University of Wisconsin–Madison
Randall Kimple, MD, PhD

Randall Kimple, MD, PhD

Assistant Professor

Department of Human Oncology

I am an assistant professor in the Department of Human Oncology with a clinical practice and research laboratory. In my clinical practice, I specialize in treating patients with malignancies of the head and neck. I am a member of the UW Multidisciplinary Head and Neck Program and work closely with head and neck surgeons, medical oncologists, radiologists, speech and swallow therapists and other specialists to best meet the individual patient’s needs.

My research laboratory is focused on understanding why cancers don’t always respond to treatment. We use patient-derived xenografts—patient tumor samples grown in animals—to test radiation, chemotherapy and combinations of therapies to understand which characteristics of a patient’s tumor may predict response to treatment. The goal of this work is to enable more personalized treatment and to find ways to combine different treatment methods to decrease treatment side effects without reducing cure rates.

In addition to my clinical and research roles, I teach undergraduate, graduate and medical students as well as residents and postdoctoral fellows. I try to give these students a sense of the breadth of opportunities in medicine and challenge them to be the best doctors and scientists they can be. I encourage my students to come to the clinic with me once in a while so they can see the patients because that’s where our work is ultimately headed. If we make great discoveries we can really make a positive difference for our patients.

Education

MD, University of North Carolina at Chapel Hill, Medicine (2005)

PhD, University of North Carolina at Chapel Hill, Pharmacology (2003)

BS, Michigan State University, Environmental Science and Management (1998)

Academic Appointments

Assistant Professor, Human Oncology (2012)

Assistant Professor, Medical Physics (2014)

Selected Honors and Awards

Postdoctoral Mentor Award, University of Wisconsin (2017)

Teacher of the Year Award, Association of Residents in Radiation Oncology (2017)

Medical Faculty Award, University of North Carolina (2005)

Graduate Fellow Award, Lineberger Comprehensive Cancer Center (2002)

Outstanding Senior Award, Michigan State University (1998)

Convocation Speaker, College of Natural Science, Michigan State University (1998)

Tower Guard Sophomore Honor Service Society, Michigan State University (1995-1996)

Boards, Advisory Committees and Professional Organizations

American Society for Pharmacology and Experimental Therapeutics (2003-pres.)

Radiological Society of North America (2006-pres.)

American Society for Radiation Oncology (2006-pres.)

University of Wisconsin Institute for Clinical and Translational Research (2010-pres.)

Radiation Research Society (2012-pres.)

American Society of Clinical Oncology Leadership Development Program (2012-2018)

Senior Associate Editor, International Journal of Radiation Oncology Biology and Physics (2014-pres.)

ASTRO Annual Meeting Scientific Track co-chair – Biology (2017-pres.)

Co-chair, Big Ten Cancer Research Consortium Head and Neck Working Group (2017-pres.)

Research Focus

Head and Neck Cancer

Kimple group shot at conference
(L-R) Gopika Senthilkumar (U), Amber Bo (U), Kwang Nickel (S), Lindsey Abel (S), Austin Maas (RI), Adam Swick (P), Jaimee Eckers (P), Amal Javaid (RI), Michael Fisher (U) U- undergrad S – scientist RI – research intern P – postdoc
In the Kimple Lab we seek to improve the care of cancer patients through our strengths in translational radiation research while providing a supportive world-class learning environment for scientists at all levels. 

Radiation therapy can be used to cure many cancer patients. Our best treatments are still not good enough for too many patients. We use powerful patient-derived model systems to study how cancers evade current treatments. Our long-term goal is to identify new ways to prevent or overcome resistance to current treatments.

Human papillomavirus related head and neck cancer

Human papillomavirus (HPV) is a ubiquitious virus that can cause multiple cancers in humans. Our lab studies HPV-related head and neck cancer and has helped define the molecular basis for the improved outcomes in patients with HPV-related head and neck cancer (in comparison to HPV-unrelated). Multiple clinical trials investigating how we can personalize treatment for patients based on the cause of their cancer are now ongoing. The role the HPV oncogenes E6 and E7 play in how these cancers respond to treatment is a common theme across the research in the Kimple Lab.

Prevalence of Human Papillomavirus in Oropharyngeal Cancer: A Systematic Review

Funding

  • NIH R00

Relevant Publications

Molecular signaling

Cells use signaling pathways to communicate both between cells and within cells. A major interest for our lab is how we can utilize drugs targeting intracellular signaling pathways to make cancers more sensitive to radiation or chemotherapy. Much of this work has focused on signaling from the cell surface (e.g., the epidermal growth factor receptor, EGFR) to multiple intracellular targets. These pathways influence cell fate, growth and differentiation and can be altered due to cancer-specific mutations, epigenetic alterations or due to the effects of viral or tumor oncogenes.

Mutational profile and patient characteristics

Funding

  • NIH R00
  • Wisconsin Partnership Program
  • UW Head and Neck SPORE, Project 4
  • American Cancer Society

Relevant publications

Resistance to standard therapy

Therapy-induced autophagy

Autophagy, or self-eating, is a pro-survival process cells use to recycle damaged parts during times of stress. Autophagy is often activated during nutrient deprivation, although many types of cell stress can activate autophagy. We are interested in how head and neck cancer cells use autophagy to respond to radiation and chemotherapy. Autophagy is activated in a dose and time dependent manner by radiation and the epidermal growth factor receptor (EGFR) inhibitor, cetuximab. A grant funded by the American Cancer Society seeks to understand the molecular mechanisms by which radiation and cetuximab activate autophagy. In addition, we will test drugs that specifically block autophagy to determine whether they can increase sensitivity to anticancer treatments.

Autophagy

Funding:

  • American Cancer Society

Cancer Stem Cells

Cancer stem cells have been identified in most cancers and are thought to form a subpopulation of cells that are resistant to therapy, capable of reconstituting a heterogeneous cancer and must be eliminated in order to cure a patient. A project supported by the Jimmy V Foundation has allowed us to identify head and neck cancer stem cells using functional assays and cell biomarkers. We have shown that these cancer cells are able to form tumors in mice, are resistant to radiation and chemotherapy and can be identified by molecular markers. We are currently focused on ways in which these cells can be killed by understanding the molecular differences between cancer stem cells and their non-stem-cell counterparts.

CSC

Relevant publications:

 Funding:

  • V Foundation for Cancer Research

Patient-Derived Xenografts

We have established one of the largest tissue repositories of head and neck cancer patient-derived xenografts (PDX) and have helped define best practices for the establishment, passage and use of these valuable resources. Patient-derived xenografts are established in mice directly from patient biopsies and are thought to better represent the biology of their human source than model cell lines grown in plastic tissue culture plates. Current work seeks to continue the characterization of this resource and to improve out ability to utilize them by understanding how they can best be used in the setting of a “humanized” mouse with a functional immune system. This would allow us to use this resource to study immunotherapy and immunomodulation.

In addition to our focus on head and neck cancer models, we have also established PDXs from brain metastases in lung cancer patients, pancreas cancer, melanoma, breast cancer and rare tumors such as adenoid cystic carcinoma and NUT midline carcinomas. We have used our PDXs to partner with several pharmaceutical companies to test novel drugs in head and neck cancers. Please contact us if you are interested in this aspect of our work.

Establishing Patient-Derived Xenografts (PDX)

 

Relevant publications

Funding

  • UW Head and Neck Cancer SPORE
  • UW Tumor Microenvironment Pilot Grant
  • DHO pilot grant

Current Team Members

  • Kwang Nickel, PhD, Research Specialist
  • Adam Swick, PhD, Postdoctoral Research Associate
  • Lindsey Abel, BS, Research Specialist
  • Jaimee Eckers, PhD, Postdoctoral Research Associate
  • Michael “Mac” Fisher, BS, Research Intern
  • Austin Maas, BS, Research Intern

Undergraduates

  • Amber Bo
  • Heather Barnwell
  • Justin Skiba
  • Aastha Pandey
  • Margot Miller
  • Gopika Senthilkumar

Alumni

Graduate/medical students (position after leaving Kimple Lab)

  • Tyler Fowler, PhD Medical Physics (Medical Physics Residency, Stanford Univ)
  • Robert Yang, MD, Medicine (Residency Otolaryngology, Univ Minnesota)
  • Julian Hong, MD MS, Medicine (Residency Radiation Oncology, Duke Univ)
  • Andy Stein, MD, Medicine (Residency Otolaryngology, Case Western Reserve University)
  • Stephanie Rice, MD Medicine (Residency Radiation Oncology, Univ of Maryland)
  • Evan Liang, MD, Medicine (Residency Radiation Oncology, Henry Ford Hospital)
  • Anirban Chatterjee, PhD, (Assistant Professor, Bolpur College, University of Burdwan, Bolpur, Birbhum, West Bengal, India)

Album

Poster presentation

Rachel Orbuch, Adam Swick and Austin Maas at AACR meeting

Kimple lab in front of Senator Johnson's office in Washington, DC

Locations

University Hospitals and Clinics

Clinical trials

I play an active role in the UW Head and Neck Cancer Disease Oriented Team, which oversees and prioritizes head and neck cancer focused clinical research activities at the University of Wisconsin. In addition, I am co-chair of the Big Ten Cancer Research Consortium Head and Neck Cancer Working Group. This group focuses on early phase multi-institutional clinical trials for patients with head and neck cancers.

 

Through clinical trials I hope to bring promising treatments to patients and integrate advanced imaging, novel molecular tests and molecular markers to match each patient to the right treatment for their individual cancer.

 

Funding

  • UW Head and Neck Cancer SPORE

Relevant Publications

  1. Is radiation dose reduction the right answer for HPV-positive head and neck cancer?
  2. Local control following single-dose intraoperative radiotherapy prior to surgical excision of early-stage breast cancer.
  3. Phase I study and biomarker analysis of lapatinib and concurrent radiation for locally advanced breast cancer.

Kimple group shot in front of White House

In addition to my clinical and research roles, I teach undergraduate, graduate and medical students as well as residents and postdoctoral fellows. I try to give these students a sense of the breadth of opportunities in medicine and challenge them to be the best doctors and scientists they can be. I encourage my students to come to the clinic with me once in a while so they can see the patients because that’s where our work is ultimately headed. If we make great discoveries we can really make a positive difference for our patients.

  • Overcoming resistance to cetuximab with honokiol, a small-molecule polyphenol. Mol Cancer Ther
    Pearson HE, Iida M, Orbuch RA, McDaniel NK, Nickel KP, Kimple RJ, Arbiser J, Wheeler DL
    2017 Oct 20; :
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      Overexpression and activation of the Epidermal Growth Factor Receptor (EGFR) have been linked to poor prognosis in several human cancers. Cetuximab is a monoclonal antibody against EGFR, that is used for the treatment in head and neck squamous cell carcinoma (HNSCC) and metastatic colorectal cancer. Unfortunately, most tumors have intrinsic or acquire resistance to cetuximab during the course of therapy. Honokiol is a natural compound found in the bark and leaves of the Chinese Magnolia tree and is established to have several anti-cancer properties without appreciable toxicity. In this study, we hypothesized that combining cetuximab and honokiol treatments could overcome acquired resistance to cetuximab. We previously developed a model of acquired resistance to cetuximab in non-small cell lung cancer cell line. Treatment of cetuximab resistant clones with honokiol and cetuximab resulted in a robust anti-proliferative response. Immunoblot analysis revealed the HER family and their signaling pathways were downregulated after combination treatment, most notably the proliferation (MAPK) and survival (AKT) pathways. Additionally, we found a decrease in phosphorylation of DRP1 and reactive oxygen species after combination treatment in cetuximab resistant clones which may signify a change in mitochondrial function. Furthermore, we utilized cetuximab resistant HNSCC patient derived xenografts (PDX) to test the benefit of combinatorial treatment in vivo. There was significant growth delay in PDX tumors after combination treatment with a subsequent down-regulation of active MAPK, AKT, and DRP1 signaling as seen in vitro. Collectively these data suggest that honokiol is a promising natural compound in overcoming acquired resistance to cetuximab.

      View details for PubMedID 29054984
  • Survival Outcomes for Patients With T3N0M0 Squamous Cell Carcinoma of the Glottic Larynx. JAMA Otolaryngol Head Neck Surg
    Ko HC, Harari PM, Chen S, Wieland AM, Yu M, Baschnagel AM, Kimple RJ, Witek ME
    2017 Oct 12; :
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      Importance: Radiotherapy (RT)-based organ preservation approaches for patients with advanced laryngeal cancer have been established stepwise through prospective randomized clinical trials. However, broad adoption of these approaches has stimulated discussion about long-term results challenging their applicability in a heterogeneous patient population, most recently for patients with T3 disease.

      Objective: To define outcomes in patients with clinical T3N0M0 glottic laryngeal cancer treated with definitive surgical and RT-based approaches.

      Design, Setting, and Participants: This retrospective cohort study included patients treated from January 1, 2004, through December 31, 2013, with a median follow-up time of 58 months (range, 0-126.6 months) in the National Cancer Database. Of the 4003 patients with T3N0M0 disease, 2622 received definitive therapy defined by the study protocol. Data were obtained from the clinical oncology database sourced from hospital registry data that are collected from more than 1500 Commission on Cancer-accredited facilities. Data were analyzed from September 14, 2016, through April 24, 2017.

      Interventions: Radiotherapy, chemoradiotherapy, surgery, surgery and RT, or surgery and chemoradiotherapy.

      Results: A total of 2622 patients (2251 men [85.9%] and 371 women [14.1%]; median age, 64 years [range, 19-90 years]) were included in the analytic cohort. In the overall patient cohort, the adjusted 5-year survival probability was 53%. No statistical differences were observed between the primary surgery (53%; 95% CI, 48%-57%) and primary RT (54%; 95% CI, 52%-57%) cohorts. In multivariate analysis, patient factors associated with decreased overall survival (OS) included age (hazard ratio [HR], 1.04; 95% CI, 1.03-1.04), insurance status (HR, 1.26; 95% CI, 1.06-1.50), and increasing comorbidity (HR, 1.20; 95% CI, 1.02-1.42).

      Conclusions and Relevance: Current management of T3N0M0 glottic laryngeal cancer relies largely on RT-based organ preservation approaches. The present study substantiates randomized clinical trial data supporting the use of RT-based organ preservation approaches for patients with T3N0M0 glottic laryngeal cancer without compromising OS.

      View details for PubMedID 29049434
  • Prognostic implications of human papillomavirus status for patients with non-oropharyngeal head and neck squamous cell carcinomas. J Cancer Res Clin Oncol
    Ko HC, Harari PM, Sacotte RM, Chen S, Wieland AM, Yu M, Baschnagel AM, Bruce JY, Kimple RJ, Witek ME
    2017 Nov; 143 (11): 2341-2350
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      PURPOSE: We examined overall survival in a large cohort of patients with human papillomavirus (HPV)-positive and HPV-negative non-oropharyngeal squamous cell carcinoma of the head and neck (non-OPSCC).

      METHODS: Patients diagnosed with non-OPSCC and known HPV status were identified in the National Cancer Database (NCDB). Multivariate logistic regression was applied to examine factors associated with HPV status. Multivariate analysis was utilized to determine factors correlated with overall survival. Propensity score-weighted Kaplan-Meier estimation was used to adjust for confounders in survival analyses. Multiple imputation method was used for sensitivity analysis.

      RESULTS: We identified 19,993 non-OPSCC patients with 5070 being positive for HPV in the NCDB. Median follow-up was 23.5 months. HPV-positive patients were more commonly male, white, with a lower comorbidity index score, presenting with T-stage <2, and N-stage ≥1. Unadjusted 3-year overall survival was 62% and 80% for HPV-negative and HPV-positive patients, respectively (p < 0.0001). On multivariate analysis, mortality was reduced for HPV-positive patients with early stage (HR = 0.68) and locally advanced disease (HR = 0.46). Adjusted 3-year overall survival was 65% for HPV-negative and 76% for HPV-positive patients (p < 0.0001). The survival advantage of HPV was maintained in all subsites and robust on sensitivity analysis.

      CONCLUSIONS: Patients with HPV-positive non-OPSCC exhibit similar characteristics as HPV-positive OPSCC. Overall survival was significantly higher for patients with HPV-positive versus HPV-negative non-OPSCC. These data reveal that HPV-positive non-OPSCC represent a favorable cohort that warrants recognition in the design of future clinical trial investigation.

      View details for PubMedID 28752235
  • Radiosensitization of Adenoid Cystic Carcinoma with MDM2 Inhibition. Clin Cancer Res
    Prabakaran PJ, Javaid AM, Swick AD, Werner LR, Nickel KP, Sampene E, Hu R, Ong IM, Bruce JY, Hartig GK, Wieland AM, Canon J, Harari PM, Kimple RJ
    2017 Oct 15; 23 (20): 6044-6053
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      Purpose: Adenoid cystic carcinoma (ACC) is a rare cancer arising from the major or minor salivary gland tissues of the head and neck. There are currently no approved systemic agents or known radiosensitizers for ACC. Unlike the more common head and neck squamous cell carcinomas that frequently harbor TP53 mutations, ACCs contain TP53 mutations at a rate of <5%, rendering them an attractive target for MDM2 inhibition.Experimental Design: We report the successful establishment and detailed characterization of a TP53-WT ACC patient-derived xenograft (PDX), which retained the histologic features of the original patient tumor. We evaluated this model for response to the MDM2 inhibitor AMG 232 as monotherapy and in combination with radiotherapy.Results: AMG 232 monotherapy induced modest tumor growth inhibition, and radiation monotherapy induced a transient tumor growth delay in a dose-dependent fashion. Strikingly, combination treatment of AMG 232 with radiotherapy (including low-dose radiotherapy of 2 Gy/fraction) induced dramatic tumor response and high local tumor control rates 3 months following treatment. Posttreatment analysis revealed that although both AMG 232 and radiotherapy alone induced TP53 tumor-suppressive activities, combination therapy amplified this response with potent induction of apoptosis after combination treatment.Conclusions: These data identify that MDM2 inhibition can provide potent radiosensitization in TP53-WT ACC. In light of the absence of effective systemic agents for ACC, the powerful response profile observed here suggests that clinical trial evaluation of this drug/radiotherapy combination may be warranted to improve local control in this challenging malignancy. Clin Cancer Res; 23(20); 6044-53. ©2017 AACR.

      View details for PubMedID 28659312
  • Small cell carcinoma of the head and neck: An analysis of the National Cancer Database. Oral Oncol
    Pointer KB, Ko HC, Brower JV, Witek ME, Kimple RJ, Lloyd RV, Harari PM, Baschnagel AM
    2017 Jun; 69: 92-98
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      PURPOSE/OBJECTIVE(S): To evaluate treatment trends and overall survival of patients with small cell carcinoma of the head and neck region.

      MATERIALS/METHODS: Patients from 2004 to 2012 were identified from the National Cancer Database. Patient demographics and overall survival were analyzed. Multivariable analysis was used to identify predictors of survival.

      RESULTS: Among 347,252 head and neck patients a total of 1042 (0.3%) patients with small cell carcinoma were identified. 17% of patients were diagnosed as stage I/II, 61% as stage III/IVA/IVB and 22% as stage IVC disease. The distribution by anatomic site was 9% oral cavity, 12% oropharynx, 35% larynx, 4% hypopharynx, 10% nasopharynx and 30% nasal cavity and paranasal sinuses. The median overall survival by anatomical site was 20.8months for oral cavity, 23.7months for oropharynx, 17.9months for larynx/hypopharynx, 15.1months for nasopharynx and 36.4months for nasal cavity primary tumors. On multivariable analysis across stage, patients with nasal cavity and paranasal sinuses tumors had the best survival and patients with nasopharynx primaries had the worst survival. In stage I/II patients, type of treatment delivered resulted in no overall survival difference (p=0.78). In patients with locally advanced disease, there was no difference in survival between those treated with combined surgery, radiotherapy and chemotherapy compared to those treated only with radiotherapy and chemotherapy (p=0.46). The addition of radiotherapy to chemotherapy in the metastatic setting did not result in improved survival (p=0.14).

      CONCLUSIONS: Small cell carcinoma of the head and neck is a rare malignancy with a poor prognosis. The addition of surgery to radiotherapy and chemotherapy did not improve survival in patients with locally advanced disease.

      View details for PubMedID 28559027
  • Clinical Outcomes and Prognostic Factors of Adenoid Cystic Carcinoma of the Head and Neck. Anticancer Res
    Jang S, Patel PN, Kimple RJ, McCulloch TM
    2017 06; 37 (6): 3045-3052
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      BACKGROUND: Adenoid cystic carcinoma (ACC) is a salivary gland malignancy with unpredictable growth and poorly understood prognostic factors.

      PATIENTS AND METHODS: A database search of patients treated at a single Institution was used to identify patients with histologically-confirmed ACC. Patient, tumor, and treatment characteristics were examined via review of medical records.

      RESULTS: Overall survival of 70 patients identified at 5, 10, and 15 years was 80.4%, 61.3%, and 29.4%, respectively. Disease recurrence was seen in 31.9%; of these, 72.7% developed distant metastasis. Older age, higher stage, skull base involvement, positive margins, and metastatic disease, but not local recurrence, predicted a worse overall survival. Higher stage and skull base disease were also associated with shorter disease-free survival. While lung metastasis was the most common, vertebral metastasis was associated with poorer survival.

      CONCLUSION: Disease stage, positive margins, skull base involvement, perineural invasion, time to recurrence, and location of metastasis, but not nodal involvement, could serve as poor prognostic factors in ACC.

      View details for PubMedID 28551643
  • Potential Mechanisms of Vascular Thrombosis after Microwave Ablation in an in Vivo Liver. J Vasc Interv Radiol
    Chiang J, Nickel K, Kimple RJ, Brace CL
    2017 Jul; 28 (7): 1053-1058
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      PURPOSE: To evaluate potential biologic and thermal mechanisms of the observed differences in thrombosis rates between hepatic vessels during microwave (MW) ablation procedures.

      MATERIALS AND METHODS: MW ablation antennae were placed in single liver lobes of 2 in vivo porcine liver models (n = 3 in each animal; N = 6 total) in the proximity of a large (> 5 mm) portal vein (PV) and hepatic veins (HVs). Each ablation was performed with 100 W for 5 minutes. Conventional ultrasound imaging and intravascular temperature probes were used to evaluate vessel patency and temperature changes during the ablation procedure. Vascular endothelium was harvested 1 hour after ablation and used to characterize genes and proteins associated with thrombosis in PVs and HVs.

      RESULTS: Targeted PVs within the MW ablation zone exhibited thrombosis at a significantly higher rate than HVs (54.5% vs 0.0%; P = .0046). There was a negligible change in intravascular temperature in PVs and HVs during the ablation procedure (0.2°C ± 0.4 vs 0.6°C ± 0.9; P = .46). PVs exhibited significantly higher gene expression than HVs in terms of fold differences in thrombomodulin (2.9 ± 2.0; P = .0001), von Willebrand factor (vWF; 7.6 ± 1.5; P = .0001), endothelial protein C receptor (3.50 ± 0.49; P = .0011), and plasminogen activator inhibitor (1.46 ± 0.05; P = .0014). Western blot analysis showed significantly higher expression of vWF (2.32 ± 0.92; P = .031) in PVs compared with HVs.

      CONCLUSIONS: Large PVs exhibit thrombosis more frequently than HVs during MW ablation procedures. Biologic differences in thrombogenicity, rather than heat transfer, between PVs and HVs may contribute to their different rates of thrombosis.

      View details for PubMedID 28456355
  • Cotargeting mTORC and EGFR Signaling as a Therapeutic Strategy in HNSCC. Mol Cancer Ther
    Swick AD, Prabakaran PJ, Miller MC, Javaid AM, Fisher MM, Sampene E, Ong IM, Hu R, Iida M, Nickel KP, Bruce JY, Wheeler DL, Kimple RJ
    2017 Jul; 16 (7): 1257-1268
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      Head and neck squamous cell carcinomas (HNSCC) are frequently altered along the PI3K/AKT/mTORC signaling axis. Despite excellent preclinical data, the use of compounds targeting this pathway as monotherapy has been underwhelming in initial clinical trials, and identification of predictive biomarkers remains challenging. To investigate mTORC-specific inhibition, we tested catalytic mTORC (AZD8055) and PI3K/mTORC (NVP-BEZ-235) inhibitors ± cetuximab in a panel of HNSCC cell lines and patient-derived xenografts (PDX). Cell lines were assayed for response to all agents and siRNA knockdown of targets by multiple approaches. All cell lines showed similar response to both drug and siRNA inhibition of both PI3K and mTORC pathways, with anti-EGFR combination producing modest additive effect. Five PDX models that presented PIK3CA mutation or intrinsic cetuximab resistance were treated with a combination of cetuximab and AZD8055. In vivo single-agent mTORC inhibition inhibited growth of one PIK3CA-mutant cancer, but had little effect on any PIK3CA(WT) or a second PIK3CA-mutant model. In all models, the combination therapy showed greater growth delay than monotherapy. The uniform ability of PI3K and mTORC inhibition to suppress the growth of HNSCC cells highlights the pathway's role in driving proliferation. Although single-agent therapy was largely ineffective in vivo, improved response of combination treatment in an array of PDXs suggests the potential for adding a catalytic mTORC inhibitor to cetuximab therapy. Overall, these results add to a growing body of evidence, suggesting that approaches that attempt to match biomarkers to the optimal therapy in HNSCC remain complex and challenging. Mol Cancer Ther; 16(7); 1257-68. ©2017 AACR.

      View details for PubMedID 28446642
  • The receptor tyrosine kinase AXL mediates nuclear translocation of the epidermal growth factor receptor. Sci Signal
    Brand TM, Iida M, Corrigan KL, Braverman CM, Coan JP, Flanigan BG, Stein AP, Salgia R, Rolff J, Kimple RJ, Wheeler DL
    2017 Jan 03; 10 (460):
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      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.

      View details for PubMedID 28049763
  • Defining the boundaries and expanding the utility of head and neck cancer patient derived xenografts. Oral Oncol
    Swick AD, Stein AP, McCulloch TM, Hartig GK, Ong IM, Sampene E, Prabakaran PJ, Liu CZ, Kimple RJ
    2017 Jan; 64: 65-72
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      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.

      View details for PubMedID 28024726
  • Online patient information from radiation oncology departments is too complex for the general population. Pract Radiat Oncol
    Rosenberg SA, Francis DM, Hullet CR, Morris ZS, Brower JV, Anderson BM, Bradley KA, Bassetti MF, Kimple RJ
    2017 Jan - Feb; 7 (1): 57-62
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      PURPOSE: Nearly two-thirds of cancer patients seek information about their diagnosis online. We assessed the readability of online patient education materials found on academic radiation oncology department Web sites to determine whether they adhered to guidelines suggesting that information be presented at a sixth-grade reading level.

      METHODS AND MATERIALS: The Association of American Medical Colleges Web site was used to identify all academic radiation oncology departments in the United States. One-third of these department Web sites were selected for analysis using a random number generator. Both general information on radiation therapy and specific information regarding various radiation modalities were collected. To test the hypothesis that the readability of these online educational materials was written at the recommended grade level, a panel of 10 common readability tests was used. A composite grade level of readability was constructed using the 8 readability measures that provide a single grade-level output.

      RESULTS: A mean of 5605 words (range, 2058-12,837) from 30 department Web sites was collected. Using the composite grade level score, the overall mean readability level was determined to be 13.36 (12.83-13.89), corresponding to a collegiate reading level. This was significantly higher than the target sixth-grade reading level (middle school, t (29) = 27.41, P < .001).

      CONCLUSIONS: Online patient educational materials from academic radiation oncology Web sites are significantly more complex than recommended by the National Institutes of Health and the Department of Health and Human Services. To improve patients' comprehension of radiation therapy and its role in their treatment, our analysis suggests that the language used in online patient information should be simplified to communicate the information at a more appropriate level.

      View details for PubMedID 27663932
  • Improved survival with dose-escalated radiotherapy in stage III non-small-cell lung cancer: analysis of the National Cancer Database. Ann Oncol
    Brower JV, Amini A, Chen S, Hullett CR, Kimple RJ, Wojcieszynski AP, Bassetti M, Witek ME, Yu M, Harari PM, Baschnagel AM
    2016 Oct; 27 (10): 1887-94
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      BACKGROUND: Concurrent chemoradiation is the standard of care in non-operable stage III non-small-cell lung cancer (NSCLC). Data have suggested a benefit of dose escalation; however, results from the randomized dose-escalation trial RTOG 0617 revealed a lower survival rate with high-dose radiation. To evaluate the impact of dose escalation on overall survival (OS) in stage III NSCLC treated with chemoradiotherapy outside the controlled setting of a randomized trial, we carried out an observational, population-based investigation of the National Cancer Database (NCDB).

      PATIENTS AND METHODS: A total of 33 566 patients with stage III NSCLC treated with chemoradiation from 2004 to 2012 and radiation doses between 59.4 and 85 Gy were included. The primary end point was OS, with median survival calculated via Kaplan-Meier. Univariate, multivariable and propensity-score matching analyses were carried out.

      RESULTS: Patients were stratified by dose with median OS of: 18.8, 19.8 and 21.6 months for cohorts receiving 59.4-60, 61-69 and ≥70 Gy, respectively (P < 0.001). Granular dose analyses were carried out demonstrating increased OS with increasing radiation dose: median survival of 18.8, 21.1, 22.0 and 21.0 months for 59.4-60, 66, 70 and ≥71 Gy, respectively. While 66, 70 and ≥71 Gy resulted in increased OS in comparison with 59.4-60 Gy, no significant difference in OS was observed when comparing 66 with ≥71 Gy (P = 0.38).

      CONCLUSIONS: Dose escalation above 60 Gy was associated with improved OS in this cohort of stage III NSCLC patients treated with chemoradiotherapy. A plateau of benefit was observed, with no additional improvement in OS with increased dose (≥71 Gy) compared with 66-70 Gy. With evidence suggesting worse OS and quality of life with increased dose, these data support investigation of the role of intermediate-dose radiation, and in the absence of randomized evidence, may be leveraged to justify utilization of intermediate-dose radiation.

      View details for PubMedID 27502703
  • Targeting the HER Family with Pan-HER Effectively Overcomes Resistance to Cetuximab. Mol Cancer Ther
    Iida M, Bahrar H, Brand TM, Pearson HE, Coan JP, Orbuch RA, Flanigan BG, Swick AD, Prabakaran PJ, Lantto J, Horak ID, Kragh M, Salgia R, Kimple RJ, Wheeler DL
    2016 Sep; 15 (9): 2175-86
    • More

      Cetuximab, an antibody against the EGFR, has shown efficacy in treating head and neck squamous cell carcinoma (HNSCC), metastatic colorectal cancer, and non-small cell lung cancer (NSCLC). Despite the clinical success of cetuximab, many patients do not respond to cetuximab. Furthermore, virtually all patients who do initially respond become refractory, highlighting both intrinsic and acquired resistance to cetuximab as significant clinical problems. To understand mechanistically how cancerous cells acquire resistance, we previously developed models of acquired resistance using the H226 NSCLC and UM-SCC1 HNSCC cell lines. Cetuximab-resistant clones showed a robust upregulation and dependency on the HER family receptors EGFR, HER2, and HER3. Here, we examined pan-HER, a mixture of six antibodies targeting these receptors on cetuximab-resistant clones. In cells exhibiting acquired or intrinsic resistance to cetuximab, pan-HER treatment decreased all three receptors' protein levels and downstream activation of AKT and MAPK. This correlated with decreased cell proliferation in cetuximab-resistant clones. To determine whether pan-HER had a therapeutic benefit in vivo, we established de novo cetuximab-resistant mouse xenografts and treated resistant tumors with pan-HER. This regimen resulted in a superior growth delay of cetuximab-resistant xenografts compared with mice continued on cetuximab. Furthermore, intrinsically cetuximab-resistant HNSCC patient-derived xenograft tumors treated with pan-HER exhibited significant growth delay compared with vehicle/cetuximab controls. These results suggest that targeting multiple HER family receptors simultaneously with pan-HER is a promising treatment strategy for tumors displaying intrinsic or acquired resistance to cetuximab. Mol Cancer Ther; 15(9); 2175-86. ©2016 AACR.

      View details for PubMedID 27422810
  • Readability of Online Patient Educational Resources Found on NCI-Designated Cancer Center Web Sites. J Natl Compr Canc Netw
    Rosenberg SA, Francis D, Hullett CR, Morris ZS, Fisher MM, Brower JV, Bradley KA, Anderson BM, Bassetti MF, Kimple RJ
    2016 Jun; 14 (6): 735-40
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      BACKGROUND: The NIH and Department of Health & Human Services recommend online patient information (OPI) be written at a sixth grade level. We used a panel of readability analyses to assess OPI from NCI-Designated Cancer Center (NCIDCC) Web sites.

      METHODS: Cancer.gov was used to identify 68 NCIDCC Web sites from which we collected both general OPI and OPI specific to breast, prostate, lung, and colon cancers. This text was analyzed by 10 commonly used readability tests: the New Dale-Chall Readability Formula, Flesch Reading Ease scale, Flesch-Kinaid Grade Level, FORCAST scale, Fry Readability Graph, Simple Measure of Gobbledygook test, Gunning Frequency of Gobbledygook index, New Fog Count, Raygor Readability Estimate Graph, and Coleman-Liau Index. We tested the hypothesis that the readability of NCIDCC OPI was written at the sixth grade level. Secondary analyses were performed to compare readability of OPI between comprehensive and noncomprehensive centers, by region, and to OPI produced by the American Cancer Society (ACS).

      RESULTS: A mean of 30,507 words from 40 comprehensive and 18 noncomprehensive NCIDCCs was analyzed (7 nonclinical and 3 without appropriate OPI were excluded). Using a composite grade level score, the mean readability score of 12.46 (ie, college level: 95% CI, 12.13-12.79) was significantly greater than the target grade level of 6 (middle-school: P<.001). No difference between comprehensive and noncomprehensive centers was identified. Regional differences were identified in 4 of the 10 readability metrics (P<.05). ACS OPI provides easier language, at the seventh to ninth grade level, across all tests (P<.01).

      CONCLUSIONS: OPI from NCIDCC Web sites is more complex than recommended for the average patient.

      View details for PubMedID 27283166
  • Radiation Promptly Alters Cancer Live Cell Metabolic Fluxes: An In Vitro Demonstration. Radiat Res
    Campos D, Peeters W, Nickel K, Burkel B, Bussink J, Kimple RJ, van der Kogel A, Eliceiri KW, Kissick MW
    2016 May; 185 (5): 496-504
    • More

      Quantitative data is presented that shows significant changes in cellular metabolism in a head and neck cancer cell line 30 min after irradiation. A head and neck cancer cell line (UM-SCC-22B) and a comparable normal cell line, normal oral keratinocyte (NOK) were each separately exposed to 10 Gy and treated with a control drug for disrupting metabolism (potassium cyanide; KCN). The metabolic changes were measured live by fluorescence lifetime imaging of the intrinsically fluorescent intermediate metabolite nicotinamide adenosine dinucleotide (NADH) fluorescence; this method is sensitive to the ratio of bound to free NADH. The results indicated a prompt shift in metabolic signature in the cancer cell line, but not in the normal cell line. Control KCN treatment demonstrated expected metabolic fluxes due to mitochondrial disruption. The detected radiation shift in the cancer cells was blunted in the presence of both a radical scavenger and a HIF-1α inhibitor. The HIF-1α abundance as detected by immunohistochemical staining also increased substantially for these cancer cells, but not for the normal cells. This type of live-cell metabolic monitoring could be helpful for future real-time studies and in designing adaptive radiotherapy approaches.

      View details for PubMedID 27128739
  • Defects in the Fanconi Anemia Pathway in Head and Neck Cancer Cells Stimulate Tumor Cell Invasion through DNA-PK and Rac1 Signaling. Clin Cancer Res
    Romick-Rosendale LE, Hoskins EE, Privette Vinnedge LM, Foglesong GD, Brusadelli MG, Potter SS, Komurov K, Brugmann SA, Lambert PF, Kimple RJ, Virts EL, Hanenberg H, Gillison ML, Wells SI
    2016 Apr 15; 22 (8): 2062-73
    • More

      PURPOSE: Head and neck squamous cell carcinoma (HNSCC) remains a devastating disease, and Fanconi anemia (FA) gene mutations and transcriptional repression are common. Invasive tumor behavior is associated with poor outcome, but relevant pathways triggering invasion are poorly understood. There is a significant need to improve our understanding of genetic pathways and molecular mechanisms driving advanced tumor phenotypes, to develop tailored therapies. Here we sought to investigate the phenotypic and molecular consequences of FA pathway loss in HNSCC cells.

      EXPERIMENTAL DESIGN: Using sporadic HNSCC cell lines with and without FA gene knockdown, we sought to characterize the phenotypic and molecular consequences of FA deficiency. FA pathway inactivation was confirmed by the detection of classic hallmarks of FA following exposure to DNA cross-linkers. Cells were subjected to RNA sequencing with qRT-PCR validation, followed by cellular adhesion and invasion assays in the presence and absence of DNA-dependent protein kinase (DNA-PK) and Rac1 inhibitors.

      RESULTS: We demonstrate that FA loss in HNSCC cells leads to cytoskeletal reorganization and invasive tumor cell behavior in the absence of proliferative gains. We further demonstrate that cellular invasion following FA loss is mediated, at least in part, through NHEJ-associated DNA-PK and downstream Rac1 GTPase activity.

      CONCLUSIONS: These findings demonstrate that FA loss stimulates HNSCC cell motility and invasion, and implicate a targetable DNA-PK/Rac1 signaling axis in advanced tumor phenotypes.

      View details for PubMedID 26603260
  • Potential role of the glycolytic oscillator in acute hypoxia in tumors. Phys Med Biol
    Fru LC, Adamson EB, Campos DD, Fain SB, Jacques SL, van der Kogel AJ, Nickel KP, Song C, Kimple RJ, Kissick MW
    2015 Dec 21; 60 (24): 9215-25
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      Tumor acute hypoxia has a dynamic component that is also, at least partially, coherent. Using blood oxygen level dependent magnetic resonance imaging, we observed coherent oscillations in hemoglobin saturation dynamics in cell line xenograft models of head and neck squamous cell carcinoma. We posit a well-established biochemical nonlinear oscillatory mechanism called the glycolytic oscillator as a potential cause of the coherent oscillations in tumors. These data suggest that metabolic changes within individual tumor cells may affect the local tumor microenvironment including oxygen availability and therefore radiosensitivity. These individual cells can synchronize the oscillations in patches of similar intermediate glucose levels. These alterations have potentially important implications for radiation therapy and are a potential target for optimizing the cancer response to radiation.

      View details for PubMedID 26576743
  • Pan-HER Inhibitor Augments Radiation Response in Human Lung and Head and Neck Cancer Models. Clin Cancer Res
    Francis DM, Huang S, Armstrong EA, Werner LR, Hullett C, Li C, Morris ZS, Swick AD, Kragh M, Lantto J, Kimple RJ, Harari PM
    2016 Feb 01; 22 (3): 633-43
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      PURPOSE: Aberrant regulation of the EGF receptor family (EGFR, HER2, HER3, HER4) contributes to tumorigenesis and metastasis in epithelial cancers. Pan-HER represents a novel molecular targeted therapeutic composed of a mixture of six monoclonal antibodies against EGFR, HER2, and HER3.

      EXPERIMENTAL DESIGN: In the current study, we examine the capacity of Pan-HER to augment radiation response across a series of human lung and head and neck cancers, including EGFR inhibitor-resistant cell lines and xenografts.

      RESULTS: Pan-HER demonstrates superior antiproliferative and radiosensitizing impact when compared with cetuximab. The mechanisms underlying these effects appear to involve attenuation of DNA damage repair, enhancement of programmed cell death, cell-cycle redistribution, and induction of cellular senescence. Combined treatment of Pan-HER with single or fractionated radiation in human tumor xenografts reveals a potent antitumor and regrowth delay impact compared with Pan-HER or radiation treatment alone.

      CONCLUSIONS: These data highlight the capacity of Pan-HER to augment radiation response in lung and head and neck cancer models and support investigation of Pan-HER combined with radiation as a promising clinical therapeutic strategy.

      View details for PubMedID 26420857
  • Modulation of therapeutic sensitivity by human papillomavirus. Radiother Oncol
    Swick AD, Chatterjee A, De Costa AM, Kimple RJ
    2015 Sep; 116 (3): 342-5
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      Human papillomaviruses (HPVs) are small double-stranded DNA viruses that pose significant public health concerns as the causative agent of approximately 5% of worldwide cancers. The HPV oncogenes E6 and E7 play key roles in carcinogenesis. In the last 15years there has been a significant increase in the incidence of HPV-related head and neck cancers arising primarily in the oropharynx. Patients with HPV-positive head and neck cancers (HNCs) have a significantly improved prognosis compared to those with HPV-negative disease. In this review we will discuss data suggesting how HPV oncogenes modulate both the intrinsic radiation sensitivity of HNCs and also have important effects upon the tumor microenvironment. Together, these findings contribute to the improved outcomes seen in patients with HPV-positive HNC.

      View details for PubMedID 26364887
  • Therapeutic combination of radiolabeled CLR1404 with external beam radiation in head and neck cancer model systems. Radiother Oncol
    Morris ZS, Weichert JP, Saker J, Armstrong EA, Besemer A, Bednarz B, Kimple RJ, Harari PM
    2015 Sep; 116 (3): 504-9
    • More

      BACKGROUND AND PURPOSE: CLR1404 is a phospholipid ether that exhibits selective uptake and retention in malignant tissues. Radiolabeled CLR1404 enables tumor-specific positron-emission tomography (PET) imaging ((124)I) and targeted delivery of ionizing radiation ((131)I). Here we describe the first preclinical studies of this diapeutic molecule in head and neck cancer (HNC) models.

      MATERIAL AND METHODS: Tumor-selective distribution of (124)I-CLR1404 and therapeutic efficacy of (131)I-CLR1404 were tested in HNC cell lines and patient-derived xenograft tumor models. Monte Carlo dose calculations and (124)I-CLR1404 PET/CT imaging were used to examine (131)I-CLR1404 dosimetry in preclinical HNC tumor models.

      RESULTS: HNC tumor xenograft studies including patient-derived xenografts demonstrate tumor-selective uptake and retention of (124)I-CLR1404 resulting in a model of highly conformal dose distribution for (131)I-CLR1404. We observe dose-dependent response to (131)I-CLR1404 with respect to HNC tumor xenograft growth inhibition and this effect is maintained together with external beam radiation.

      CONCLUSIONS: We confirm the utility of CLR1404 for tumor imaging and treatment of HNC. This promising agent warrants further investigation in a developing phase I trial combining (131)I-CLR1404 with reduced-dose external beam radiation in patients with loco-regionally recurrent HNC.

      View details for PubMedID 26123834
  • Merkel Cell Carcinoma Analysis of Outcomes: A 30-Year Experience. PLoS One
    Liang E, Brower JV, Rice SR, Buehler DG, Saha S, Kimple RJ
    2015; 10 (6): e0129476
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      BACKGROUND: Merkel cell carcinoma (MCC) is an aggressive cutaneous malignancy with poor prognosis. Limited data exists to guide treatment decisions. Here we report on our institutional experience and outcomes treating patients with MCC.

      METHODS: A database search (1984-2014) of patients treated at the University of Wisconsin Hospital and Clinics was used to identify patients with histologically confirmed MCC. Patient, tumor, and treatment characteristics were examined via review of medical records. Statistical analyses were performed to assess outcomes and associated prognostic factors.

      RESULTS: A total of 87 patients with MCC were identified with a median follow-up of 17 months (mean: 38, range: 0-210 months). Two and five-year overall survival rates were 53.9% and 32.8%, respectively. Recurrence was documented in 31.0% of patients (85.2% locoregional, 48.1% distant and 33.3% both). Patients with a history of immunosuppression exhibited significantly worse survival (hazard ratio, 2.01; 95% CI, 1.1-3.7) when compared to immune-competent individuals. The head and neck region was the most common location of primary lesion (N=49) followed by the extremities (N=31). Upper extremity primaries predicted significantly better overall survival (hazard ratio, 0.48; 95% CI, 0.23-0.99) while lower extremity primaries did not have significantly better results (hazard ratio, 0.5; 95% CI, 0.21-1.2) in comparison to head and neck site of primary. Nodal involvement (hazard ratio, 2.95; 95% CI, 1.5-5.79) was also a negative prognostic factor associated with poor overall survival when compared with clinically node negative patients. Primary tumor size > 2 cm (hazard ratio, 1.76; 95% CI, 0.91-3.4) was not associated with survival.

      CONCLUSIONS: This study highlights the role of various factors in determining prognosis of Merkel cell carcinoma; history of immunosuppression, nodal involvement, and head/neck primary predicted worse overall survival. These findings suggest that improvements in both distant and locoregionally directed therapies might play an important role in control of MCC and identify areas for future study.

      View details for PubMedID 26053480
  • Prevalence of Human Papillomavirus in Oropharyngeal Cancer: A Systematic Review. Cancer J
    Stein AP, Saha S, Kraninger JL, Swick AD, Yu M, Lambert PF, Kimple RJ
    2015 May-Jun; 21 (3): 138-46
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      PURPOSE: The 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.

      METHODS: A 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.

      RESULTS: Our 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).

      CONCLUSIONS: Interestingly, 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.

      View details for PubMedID 26049691
  • The prognostic value of HPV in head and neck cancer patients undergoing postoperative chemoradiotherapy. Ann Transl Med
    Kimple RJ, Harari PM
    2015 May; 3 (Suppl 1): S14
  • Human papillomavirus and head and neck cancer. Int J Radiat Oncol Biol Phys
    Kimple RJ, Sher DJ
    2015 Jun 01; 92 (2): 196-9
  • Acute Toxicity From Breast Cancer Radiation Using Helical Tomotherapy With a Simultaneous Integrated Boost. Technol Cancer Res Treat
    Wojcieszynski AP, Olson AK, Rong Y, Kimple RJ, Yadav P
    2016 Apr; 15 (2): 257-65
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      PURPOSE: To evaluate 2 simultaneous integrated boost treatment planning techniques using helical tomotherapy for breast conserving therapy with regard to acute skin toxicity and dosimetry.

      METHODS: Thirty-two patients were studied. The original approach was for 16 patients and incorporated a directional block of the ipsilateral lung and breast. An additional 16 patients were planned for using a modified approach that incorporates a full block of the ipsilateral lung exclusive of 4 cm around the breast. Dose-volume histograms of targets and critical structures were evaluated. Skin toxicity monitoring was performed throughout treatment and follow-up using the Common Terminology Criteria for Adverse Events.

      RESULTS: Treatment was well tolerated with patients receiving a median dose of 59.36 Gy. Of the 16 patients in both groups, 8 had grade 2 erythema immediately after radiation. On 3-week follow-up, 10 and 7 patients in the original and modified groups showed grade 1 erythema. On 3- and 6-month follow-up, both groups had minimal erythema, with all patients having either grade 0 or 1 symptoms. No grade 2 or 3 toxicities were reported. Mean treatment time was 7.5 and 10.4 minutes using the original and modified methods. Adequate dose coverage was achieved using both methods (V95 = 99.5% and 98%). Mean dose to the heart was 10.5 and 1.8 Gy, respectively (P < .01). For right-sided tumors, the original and modified plans yielded a mean of 8.8 and 1.1 Gy (P < .01) versus 11.7 and 2.4 Gy for left-sided tumors (P < .01). The mean dose to the ipsilateral lung was also significantly lower in the modified plans (11.8 vs. 5.0 Gy, P < .01).

      CONCLUSIONS: Tomotherapy is capable of delivering homogeneous treatment plans to the whole breast and lumpectomy cavity using simultaneous integrated boost treatment. Using the treatment methods described herein, extremely low doses to critical structures can be achieved without compromising acute skin toxicity.

      View details for PubMedID 25780060
  • AXL Is a Logical Molecular Target in Head and Neck Squamous Cell Carcinoma. Clin Cancer Res
    Brand TM, Iida M, Stein AP, Corrigan KL, Braverman CM, Coan JP, Pearson HE, Bahrar H, Fowler TL, Bednarz BP, Saha S, Yang D, Gill PS, Lingen MW, Saloura V, Villaflor VM, Salgia R, Kimple RJ, Wheeler DL
    2015 Jun 01; 21 (11): 2601-12
    • More

      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.

      View details for PubMedID 25767293
  • Xenograft assessment of predictive biomarkers for standard head and neck cancer therapies. Cancer Med
    Stein AP, Swick AD, Smith MA, Blitzer GC, Yang RZ, Saha S, Harari PM, Lambert PF, Liu CZ, Kimple RJ
    2015 May; 4 (5): 699-712
    • More

      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.

      View details for PubMedID 25619980
  • High-throughput detection of DNA double-strand breaks using image cytometry. Biotechniques
    Fowler TL, Bailey AM, Bednarz BP, Kimple RJ
    2015 Jan; 58 (1): 37-9
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      Assessment of γH2AX expression for studying DNA double-strand break formation is often performed by manual counting of foci using immunofluorescence microscopy, an approach that is laborious and subject to significant foci selection bias. Here we present a novel high-throughput method for detecting DNA double-strand breaks using automated image cytometry assessment of cell average γH2AX immunofluorescence. Our technique provides an expedient, high-throughput, objective, and cost-effective method for γH2AX analysis.

      View details for PubMedID 25605579
  • Loss of Trop2 causes ErbB3 activation through a neuregulin-1-dependent mechanism in the mesenchymal subtype of HNSCC. Oncotarget
    Zhang K, Jones L, Lim S, Maher CA, Adkins D, Lewis J, Kimple RJ, Fertig EJ, Chung CH, Van Tine BA, Ellis MJ, Herrlich A, Michel LS
    2014 Oct 15; 5 (19): 9281-94
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      In head and neck squamous cell cancer (HNSCC), four intrinsic subtypes (or groups) have been identified, and each one possesses a unique biology that will require specific treatment strategies. We previously reported that mesenchymal (group 2) tumors exhibit reduced levels of Trop2 expression. In this study, we investigated the functional role of Trop2 in HNSCC and find that loss results in autocrine activation of the EGFR family member ErbB3 via neuregulin-1. Trop2 localizes to both the cell surface and cytosol of HNSCC cells and forms a complex with neuregulin-1, which is predominantly cytosolic. Inactivation of Trop2 increases the concentration of neuregulin-1 at the cell surface where it is cleaved to activate ErbB3. In primary HNSCC, detection of ErbB3 activation was limited to Trop2 negative tumors. An analysis of the Cancer Genome Atlas (TCGA) HNSCC dataset confirms enrichment for ErbB3 activity in mesenchymal tumors. Notably, Trop2 loss triggers sensitivity to anti-ErbB3 antibodies, which results in reduced proliferation and tumorigenic growth of Trop2 negative HNSCC cancer cells. These results uncover a molecular mechanism by which tumor cells control the amount of cell-surface neuregulin-1 available for cleavage and ErbB3 activation. Moreover, we demonstrate that Trop2 is a potential surrogate biomarker to identify tumors with ErbB3 activation and may therefore respond to anti-ErbB3 therapeutics.

      View details for PubMedID 25238142
  • Human papillomavirus type 16 E7 oncoprotein causes a delay in repair of DNA damage. Radiother Oncol
    Park JW, Nickel KP, Torres AD, Lee D, Lambert PF, Kimple RJ
    2014 Dec; 113 (3): 337-44
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      BACKGROUND AND PURPOSE: Patients with human papillomavirus related (HPV+) head and neck cancers (HNCs) demonstrate improved clinical outcomes compared to traditional HPV negative (HPV-) HNC patients. We have recently shown that HPV+ HNC cells are more sensitive to radiation than HPV- HNC cells. However, roles of HPV oncogenes in regulating the response of DNA damage repair remain unknown.

      MATERIAL AND METHODS: Using immortalized normal oral epithelial cell lines, HPV+ HNC derived cell lines, and HPV16 E7-transgenic mice we assessed the repair of DNA damage using γ-H2AX foci, single and split dose clonogenic survival assays, and immunoblot. The ability of E7 to modulate expression of proteins associated with DNA repair pathways was assessed by immunoblot.

      RESULTS: HPV16 E7 increased retention of γ-H2AX nuclear foci and significantly decreased sublethal DNA damage repair. While phospho-ATM, phospho-ATR, Ku70, and Ku80 expressions were not altered by E7, Rad51 was induced by E7. Correspondingly, HPV+ HNC cell lines showed retention of Rad51 after γ-radiation.

      CONCLUSIONS: Our findings provide further understanding as to how HPV16 E7 manipulates cellular DNA damage responses that may underlie its oncogenic potential and influence the altered sensitivity to radiation seen in HPV+ HNC as compared to HPV- HNC.

      View details for PubMedID 25216575
  • AXL mediates resistance to cetuximab therapy. Cancer Res
    Brand TM, Iida M, Stein AP, Corrigan KL, Braverman CM, Luthar N, Toulany M, Gill PS, Salgia R, Kimple RJ, Wheeler DL
    2014 Sep 15; 74 (18): 5152-64
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      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.

      View details for PubMedID 25136066
  • Wetting the whistle: neurotropic factor improves salivary function. J Clin Invest
    Swick A, Kimple RJ
    2014 Aug; 124 (8): 3282-4
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      Xerostomia, or dry mouth, is a common side effect of head and neck radiotherapy, Sjögren syndrome, diabetes, old age, and numerous medications. In this issue of the JCI, Xiao and colleagues identified glial cell line-derived neurotrophic factor (GDNF) as a potential stimulus for salivary stem cell growth. Due to its ability to promote neuronal growth, differentiation, and survival, GDNF is currently being used in clinical trials as a treatment for Parkinson disease; therefore, the findings of Xiao and colleagues may initiate a potential treatment for the millions of patients who suffer from xerostomia each year.

      View details for PubMedID 25036702
  • Influence of handling conditions on the establishment and propagation of head and neck cancer patient derived xenografts. PLoS One
    Stein AP, Saha S, Liu CZ, Hartig GK, Lambert PF, Kimple RJ
    2014; 9 (6): e100995
    • More

      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.

      View details for PubMedID 24967635
  • Prevalence of human papillomavirus in oropharyngeal squamous cell carcinoma in the United States across time. Chem Res Toxicol
    Stein AP, Saha S, Yu M, Kimple RJ, Lambert PF
    2014 Apr 21; 27 (4): 462-9
    • More

      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.

      View details for PubMedID 24641254
  • DEK promotes HPV-positive and -negative head and neck cancer cell proliferation. Oncogene
    Adams AK, Hallenbeck GE, Casper KA, Patil YJ, Wilson KM, Kimple RJ, Lambert PF, Witte DP, Xiao W, Gillison ML, Wikenheiser-Brokamp KA, Wise-Draper TM, Wells SI
    2015 Feb 12; 34 (7): 868-77
    • More

      Head and neck squamous cell carcinoma (HNSCC) is the sixth most common malignancy worldwide, and patient outcomes using current treatments remain poor. Tumor development is etiologically associated with tobacco or alcohol use and/or human papillomavirus (HPV) infection. HPV-positive HNSCCs, which frequently harbor wild-type p53, carry a more favorable prognosis and are a biologically distinct subgroup when compared with their HPV-negative counterparts. HPV E7 induces expression of the human DEK gene, both in vitro and in vivo. In keratinocytes, DEK overexpression is sufficient for causing oncogenic phenotypes in the absence of E7. Conversely, DEK loss results in cell death in HPV-positive cervical cancer cells at least in part through p53 activation, and Dek knockout mice are relatively resistant to the development of chemically induced skin papillomas. Despite the established oncogenic role of DEK in HPV-associated cervical cancer cell lines and keratinocytes, a functional role of DEK has not yet been explored in HNSCC. Using an established transgenic mouse model of HPV16 E7-induced HNSCC, we demonstrate that Dek is required for optimal proliferation of E7-transgenic epidermal cells and for the growth of HNSCC tumors. Importantly, these studies also demonstrate that DEK protein is universally upregulated in both HPV-positive and -negative human HNSCC tumors relative to adjacent normal tissue. Furthermore, DEK knockdown inhibited the proliferation of HPV-positive and -negative HNSCC cells, establishing a functional role for DEK in human disease. Mechanistic studies reveal that attenuated HNSCC cell growth in response to DEK loss was associated with reduced expression of the oncogenic p53 family member, ΔNp63. Exogenous ΔNp63 expression rescued the proliferative defect in the absence of DEK, thereby establishing a functional DEK-ΔNp63 oncogenic pathway that promotes HNSCC. Taken together, our data demonstrate that DEK stimulates HNSCC cellular growth and identify ΔNp63 as a novel DEK effector.

      View details for PubMedID 24608431
  • Review of the clinical and biologic aspects of human papillomavirus-positive squamous cell carcinomas of the head and neck. Int J Radiat Oncol Biol Phys
    Blitzer GC, Smith MA, Harris SL, Kimple RJ
    2014 Mar 15; 88 (4): 761-70
    • More

      Human papillomavirus (HPV), a known etiology of a subset of head-and-neck squamous cell carcinomas (HNCs), causes numerous alterations in normal cellular functions. This article reviews the biology, detection, and treatment of HPV-positive HNC. The role of HPV oncoproteins in tumor development, the natural history of HPV infection, and risk factors for and prevention of transmission of oral HPV are considered. Commonly used methods for detecting HPV infection, including limitations of these methods, are discussed to aid the practicing clinician in using these tests in their clinical practice. Clinical characteristics of HPV-positive HNC, including potential explanations for the improved outcomes seen in patients with HPV-positive HNC, are assessed. Ongoing clinical trials specific for patients with HPV-positive HNC are described, and areas in need of additional research are summarized. Until the results of ongoing trials are known, treatment of HPV-positive HNC should not differ in clinical practice from treatment of similar non-HPV related cancers.

      View details for PubMedID 24606845
  • A novel high-throughput irradiator for in vitro radiation sensitivity bioassays. Phys Med Biol
    Fowler TL, Fulkerson RK, Micka JA, Kimple RJ, Bednarz BP
    2014 Mar 21; 59 (6): 1459-70
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      This paper describes the development and characterization of a fully automated in vitro cell irradiator using an electronic brachytherapy source to perform radiation sensitivity bioassays. This novel irradiator allows complex variable dose and dose rate schemes to be delivered to multiple wells of 96-well culture plates used in standard biological assays. The Xoft Axxent® eBx™ was chosen as the x-ray source due to its ability to vary tube current up to 300 µA for a 50 kVp spectrum using clinical surface applicators. Translation of the multiwell plate across the fixed radiation field is achieved using a precision motor driven computer controlled positioning system. A series of measurements was performed to characterize dosimetric performance of the system. Measurements have shown that the radiation output measured with an end window ionization chamber is stable between operating currents of 50-300 µA. In addition, radiochromic film was used to characterize the field flatness and symmetry. The average field flatness in the in-plane and cross-plane direction was 2.9 ± 1.0% and 4.0 ± 1.7%, respectively. The average symmetry in the in-plane and cross-plane direction was 1.8 ± 0.9% and 1.6 ± 0.5%, respectively. The optimal focal spot resolution at the cellular plane was determined by measuring sequential irradiations on radiochromic film for three different well spacing schemes. It was determined that the current system can irradiate every other well with negligible impact on the radiation field characteristics. Finally, a performance comparison between this system and a common cabinet irradiator is presented.

      View details for PubMedID 24584120
  • Is radiation dose reduction the right answer for HPV-positive head and neck cancer? Oral Oncol
    Kimple RJ, Harari PM
    2014 Jun; 50 (6): 560-4
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      Patients with head and neck squamous cell carcinoma (HNC) related to human papillomavirus (HPV) represent a growing and distinct patient cohort with unique molecular and epidemiologic characteristics. These patients have markedly improved survival outcomes compared to those with traditional HNC, leading some to advocate for treatment dose reduction. In this article, we review ongoing clinical trials investigating several ways to reduce therapeutic intensity for patients with HPV-positive HNC, discuss the risks and benefits associated with these trials, and summarize the data underlying the advancement of dose reduction trials for patients with HPV-positive HNC.

      View details for PubMedID 24134946
  • Enhanced radiation sensitivity in HPV-positive head and neck cancer. Cancer Res
    Kimple RJ, Smith MA, Blitzer GC, Torres AD, Martin JA, Yang RZ, Peet CR, Lorenz LD, Nickel KP, Klingelhutz AJ, Lambert PF, Harari PM
    2013 Aug 01; 73 (15): 4791-800
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      Patients with human papillomavirus (HPV+)-associated head and neck cancer (HNC) show significantly improved survival outcome compared with those with HPV-negative (HPV-) tumors. Published data examining this difference offers conflicting results to date. We systematically investigated the radiation sensitivity of all available validated HPV+ HNC cell lines and a series of HPV- HNC cell lines using in vitro and in vivo techniques. HPV+ HNCs exhibited greater intrinsic radiation sensitivity (average SF2 HPV-: 0.59 vs. HPV+: 0.22; P < 0.0001), corresponding with a prolonged G2-M cell-cycle arrest and increased apoptosis following radiation exposure (percent change 0% vs. 85%; P = 0.002). A genome-wide microarray was used to compare gene expression 24 hours following radiation between HPV+ and HPV- cell lines. Multiple genes in TP53 pathway were upregulated in HPV+ cells (Z score 4.90), including a 4.6-fold increase in TP53 (P < 0.0001). Using immortalized human tonsillar epithelial (HTE) cells, increased radiation sensitivity was seen in cell expressing HPV-16 E6 despite the effect of E6 to degrade p53. This suggested that low levels of normally functioning p53 in HPV+ HNC cells could be activated by radiation, leading to cell death. Consistent with this, more complete knockdown of TP53 by siRNA resulted in radiation resistance. These results provide clear evidence, and a supporting mechanism, for increased radiation sensitivity in HPV+ HNC relative to HPV- HNC. This issue is under active investigation in a series of clinical trials attempting to de-escalate radiation (and chemotherapy) in selected patients with HPV+ HNC in light of their favorable overall survival outcome.

      View details for PubMedID 23749640
  • Applicability of randomized trials in radiation oncology to standard clinical practice. Cancer
    Apisarnthanarax S, Swisher-McClure S, Chiu WK, Kimple RJ, Harris SL, Morris DE, Tepper JE
    2013 Aug 15; 119 (16): 3092-9
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      BACKGROUND: Randomized controlled trials (RCTs) are commonly used to inform clinical practice; however, it is unclear how generalizable RCT data are to patients in routine clinical practice. The authors of this report assessed the availability and applicability of randomized evidence guiding medical decisions in a cohort of patients who were evaluated for consideration of definitive management in a radiation oncology clinic.

      METHODS: The medical records of consecutive, new patient consultations between January and March 2007 were reviewed. Patient medical decisions were classified as those with (Group 1) or without (Group 2) available, relevant level I evidence (phase 3 RCT) supporting recommended treatments. Group 1 medical decisions were further divided into 3 groups based on the extent of fulfilling eligibility criteria for each RCT: Group 1A included decisions that fulfilled all eligibility criteria; Group 1B, decisions that did not fulfill at least 1 minor eligibility criteria; or Group 1C, decisions that did not fulfill at least 1 major eligibility criteria. Patient and clinical characteristics were tested for correlations with the availability of evidence.

      RESULTS: Of the 393 evaluable patients, malignancies of the breast (30%), head and neck (18%), and genitourinary system (14%) were the most common presenting primary disease sites. Forty-seven percent of all medical decisions (n = 451) were made without available (36%) or applicable (11%) randomized evidence to inform clinical decision making. Primary tumor diagnosis was significantly associated with the availability of evidence (P < .0001).

      CONCLUSIONS: A significant proportion of medical decisions in an academic radiation oncology clinic were made without available or applicable level I evidence, underscoring the limitations of relying solely on RCTs for the development of evidence-based health care.

      View details for PubMedID 23674290
  • Phase 2 study of pre-excision single-dose intraoperative radiation therapy for early-stage breast cancers: six-year update with application of the ASTRO accelerated partial breast irradiation consensus statement criteria. Cancer
    Vanderwalde NA, Jones EL, Kimple RJ, Moore DT, Klauber-Demore N, Sartor CI, Ollila DW
    2013 May 01; 119 (9): 1736-43
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      BACKGROUND: Intraoperative radiation therapy (IORT) allows delivery of high-dose radiation at the time of lumpectomy, potentially sparing adjuvant daily radiation. A phase 2 study of pre-excision IORT was performed for early-stage breast cancer.

      METHODS: Patients ≥ 48 years of age with invasive ductal carcinoma, ≤ 3 cm, and clinically node-negative were eligible for this study, which was approved by institutional review board. Ultrasound was used to select electron energy and cone size to cover the tumor plus 1.5- to 2.0-cm lateral margins and 1-cm-deep margins (90% isodose). Fifteen Gy was delivered with a Mobetron irradiator, and immediate needle-localized partial mastectomy followed. Local event results were updated using the Kaplan-Meier method.

      RESULTS: A total of 53 patients received IORT alone. Median age was 63 years, and median tumor size was 1.2 cm. Of these, 81% were positive for estrogen receptor or progesterone receptor, 11% were positive for human epidermal growth factor receptor 2, and 15% were triple-negative. Also, 42%, 49%, and 9% would have fallen into the Suitable, Cautionary, and Unsuitable groups, respectively, of the American Society of Therapeutic Radiation Oncology consensus statement for accelerated partial breast irradiation. Median follow-up was 69 months. Ipsilateral events occurred in 8 of 53 patients. The 6-year actuarial rate of ipsilateral events was 15% (95% confidence interval = 7%-29%). The crude event rate for Suitable and Cautionary groups was 1 of 22 (5%) and 7 of 26 (27%), respectively. Overall survival was 94.4%, and breast cancer-specific survival was 100%.

      CONCLUSIONS: The rate of local events in this study is a matter of concern, especially in the Cautionary group. On the basis of these findings, pre-excision IORT, as delivered in this study, may not provide adequate local control for less favorable early-stage breast cancers.

      View details for PubMedID 23361892
  • Development and characterization of HPV-positive and HPV-negative head and neck squamous cell carcinoma tumorgrafts. Clin Cancer Res
    Kimple RJ, Harari PM, Torres AD, Yang RZ, Soriano BJ, Yu M, Armstrong EA, Blitzer GC, Smith MA, Lorenz LD, Lee D, Yang DT, McCulloch TM, Hartig GK, Lambert PF
    2013 Feb 15; 19 (4): 855-64
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      PURPOSE: To develop a clinically relevant model system to study head and neck squamous cell carcinoma (HNSCC), we have established and characterized a direct-from-patient tumorgraft model of human papillomavirus (HPV)-positive and HPV-negative cancers.

      EXPERIMENTAL DESIGN: Patients with newly diagnosed or recurrent HNSCC were consented for donation of tumor specimens. Surgically obtained tissue was implanted subcutaneously into immunodeficient mice. During subsequent passages, both formalin-fixed/paraffin-embedded as well as flash-frozen tissues were harvested. Tumors were analyzed for a variety of relevant tumor markers. Tumor growth rates and response to radiation, cisplatin, or cetuximab were assessed and early passage cell strains were developed for rapid testing of drug sensitivity.

      RESULTS: Tumorgrafts have been established in 22 of 26 patients to date. Significant diversity in tumorgraft tumor differentiation was observed with good agreement in degree of differentiation between patient tumor and tumorgraft (Kappa 0.72). Six tumorgrafts were HPV-positive on the basis of p16 staining. A strong inverse correlation between tumorgraft p16 and p53 or Rb was identified (Spearman correlations P = 0.085 and P = 0.002, respectively). Significant growth inhibition of representative tumorgrafts was shown with cisplatin, cetuximab, or radiation treatment delivered over a two-week period. Early passage cell strains showed high consistency in response to cancer therapy between tumorgraft and cell strain.

      CONCLUSIONS: We have established a robust human tumorgraft model system for investigating HPV-positive and HPV-negative HNSCC. These tumorgrafts show strong correlation with the original tumor specimens and provide a powerful resource for investigating mechanisms of therapeutic response as well as preclinical testing.

      View details for PubMedID 23251001
  • Phase I study and biomarker analysis of lapatinib and concurrent radiation for locally advanced breast cancer. Oncologist
    Kimple RJ, Horton JK, Livasy CA, Shields JM, Lawrence JA, Chiu WM, Ivanova A, Ollila DW, Carey LA, Halle JS, Sartor CI, Dees EC
    2012; 17 (12): 1496-503
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      PURPOSE: This phase I study assessed the toxicity and safety of combining daily lapatinib with radiation therapy. Sequential tumor biopsies were obtained to evaluate changes in biomarkers, such as epidermal growth factor receptor (EGFR) and human EGFR-2 (HER2) signaling pathways.

      METHODS: Eligibility for this dose-escalation study included unresectable and locally recurrent or chemotherapy-refractory and locally advanced breast cancer, and adequate organ function. Patients underwent three serial biopsies: at baseline, after 1 week of lapatinib alone, and after 1 week of lapatinib and radiation. Endpoints included determination of toxicity, maximum tolerated dose, and analysis of the effect of lapatinib with or without radiation on EGFR and HER2 signaling pathways by immunohistochemistry.

      RESULTS: Doses of lapatinib up to 1,500 mg/day were well tolerated. Toxicity of grade 3 or more was limited to radiation dermatitis and pain. Out of 19 patients treated, in field responses per response evaluation criteria in solid tumors criteria were complete in four patients and partial in six patients. Serial biopsies were obtained in 16 patients with no complications. Total Her2 was relatively unchanged while phospho-Her2, phospho-Akt, and phospho-ERK showed variable responses to both lapatinib alone and dual therapy with lapatinib and radiation.

      CONCLUSIONS: The combination of lapatinib and radiation was well tolerated in this patient cohort. Overall local response rates were comparable to those reported in other studies in this patient population. Biopsies were safely performed at all time points. Inhibition of HER2 and downstream signaling pathways was identified, although no strong correlation with response was seen.

      View details for PubMedID 23006498
  • A 10-year analysis of American Society For Radiation Oncology Junior Faculty Career Development Awards. Int J Radiat Oncol Biol Phys
    Kimple RJ, Kao GD, Research Evaluation Committee of the ASTRO Research Council
    2013 Mar 15; 85 (4): 924-8
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      PURPOSE: Between 2000 and 2010, the American Society for Radiation Oncology (ASTRO) awarded 22 Junior Faculty Career Development Awards (JFA) totaling $4.4 million. This study aimed to evaluate the impact of these awards on the grantees' career development, including current position, publications, and subsequent independent grant funding.

      METHODS: Each awardee was requested via email and telephone to provide an updated curriculum vitae, a National Institutes of Health (NIH) biosketch, and information regarding current position of employment. Twenty-one of the 22 JFA recipients complied. Reported grant funding was extracted from each candidate's CV, and the amounts of NIH grants obtained were confirmed via NIH REPORTER. Reported publications were confirmed via PubMed.

      RESULTS: All survey respondents (21 of 21) have remained in academic positions. Subsequent aggregate grant funding totaled more than $25 million (range, $0-$4.1 million), 5.9 times the initial investment. NIH grant funding totaled almost $15 million, 3 times the initial investment. Awardees have published an average of 34.6 publications (range, 0-123) for an overall rate of 4.5 papers/year (range, 1-11).

      CONCLUSIONS: ASTRO JFAs over the past decade have been strongly associated with grantees remaining in academic positions, success in attracting private and NIH grants, and publication productivity. In an era of dwindling federal research funding, the support provided by the ASTRO JFA may be especially helpful to support the research careers of promising junior faculty members.

      View details for PubMedID 22929862
  • HPV-associated head and neck cancer: molecular and nano-scale markers for prognosis and therapeutic stratification. Sensors (Basel)
    Kimple AJ, Torres AD, Yang RZ, Kimple RJ
    2012; 12 (4): 5159-69
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      Over the last 10 years, it has become clear that patients with head and neck cancer can be stratified into two distinct subgroups on the basis of the etiology of their disease. Patients with human papillomavirus-related cancers have significantly better survival rates and may necessitate different therapeutic approaches than those with tobacco and/or alcohol related cancers. This review discusses the various biomarkers currently in use for identification of patients with HPV-positive cancers with a focus on the advantages and limitations of molecular and nano-scale markers.

      View details for PubMedID 22666080
  • The role of chemoradiation for patients with resectable or potentially resectable pancreatic cancer. Expert Rev Anticancer Ther
    Kimple RJ, Russo S, Monjazeb A, Blackstock AW
    2012 Apr; 12 (4): 469-80
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      Conflicting data and substantial controversy exist regarding optimal adjuvant treatment for those patients with resectable or potentially resectable adenocarcinoma of the pancreas. Despite improvements in short-term surgical outcomes, the use of newer chemotherapeutic agents, development of targeted agents and more precise delivery of radiation, the 5-year survival rates for early-stage patients remains less than 25%. This article critically reviews the existing data for various adjuvant treatment approaches for patients with surgically resectable pancreatic cancer. Our review confirms that despite several randomized clinical trials, the optimal adjuvant treatment approach for these patients remains unclear.

      View details for PubMedID 22500684
  • Association of p16(INK4a) overexpression with improved outcomes in young patients with squamous cell cancers of the oral tongue. Head Neck
    Harris SL, Thorne LB, Seaman WT, Hayes DN, Couch ME, Kimple RJ
    2011 Nov; 33 (11): 1622-7
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      BACKGROUND: The aim of this study was to examine biomolecular profiles in a cohort of young adults with squamous cell cancers (SCCs) of the oral tongue.

      METHODS: We identified all patients aged 18 to 39 years diagnosed with SCC of the oral tongue at our institution. Immunohistochemical (IHC) staining was performed for p16(INK4a) , epidermal growth factor receptor (EGFR), phosphorylated-EGFR (pEGFR), p53, and ERCC1. Human papillomavirus (HPV) testing was performed using in situ hybridization (ISH) and polymerase chain reaction (PCR). Biomarker expression and HPV status were correlated with outcomes.

      RESULTS: We identified 25 patients with sufficient tumor samples. Median age at diagnosis was 30 years (range, 20-39 years). p16(INK4a) overexpression was observed in 11 of 25 patients, whereas HPV-16 positivity was observed in none of the tumor samples by ISH and 2 of the tumor samples by PCR. p16(INK4a) positivity was correlated with improved relapse-free survival (hazard ratio [HR] = 0.23, p = .01) and overall survival (HR = 0.28, p = .05). Neither EGFR, pEGFR, p53, nor excision repair cross-complementing rodent repair deficiency, complementation group 1 (ERCC1) expression correlated with outcome on univariate analysis.

      CONCLUSIONS: p16(INK4a) overexpression was common and was a marker of favorable prognosis. p16(INK4a) overexpression was not a reliable predictor of HPV positivity in our cohort.

      View details for PubMedID 21990227
  • Melanoma cells show a heterogeneous range of sensitivity to ionizing radiation and are radiosensitized by inhibition of B-RAF with PLX-4032. Radiother Oncol
    Sambade MJ, Peters EC, Thomas NE, Kaufmann WK, Kimple RJ, Shields JM
    2011 Mar; 98 (3): 394-9
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      PURPOSE: To assess the relative radiosensitivities of a large collection of melanoma cell lines and to determine whether pharmacologic inhibition of mutant B-RAF with PLX-4032 can radiosensitize B-Raf+ melanoma cells.

      MATERIALS AND METHODS: A large collection of melanoma cell lines (n=37) were treated with 0-8Gy IR and clonogenic survival assays used to generate survival curves to rank relative radiosensitivities among the cell lines. The ability of a B-RAF inhibitor, PLX-4032, to radiosensitize highly radioresistant B-Raf+ cells was also assessed by clonogenic cell survival and spheroid invasion assays and the effects of treatment on the cell cycle assessed by FACS.

      RESULTS: Melanoma cell lines displayed a very large, heterogeneous range of SF2 values (1.002-0.053) with a mean of 0.51. Cell lines with surviving fractions of 0.29 or less at SF2 and SF4 were observed at a high frequency of 18.9% and 70.2%, respectively. Treatment of B-Raf+ cells with the B-RAF inhibitor PLX-4032 in combination with radiation provided enhanced inhibition of both colony formation and invasion, and radiosensitized cells through an increase in G(1) arrest.

      CONCLUSIONS: Our data suggest that melanomas are not uniformly radioresistant with a significant subset displaying inherent radiosensitivity. Pharmacologic inhibition of B-RAF with PLX-4032 effectively radiosensitized B-Raf+ melanoma cells suggesting that this combination approach could provide improved radiotherapeutic response in B-Raf+ melanoma patients.

      View details for PubMedID 21295875
  • Strategizing the clone wars: pharmacological control of cellular sensitivity to radiation. Mol Interv
    Kimple RJ
    2010 Dec; 10 (6): 341-53
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      The combined administration of ionizing radiation and systemic chemotherapy is an accepted standard of care for numerous cancers. Improved efficacy through the combination of therapies reflects several interrelated processes, including DNA damage, inhibition of DNA synthesis, alteration of cell cycle distribution, and impaired DNA repair. Insights into cellular responses to radiation have led to the use of drugs that target specific intracellular signaling pathways to sensitize cells to radiation. Combinations of chemotherapy and radiation continue to be optimized, based on preclinical and early-phase clinical data that indicate the ideal sequencing of therapies, the best combinations of agents (including radiosensitizers), and the most reliable biological markers for predicting patient responsiveness. This review summarizes our current understanding of radiosensitization as it relates to preclinical drug development and discusses the potential benefits of judiciously incorporating both traditional and targeted chemotherapy into radiation regimens.

      View details for PubMedID 21263160
  • First use of electromagnetic setup and real-time tracking in a pediatric patient with vaginal rhabdomyosarcoma. Pract Radiat Oncol
    Kimple RJ, Wallen KE, Person T, Erwin RN, Helmrath MA, Gold S, Morris DE
    2011 Jan-Mar; 1 (1): 47-51
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      We report the first use of the Calypso system (Calypso Medical, Seattle, WA) in a pediatric patient with group III vaginal rhabdomyosarcoma. The Calypso system was used to improve patient setup, to limit anesthesia, to provide for real-time tracking of target location, and to minimize the need for daily portal imaging studies and their associated extraneous radiation dose.

      View details for PubMedID 24673871
  • Local control following single-dose intraoperative radiotherapy prior to surgical excision of early-stage breast cancer. Ann Surg Oncol
    Kimple RJ, Klauber-DeMore N, Kuzmiak CM, Pavic D, Lian J, Livasy CA, Chiu WM, Moore DT, Sartor CI, Ollila DW
    2011 Apr; 18 (4): 939-45
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      BACKGROUND: Multiple partial breast radiotherapy techniques are available. We have previously presented the technical details of our procedure of delivering partial breast irradiation with a single fraction of intraoperative radiotherapy (IORT) targeting the tumor in situ prior to partial mastectomy. This study details our completed, single-institution trial.

      MATERIALS AND METHODS: An IRB-approved, DSMB-monitored phase II trial was performed with the following inclusion criteria: women age ≥48, ultrasound-visible invasive ductal cancers <3 cm, clinically negative axillary nodes. IORT was delivered using mobile electron irradiator, at least a 1.5-cm radial and 1-cm deep margin; patients received 15 Gy and immediately underwent partial mastectomy. Ipsilateral breast recurrence was classified as true/marginal, elsewhere in the breast or nodal basin. Kaplan-Meier methods were used to estimate survival functions and exact 95% confidence intervals are reported.

      RESULTS: Between 2003 and 2007, 71 women underwent IORT (median follow-up: 3.5 years). For patients with tumor-involved or close margins, additional therapy was required: 7 patients, total mastectomy; 11, whole breast radiation. Four women experienced invasive ipsilateral breast failures (1 new primary, 3 margin recurrences) for a 3-year local control rate of 49 of 53 (94.8%; 95% confidence interval 92.4% [95% CI] 84.2–98.3%), actuarial three-year in breast recurrence was 8% (95% CI 2–18%), and breast cancer-specific survival was 100%.

      CONCLUSIONS: Intraoperative radiotherapy delivered to an in situ tumor is feasible, but our local control rate at 3.5 years is concerning. Possible changes to this technique to improve local control rates include better preoperative imaging (MRI), routine intraoperative ultrasound, and improved IORT delivery (larger cone, increased dose).

      View details for PubMedID 21061074
  • Lapatinib in combination with radiation diminishes tumor regrowth in HER2+ and basal-like/EGFR+ breast tumor xenografts. Int J Radiat Oncol Biol Phys
    Sambade MJ, Kimple RJ, Camp JT, Peters E, Livasy CA, Sartor CI, Shields JM
    2010 Jun 01; 77 (2): 575-81
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      PURPOSE: To determine whether lapatinib, a dual epidermal growth factor receptor (EGFR)/HER2 kinase inhibitor, can radiosensitize EGFR+ or HER2+ breast cancer xenografts.

      METHODS AND MATERIALS: Mice bearing xenografts of basal-like/EGFR+ SUM149 and HER2+ SUM225 breast cancer cells were treated with lapatinib and fractionated radiotherapy and tumor growth inhibition correlated with alterations in ERK1 and AKT activation by immunohistochemistry.

      RESULTS: Basal-like/EGFR+ SUM149 breast cancer tumors were completely resistant to treatment with lapatinib alone but highly growth impaired with lapatinib plus radiotherapy, exhibiting an enhancement ratio average of 2.75 and a fractional tumor product ratio average of 2.20 during the study period. In contrast, HER2+ SUM225 breast cancer tumors were highly responsive to treatment with lapatinib alone and yielded a relatively lower enhancement ratio average of 1.25 during the study period with lapatinib plus radiotherapy. Durable tumor control in the HER2+ SUM225 model was more effective with the combination treatment than either lapatinib or radiotherapy alone. Immunohistochemical analyses demonstrated that radiosensitization by lapatinib correlated with ERK1/2 inhibition in the EGFR+ SUM149 model and with AKT inhibition in the HER2+ SUM225 model.

      CONCLUSION: Our data suggest that lapatinib combined with fractionated radiotherapy may be useful against EGFR+ and HER2+ breast cancers and that inhibition of downstream signaling to ERK1/2 and AKT correlates with sensitization in EGFR+ and HER2+ cells, respectively.

      View details for PubMedID 20457354
  • Cosmetic outcomes for accelerated partial breast irradiation before surgical excision of early-stage breast cancer using single-dose intraoperative radiotherapy. Int J Radiat Oncol Biol Phys
    Kimple RJ, Klauber-DeMore N, Kuzmiak CM, Pavic D, Lian J, Livasy CA, Esler L, Moore DT, Sartor CI, Ollila DW
    2011 Feb 01; 79 (2): 400-7
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      PURPOSE: Determine cosmetic outcome and toxicity profile of intraoperative radiation delivered before tumor excision for patients with early-stage breast cancer.

      METHODS AND MATERIALS: Patients age 48 or older with ultrasound-visible invasive ductal cancers <3 cm and clinically negative lymph nodes were eligible for treatment on this institutional review board-approved Phase II clinical trial. Treatment planning ultrasound was used to select an electron energy and cone size sufficient to cover the tumor plus a 1.5- to 2.0-cm circumferential margin laterally and a 1-cm-deep margin with the 90% isodose line. The dose was prescribed to a nominal 15 Gy and delivered using a Mobetron electron irradiator before tumor excision by segmental mastectomy. Physician- and patient-assessed cosmetic outcome and patient satisfaction were determined by questionnaire.

      RESULTS: From March 2003 to July 2007, 71 patients were treated with intraoperative radiation therapy. Of those, 56 patients were evaluable, with a median follow-up of 3.1 years (minimum 1 year). Physician and patient assessment of cosmesis was "good or excellent" (Radiation Therapy Oncology Group cosmesis scale) in 45/56 (80%) and 32/42 (76%) of all patients, respectively. Eleven patients who received additional whole breast radiation had similar rates of good or excellent cosmesis: 40/48 (83%) and 29/36 (81%), respectively). Grade 1 or 2 acute toxicities were seen in 4/71 (6%) patients. No Grade 3 or 4 toxicities or serious adverse events have been seen.

      CONCLUSION: Intraoperative radiotherapy delivered to an in situ tumor is feasible with acceptable acute tolerance. Patient and physician assessment of the cosmetic outcome is good to excellent.

      View details for PubMedID 20395062
  • High-affinity immobilization of proteins using biotin- and GST-based coupling strategies. Methods Mol Biol
    Hutsell SQ, Kimple RJ, Siderovski DP, Willard FS, Kimple AJ
    2010; 627: 75-90
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      Surface plasmon resonance (SPR) is a highly sensitive method for the detection of molecular interactions. One interacting partner is immobilized on the sensor chip surface while the other is injected across the sensor surface. This chapter focuses on high-affinity immobilization of protein substrates for affinity and kinetic analyses using biotin/streptavidin interaction and GST/anti-GST-antibody interaction.

      View details for PubMedID 20217614
  • A simple algorithm to assess patient suitability for Calypso-seed implantation for four-dimensional prostate localization. J Appl Clin Med Phys
    Kimple RJ, Wallen EM, Pruthi R, Marks LB
    2009 Dec 03; 11 (1): 3107
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      To retrospectively determine the proportion of prostate cancer patients who are appropriate candidates for prostate localization with Calypso (Calypso Medical, Seattle, WA); to assess the accuracy of surface anatomy in predicting prostate depth; and to describe a simple clinical algorithm predicting patient's appropriateness for Calypso localization. Medical records and archived CT scans of all patients treated for localized prostate cancer at our institution between 2006 and 2007 were reviewed. Association between the feasibility of Calypso use, the depth of the prostate from the anterior torso, and a variety of anatomic factors were assessed (ANOVA, linear regression, and ROC). Patients were appropriate for the Calypso system in 91% of cases (localize and track, 52%; localize only, 39%). Strong correlation between greater trochanter location and the posterior prostate was seen (r 2 = 0.91, mean difference 0.6 cm). The negative predictive value of the greater trochanter measurements was 31%. Thirty-one out of forty-five patients (69%) who were deemed inappropriate for Calypso based on greater trochanter to anterior torso measurements were eligible on the basis of CT-based measurements of prostate depth. Weight, BMI, waist circumference, and hip circumference correlated with distance from the prostate to the anterior torso and were predictive of Calypso appropriateness. All patients with weight <or= 100 kg, BMI <or= 30, or waist/hip circumference <or= 100 cm, were eligible for Calypso. Most prostate cancer patients are candidates for Calypso localization +/- tracking. The greater trochanter to anterior torso distance underestimates the number of eligible patients. Weight, BMI and waist/hip circumference are good predictors for Calypso appropriateness.

      View details for PubMedID 20160683
  • Radiosensitization of epidermal growth factor receptor/HER2-positive pancreatic cancer is mediated by inhibition of Akt independent of ras mutational status. Clin Cancer Res
    Kimple RJ, Vaseva AV, Cox AD, Baerman KM, Calvo BF, Tepper JE, Shields JM, Sartor CI
    2010 Feb 01; 16 (3): 912-23
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      PURPOSE: Epidermal growth factor receptor (EGFR) family members (e.g., EGFR, HER2, HER3, and HER4) are commonly overexpressed in pancreatic cancer. We investigated the effects of inhibition of EGFR/HER2 signaling on pancreatic cancer to elucidate the role(s) of EGFR/HER2 in radiosensitization and to provide evidence in support of further clinical investigations.

      EXPERIMENTAL DESIGN: Expression of EGFR family members in pancreatic cancer lines was assessed by quantitative reverse transcription-PCR. Cell growth inhibition was determined by MTS assay. The effects of inhibition of EGFR family receptors and downstream signaling pathways on in vitro radiosensitivity were evaluated using clonogenic assays. Growth delay was used to evaluate the effects of nelfinavir on in vivo tumor radiosensitivity.

      RESULTS: Lapatinib inhibited cell growth in four pancreatic cancer cell lines, but radiosensitized only wild-type K-ras-expressing T3M4 cells. Akt activation was blocked in a wild-type K-ras cell line, whereas constitutive phosphorylation of Akt and extracellular signal-regulated kinase (ERK) was seen in lines expressing mutant K-ras. Overexpression of constitutively active K-ras (G12V) abrogated lapatinib-mediated inhibition of both Akt phosphorylation and radiosensitization. Inhibition of MAP/ERK kinase/ERK signaling with U0126 had no effect on radiosensitization, whereas inhibition of activated Akt with LY294002 (enhancement ratio, 1.2-1.8) or nelfinavir (enhancement ratio, 1.2-1.4) radiosensitized cells regardless of K-ras mutation status. Oral nelfinavir administration to mice bearing mutant K-ras-containing Capan-2 xenografts resulted in a greater than additive increase in radiation-mediated tumor growth delay (synergy assessment ratio of 1.5).

      CONCLUSIONS: Inhibition of EGFR/HER2 enhances radiosensitivity in wild-type K-ras pancreatic cancer. Nelfinavir, and other phosphoinositide 3-kinase/Akt inhibitors, are effective pancreatic radiosensitizers regardless of K-ras mutation status.

      View details for PubMedID 20103665
  • Mechanism of lapatinib-mediated radiosensitization of breast cancer cells is primarily by inhibition of the Raf>MEK>ERK mitogen-activated protein kinase cascade and radiosensitization of lapatinib-resistant cells restored by direct inhibition of MEK. Radiother Oncol
    Sambade MJ, Camp JT, Kimple RJ, Sartor CI, Shields JM
    2009 Dec; 93 (3): 639-44
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      BACKGROUND AND PURPOSE: We recently showed that lapatinib, an EGFR/HER2 inhibitor, radiosensitized breast cancer cells of the basal and HER2+ subtypes. The purpose of this study was to identify the downstream signaling pathways responsible for lapatinib-mediated radiosensitization in breast cancer.

      MATERIALS AND METHODS: Response of EGFR downstream signaling pathways was assessed by Western blot and clonogenic cell survival assays in breast tumor cells after irradiation (5Gy), lapatinib, CI-1040, or combined treatment.

      RESULTS: In SUM102 cells, an EGFR+ basal breast cancer cell line, exposure to ionizing radiation elicited strong activation of ERK1/2 and JNK, which was blocked by lapatinib, and weak/no activation of p38, AKT or STAT3. Direct inhibition of MEK1 with CI-1040 resulted in 95% inhibition of surviving colonies when combined with radiation while inhibition of JNK with SP600125 had no effect. Lapatinib-mediated radiosensitization of SUM102 cells was completely abrogated with expression of constitutively active Raf. Treatment of lapatinib-resistant SUM185 cells with CI-1040 restored radiosensitization with 45% fewer surviving colonies when combined with radiation.

      CONCLUSIONS: These data suggest that radiosensitization by lapatinib is mediated largely through inhibition of MEK/ERK and that direct inhibition of this pathway may provide an additional avenue of radiosensitization in EGFR+ or HER2+ breast cancers.

      View details for PubMedID 19853943
  • Concurrent temozolomide and radiation, a reasonable option for elderly patients with glioblastoma multiforme? Am J Clin Oncol
    Kimple RJ, Grabowski S, Papez M, Collichio F, Ewend MG, Morris DE
    2010 Jun; 33 (3): 265-70
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      OBJECTIVES: There is no accepted standard of care for patients over age 70 with glioblastoma (GBM). We began this study to describe our results and toxicities in the over 70 population treated with concurrent temozolomide and radiation for GBM, and to describe our outcomes treating elderly patients with GBM regardless of therapy.

      METHODS: We reviewed the records of all patients aged 70 or older who were diagnosed with glioblastoma since 2002 at the University of North Carolina to determine age at diagnosis, performance status, neurologic status, recursive partitioning analysis class, treatment received, and toxicity. Median survival was calculated according to the Kaplan-Meier method and compared by the Log-rank test.

      RESULTS: Thirty-one patients were identified with a median age of 76 years and a median survival of 20.6 weeks. Thirteen patients received best supportive care, 4 patients were treated with radiation alone, and 14 with radiation and concurrent temozolomide. The median survival for each group was 8.4, 28.2, and 50.5 weeks, respectively. Grade 1/2 toxicity was seen in 20% of patients, whereas only 1 patient had grade 3 toxicity. Neurologic status (P = 0.0028), performance status (P = 0.0096), and recursive partitioning analysis class (P = 0.0033) retained their prognostic significance.

      CONCLUSIONS: Concomitant daily temozolomide and radiation followed by adjuvant temozolomide is a tolerable and reasonable treatment option and has a good performance status for elderly patients diagnosed with glioblastoma.

      View details for PubMedID 19823072
  • Never-smokers, never-drinkers: unique clinical subgroup of young patients with head and neck squamous cell cancers. Head Neck
    Harris SL, Kimple RJ, Hayes DN, Couch ME, Rosenman JG
    2010 Apr; 32 (4): 499-503
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      BACKGROUND: Young patients represent an increasing subgroup with head and neck cancer.

      METHODS: Patients between 18 and 39 years of age with newly diagnosed and previously untreated squamous cell cancers were identified.

      RESULTS: Seventy-eight patients met the selection criteria: 28 patients were never-smokers/never-drinkers (NSNDs), and 50 patients reported tobacco or alcohol abuse (smokers and/or drinkers [SD]). NSND patients were diagnosed at a younger median age (31.5 years vs 35.5 years, p = .007), were more likely to be female (75% vs 30%, p < .001) and white (89% vs 60%, p = .006), and were more likely to have tumors of the oral tongue (57% vs 24%, p = .003) and T1 disease (47% vs 20%, p = .01). There was no difference in 10-year relapse-free survival, but a suggestion of improved 10-year overall survival for NSND patients (71% vs 46%, p = .10).

      CONCLUSIONS: Young patients with head and neck squamous cell carcinoma (HNSCC) appear to have unique clinical profiles based on history of alcohol and tobacco abuse.

      View details for PubMedID 19691028
  • Normal tissue tolerance for high-grade gliomas: is it an issue? Semin Radiat Oncol
    Morris DE, Kimple RJ
    2009 Jul; 19 (3): 187-92
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      In this article, we address the currently accepted dose tolerance parameters for the treatment of high-grade gliomas. The issue of normal tissue tolerance is becoming increasingly important because of the long-term survival of a significant subset of young, good performance status patients and the use of hypofractionated regimens for elderly patients with poor performance status. In addition, we address relevant clinical endpoints including clinical, pathologic, and radiographic changes and highlight the difficulty in discriminating between tumor-related and treatment-related effects. Finally, we review relevant clinical trials addressing issues of dose and/or volume parameters. Future trials for patients with high-grade gliomas should consider the inclusion of a prospective evaluation of neurocognitive function and imaging correlates of the brain to assist in the prediction, prevention, and treatment of radiation-induced damage of normal brain tissue.

      View details for PubMedID 19464634
  • Regulator of G-protein signaling 14 (RGS14) is a selective H-Ras effector. PLoS One
    Willard FS, Willard MD, Kimple AJ, Soundararajan M, Oestreich EA, Li X, Sowa NA, Kimple RJ, Doyle DA, Der CJ, Zylka MJ, Snider WD, Siderovski DP
    2009; 4 (3): e4884
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      BACKGROUND: Regulator of G-protein signaling (RGS) proteins have been well-described as accelerators of Galpha-mediated GTP hydrolysis ("GTPase-accelerating proteins" or GAPs). However, RGS proteins with complex domain architectures are now known to regulate much more than Galpha GTPase activity. RGS14 contains tandem Ras-binding domains that have been reported to bind to Rap- but not Ras GTPases in vitro, leading to the suggestion that RGS14 is a Rap-specific effector. However, more recent data from mammals and Drosophila imply that, in vivo, RGS14 may instead be an effector of Ras.

      METHODOLOGY/PRINCIPAL FINDINGS: Full-length and truncated forms of purified RGS14 protein were found to bind indiscriminately in vitro to both Rap- and Ras-family GTPases, consistent with prior literature reports. In stark contrast, however, we found that in a cellular context RGS14 selectively binds to activated H-Ras and not to Rap isoforms. Co-transfection / co-immunoprecipitation experiments demonstrated the ability of full-length RGS14 to assemble a multiprotein complex with components of the ERK MAPK pathway in a manner dependent on activated H-Ras. Small interfering RNA-mediated knockdown of RGS14 inhibited both nerve growth factor- and basic fibrobast growth factor-mediated neuronal differentiation of PC12 cells, a process which is known to be dependent on Ras-ERK signaling.

      CONCLUSIONS/SIGNIFICANCE: In cells, RGS14 facilitates the formation of a selective Ras.GTP-Raf-MEK-ERK multiprotein complex to promote sustained ERK activation and regulate H-Ras-dependent neuritogenesis. This cellular function for RGS14 is similar but distinct from that recently described for its closely-related paralogue, RGS12, which shares the tandem Ras-binding domain architecture with RGS14.

      View details for PubMedID 19319189
  • Structure-based protocol for identifying mutations that enhance protein-protein binding affinities. J Mol Biol
    Sammond DW, Eletr ZM, Purbeck C, Kimple RJ, Siderovski DP, Kuhlman B
    2007 Aug 31; 371 (5): 1392-404
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      The ability to manipulate protein binding affinities is important for the development of proteins as biosensors, industrial reagents, and therapeutics. We have developed a structure-based method to rationally predict single mutations at protein-protein interfaces that enhance binding affinities. The protocol is based on the premise that increasing buried hydrophobic surface area and/or reducing buried hydrophilic surface area will generally lead to enhanced affinity if large steric clashes are not introduced and buried polar groups are not left without a hydrogen bond partner. The procedure selects affinity enhancing point mutations at the protein-protein interface using three criteria: (1) the mutation must be from a polar amino acid to a non-polar amino acid or from a non-polar amino acid to a larger non-polar amino acid, (2) the free energy of binding as calculated with the Rosetta protein modeling program should be more favorable than the free energy of binding calculated for the wild-type complex and (3) the mutation should not be predicted to significantly destabilize the monomers. The performance of the computational protocol was experimentally tested on two separate protein complexes; Galpha(i1) from the heterotrimeric G-protein system bound to the RGS14 GoLoco motif, and the E2, UbcH7, bound to the E3, E6AP from the ubiquitin pathway. Twelve single-site mutations that were predicted to be stabilizing were synthesized and characterized in the laboratory. Nine of the 12 mutations successfully increased binding affinity with five of these increasing binding by over 1.0 kcal/mol. To further assess our approach we searched the literature for point mutations that pass our criteria and have experimentally determined binding affinities. Of the eight mutations identified, five were accurately predicted to increase binding affinity, further validating the method as a useful tool to increase protein-protein binding affinities.

      View details for PubMedID 17603074
  • G alpha selectivity and inhibitor function of the multiple GoLoco motif protein GPSM2/LGN. Biochim Biophys Acta
    McCudden CR, Willard FS, Kimple RJ, Johnston CA, Hains MD, Jones MB, Siderovski DP
    2005 Sep 10; 1745 (2): 254-64
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      GPSM2 (G-protein signalling modulator 2; also known as LGN or mammalian Pins) is a protein that regulates mitotic spindle organization and cell division. GPSM2 contains seven tetratricopeptide repeats (TPR) and four Galpha(i/o)-Loco (GoLoco) motifs. GPSM2 has guanine nucleotide dissociation inhibitor (GDI) activity towards both Galpha(o)- and Galpha(i)-subunits; however, a systematic analysis of its individual GoLoco motifs has not been described. We analyzed each of the four individual GoLoco motifs from GPSM2, assessing their relative binding affinities and GDI potencies for Galpha(i1), Galpha(i2), and Galpha(i3) and Galpha(o). Each of the four GPSM2 GoLoco motifs (36-43 amino acids in length) was expressed in bacteria as a GST-fusion protein and purified to homogeneity. The binding of each of the four GST-GoLoco motifs to Galpha(i1)-, Galpha(o)-, and Galpha(s)-subunits was assessed by surface plasmon resonance; all of the motifs bound Galpha(i1), but exhibited low affinity towards Galpha(o). GDI activity was assessed by a fluorescence-based nucleotide-binding assay, revealing that all four GoLoco motifs are functional as GDIs for Galpha(i1), Galpha(i2), and Galpha(i3). Consistent with our binding studies, the GDI activity of GPSM2 GoLoco motifs on Galpha(o) was significantly lower than that toward Galpha(i1), suggesting that the in vivo targets of GPSM2 are most likely to be Galpha(i)-subunits.

      View details for PubMedID 15946753
  • D2 dopamine receptor activation of potassium channels is selectively decoupled by Galpha-specific GoLoco motif peptides. J Neurochem
    Webb CK, McCudden CR, Willard FS, Kimple RJ, Siderovski DP, Oxford GS
    2005 Mar; 92 (6): 1408-18
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      The GoLoco motif is a short polypeptide sequence found in G-protein signaling regulators such as regulator of G-protein signaling proteins type 12 and 14 and activator of G-protein signaling protein type 3. A unique property of the GoLoco motifs from these three proteins is their preferential interaction with guanosine diphosphate (GDP)-bound Galpha(i1), Galpha(i3) and, sometimes, Galpha(i2) subunits over Galpha(o) subunits. This interaction prevents both spontaneous guanine nucleotide release and reassociation of Galpha(i)-GDP with Gbetagamma. We utilized this property of the GoLoco motif to examine dopamine (D2 and D3) and somatostatin receptor coupling to G-protein-regulated inwardly rectifying potassium (GIRK) channels in mouse AtT20 cells. GoLoco motif peptides had no effect on either basal channel activity or the initial responses to agonists, suggesting that the GoLoco motif cannot disrupt pre-formed G-protein heterotrimers. GoLoco motif peptides did, however, interfere with human D2((short)) receptor coupling to GIRK channels as demonstrated by the progressively diminished responses after repeated agonist application. This behavior is consistent with some form of compartmentalization of D2 receptors and GIRK channels such that Gbetagamma subunits, freed by local receptor activation and prevented from reforming a heterotrimeric complex, are not functionally constrained within the receptor-channel complex and thus are unable to exert a persistent activating effect. In contrast, GoLoco motif peptides had no effect on either D3 or somatostatin coupling to GIRK channels. Our results suggest that GoLoco motif-based peptides will be useful tools in examining the specificity of G-protein-coupled receptor-effector coupling.

      View details for PubMedID 15748159
  • G-protein signaling: back to the future. Cell Mol Life Sci
    McCudden CR, Hains MD, Kimple RJ, Siderovski DP, Willard FS
    2005 Mar; 62 (5): 551-77
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      Heterotrimeric G-proteins are intracellular partners of G-protein-coupled receptors (GPCRs). GPCRs act on inactive Galpha.GDP/Gbetagamma heterotrimers to promote GDP release and GTP binding, resulting in liberation of Galpha from Gbetagamma. Galpha.GTP and Gbetagamma target effectors including adenylyl cyclases, phospholipases and ion channels. Signaling is terminated by intrinsic GTPase activity of Galpha and heterotrimer reformation - a cycle accelerated by 'regulators of G-protein signaling' (RGS proteins). Recent studies have identified several unconventional G-protein signaling pathways that diverge from this standard model. Whereas phospholipase C (PLC) beta is activated by Galpha(q) and Gbetagamma, novel PLC isoforms are regulated by both heterotrimeric and Ras-superfamily G-proteins. An Arabidopsis protein has been discovered containing both GPCR and RGS domains within the same protein. Most surprisingly, a receptor-independent Galpha nucleotide cycle that regulates cell division has been delineated in both Caenorhabditis elegans and Drosophila melanogaster. Here, we revisit classical heterotrimeric G-protein signaling and explore these new, non-canonical G-protein signaling pathways.

      View details for PubMedID 15747061
  • RGS14 is a mitotic spindle protein essential from the first division of the mammalian zygote. Dev Cell
    Martin-McCaffrey L, Willard FS, Oliveira-dos-Santos AJ, Natale DR, Snow BE, Kimple RJ, Pajak A, Watson AJ, Dagnino L, Penninger JM, Siderovski DP, D'Souza SJ
    2004 Nov; 7 (5): 763-9
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      Heterotrimeric G protein alpha subunits, RGS proteins, and GoLoco motif proteins have been recently implicated in the control of mitotic spindle dynamics in C. elegans and D. melanogaster. Here we show that "regulator of G protein signaling-14" (RGS14) is expressed by the mouse embryonic genome immediately prior to the first mitosis, where it colocalizes with the anastral mitotic apparatus of the mouse zygote. Loss of Rgs14 expression in the mouse zygote results in cytofragmentation and failure to progress to the 2-cell stage. RGS14 is found in all tissues and segregates to the nucleus in interphase and to the mitotic spindle and centrioles during mitosis. Alteration of RGS14 levels in exponentially proliferating cells leads to cell growth arrest. Our results indicate that RGS14 is one of the earliest essential product of the mammalian embryonic genome yet described and has a general role in mitosis.

      View details for PubMedID 15525537
  • Purification and in vitro functional analyses of RGS12 and RGS14 GoLoco motif peptides. Methods Enzymol
    Kimple RJ, Willard FS, Siderovski DP
    2004; 390: 416-36
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      The GoLoco motif is a short polypeptide sequence that binds to heterotrimeric G-protein alpha subunits of the adenylyl cyclase-inhibitory (Galpha(i/o)) subclass in a nucleotide-dependent manner (i.e., solely to the GDP-bound ground state). This article describes methods used for the expression, purification, and in vitro evaluation of membrane-permeant tag fusion peptides derived from the GoLoco motif regions of "regulator of G-protein signaling" proteins type 12 (RGS12) and 14 (RGS14) and a consensus GoLoco sequence from the multiple GoLoco motif protein AGS3. Three different fluorescence-based assays are described for evaluating the in vitro function of these GoLoco peptides as guanine nucleotide dissociation inhibitors, including measurements of GTPgammaS binding and Galpha subunit activation by the planar ion aluminum tetrafluoride.

      View details for PubMedID 15488192
  • What do surgery residents do on their call nights? Am J Surg
    Morton JM, Baker CC, Farrell TM, Yohe ME, Kimple RJ, Herman DC, Udekwu P, Galanko JA, Behrns KE, Meyer AA
    2004 Sep; 188 (3): 225-9
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      BACKGROUND: Surgical resident education is entering a critical era of achieving core competencies despite work hour restrictions. An assessment of on-call activity is needed to maximize educational merit.

      METHODS: A time-motion study of resident on-call activity was performed at a university medical center and an urban affiliate hospital. Residents were followed by "shadow" residents who concurrently recorded resident activity.

      RESULTS: Activities of daily living and patient evaluation comprised the majority of on-call activity. Residents slept a median of 200 minutes per night. Cross-coverage activities accounted for 41% of pages and 19% of patient evaluation. Direct patient contact comprised only 7% of call night duties. Communication activity occupied 15% of total minutes, and a mean of 16 pages were received nightly. Significant differences in activities existed between resident levels and hospitals.

      CONCLUSIONS: Call activity consists primarily of activities of daily living, patient evaluation, and communication. Sleep accounts for nearly one third of all on-call activity. These data may be useful in improving both patient care and resident call experience.

      View details for PubMedID 15450824
  • Fluorescence-based assays for RGS box function. Methods Enzymol
    Willard FS, Kimple RJ, Kimple AJ, Johnston CA, Siderovski DP
    2004; 389: 56-71
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      Ligand-activated, seven transmembrane-spanning receptors interact with inactive G-protein heterotrimers (Galphabetagamma) to catalyze GTP loading and, consequently, activation of Galpha subunits and the liberation of Gbetagamma. Galpha.GTP and Gbetagamma are then competent to regulate independent effector pathways. The duration of heterotrimeric G-protein signaling is determined by the lifetime of the Galpha subunit in the GTP-bound state. Signal termination is facilitated by the intrinsic guanosine triphosphatase (GTPase) activity of Galpha and subsequent reformation of the inactive heterotrimer. Regulators of G-protein signaling (RGS) proteins act enzymatically, via their hallmark "RGS box," as GTPase-accelerating proteins (GAPs) for Galpha subunits and thus function as negative regulators of G-protein signaling in vitro and in vivo. This article describes the use of fluorescence resonance energy transfer (FRET) to monitor the interaction between a Galpha subunit and an RGS box protein. Furthermore, this article describes optimization of this assay for high-throughput screening and the evaluation of mutant RGS box and Galpha proteins. Finally, this article describes the novel application of this FRET technique to measure the activity of RGS protein-derived GoLoco peptides that modulate Galpha activation by aluminum tetrafluoride.

      View details for PubMedID 15313559
  • Return of the GDI: the GoLoco motif in cell division. Annu Rev Biochem
    Willard FS, Kimple RJ, Siderovski DP
    2004; 73: 925-51
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      The GoLoco motif is a 19-amino-acid sequence with guanine nucleotide dissociation inhibitor activity against G-alpha subunits of the adenylyl-cyclase-inhibitory subclass. The GoLoco motif is present as an independent element within multidomain signaling regulators, such as Loco, RGS12, RGS14, and Rap1GAP, as well as in tandem arrays in proteins, such as AGS3, G18, LGN, Pcp-2/L7, and Partner of Inscuteable (Pins/Rapsynoid). Here we discuss the biochemical mechanisms of GoLoco motif action on G-alpha subunits in light of the recent crystal structure of G-alpha-i1 bound to the RGS14 GoLoco motif. Currently, there is sparse evidence for GoLoco motif regulation of canonical G-protein-coupled receptor signaling. Rather, studies of asymmetric cell division in Drosophila and Caenorhabditis elegans, as well as mammalian mitosis, implicate GoLoco proteins, such as Pins, GPR-1/GPR-2, LGN, and RGS14, in mitotic spindle organization and force generation. We discuss potential mechanisms by which GoLoco/Galpha complexes might modulate spindle dynamics.

      View details for PubMedID 15189163
  • The GoLoco motif: heralding a new tango between G protein signaling and cell division. Mol Interv
    Kimple RJ, Willard FS, Siderovski DP
    2002 Apr; 2 (2): 88-100
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      The Galpha and Gbetagamma components of heterotrimeric G proteins, typically associated with cell-surface receptor signaling, also partake in the macromolecular interactions that underlie cell polarity and cell division. Proteins with Galpha-binding GoLoco motifs, such as Drosophila melanogaster Pins (for Partner of Inscuteable) and its mammalian counterpart LGN, participate in multi-protein complexes that maintain cellular asymmetry and orderly segregation of chromosomal content and daughter cell bodies. The GoLoco motif was recently identified as a selective Galpha-binding partner: the GoLoco-Galpha interaction can displace Gbetagamma and inhibit guanine nucleotide release from the bound Galpha subunit. Recent x-ray crystallographic studies suggest ways in which GoLoco-motif peptides may modulate heterotrimeric G protein signaling. Such peptides could be exploited to help dissect the signals that underpin cell polarity and cell division processes.

      View details for PubMedID 14993354
  • Guanine nucleotide dissociation inhibitor activity of the triple GoLoco motif protein G18: alanine-to-aspartate mutation restores function to an inactive second GoLoco motif. Biochem J
    Kimple RJ, Willard FS, Hains MD, Jones MB, Nweke GK, Siderovski DP
    2004 Mar 15; 378 (Pt 3): 801-8
    • More

      GoLoco ('Galpha(i/o)-Loco' interaction) motif proteins have recently been identified as novel GDIs (guanine nucleotide dissociation inhibitors) for heterotrimeric G-protein alpha subunits. G18 is a member of the mammalian GoLoco-motif gene family and was uncovered by analyses of human and mouse genomes for anonymous open-reading frames. The encoded G18 polypeptide is predicted to contain three 19-amino-acid GoLoco motifs, which have been shown in other proteins to bind Galpha subunits and inhibit spontaneous nucleotide release. However, the G18 protein has thus far not been characterized biochemically. Here, we have cloned and expressed the G18 protein and assessed its ability to act as a GDI. G18 is capable of simultaneously binding more than one Galpha(i1) subunit. In binding assays with the non-hydrolysable GTP analogue guanosine 5'-[gamma-thio]triphosphate, G18 exhibits GDI activity, slowing the exchange of GDP for GTP by Galpha(i1). Only the first and third GoLoco motifs within G18 are capable of interacting with Galpha subunits, and these bind with low micromolar affinity only to Galpha(i1) in the GDP-bound form, and not to Galpha(o), Galpha(q), Galpha(s) or Galpha12. Mutation of Ala-121 to aspartate in the inactive second GoLoco motif of G18, to restore the signature acidic-glutamine-arginine tripeptide that forms critical contacts with Galpha and its bound nucleotide [Kimple, Kimple, Betts, Sondek and Siderovski (2002) Nature (London) 416, 878-881], results in gain-of-function with respect to Galpha binding and GDI activity.

      View details for PubMedID 14656218
  • Established and emerging fluorescence-based assays for G-protein function: Ras-superfamily GTPases. Comb Chem High Throughput Screen
    Rojas RJ, Kimple RJ, Rossman KL, Siderovski DP, Sondek J
    2003 Jun; 6 (4): 409-18
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      Ras and Rho GTPases are signaling proteins that regulate a variety of physiological events and are intimately linked to the progression of cancer. Recently, a variety of fluorescence-based assays have been refined to monitor activation of these GTPases. This review summarizes current fluorescence-based techniques for studying Ras superfamily GTPases with an emphasis on practical examples and high-throughput applications. These techniques are not only useful for biochemical characterization of Ras superfamily members, but will also facilitate the discovery of small molecule therapeutics designed to inhibit signal transduction mediated by GTPases.

      View details for PubMedID 12769685
  • Established and emerging fluorescence-based assays for G-protein function: heterotrimeric G-protein alpha subunits and regulator of G-protein signaling (RGS) proteins. Comb Chem High Throughput Screen
    Kimple RJ, Jones MB, Shutes A, Yerxa BR, Siderovski DP, Willard FS
    2003 Jun; 6 (4): 399-407
    • More

      Heterotrimeric G-proteins are molecular switches that couple serpentine receptors to intracellular effector pathways and the regulation of cell physiology. Ligand-bound receptors cause G-protein alpha subunits to bind guanosine 5'-triphosphate (GTP) and activate effector pathways. Signal termination is facilitated by the intrinsic GTPase activity of G-protein alpha subunits. Regulators of G-protein signaling (RGS) proteins accelerate the GTPase activity of the G-protein alpha subunit, and thus negatively regulate G-protein-mediated signal transduction. In vitro biochemical assays of heterotrimeric G-proteins commonly include measurements of nucleotide binding, GTPase activity, and interaction with RGS proteins. However, the conventional assays for most of these processes involve radiolabeled guanine nucleotide analogues and scintillation counting. In this article, we focus on fluorescence-based methodologies to study heterotrimeric G-protein alpha subunit regulation in vitro. Furthermore, we consider the potential of such techniques in high-throughput screening and drug discovery.

      View details for PubMedID 12769684
  • Translation of polarity cues into asymmetric spindle positioning in Caenorhabditis elegans embryos. Science
    Colombo K, Grill SW, Kimple RJ, Willard FS, Siderovski DP, Gönczy P
    2003 Jun 20; 300 (5627): 1957-61
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      Asymmetric divisions are crucial for generating cell diversity; they rely on coupling between polarity cues and spindle positioning, but how this coupling is achieved is poorly understood. In one-cell stage Caenorhabditis elegans embryos, polarity cues set by the PAR proteins mediate asymmetric spindle positioning by governing an imbalance of net pulling forces acting on spindle poles. We found that the GoLoco-containing proteins GPR-1 and GPR-2, as well as the Galpha subunits GOA-1 and GPA-16, were essential for generation of proper pulling forces. GPR-1/2 interacted with guanosine diphosphate-bound GOA-1 and were enriched on the posterior cortex in a par-3- and par-2-dependent manner. Thus, the extent of net pulling forces may depend on cortical Galpha activity, which is regulated by anterior-posterior polarity cues through GPR-1/2.

      View details for PubMedID 12750478
  • Structural determinants for GoLoco-induced inhibition of nucleotide release by Galpha subunits. Nature
    Kimple RJ, Kimple ME, Betts L, Sondek J, Siderovski DP
    2002 Apr 25; 416 (6883): 878-81
    • More

      Heterotrimeric G-proteins bind to cell-surface receptors and are integral in transmission of signals from outside the cell. Upon activation of the Galpha subunit by binding of GTP, the Galpha and Gbetagamma subunits dissociate and interact with effector proteins for signal transduction. Regulatory proteins with the 19-amino-acid GoLoco motif can bind to Galpha subunits and maintain G-protein subunit dissociation in the absence of Galpha activation. Here we describe the structural determinants of GoLoco activity as revealed by the crystal structure of Galpha(i1) GDP bound to the GoLoco region of the 'regulator of G-protein signalling' protein RGS14. Key contacts are described between the GoLoco motif and Galpha protein, including the extension of GoLoco's highly conserved Asp/Glu-Gln-Arg triad into the nucleotide-binding pocket of Galpha to make direct contact with the GDP alpha- and beta-phosphates. The structural organization of the GoLoco Galpha(i1) complex, when combined with supporting data from domain-swapping experiments, suggests that the Galpha all-helical domain and GoLoco-region carboxy-terminal residues control the specificity of GoLoco Galpha interactions.

      View details for PubMedID 11976690
  • RGS12 and RGS14 GoLoco motifs are G alpha(i) interaction sites with guanine nucleotide dissociation inhibitor Activity. J Biol Chem
    Kimple RJ, De Vries L, Tronchère H, Behe CI, Morris RA, Gist Farquhar M, Siderovski DP
    2001 Aug 03; 276 (31): 29275-81
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      The regulators of G-protein signaling (RGS) proteins accelerate the intrinsic guanosine triphosphatase activity of heterotrimeric G-protein alpha subunits and are thus recognized as key modulators of G-protein-coupled receptor signaling. RGS12 and RGS14 contain not only the hallmark RGS box responsible for GTPase-accelerating activity but also a single G alpha(i/o)-Loco (GoLoco) motif predicted to represent a second G alpha interaction site. Here, we describe functional characterization of the GoLoco motif regions of RGS12 and RGS14. Both regions interact exclusively with G alpha(i1), G alpha(i2), and G alpha(i3) in their GDP-bound forms. In GTP gamma S binding assays, both regions exhibit guanine nucleotide dissociation inhibitor (GDI) activity, inhibiting the rate of exchange of GDP for GTP by G alpha(i1). Both regions also stabilize G alpha(i1) in its GDP-bound form, inhibiting the increase in intrinsic tryptophan fluorescence stimulated by AlF(4)(-). Our results indicate that both RGS12 and RGS14 harbor two distinctly different G alpha interaction sites: a previously recognized N-terminal RGS box possessing G alpha(i/o) GAP activity and a C-terminal GoLoco region exhibiting G alpha(i) GDI activity. The presence of two, independent G alpha interaction sites suggests that RGS12 and RGS14 participate in a complex coordination of G-protein signaling beyond simple G alpha GAP activity.

      View details for PubMedID 11387333
  • The Future of Radiobiology. J Natl Cancer Inst
    Kirsch DG, Diehn M, Kesarwala AH, Maity A, Morgan MA, Schwarz JK, Bristow R, Demaria S, Eke I, Griffin RJ, Haas-Kogan D, Higgins GS, Kimmelman AC, Kimple RJ, Lombaert IM, Ma L, Marples B, Pajonk F, Park CC, Schaue D, Bernhard EJ
    2017 Nov 03; :
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      Innovation and progress in radiation oncology depend on discovery and insights realized through research in radiation biology. Radiobiology research has led to fundamental scientific insights, from the discovery of stem/progenitor cells to the definition of signal transduction pathways activated by ionizing radiation that are now recognized as integral to the DNA damage response (DDR). Radiobiological discoveries are guiding clinical trials that test radiation therapy combined with inhibitors of the DDR kinases DNA-dependent protein kinase (DNA-PK), ataxia telangiectasia mutated (ATM), ataxia telangiectasia related (ATR), and immune or cell cycle checkpoint inhibitors. To maintain scientific and clinical relevance, the field of radiation biology must overcome challenges in research workforce, training, and funding. The National Cancer Institute convened a workshop to discuss the role of radiobiology research and radiation biologists in the future scientific enterprise. Here, we review the discussions of current radiation oncology research approaches and areas of scientific focus considered important for rapid progress in radiation sciences and the continued contribution of radiobiology to radiation oncology and the broader biomedical research community.

      View details for PubMedID 29126306
  • The Second Stain: A Viral Whodunnit. Int J Radiat Oncol Biol Phys
    Rosenberg SA, Kimple RJ
    2017 Dec 01; 99 (5): 1061

Contact Information

Randall Kimple, MD, PhD

1111 Highland Avenue,
3107 WIMR
Madison, WI 53705-2275
Email