinical benefit in 40-50% of patients with relapsed and/or metastatic head and neck squamous cell carcinoma (RM-HNSCC). Selection of anti- PD-1 therapy is typically based on patient PD-L1 immunohistochemistry (IHC) which has low specificity for predicting disease control. Therefore, there is a critical need for a clinical biomarker that will predict clinical benefit to anti-PD-1 treatment with high specificity. METHODS: Clinical treatment and outcomes data for 103 RM-HNSCC patients were paired with RNA-sequencing data from formalin-fixed patient samples. Using logistic regression methods, we developed a novel biomarker classifier based on expression patterns in the tumor immune microenvironment to predict disease control with monotherapy PD-1 inhibitors (pembrolizumab and nivolumab). The performance of the biomarker was internally validated using out-of-bag methods. RESULTS: The biomarker significantly predicted disease control (65% in predicted non-progressors vs. 17% in predicted progressors, p < 0.001) and was significantly correlated with overall survival (OS; p = 0.004). In addition, the biomarker outperformed PD-L1 IHC across numerous metrics including sensitivity (0.79 vs 0.64, respectively; p = 0.005) and specificity (0.70 vs 0.61, respectively; p = 0.009). CONCLUSION: This novel assay uses tumor immune microenvironment expression data to predict disease control and OS with high sensitivity and specificity in patients with RM-HNSCC treated with anti-PD-1 monotherapy.

Costs of Definitive Chemoradiation, Surgery, and Adjuvant Radiation Versus De-Escalated Adjuvant Radiation per MC1273 in HPV+ Cancer of the Oropharynx.

PURPOSE: De-escalated treatment for human papillomavirus (HPV)+ oropharynx squamous cell carcinoma (OPSCC) has shown promising initial results. Health-care policy is increasingly focusing on high-value care. This analysis compares the cost of care for HPV+ OPSCC treated with definitive chemoradiation (CRT), surgery and adjuvant radiation (RT), and surgery and de-escalated CRT on MC1273. METHODS AND MATERIALS: MC1273 is a prospective, phase 2 study evaluating adjuvant CRT to 30 to 36 Gy plus docetaxel for HPV+ OPSCC after surgery for high-risk patients. Matched standard-of-care control groups were retrospectively identified for patients treated with definitive CRT or adjuvant RT. Standardized costs were evaluated before radiation, during treatment (during RT), and at short-term (6 month) and long-term (7-24 month) follow-up periods. RESULTS: A total of 56 definitive CRT, 101 adjuvant RT, and 66 MC1273 patients were included. The CRT arm had more T3-4 disease (63% vs 17-21%) and higher N2c-N3 disease (52% vs 20-24%) vs both other groups. The total treatment costs in the CRT, adjuvant RT, and MC1273 groups were $47,763 (standard deviation [SD], $19,060], $57,845 (SD, $17,480), and $46,007 (SD, $9019), respectively, and the chemotherapy and/or RT costs were $39,936 (SD, $18,480), $26,603 (SD, $12,542), and $17,864 (SD, $3288), respectively. The per-patient, per-month, average short-term follow-up costs were $3860 (SD, $10,525), $1072 (SD, $996), and $972 (SD, $833), respectively, and the long-term costs were $978 (SD, $2294), $485 (SD, $1156), and $653 (SD, $1107), respectively. After adjustment for age, T-stage, and N-stage, treatment costs remained lower for CRT and MC1273 versus adjuvant RT ($45,450 and $47,114 vs $58,590, respectively; P < .001), whereas the total per-patient, per-month follow-up costs were lower in the MC1273 study group and adjuvant RT versus CRT ($853 and $866 vs $2030, respectively; P = .03). CONCLUSIONS: MC1273 resulted in 10% and 20% reductions in global costs compared with standard-of-care adjuvant RT and definitive CRT treatments. Substantial cost savings may be an added benefit to the already noted low toxicity and maintained quality of life of treatment per MC1273.

Intracellular distribution of fluorescent copper and zinc bis(thiosemicarbazonato) complexes measured with fluorescence lifetime spectroscopy.

The intracellular distribution of fluorescently labeled copper and zinc bis(thiosemicarbazonato) complexes was investigated in M17 neuroblastoma cells and primary cortical neurons with a view to providing insights into the neuroprotective activity of a copper bis(thiosemicarbazonato) complex known as Cu(II)(atsm). Time-resolved fluorescence measurements allowed the identification of the Cu(II) and Zn(II) complexes as well as the free ligand inside the cells by virtue of the distinct fluorescence lifetime of each species. Confocal fluorescent microscopy of cells treated with the fluorescent copper(II)bis(thiosemicarbazonato) complex revealed significant fluorescence associated with cytoplasmic puncta that were identified to be lysosomes in primary cortical neurons and both lipid droplets and lysosomes in M17 neuroblastoma cells. Fluorescence lifetime imaging microscopy confirmed that the fluorescence signal emanating from the lipid droplets could be attributed to the copper(II) complex but also that some degree of loss of the metal ion led to diffuse cytosolic fluorescence that could be attributed to the metal-free ligand. The accumulation of the copper(II) complex in lipid droplets could be relevant to the neuroprotective activity of Cu(II)(atsm) in models of amyotrophic lateral sclerosis and Parkinson's disease.

Copper and zinc bis(thiosemicarbazonato) complexes with a fluorescent tag: synthesis, radiolabelling with copper-64, cell uptake and fluorescence studies.

The synthesis of new copper(II) bis(thiosemicarbazonato) complexes with an appended pyrene chromophore and their zinc(II) analogues is reported. The new proligands and their copper(II) and zinc(II) complexes were characterised by a combination of NMR, EPR, high performance liquid chromatography, mass spectrometry, electronic spectroscopy and electrochemical measurements. The new copper(II) complexes are fluorescent as a consequence of an appended pyrene substituent that is separated from the sulphur coordinating to the metal ion by five bonds. The emission from the pyrene substituent is concentration- and solvent-dependent with characteristic formation of excimer aggregates. A radioactive (64)Cu complex has been prepared. Cell permeability, intracellular distribution and importantly the ability to cross the nuclear membrane to target DNA were investigated using confocal fluorescence microscopy in a human cancer cell line under normal oxygen conditions and hypoxic conditions. In both cases, there was no evidence of uptake of the copper(II) bis(thiosemicarbazonato) complexes in the area of the cell nucleus.

Activation of epidermal growth factor receptor by metal-ligand complexes decreases levels of extracellular amyloid beta peptide.

The epidermal growth factor receptor is a receptor tyrosine kinase expressed in a range of tissues and cell-types. Activation of the epidermal growth factor receptor by a number of ligands induces downstream signalling that modulates critical cell functions including growth, survival and differentiation. Abnormal epidermal growth factor receptor expression and activation is also involved in a number of cancers. In addition to its cognate ligands, the epidermal growth factor receptor can be activated by metals such as zinc (Zn) and copper (Cu). Due to the important role of these metals in a number of diseases including neurodegenerative disorders, therapeutic approaches are being developed based on the use of lipid permeable metal-complexing molecules. While these agents are showing promising results in animal models and clinical trials, little is known about the effects of metal-ligand complexes on cell signalling pathways. In this study, we investigated the effects of clioquinol (CQ)-metal complexes on activation of epidermal growth factor receptor. We show here that CQ-Cu complexes induced potent epidermal growth factor receptor phosphorylation resulting in downstream activation of extracellular signal-regulated kinase. Similar levels of epidermal growth factor receptor activation were observed with alternative lipid permeable metal-ligands including neocuproine and pyrrolidine dithiocarbamate. We found that CQ-Cu complexes induced a significant reduction in the level of extracellular Abeta1-40 in cell culture. Inhibition of epidermal growth factor receptor activation by PD153035 blocked extracellular signal-regulated kinase phosphorylation and restored Abeta1-40 levels. Activation of the epidermal growth factor receptor by CQ-Cu was mediated through up-regulation of src kinase activity by a cognate ligand-independent process involving membrane integrins. These findings provide the first evidence that metal-ligand complexes can activate the epidermal growth factor receptor with potentially neuroprotective effects.

The role of metals in modulating metalloprotease activity in the AD brain.

Biometals such as copper and zinc have an important role in Alzheimer's disease (AD). Accumulating evidence indicates that copper homeostasis is altered in AD brain with elevated extracellular and low intracellular copper levels. Studies in animals and cell cultures have suggested that increasing intracellular copper can ameliorate AD-like pathology including amyloid deposition and tau phosphorylation. Modulating copper homeostasis can also improve cognitive function in animal models of AD. Treatments are now being developed that may result in redistribution of copper within the brain. Metal ligands such as clioquinol (CQ), DP-109 or pyrrolidine dithiocarbamate (PDTC) have shown promising results in animal models of AD, however, the actual mode of action in vivo has not been fully determined. We previously reported that CQ-metal complexes were able to increase intracellular copper levels in vitro. This resulted in stimulation of phosphoinositol-3-kinase activity and mitogen activated protein kinases (MAPK). Increased kinase activity resulted in up-regulated matrix metalloprotease (MMP2 and MMP3) activity resulting in enhanced degradation of secreted A beta. These findings are consistent with previous studies reporting metal-mediated activation of MAPKs and MMPs. How this activation occurs is unknown but evidence suggests that copper may be able to activate membrane receptors such as the epidermal growth factor receptor (EGFR) and result in downstream activation of MAPK pathways. This has been supported by studies showing metal-mediated activation of EGFR through ligand-independent processes in a number of cell-types. Our initial studies reveal that copper complexes can in fact activate EGFR. However, further studies are necessary to determine if metal complexes such as CQ-copper induce up-regulation of A beta-degrading MMP activity through this mechanism. Elucidation of this pathway may have important implications for the development of metal ligand based therapeutics for treatment of AD and other neurodegenerative disorders.