Search

Epithermal Growth Factor Receptor (EGFR) is overexpressed in up to 90% of head and neck squamous cell carcinoma (HNSCC). Cetuximab is the first and the only anti-EGFR monoclonal antibody which received approval from FDA for the treatment of HNSCC. However, most patients either do not respond to cetuximab or develop acquired resistance. The aim of my D.Phil. study was to investigate the role of ADAM10 and 17 in resistance mechanisms of cetuximab in HNSCC. Chronic exposure to cetuximab led to an activation of HER receptors and downstream signalling pathways in HNSCC cell lines. Higher levels of ADAM10 and 17 and their substrates, BTC and NRG-1 were found in cetuximab resistant cells compared to their parental cells, suggesting the involvement of ADAM-mediated ligands’ release in reactivation of HER receptors. Inhibition or knockdown of ADAM10 and 17 enhanced cetuximab response and reversed cetuximab resistance in HNSCC cells. In addition, results from this study showed that the combination of cetuximab with EGFR-TKIs had greater effect in parental cells and reversed cetuximab resistance in HNSCC cells. Upregulation of ADAM10 and 17 also was observed in HNSCC TMAs compared to normal head and neck TMAs. High nuclear ADAM10 and cytoplasmic ADAM17 expression levels were associated with shorter DFS. By evaluating tumour excision samples from HNSCC patients who underwent a cetuximab window study high ADAM10 and 17 expression levels were found to be associated with poor response to cetuximab. In conclusion cetuximab-induced ADAM-mediated ligands’ release is a potential mechanism of resistance to cetuximab in HNSCC. Thus, targeting ADAM10/17 or subsequent HER activations may represent an important strategy in overcoming resistance to cetuximab in HNSCC. Results from this study also suggest the implication of ADAM10 and 17 as potential prognostic and predictive biomarkers in HNSCC although further validation is required.

Total tumor size (TS) metrics used in TS models in oncology do not consider tumor heterogeneity, which could help to better predict drug efficacy. We analyzed individual target lesions (iTLs) of patients with metastatic colorectal carcinoma (mCRC) to determine differences in TS dynamics by using the ClassIfication Clustering of Individual Lesions (CICIL) methodology. Results from subgroup analyses comparing genetic mutations and TS metrics were assessed and applied to survival analyses. Data from four mCRC clinical studies were analyzed (1781 patients, 6369 iTLs). CICIL was used to assess differences in lesion TS dynamics within a tissue (intra-class) or across different tissues (inter-class). First, lesions were automatically classified based on their location. Cross-correlation coefficients (CCs) determined if each pair of lesions followed similar or opposite dynamics. Finally, CCs were grouped by using the K-means clustering method. Heterogeneity in tumor dynamics was lower in the intra-class analysis than in the inter-class analysis for patients receiving cetuximab. More tumor heterogeneity was found in KRAS mutated patients compared to KRAS wild-type (KRASwt) patients and when using sum of longest diameters versus sum of products of diameters. Tumor heterogeneity quantified as the median patient’s CC was found to be a predictor of overall survival (OS) (HR = 1.44, 95% CI 1.08–1.92), especially in KRASwt patients. Intra- and inter-tumor tissue heterogeneities were assessed with CICIL. Derived metrics of heterogeneity were found to be a predictor of OS time. Considering differences between lesions’ TS dynamics could improve oncology models in favor of a better prediction of OS.

The epidermal growth factor receptor inhibitor cetuximab is the only oncogene-targeted agent that has been FDA approved for the treatment of head and neck squamous cell carcinoma (HNSCC). Currently, there are no biomarkers used in the clinic to predict which HNSCC tumors will respond to cetuximab, and even in tumors that regress with treatment, acquired resistance occurs in the majority of cases. Though a number of mechanisms of acquired resistance to cetuximab have been identified in preclinical studies, no therapies targeting these resistance pathways have yet been effectively translated into the clinic. To address this unmet need, we examined the role of the cytokine interleukin 6 (IL-6) in acquired cetuximab resistance in preclinical models of HNSCC. We found that IL-6 secretion was increased in PE/CA-PJ49 cells that had acquired resistance to cetuximab compared to the parental cells from which they were derived. However, addition of exogenous IL-6 to parental cells did not promote cetuximab resistance, and inhibition of the IL-6 pathway did not restore cetuximab sensitivity in the cetuximab-resistant cells. Further examination of the IL-6 pathway revealed that expression of IL6R, which encodes a component of the IL-6 receptor, was decreased in cetuximab-resistant cells compared to parental cells, and that treatment of the cetuximab-resistant cells with exogenous IL-6 did not induce phosphorylation of signal transducer and activator of transcription 3, suggesting that the IL-6 pathway was functionally impaired in the cetuximab-resistant cells. These findings demonstrate that, even if IL-6 is increased in the context of cetuximab resistance, it is not necessarily required for maintenance of the resistant phenotype, and that targeting the IL-6 pathway may not restore sensitivity to cetuximab in cetuximab-refractory HNSCC.

Enhancement of antibody-dependent cellular cytotoxicity (ADCC) may potentiate the antitumor efficacy of tumor-targeted monoclonal antibodies. Increasing the numbers and antitumor activity of NK cells is a promising strategy to maximize the ADCC of standard-of-care tumor-targeted antibodies. For this purpose, we have preclinically tested a recombinant chimeric protein encompassing the sushi domain of the IL15Rα, IL-15, and apolipoprotein A-I (Sushi-IL15-Apo) as produced in CHO cells. The size-exclusion purified monomeric fraction of this chimeric protein was stable and retained the IL-15 and the sushi domain bioactivity as measured by CTLL-2 and Mo-7e cell proliferation and STAT5 phosphorylation in freshly isolated human NK and CD8+ T cells. On cell cultures, Sushi-IL15-Apo increases NK cell proliferation and survival as well as spontaneous and antibody-mediated cytotoxicity. Scavenger receptor class B type I (SR-B1) is the receptor for ApoA-I and is expressed on the surface of tumor cells. SR-B1 can adsorb the chimeric protein on tumor cells and can transpresent IL-15 to NK and CD8+ T cells. A transient NK-humanized murine model was developed to test the increase of ADCC attained by the chimeric protein in vivo. The EGFR+ human colon cancer cell line HT-29 was intraperitoneally inoculated in immune-deficient Rag2-/-γc-/- mice that were reconstituted with freshly isolated PBMCs and treated with the anti-EGFR mAb cetuximab. The combination of the Sushi-IL15-Apo protein and cetuximab reduced the number of remaining tumor cells in the peritoneal cavity and delayed tumor engraftment in the peritoneum. Furthermore, Sushi-IL15-Apo increased the anti-tumor effect of a murine anti-EGFR mAb in Rag1-/- mice bearing subcutaneous MC38 colon cancer transfected to express EGFR. Thus, Sushi-IL15-Apo is a potent tool to increase the number and the activation of NK cells to promote the ADCC activity of antibodies targeting tumor antigens.

Human papillomaviruses (HPVs) are the most common sexually transmitted infectious agents. They are responsible for >99% of all cervical cancers as well as subsets of other anogenital cancers. HPVs are also the causative agents of a growing number of head and neck cancers. As such, they present a significant health problem both in the U.S. and developing countries. Much is still unknown regarding the factors underlying progression from HPV infection to cancer or maintenance of viral oncoprotein expression following oncogenic transformation. Previous studies reported significant interplay between the epidermal growth factor (EGFR) pathway and HPV oncoproteins. HPV oncoproteins E5, E6, and E7 have been implicated in upregulation of EGFR signaling and expression. Additionally, downstream effectors of EGFR signaling were shown to upregulate HPV early gene expression. These reports led us to hypothesize that, in infected cells, HPV establishes a positive feedback loop with the EGFR pathway, wherein viral proteins upregulate EGFR signaling, which then leads to enhanced viral oncogene expression. We further postulated that interruption of this feedback loop, via inhibitors of the EGFR signaling pathway, would result in decreased viral oncoprotein levels and increased sensitivity to apoptotic stimuli. In a model of early, persistent infection, we found that EGFR signaling modulated HPV early gene expression, including upregulation of viral oncogene expression upon EGFR stimulation. We discovered that EGFR inhibition by cetuximab, a monoclonal antibody targeting EGFR, had antiviral effects including downregulation of HPV oncogene expression levels in this model. Furthermore, in cells harboring episomal viral genomes, inhibition of this pathway led to reduced viral genome burden and sensitization to apoptotic stimuli. Our study further reveals that EGFR/MEK inhibition can lead to downregulation of HPV oncogene expression in vivo, in subsets of HPV-positive xenografts, concomitant with delayed tumor growth. These results suggest a possible role for antiviral effects in the therapeutic outcomes observed following EGFR-inhibitor treatment of HPV-associated diseases in the clinic. Together, these data indicate that use of EGFR/MEK inhibitors may be beneficial in the treatment of HPV-associated diseases. Furthermore, the results of this study set the frame work to better understand the role of growth factor signaling in the HPV lifecycle.

Oxaliplatin (L-OH), a platinum derivative with good tolerability is currently combined with Cetuximab (CTX), a monoclonal antibody (mAb), for the treatment of certain (wild-type KRAS) metastatic colorectal cancer (CRC) expressing epidermal growth factor receptor (EGFR). Improvement of L-OH pharmacokinetics (PK) can be provided by its encapsulation into liposomes, allowing a more selective accumulation and delivery to the tumor. Here, we aim to associate both agents in a novel liposomal targeted therapy by linking CTX to the drug-loaded liposomes. These EGFR-targeted liposomes potentially combine the therapeutic activity and selectivity of CTX with tumor-cell delivery of L-OH in a single therapeutic approach. L-OH liposomes carrying whole CTX or CTX-Fab’ fragments on their surface were designed and characterized. Their functionality was tested in vitro using four human CRC cell lines, expressing different levels of EGFR to investigate the role of CTX-EGFR interactions in the cellular binding and uptake of the nanocarriers and encapsulated drug. Next, those formulations were evaluated in vivo in a colorectal cancer xenograft model with regard to tumor drug accumulation, toxicity and therapeutic activity. In EGFR-overexpressing cell lines, intracellular drug delivery by targeted liposomes increased with receptor density reaching up to 3-fold higher levels than with non-targeted liposomes. Receptor specific uptake was demonstrated by competition with free CTX, which reduced internalization to levels similar to non-targeted liposomes. In a CRC xenograft model, drug delivery was strongly enhanced upon treatment with targeted formulations. Liposomes conjugated with monovalent CTX-Fab’ fragments showed superior drug accumulation in tumor tissue (2916.0 ± 507.84 ng/g) compared to CTX liposomes (1546.02 ± 362.41 ng/g) or non-targeted liposomes (891.06 ± 155.1 ng/g). Concomitantly, CTX-Fab’ targeted L-OH liposomes outperformed CTX-liposomes, which on its turn was still more efficacious than non-targeted liposomes and free drug treatment in CRC bearing mice. These results show that site-directed conjugation of monovalent CTX-Fab’ provides targeted L-OH liposomes that display an increased tumor drug delivery and efficacy over a formulation with CTX and non-targeted liposomes.

Introduction: Current treatment recommendations for advanced SCCHN include the use of combined modality therapy (e.g., radiation plus chemotherapy/biologic therapy). The new biologic agent, cetuximab, is considered a primary cost driver for SCCHN management. Cetuximab’s impact, however, has not been factored into SCCHN cost estimates; furthermore, costs have not been delineated for diagnostic, treatment, and end-of-life segments of advanced SCCHN management. We aim to characterize SCCHN segmented and overall costs, before and after cetuximab’s approval. Methods: This was a retrospective analysis of the PharMetrics Choice database. Patients >20 years of age with ICD-9-CM codes suggestive of advanced SCCHN diagnosed between 3/1/2003 and 3/1/2008 were included. Patients were divided into cohorts by diagnosis date: “pre-biologic” and “post-biologic.” Descriptive statistics were used to summarize patient characteristics, monthly and total medical costs, and cost drivers. The Mann-Whitney U test was used to compare costs between segments and cohorts; whereas, least squares regression was used to ascertain the impact of covariates. Results: A total of 365 patients met study criteria. Patients were predominately male (78%), with a median age of 57 years. Median monthly costs were: diagnosis ($2,199), treatment ($4,161), end-of-life ($6,614), and total ($4,167). Total direct medical costs were primarily driven by outpatient costs (23%). Patient age and length of follow-up had a significant impact on total costs, with older age associated with lower costs. When treatment segment was isolated, median monthly costs were lower in the “pre-biologic” as compared to the “post-biologic” era ($3,301 vs. $4,381, p=0.0024); biologic therapy accounted for 4.2% of total cost in the study period. In those patients experiencing all segments of care (“benchmark group”), median monthly costs were: diagnosis ($1,733), treatment ($8,265), end-of-life ($6,614), and total ($7,817). There were no significant differences in monthly medical costs between cohorts for the benchmark group. Conclusions: Median total cost exceeded $4,000/month for SCCHN patients; treatment and end-of-life segments incurred higher costs. Outpatient costs were the biggest cost-driver. Median monthly cost for the treatment segment increased by approximately $1,000 following cetuximab’s approval; however, this difference dissipated when the analysis was limited to those patients who experienced all segments of care.

Current methods to diagnose and treat cancer often involve expensive, time-consuming equipment and materials that may lead to unwanted side effects and may not even increase a patient’s chance of survival. Thus, for a while now, a large part of the research community has focused on developing improved methods to detect, diagnose, and treat cancer on the molecular scale. One of the most recently discovered methods of cancer therapy is targeted therapy. These targeted therapies have potential to provide a patient with a form of personalized medicine because these therapies are biological molecules that specifically target other molecules involved with a cancer’s growth. Past trials using these therapeutic molecules, however, have led to controversial results, where certain patients responded better than others to the therapy for unknown reasons. Elucidating the reason behind these mixed results can be accomplished using metal nanoparticle technologies which could provide a bright signal to monitor the path that these therapeutic molecules take in vivo as well as enhance the molecule’s efficacy. Literature has shown that presenting targeting molecules in a dense manner to their target will increase these molecules’ binding affinity. This concept has been explored here to increase binding affinity of therapeutic molecules by attaching these molecules in a dense manner on the surface of gold nanoparticles, and correlating this increased affinity with therapeutic efficacy. Additionally, gold nanoparticles provide an easy surface for molecules to be functionalized on and have shown to be effective imaging, x-ray, and photothermal therapy agents. A major roadblock to using these gold nanoparticles clinically is their non-degradability and thus potential to cause long-term negative side effects in vivo. A platform for developing biodegradable gold nanoparticles is also explored here to take advantage of the gold nanoparticles’ excellent imaging and drug delivery capabilities while still allowing them to be used safely in the long term.