Andrew P. Stein

AXL Mediates Resistance to Cetuximab Therapy

The EGFR antibody cetuximab is used to treat numerous cancers, but intrinsic and acquired resistance to this agent is a common clinical outcome. In this study, we show that overexpression of the oncogenic receptor tyrosine kinase AXL is sufficient to mediate acquired resistance to cetuximab in models of non-small cell lung cancer (NSCLC) and head and neck squamous cell carcinoma (HNSCC), where AXL was overexpressed, activated, and tightly associated with EGFR expression in cells resistant to cetuximab (Ctx(R) cells). Using RNAi methods and novel AXL-targeting agents, we found that AXL activation stimulated cell proliferation, EGFR activation, and MAPK signaling in Ctx(R) cells. Notably, EGFR directly regulated the expression of AXL mRNA through MAPK signaling and the transcription factor c-Jun in Ctx(R) cells, creating a positive feedback loop that maintained EGFR activation by AXL. Cetuximab-sensitive parental cells were rendered resistant to cetuximab by stable overexpression of AXL or stimulation with EGFR ligands, the latter of which increased AXL activity and association with the EGFR. In tumor xenograft models, the development of resistance following prolonged treatment with cetuximab was associated with AXL hyperactivation and EGFR association. Furthermore, in an examination of patient-derived xenografts established from surgically resected HNSCCs, AXL was overexpressed and activated in tumors that displayed intrinsic resistance to cetuximab. Collectively, our results identify AXL as a key mediator of cetuximab resistance, providing a rationale for clinical evaluation of AXL-targeting drugs to treat cetuximab-resistant cancers. Cancer Res; 74(18); 5152-64. ©2014 AACR.

Prevalence of Human Papillomavirus in Oropharyngeal Squamous Cell Carcinoma in the United States Across Time

Human papillomaviruses (HPVs) are involved in approximately 5% of all human cancer. Although initially recognized for causing nearly all cases of cervical carcinoma, much data has now emerged implicating HPVs as a causal factor in other anogenital cancers as well as a subset of head and neck squamous cell carcinomas (HNSCCs), most commonly oropharyngeal cancers. Numerous clinical trials have demonstrated that patients with HPV+ oropharyngeal squamous cell carcinoma (OPSCC) have improved survival compared to patients with HPV– cancers. Furthermore, epidemiological evidence shows the incidence of OPSCC has been steadily rising over time in the United States. It has been proposed that an increase in HPV-related OPSCCs is the driving force behind the increasing rate of OPSCC. Although some studies have revealed an increase in HPV+ head and neck malignancies over time in specific regions of the United States, there has not been a comprehensive study validating this trend across the entire country. Therefore, we undertook this meta-analysis to assess all literature through August 2013 that reported on the prevalence of HPV in OPSCC for patient populations within the United States. The results show an increase in the prevalence of HPV+ OPSCC from 20.9% in the pre-1990 time period to 51.4% in 1990–1999 and finally to 65.4% for 2000–present. In this manner, our study provides further evidence to support the hypothesis that HPV-associated OPSCCs are driving the increasing incidence of OPSCC over time in the United States.

Potential role of mesenchymal stromal cells in pancreatic islet transplantation

Pancreatic islet transplantation is an attractive option for treatment of type 1 diabetes mellitus but maintaining long term islet function remains challenging. Mesenchymal stromal cells (MSCs), derived from bone marrow or other sources, are being extensively investigated in the clinical setting for their immunomodulatory and tissue regenerative properties. Indeed, MSCs have been already tested in some feasibility studies in the context of islet transplantation. MSCs could be utilized to improve engraftment of pancreatic islets by suppressing inflammatory damage and immune mediated rejection. In addition to their immunomodulatory effects, MSCs are known to provide a supportive microenvironmental niche by secreting paracrine factors and depositing extracellular matrix. These properties could be used for in vivo co-transplantation to improve islet engraftment, or for in vitro co-culture to prime freshly isolated islets prior to implantation. Further, tissue specific pancreatic islet derived MSCs may open new opportunities for its use in islet transplantation as those cells might be more physiological to pancreatic islets.

Prevalence of Human Papillomavirus in Oropharyngeal Cancer: A Systematic Review.

PurposeThe global incidence of oropharyngeal squamous cell carcinoma (OPSCC) has been increasing, and it has been proposed that a rising rate of human papillomavirus (HPV)–associated cancers is driving the observed changes in OPSCC incidence. We carried out this systematic review to further examine the prevalence of HPV in OPSCC over time worldwide. MethodsA systematic literature search was performed to identify all articles through January 31, 2014, which reported on the prevalence of HPV in OPSCC. Articles that met the inclusion criteria were divided into 4 time frames (pre-1995, 1995–1999, 2000–2004, and 2005 to present) based on the median year of the study’s sample collection period. Using a weighted analysis of variance model, we examined the trends of HPV-positivity over time worldwide, in North America, and in Europe. ResultsOur literature search identified 699 unique articles. One hundred seventy-five underwent review of the entire study, and 105 met the inclusion criteria. These 105 articles reported on the HPV prevalence in 9541 OPSCC specimens across 23 nations. We demonstrated significant increases in the percentage change of HPV-positive OPSCCs from pre-1995 to present: 20.6% worldwide (P for trend: P < 0.001), 21.6% in North America (P = 0.013), and 21.5% in Europe (P = 0.033). ConclusionsInterestingly, whereas in Europe there was a steady increase in HPV prevalence across all time frames, reaching nearly 50% most recently, in North America HPV prevalence appears to have plateaued over the past decade at about 65%. These findings may have important implications regarding predictions for the future incidence of OPSCC.

AXL Is a Logical Molecular Target in Head and Neck Squamous Cell Carcinoma

Purpose: Head and neck squamous cell carcinoma (HNSCC) represents the eighth most common malignancy worldwide. Standard-of-care treatments for patients with HNSCC include surgery, radiation, and chemotherapy. In addition, the anti-EGFR monoclonal antibody cetuximab is often used in combination with these treatment modalities. Despite clinical success with these therapeutics, HNSCC remains a difficult malignancy to treat. Thus, identification of new molecular targets is critical. Experimental Design: In the current study, the receptor tyrosine kinase AXL was investigated as a molecular target in HNSCC using established cell lines, HNSCC patient-derived xenografts (PDX), and human tumors. HNSCC dependency on AXL was evaluated with both anti-AXL siRNAs and the small-molecule AXL inhibitor R428. Furthermore, AXL inhibition was evaluated with standard-of-care treatment regimens used in HNSCC. Results: AXL was found to be highly overexpressed in several models of HNSCC, where AXL was significantly associated with higher pathologic grade, presence of distant metastases, and shorter relapse-free survival in patients with HNSCC. Further investigations indicated that HNSCC cells were reliant on AXL for cellular proliferation, migration, and invasion. In addition, targeting AXL increased HNSCC cell line sensitivity to chemotherapy, cetuximab, and radiation. Moreover, radiation-resistant HNSCC cell line xenografts and PDXs expressed elevated levels of both total and activated AXL, indicating a role for AXL in radiation resistance. Conclusions: This study provides evidence for the role of AXL in HNSCC pathogenesis and supports further preclinical and clinical evaluation of anti-AXL therapeutics for the treatment of patients with HNSCC. Clin Cancer Res; 21(11); 2601–12. ©2015 AACR.

Biologic and immunomodulatory properties of mesenchymal stromal cells derived from human pancreatic islets

BACKGROUND AIMS Mesenchymal stromal cells (MSC) have now been shown to reside in numerous tissues throughout the body, including the pancreas. Ex vivo culture-expanded MSC derived from many tissues display important interactions with different types of immune cells in vitro and potentially play a significant role in tissue homeostasis in vivo. In this study, we investigated the biologic and immunomodulatory properties of human pancreatic islet-derived MSC. METHODS We culture-expanded MSC from cadaveric human pancreatic islets and characterized them using flow cytometry, differentiation assays and nuclear magnetic resonance-based metabolomics. We also investigated the immunologic properties of pancreatic islet-derived MSC compared with bone marrow (BM) MSC. RESULTS Pancreatic islet and BM-derived MSC expressed the same cell-surface markers by flow cytometry, and both could differentiate into bone, fat and cartilage. Metabolomics analysis of MSC from BM and pancreatic islets also showed a similar set of metabolic markers but quantitative polymerase chain reactions showed that pancreatic islet MSC expressed more interleukin(IL)-1b, IL-6, STAT3 and FGF9 compared with BM MSC, and less IL-10. However, similar to BM MSC, pancreatic islet MSC were able to suppress proliferation of allogeneic T lymphocytes stimulated with anti-CD3 and anti-CD28 antibodies. CONCLUSIONS Our in vitro analysis shows pancreatic islet-derived MSC have phenotypic, biologic and immunomodulatory characteristics similar, but not identical, to BM-derived MSC. We propose that pancreatic islet-derived MSC could potentially play an important role in improving the outcome of pancreatic islet transplantation by promoting engraftment and creating a favorable immune environment for long-term survival of islet allografts.

Influence of handling conditions on the establishment and propagation of head and neck cancer patient derived xenografts.

Background Patient derived xenografts (PDXs) for head and neck cancer (HNC) and other cancers represent powerful research platforms. Most groups implant patient tissue into immunodeficient mice immediately although the significance of this time interval is anecdotal. We tested the hypothesis that the time from tumor excision to implantation is crucial for PDX passaging and establishment. Methods We examined whether time or storage medium affected PDX viability for passaging two established HNC PDXs (UW-SCC34, UW-SCC52). Tumors were harvested, stored in ice-cold media or saline for 0–48 hours, and implanted into new mice. Tumor growth was compared by two-way ANOVA with respect to time and storage condition. Three new HNC PDXs (UW-SCC63-65) were generated by implanting patient tissue into mice immediately (Time 0) and 24 hours after receiving tissue from the operating room. Results Similar quantities of tumor were implanted into each mouse. At the end of the experiment, no significant difference was seen in mean tumor weight between the media and saline storage conditions for UW-SCC34 or UW-SCC52 (p = 0.650 and p = 0.177, respectively). No difference in tumor formation prevalence was seen on the basis of time from harvest to implantation (≥13 of 16 tumors grew at every time point). Histological analysis showed strong similarity to the initial tumor across all groups. Tumors developed at both Time 0 and 24 hours for UW-SCC63 and UW-SCC64. Conclusions We demonstrated that neither storage medium nor time from tumor excision to implantation (up to 48 hours) affected viability or histological differentiation in a subsequent passage for two HNC PDXs. Moreover, we revealed that fresh patient tissue is viable up to 24 hours post-resection. This information is important as it applies to the development and sharing of PDXs.

The receptor tyrosine kinase AXL mediates nuclear translocation of the epidermal growth factor receptor

The kinase AXL promotes cetuximab resistance by promoting the nuclear accumulation of EGFR. AXL sends EGFR to the nucleus The receptor tyrosine kinase (RTK) EGFR drives the growth of various cancers. Although a transmembrane protein that detects extracellular signals, EGFR accumulates at the nucleus in advanced and aggressive tumors. The abundance and activity of another RTK, AXL, is correlated with EGFR activation and drug resistance. Using patient-derived xenografts and lung cancer cell lines that were resistant or sensitive to cetuximab (an antibody that inhibits EGFR activity), Brand et al. found that AXL increases the expression of genes encoding the EGFR family ligand neuregulin-1 and two non-RTKs, YES and LYN. In the absence of AXL, nuclear accumulation of EGFR was blocked but was restored by overexpressing YES or LYN or adding neuregulin-1 to the cultures. Thus, ligand-mediated activation of EGFR in the context of enhanced non-RTK activity triggers the nuclear accumulation of EGFR, providing resistance to antibody therapies that target the extracellular part of EGFR. This study reveals a connection between an RTK and non-RTKs in resistance to rational targeted cancer therapies. The epidermal growth factor receptor (EGFR) is a therapeutic target in patients with various cancers. Unfortunately, resistance to EGFR-targeted therapeutics is common. Previous studies identified two mechanisms of resistance to the EGFR monoclonal antibody cetuximab. Nuclear translocation of EGFR bypasses the inhibitory effects of cetuximab, and the receptor tyrosine kinase AXL mediates cetuximab resistance by maintaining EGFR activation and downstream signaling. Thus, we hypothesized that AXL mediated the nuclear translocation of EGFR in the setting of cetuximab resistance. Cetuximab-resistant clones of non–small cell lung cancer in culture and patient-derived xenografts in mice had increased abundance of AXL and nuclear EGFR (nEGFR). Cellular fractionation analysis, super-resolution microscopy, and electron microscopy revealed that genetic loss of AXL reduced the accumulation of nEGFR. SRC family kinases (SFKs) and HER family ligands promote the nuclear translocation of EGFR. We found that AXL knockdown reduced the expression of the genes encoding the SFK family members YES and LYN and the ligand neuregulin-1 (NRG1). AXL knockdown also decreased the interaction between EGFR and the related receptor HER3 and accumulation of HER3 in the nucleus. Overexpression of LYN and NRG1 in cells depleted of AXL resulted in accumulation of nEGFR, rescuing the deficit induced by lack of AXL. Collectively, these data uncover a previously unrecognized role for AXL in regulating the nuclear translocation of EGFR and suggest that AXL-mediated SFK and NRG1 expression promote this process.

Xenograft assessment of predictive biomarkers for standard head and neck cancer therapies

Head and neck squamous cell carcinoma (HNSCC) remains a challenging cancer to treat with overall 5‐year survival on the order of 50–60%. Therefore, predictive biomarkers for this disease would be valuable to provide more effective and individualized therapeutic approaches for these patients. While prognostic biomarkers such as p16 expression correlate with outcome; to date, no predictive biomarkers have been clinically validated for HNSCC. We generated xenografts in immunocompromised mice from six established HNSCC cell lines and evaluated response to cisplatin, cetuximab, and radiation. Tissue microarrays were constructed from pre‐ and posttreatment tumor samples derived from each xenograft experiment. Quantitative immunohistochemistry was performed using a semiautomated imaging and analysis platform to determine the relative expression of five potential predictive biomarkers: epidermal growth factor receptor (EGFR), phospho‐EGFR, phospho‐Akt, phospho‐ERK, and excision repair cross‐complementation group 1 (ERCC1). Biomarker levels were compared between xenografts that were sensitive versus resistant to a specific therapy utilizing a two‐sample t‐test with equal standard deviations. Indeed the xenografts displayed heterogeneous responses to each treatment, and we linked a number of baseline biomarker levels to response. This included low ERCC1 being associated with cisplatin sensitivity, low phospho‐Akt correlated with cetuximab sensitivity, and high total EGFR was related to radiation resistance. Overall, we developed a systematic approach to identifying predictive biomarkers and demonstrated several connections between biomarker levels and treatment response. Despite these promising initial results, this work requires additional preclinical validation, likely involving the use of patient‐derived xenografts, prior to moving into the clinical realm for confirmation among patients with HNSCC.

Defining the boundaries and expanding the utility of head and neck cancer patient derived xenografts

BACKGROUND Patient derived xenografts (PDXs) represent an essential tool in oncologic research, and we sought to further expand our repertoire of head and neck squamous cell carcinoma (HNSCC) while determining potential boundaries for this system. METHODS We consented new patients for PDX development and determined if a 24-h time delay from tumor excision to xenograft implantation affected PDX establishment. We developed a tissue microarray (TMA) from formalin fixed, paraffin embedded PDXs and their subsequent passages and carried out quantitative immunohistochemistry for EGFR, pEGFR, pAkt, pERK and ERCC1. First and last passaged PDXs were compared via a paired t-test to examine for the stability of protein expression across passages. We performed a similar comparison of the mutational profile of the patient tumor and resulting xenografts using a targeted sequencing approach. RESULTS No patient/tumor characteristics influenced PDX take rate and the 24-h time delay from tumor excision to xenograft implantation did not affect PDX establishment, growth or histology. There was no significant difference in biomarker expression between the first and last passaged PDXs for EGFR, pEGFR, pAkt, and ERCC1. For pERK there was a significant difference (p=0.002), but further analysis demonstrated this only arose in three of 15 PDXs. Targeted sequencing revealed striking stability of passenger and likely driver mutations from patient to xenograft. CONCLUSIONS The stability of protein expression across PDX passages will hopefully allow greater investigation of predictive biomarkers in order to identify ones for further pre-clinical and clinical investigation.