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1.  Estimating Functional Liver Reserve Following Hepatic Irradiation: Adaptive Normal Tissue Response Models 
Purpose
To estimate the limit of functional liver reserve for safe application of hepatic irradiation using changes in indocyanine green, an established assay of liver function.
Materials and Methods
From 2005–2011, 60 patients undergoing hepatic irradiation were enrolled in a prospective study assessing the plasma retention fraction of indocyanine green at 15-min (ICG-R15) prior to, during (at 60% of planned dose), and after radiotherapy (RT). The limit of functional liver reserve was estimated from the damage fraction of functional liver (DFL) post-RT [1−(ICG-R15pre-RT/ICG-R15post-RT)] where no toxicity was observed using a beta distribution function.
Results
Of 48 evaluable patients, 3 (6%) developed RILD, all within 2.5 months of completing RT. The mean ICG-R15 for non-RILD patients pre-RT, during-RT and 1-month post-RT was 20.3%(SE 2.6), 22.0%(3.0), and 27.5%(2.8), and for RILD patients was 6.3%(4.3), 10.8%(2.7), and 47.6%(8.8). RILD was observed at post-RT damage fractions of ≥78%. Both DFL assessed by during-RT ICG and MLD predicted for DFL post-RT (p<0.0001). Limiting the post-RT DFL to 50%, predicted a 99% probability of a true complication rate <15%.
Conclusion
The DFL as assessed by changes in ICG during treatment serves as an early indicator of a patient’s tolerance to hepatic irradiation.
doi:10.1016/j.radonc.2014.04.007
PMCID: PMC4119482  PMID: 24813090
liver; radiotherapy; indocyanine green; adaptive radiotherapy
2.  Response-driven Imaging Biomarkers for Predicting Radiation Necrosis of the Brain 
Physics in medicine and biology  2014;59(10):2535-2547.
Purpose
Radiation necrosis is an uncommon but severe adverse effect of brain radiation therapy. Current predictive models based on radiation dose have limited accuracy. We aimed to identify early individual response biomarkers based upon diffusion tensor (DT) imaging and incorporated them into a response model for prediction of radiation necrosis.
Methods and Materials
Twenty-nine patients with glioblastoma received six weeks of intensity modulated radiation therapy (RT) and concurrent temozolamide. Patients underwent DT-MRI scans before treatment, at three weeks during RT, and one, three, and six months after RT. Cases with radiation necrosis were classified based on generalized equivalent uniform dose (gEUD) of whole brain and DT index early changes in the corpus callosum and its substructures. Significant covariates were used to develop normal tissue complication probability models using binary logistic regression.
Results
Seven patients developed radiation necrosis. Percentage changes of radial diffusivity (RD) in the splenium at three weeks during RT and at six months after RT differed significantly between the patients with and without necrosis (p=0.05 and p=0.01). Percentage change of RD at three weeks during RT in the 30 Gy dose-volume of the splenium and brain gEUD combined yielded the best-fit logistic regression model.
Conclusions
Our findings indicate that early individual response during the course of RT, assessed by radial diffusivity, has the potential to aid in predicting delayed radiation necrosis, which could provide guidance in dose-escalation trials.
doi:10.1088/0031-9155/59/10/2535
PMCID: PMC4084934  PMID: 24778364
3.  Development and Validation of a Novel Platform-Independent Metastasis Signature in Human Breast Cancer 
PLoS ONE  2015;10(5):e0126631.
Purpose
The molecular drivers of metastasis in breast cancer are not well understood. Therefore, we sought to identify the biological processes underlying distant progression and define a prognostic signature for metastatic potential in breast cancer.
Experimental design
In vivo screening for metastases was performed using Chick Chorioallantoic Membrane assays in 21 preclinical breast cancer models. Expressed genes associated with metastatic potential were identified using high-throughput analysis. Correlations with biological function were determined using the Database for Annotation, Visualization and Integrated Discovery.
Results
We identified a broad range of metastatic potential that was independent of intrinsic breast cancer subtypes. 146 genes were significantly associated with metastasis progression and were linked to cancer-related biological functions, including cell migration/adhesion, Jak-STAT, TGF-beta, and Wnt signaling. These genes were used to develop a platform-independent gene expression signature (M-Sig), which was trained and subsequently validated on 5 independent cohorts totaling nearly 1800 breast cancer patients with all p-values < 0.005 and hazard ratios ranging from approximately 2.5 to 3. On multivariate analysis accounting for standard clinicopathologic prognostic variables, M-Sig remained the strongest prognostic factor for metastatic progression, with p-values < 0.001 and hazard ratios > 2 in three different cohorts.
Conclusion
M-Sig is strongly prognostic for metastatic progression, and may provide clinical utility in combination with treatment prediction tools to better guide patient care. In addition, the platform-independent nature of the signature makes it an excellent research tool as it can be directly applied onto existing, and future, datasets.
doi:10.1371/journal.pone.0126631
PMCID: PMC4431866  PMID: 25974184
4.  Arterial Perfusion Imaging-Defined Subvolume of Intrahepatic Cancer 
Purpose
To assess whether an increase in a subvolume of intrahepatic tumor with elevated arterial perfusion during radiation therapy (RT) predicts tumor progression post RT.
Methods and Materials
Twenty patients with unresectable intrahepatic cancers undergoing RT were enrolled in a prospective IRB-approved study. Dynamic contrast-enhanced magnetic resonance imaging (DCE MRI) were performed prior to RT (pre-RT), after delivering ~60% of the planned dose (mid-RT) and one month after completion of RT to quantify hepatic arterial perfusion. The arterial perfusions of the tumors at pre-RT were clustered into low-normal and elevated perfusion by a fuzzy clustering-based method, and the tumor subvolumes with elevated arterial perfusion were extracted from the hepatic arterial perfusion images. The percentage changes in the tumor subvolumes and means of arterial perfusion over the tumors from pre-RT to mid-RT were evaluated for predicting tumor progression post-RT.
Results
Of the 24 tumors, 6 tumors in 5 patients progressed 5–21 months after RT completion. Neither tumor volumes nor means of tumor arterial perfusion at pre-RT were predictive of treatment outcome. The mean arterial perfusion over the tumors increased significantly at mid-RT in progressive tumors comparing to the responsive ones (p=0.006). From pre-RT to mid-RT, the responsive tumors had a decrease in the tumor subvolumes with elevated arterial perfusion (median: −14%, range: −75% – 65%), while the progressing tumors had an increase of the subvolumes (median: 57%, range: −7% – 165%) (p=0.003). Receiver operating characteristic (ROC) analysis of the percentage change in the subvolume for predicting tumor progression post-RT had an area under the curve (AUC) of 0.90.
Conclusion
The increase in the subvolume of the intrahepatic tumor with elevated arterial perfusion during RT has the potential to be a predictor for tumor progression post-RT. The tumor subvolume could be a radiation boost candidate for response-driven adaptive RT.
doi:10.1016/j.ijrobp.2014.01.040
PMCID: PMC3988211  PMID: 24613814
Intrahepatic cancer; Tumor subvolume; Dose boosting; Hepatic Arterial perfusion
5.  Antibody against CD44s Inhibits Pancreatic Tumor Initiation and Post-Radiation Recurrence in Mice 
Gastroenterology  2014;146(4):1108-1118.e12.
Background & Aims
CD44s is a surface marker of tumor-initiating cells (TICs); high tumor levels correlate with metastasis and recurrence, as well as poor outcomes of patients. Monoclonal antibodies against CD44s might eliminate TICs with minimal toxicity. This strategy is unclear for treatment of pancreatic cancer, and little is known about how anti-CD44s affect pancreatic cancer initiation or recurrence after radiotherapy.
Methods
192 pairs of human pancreatic adenocarcinoma and adjacent non-tumor pancreatic tissues were collected from patients undergoing surgery. We measured CD44s levels in tissue samples and pancreatic cancer cell lines by immunohistochemistry, real-time PCR and immunoblot; levels were correlated with patient survival times. We studied the effects of anti-CD44s in mice with human pancreatic tumor xenografts, and used flow cytometry to determine effects on TICs. Changes in CD44s signaling were examined by real-time PCR, immunoblot, reporter assay, and in vitro tumorsphere formation assays.
Results
Levels of CD44s were significantly higher in pancreatic cancer than adjacent non-tumor tissues. Patients whose tumors expressed high levels of CD44s had a median survival of 10 months, compared to 43 months for those with low levels. Anti-CD44s reduced growth, metastasis, and post-radiation recurrence of pancreatic xenograft tumors in mice. The antibody reduced the number of TICs in cultured pancreatic cancer cells and in xenograft tumors, as well as their tumorigenicity. In cultured pancreatic cancer cell lines, anti-CD44s downregulated the stem cell self-renewal genes Nanog, Sox-2, and Rex-1 and inhibited STAT3-mediated cell proliferation and survival signaling.
Conclusions
The TIC marker CD44s is upregulated in human pancreatic tumors and associated with patient survival time. CD44s is required for initiation, growth, metastasis, and post-radiation recurrence of xenograft tumors in mice. Anti-CD44s eliminated bulk tumor cells as well as TICs from the tumors. Strategies to target CD44s might be developed to block pancreatic tumor formation and post-radiotherapy recurrence in patients.
doi:10.1053/j.gastro.2013.12.035
PMCID: PMC3982149  PMID: 24397969
cancer stem cell; H4C4; tumorigenesis; progression
6.  Personalized dose selection in radiation therapy using statistical models for toxicity and efficacy with dose and biomarkers as covariates 
Statistics in medicine  2014;33(30):5330-5339.
Selection of dose for cancer patients treated with radiation therapy (RT) must balance the increased efficacy with the increased toxicity associated with higher dose. Historically, a single dose has been selected for a population of patients (e.g., all stage III non-small cell lung cancer). However, the availability of new biologic markers for toxicity and efficacy allows the possibility of selecting a more personalized dose. We consider the use of statistical models for toxicity and efficacy as a function of RT dose and biomarkers to select an optimal dose for an individual patient, defined as the dose that maximizes the probability of efficacy minus the sum of weighted toxicity probabilities. This function can be shown to be equal to the expected value of the utility derived from a particular family of bivariate outcome utility matrices. We show that if dose is linearly related to the probability of toxicity and efficacy, then any marker that only acts additively with dose cannot improve efficacy, without also increasing toxicity. Using a dataset of lung cancer patients treated with RT, we illustrate this approach and compare it to non-marker-based dose selection. Because typical metrics used in evaluating new markers (e.g., area under the ROC curve) do not directly address the ability of a marker to improve efficacy at a fixed probability of toxicity, we utilize a simulation study to assess the effects of marker-based dose selection on toxicity and efficacy outcomes.
doi:10.1002/sim.6285
PMCID: PMC4367186  PMID: 25164860
dose finding; phase I; biomarkers; radiation therapy; utilities
7.  The effect of erlotinib on EGFR and downstream signaling in oral cavity squamous cell carcinoma 
Head & neck  2012;35(9):1323-1330.
Purpose
To determine if there are differences in biomarker modulation and EGFR degradation between tumor and the normal mucosa following treatment with an EGFR inhibitor, erlotinib, in head and neck cancer.
Methods
Patients with primary oral cavity squamous cell cancers received a course of erlotinib, 150 mg qd for 7 days prior to surgical resection. Tumor and normal mucosa biopsies were obtained both pre and post erlotinib. Changes in known markers of EGFR activity (phospho, AKT, STAT3) were measured by immunoblotting, while changes in tissue distribution were analyzed by immunohistochemical analysis.
Results
Twelve patients were enrolled; seven had evaluable paired tumor and normal mucosa biopsies pre and post treatment. Expression of EGFR was higher in tumor compared to the normal mucosa (p=0.005). Erlotinib administration was associated with marked inhibition of pEGFR and reduction in total EGFR protein (p=0.004, p=0.007) in tumors while there was heterogeneity in EGFR inhibition in the normal mucosa (p=0.1 (pEGFR), and p=0.07 (EGFR). Reduced levels of pSrc and pSTAT3 and enhanced p27 levels were noted in tumors following erlotinib. Cell culture studies confirmed that EGFR is degraded in tumor cells after prolonged treatment with erlotinib.
Conclusion
Our results show that EGFR inhibition by erlotinib led to a marked reduction in EGFR protein levels in patients. Differential effects of erlotinib on tumor compared to the normal mucosa suggest there may be individual patient heterogeneity. These preliminary data suggest EGFR degradation should be further analyzed as a potential biomarker in selecting patients likely to benefit from EFGR inhibitors.
doi:10.1002/hed.23128
PMCID: PMC4364542  PMID: 22907806
8.  ATDC/TRIM29 phosphorylation by ATM/MAPKAP kinase 2 mediates radioresistance in pancreatic cancer cells 
Cancer research  2014;74(6):1778-1788.
Pancreatic ductal adenocarcinoma (PDAC) is characterized by therapeutic resistance for which the basis is poorly understood. Here we report that the DNA and p53 binding protein ATDC/TRIM29, which is highly expressed in PDAC, plays a critical role in DNA damage signaling and radioresistance in pancreatic cancer cells. ATDC mediated resistance to ionizing radiation in vitro and in vivo in mouse xenograft assays. ATDC was phosphorylated directly by MAPKAP kinase 2 (MK2) at Ser550 in an ATM-dependent manner. Phosphorylation at Ser-550 by MK2 was required for the radioprotective function of ATDC. Our results identify a DNA repair pathway leading from MK2 and ATM to ATDC, suggesting its candidacy as a therapeutic target to radiosensitize PDAC and improve the efficacy of DNA-damaging treatment.
doi:10.1158/0008-5472.CAN-13-2289
PMCID: PMC3961828  PMID: 24469230
pancreatic cancer; ATM; MK2; DNA repair; DNA damage response
9.  PCAT-1, a long noncoding RNA, regulates BRCA2 and controls homologous recombination in cancer 
Cancer research  2014;74(6):1651-1660.
Impairment of double-stranded DNA break (DSB) repair is essential to many cancers. However, while mutations in DSB repair proteins are common in hereditary cancers, mechanisms of impaired DSB repair in sporadic cancers remain incompletely understood. Here, we describe the first role for a long noncoding RNA (lncRNA) in DSB repair in prostate cancer. We identify PCAT-1, a prostate cancer outlier lncRNA, which regulates cell response to genotoxic stress. PCAT-1 expression produces a functional deficiency in homologous recombination (HR) through its repression of the BRCA2 tumor suppressor, which, in turn, imparts a high sensitivity to small molecule inhibitors of PARP1. These effects reflected a post-transcriptional repression of the BRCA2 3′UTR by PCAT-1. Our observations thus offer a novel mechanism of “BRCA-ness” in sporadic cancers.
doi:10.1158/0008-5472.CAN-13-3159
PMCID: PMC4009928  PMID: 24473064
prostate cancer; long noncoding RNA; homologous recombination
10.  Targeting Mcl-1 for Radiosensitization of Pancreatic Cancers12 
Translational Oncology  2015;8(1):47-54.
In order to identify targets whose inhibition may enhance the efficacy of chemoradiation in pancreatic cancer, we previously conducted an RNAi library screen of 8,800 genes. We identified Mcl-1 (myeloid cell leukemia-1), an anti-apoptotic member of the Bcl-2 family, as a target for sensitizing pancreatic cancer cells to chemoradiation. In the present study we investigated Mcl-1 inhibition by either genetic or pharmacological approaches as a radiosensitizing strategy in pancreatic cancer cells. Mcl-1 depletion by siRNA produced significant radiosensitization in BxPC-3 and Panc-1 cells in association with Caspase-3 activation and PARP cleavage, but only minimal radiosensitization in MiaPaCa-2 cells. We next tested the ability of the recently identified, selective, small molecule inhibitor of Mcl-1, UMI77, to radiosensitize in pancreatic cancer cells. UMI77 caused dissociation of Mcl-1 from the pro-apoptotic protein Bak and produced significant radiosensitization in BxPC-3 and Panc-1 cells, but minimal radiosensitization in MiaPaCa-2 cells. Radiosensitization by UMI77 was associated with Caspase-3 activation and PARP cleavage. Importantly, UMI77 did not radiosensitize normal small intestinal cells. In contrast, ABT-737, an established inhibitor of Bcl-2, Bcl-XL, and Bcl-w, failed to radiosensitize pancreatic cancer cells suggesting the unique importance of Mcl-1 relative to other Bcl-2 family members to radiation survival in pancreatic cancer cells. Taken together, these results validate Mcl-1 as a target for radiosensitization of pancreatic cancer cells and demonstrate the ability of small molecules which bind the canonical BH3 groove of Mcl-1, causing displacement of Mcl-1 from Bak, to selectively radiosensitize pancreatic cancer cells.
doi:10.1016/j.tranon.2014.12.004
PMCID: PMC4350640  PMID: 25749177
11.  Investigation of the diffusion abnormality index as a new imaging biomarker for early assessment of brain tumor response to radiation therapy 
Neuro-Oncology  2013;16(1):131-139.
Background
Diffusion MRI, although having the potential to be a biomarker for early assessment of tumor response to therapy, could be confounded by edema and necrosis in or near the brain tumors. This study aimed to develop and investigate the ability of the diffusion abnormality index (DAI) to be a new imaging biomarker for early assessment of brain metastasis response to radiation therapy (RT).
Methods
Patients with either radiosensitive or radioresistant brain metastases that were treated by whole brain RT alone or combined with bortezomib as a radiation sensitizer had diffusion-weighted (DW) MRI pre-RT and 2 weeks (2W) after starting RT. A patient-specific diffusion abnormality probability function (DAProF) was created to account for abnormal low and high apparent diffusion coefficients differently, reflecting respective high cellularity and edema/necrosis. The DAI of a lesion was then calculated by the integral of DAProF-weighted tumor apparent diffusion coefficient histogram. The changes in DAI from pre-RT to 2W were evaluated for differentiating the responsive, stable, and progressive tumors and compared with the changes in gross tumor volume and conventional diffusion metrics during the same time interval.
Results
In lesions treated with whole brain RT, the DAI performed the best among all metrics in predicting the posttreatment response of brain metastases to RT. In lesions treated with whole brain RT + bortezomib, although DAI was the best predictor, the performance of all metrics worsened compared with the first group.
Conclusions
The ability of DAI for early assessment of brain metastasis response to RT depends upon treatment regimes.
doi:10.1093/neuonc/not153
PMCID: PMC3870818  PMID: 24327584
cancer; diffusion abnormality index; DW-MRI; imaging biomarker
12.  Neoadjuvant Stereotactic Body Radiation Therapy (SBRT), Capecitabine, and Liver Transplantation for Unresectable Hilar Cholangiocarcinoma 
Hilar cholangiocarcinoma (CCA) is a difficult malignancy to treat surgically given its anatomical location and its frequent association with primary sclerosing cholangitis (PSC). Neoadjuvant chemoradiotherapy followed by liver transplantation in lymph node negative patients has been advanced by select liver transplant centers for treatment of patients with unresectable disease. This approach has most commonly used external beam radiotherapy combined with biliary brachytherapy and 5-FU based chemotherapy. Our center has recently embarked on a protocol utilizing stereotactic body radiation therapy (SBRT) followed by capecitabine in lymph node negative patients until liver transplantation. We therefore retrospectively determined tolerability and pathologic response in this pilot study. Over a three year period 17 patients with unresectable hilar CCA were evaluated for treatment under this protocol. In all, 12 patients qualified for neoadjuvant therapy and were treated with SBRT (50–60 Gy, 3–5 fractions over two weeks). Following one week of rest, capecitabine was initiated at 1330 mg/m2/day and continued until liver transplantation. During neoadjuvant therapy, there were a total of 35 adverse events with cholangitis and palmar/plantar erythrodysesthesia being the most common. Capecitabine dose reductions were required on 5 occasions. Ultimately, 9 patients were listed for transplant and 6 patients received a liver transplant. Explant pathology of hilar tumors showed at least a partial treatment response in five patients with extensive tumor necrosis and fibrosis noted. Additionally, high apoptotic and low proliferative indices were measured on histological examination. Eleven transplant-related complications occurred, and one-year survival after transplant was 83%. In this pilot study, neoadjuvant therapy with SBRT, capecitabine, and liver transplantation for unresectable CCA demonstrated acceptable tolerability. Further studies will determine the overall future efficacy of this therapy.
doi:10.1002/lt.23757
PMCID: PMC4185427  PMID: 24115315
Malignancy/liver transplantation; sclerosing cholangitis; bile duct cancer; biliary neoplasms; external beam radiotherapy
13.  HAb18G/CD147 Promotes pSTAT3-Mediated Pancreatic Cancer Development via CD44s †, ‡ 
Purpose
STAT3 plays a critical role in initiation and progression of pancreatic cancer. However, therapeutically targeting STAT3 is failure in clinic. We previously identified HAb18G/CD147 as an effective target for cancer treatment. In this study, we aimed to investigate potential role of HAb18G/CD147 in STAT3-involved pancreatic tumorigenesis in vitro and in vivo.
Experimental Design
The expression of HAb18G/CD147, pSTAT3 and CD44s were determined in tissue microarrays. The tumorigenic function and molecular signaling mechanism of HAb18G/CD147 was assessed by in vitro cellular and clonogenic growth, reporter assay, immunoblot, immunofluorescence staining, immunoprecipitation, and in vivo tumor formationusing loss or gain-of-function strategies.
Results
Highly expressed HAb18G/CD147 promoted cellular and clonogenic growth in vitro and tumorigenicity in vivo. CyPA, a ligand of CD147, stimulated STAT3 phosphorylation and its downstream genes cyclin D1/survivin through HAb18G/CD147 dependent mechanisms. HAb18G/CD147 was associated and co-localized with cancer stem cell marker CD44s in lipid rafts. The inhibitors of STAT3 and survivin, as well as CD44s neutralizing antibodies suppressed the HAb18G/CD147-induced cell growth. High HAb18G/CD147 expression in pancreatic cancer was significantly correlated with the poor tumor differentiation, and the high co-expression of HAb18G/CD147-CD44s-STAT3 associated with poor survival of patients with pancreatic cancer.
Conclusions
We identified HAb18G/CD147 as a novel upstream activator of STAT3 via interacts with CD44s and plays a critical role in the development of pancreatic cancer. The data suggest HAb18G/CD147 could be a promising therapeutic target for highly aggressive pancreatic cancer and a surrogate marker in the STAT3-targeted molecular therapies.
doi:10.1158/1078-0432.CCR-13-0621
PMCID: PMC3873783  PMID: 24132924
Pancreatic cancer; HAb18G/CD147; CD44; STAT3; tumor development
14.  The Long Non-Coding RNA PCAT-1 Promotes Prostate Cancer Cell Proliferation through cMyc12 
Neoplasia (New York, N.Y.)  2014;16(11):900-908.
Long non-coding RNAs (lncRNAs) represent an emerging layer of cancer biology, contributing to tumor proliferation, invasion, and metastasis. Here, we describe a role for the oncogenic lncRNA PCAT-1 in prostate cancer proliferation through cMyc. We find that PCAT-1–mediated proliferation is dependent on cMyc protein stabilization, and using expression profiling, we observed that cMyc is required for a subset of PCAT-1–induced expression changes. The PCAT-1–cMyc relationship is mediated through the post-transcriptional activity of the MYC 3′ untranslated region, and we characterize a role for PCAT-1 in the disruption of MYC-targeting microRNAs. To further elucidate a role for post-transcriptional regulation, we demonstrate that targeting PCAT-1 with miR-3667-3p, which does not target MYC, is able to reverse the stabilization of cMyc by PCAT-1. This work establishes a basis for the oncogenic role of PCAT-1 in cancer cell proliferation and is the first study to implicate lncRNAs in the regulation of cMyc in prostate cancer.
doi:10.1016/j.neo.2014.09.001
PMCID: PMC4240923  PMID: 25425964
PCAT-1; lncRNA; cell proliferation; 3’UTR; cMyc
15.  A Pilot Study of Diffusion-Weighted MRI in Patients Undergoing Neoadjuvant Chemoradiation for Pancreatic Cancer 
Translational Oncology  2014;7(5):644-649.
PURPOSE: In the current study we examined the ability of diffusion MRI (dMRI) to predict pathologic response in pancreatic cancer patients receiving neoadjuvant chemoradiation. METHODS: We performed a prospective pilot study of dMRI in patients with resectable pancreatic cancer. Patients underwent dMRI prior to neoadjuvant chemoradiation. Surgical specimens were graded according to the percent tumor cell destruction. Apparent diffusion coefficient (ADC) maps were used to generate whole-tumor derived ADC histogram distributions and mean ADC values. The primary objective of the study was to correlate ADC parameters with pathologic and CT response. RESULTS: Ten of the 12 patients enrolled on the study completed chemoradiation and had surgery. Three were found to be unresectable at the time of surgery and no specimen was obtained. Out of the 7 patients who underwent pancreaticoduodenectomy, 3 had a grade III histopathologic response (> 90% tumor cell destruction), 2 had a grade IIB response (51% to 90% tumor cell destruction), 1 had a grade IIA response (11% to 50% tumor cell destruction), and 1 had a grade I response (> 90% viable tumor). Median survival for patients with a grade III response, grade I-II response, and unresectable disease were 25.6, 18.7, and 6.1 months, respectively. There was a significant correlation between pre-treatment mean tumor ADC values and the amount of tumor cell destruction after chemoradiation with a Pearson correlation coefficient of 0.94 (P = .001). Mean pre-treatment ADC was 161 × 10− 5 mm2/s (n = 3) in responding patients (> 90% tumor cell destruction) compared to 125 × 10− 5 mm2/s (n = 4) in non-responding patients (> 10% viable tumor). CT imaging showed no significant change in tumor size in responders or non-responders. CONCLUSIONS: dMRI may be useful to predict response to chemoradiation in pancreatic cancer. In our study, tumors with a low ADC mean value at baseline responded poorly to standard chemoradiation and would be candidates for intensified therapy.
doi:10.1016/j.tranon.2014.07.005
PMCID: PMC4225651  PMID: 25389460
16.  A Multi-Institutional Phase II Study of Neoadjuvant Gemcitabine and Oxaliplatin with Radiation Therapy in Patients with Pancreatic Cancer 
Cancer  2013;119(15):2692-2700.
Background
The purpose of this study was to evaluate preoperative treatment with full-dose gemcitabine, oxaliplatin and radiation (RT) in localized pancreatic cancer.
Methods
Eligibility included confirmation of adenocarcinoma, resectable or borderline resectable disease, PS≤2, and adequate organ function. Treatment consisted of two, 28 day cycles of gemcitabine (1g/m2 over 30 minutes days 1, 8, 15) and oxaliplatin (85mg/m2 days 1, 15) with RT during cycle 1 (30Gy in 2Gy fractions). Patients were evaluated for surgery following cycle 2. Resected patients received 2 cycles of adjuvant chemotherapy.
Results
Sixty-eight evaluable patients were treated at 4 centers. By central radiology review, 23 patients were resectable, 39 borderline resectable, and 6 unresectable. Sixty-six patients (97%) completed cycle 1/RT and 61 patients (90%) cycle 2. Adverse events ≥ grade 3 during preoperative therapy included neutropenia (32%), thrombocytopenia (25%), and biliary obstruction/cholangitis (14%). Forty-three patients were resected (63%) with R0 resection in 36 (84%). Median overall survival for all patients was 18.2 months (95%CI 13–26.9), those resected 27.1 months (95%CI 21.2–47.1) and those not resected 10.9 months (95%CI 6.1–12.6). A decrease in CA19-9 following neoadjuvant therapy was associated with R0 resection (p=0.02) which resulted in a median survival of 34.6 months (95% CI 20.3–47.1). Fourteen patients (21%) are alive and disease-free at a median follow-up time of 31.4 months (range, 24–47.6).
Conclusion
Preoperative therapy with full dose gemcitabine, oxaliplatin and RT was feasible and resulted in a high percentage of R0 resections. Results are particularly encouraging given a majority of patients with borderline resectable disease.
doi:10.1002/cncr.28117
PMCID: PMC4174603  PMID: 23720019
pancreatic cancer; neoadjuvant; gemcitabine; oxaliplatin
17.  Improving the efficacy of chemoradiation with targeted agents 
Cancer discovery  2014;4(3):280-291.
Chemoradiation is the standard therapy for the majority of inoperable, locally advanced cancers. While there is a need to improve chemoradiation efficacy, normal tissue toxicity limits our ability to give additional chemotherapy or higher doses of radiation. Thus, there is excitement about the addition of molecularly targeted agents, which tend to be less toxic than chemotherapy, to chemoradiation regimens. Unfortunately, initial empiric attempts have not been successful. This review will focus on the evidence that supports rational combinations of targeted agents with chemoradiation, with an emphasis on agents that target the DNA damage response and radiation-induced membrane signaling.
doi:10.1158/2159-8290.CD-13-0337
PMCID: PMC3947675  PMID: 24550033
chemoradiation; homologous recombination; targeted agents; sensitization; locally advanced cancer
18.  Phase I Trial of Radiotherapy Concurrent with Twice-Weekly Gemcitabine for Head and Neck Cancer: Translation From Preclinical Investigations Aiming to Improve the Therapeutic Ratio12 
Translational Oncology  2014;7(4):479-483.
BACKGROUND: Once-weekly gemcitabine concurrent with radiotherapy was highly effective in the treatment of head and neck cancer (HNC) but limited by high mucosal toxicity. Pre-clinical investigations suggested that delivering gemcitabine at substantially lower doses twice weekly during radiotherapy improved the therapeutic ratio. We sought to translated these preclinical findings to a phase I trial. METHODS: Twenty-five patients with non-resectable HNC were scheduled to receive gemcitabine twice weekly during the last 2 weeks (total 5 infusions) of hyperfractionated radiotherapy delivering 1.2 Gy twice daily to total 76.8 Gy. Tumor biopsies to measure active intracellular (phosphorylated) gemcitabine were planned after the first drug delivery. Patients were assigned to escalating dose cohorts using the Continuous Reassessment Method. RESULTS: Twenty-one patients evaluable for toxicity were divided into cohorts receiving twice weekly treatment with 10, 20, 33, or 50 mg/m2 gemcitabine. Dose-limiting toxicity was grade 3-4 confluent mucositis/pharyngitis, and the maximally tolerated dose (MTD) was 20 mg/m2. Median survival was 20 months, with no difference between cohorts receiving lower (10, 20 mg/m2) or higher (33, 50 mg/m2) gemcitabine doses. Tumor biopsies after the first drug delivery showed only a minority of tumor cells in the specimens. CONCLUSION: These findings validate preclinical models that show that gemcitabine is radiation sensitizer at doses far below those used for systemic chemotherapy. However, the improvement in the therapeutic ratio predicted from the preclinical study did not translate into a substantial relative increase in the MTD of the drug in the clinical phase I trial.
doi:10.1016/j.tranon.2014.04.016
PMCID: PMC4202797  PMID: 25171890
19.  Sensitization of pancreatic cancer to chemoradiation by the Chk1 inhibitor, MK8776 
Purpose
The combination of radiation with chemotherapy is the most effective therapy for unresectable pancreatic cancer. To improve upon this regimen, we combined the selective Chk1 inhibitor, MK8776 with gemcitabine-based chemoradiation in preclinical pancreatic cancer models.
Experimental Design
We tested the ability of MK8776 to sensitize to gemcitabine-radiation in homologous recombination repair (HRR)- proficient and deficient pancreatic cancer cells and assessed Rad51 focus formation. In vivo, we investigated the efficacy, tumor cell selectivity, and pharmacodynamic biomarkers of sensitization by MK8776.
Results
We found that MK8776 significantly sensitized HRR-proficient (AsPC-1, MiaPaCa-2, BxPC-3) but not deficient (Capan-1) pancreatic cancer cells to gemcitabine-radiation and inhibited Rad51 focus formation in HRR-proficient cells. In vivo, MiaPaCa-2 xenografts were significantly sensitized to gemcitabine-radiation by MK8776 without significant weight loss or observable toxicity in the small intestine, the dose limiting organ for chemoradiation therapy in pancreatic cancer. We also assessed pChk1 (S345), a pharmacodynamic biomarker of DNA damage in response to Chk1 inhibition in both tumor and small intestine and found that MK8776 combined with gemcitabine or gemcitabine-radiation produced a significantly greater increase in pChk1 (S345) in tumor relative to small intestine, suggesting greater DNA damage in tumor than in normal tissue. Furthermore, we demonstrated the utility of an ex vivo platform for assessment of pharmacodynamic biomarkers of Chk1 inhibition in pancreatic cancer.
Conclusions
Together, our results suggest that MK8776 selectively sensitizes HRR-proficient pancreatic cancer cells and xenografts to gemcitabine-radiation and support the clinical investigation of MK8776 in combination with gemcitabine-radiation in locally advanced pancreatic cancer.
doi:10.1158/1078-0432.CCR-12-3748
PMCID: PMC3745540  PMID: 23804422
pancreatic cancer; Chk1; radiosensitization; homologous recombination repair; gemcitabine; cell cycle checkpoint
20.  Inhibition of protein phosphatase 2A radiosensitizes pancreatic cancers by modulating CDC25C/CDK1 and homologous recombination repair 
Purpose
To identify targets whose inhibition may enhance the efficacy of chemoradiation in pancreatic cancer and thus improve survival, we performed an siRNA library screen in pancreatic cancer cells. We investigated PPP2R1A, a scaffolding subunit of protein phosphatase 2A (PP2A) as a lead radiosensitizing target.
Experimental Design
We determined the effect of PP2A inhibition by genetic (PPP2R1A siRNA) and pharmacological (LB100, a small molecule entering Phase I clinical trials) approaches on radiosensitization of Panc-1 and MiaPaCa-2 pancreatic cancer cells both in vitro and in vivo.
Results
PPP2R1A depletion by siRNA radiosensitized Panc-1 and MiaPaCa-2 cells, with radiation enhancement ratios of 1.4 (P<0.05). Likewise, LB100 produced similar radiosensitization in pancreatic cancer cells, but minimal radiosensitization in normal small intestinal cells. Mechanistically, PPP2R1A siRNA or LB100 caused aberrant CDK1 activation, likely resulting from accumulation of the active forms of PLK1 (pPLK1 T210) and CDC25C (pCDC25C T130). Furthermore, LB100 inhibited radiation-induced Rad51 focus formation and homologous recombination repair (HRR), ultimately leading to persistent radiation-induced DNA damage, as reflected by γH2AX expression. Finally, we identified CDC25C as a key PP2A substrate involved in LB100-mediated radiosensitization as depletion of CDC25C partially reversed LB100-mediated radiosensitization. In a mouse xenograft model of human pancreatic cancer, LB100 produced significant radiosensitization with minimal weight loss.
Conclusions
Collectively, our data demonstrate that PP2A inhibition radiosensitizes pancreatic cancer both in vitro and in vivo via activation of CDC25C/CDK1 and inhibition of HRR, and provide proof-of-concept evidence that PP2A is a promising target for the improvement of local therapy in pancreatic cancer.
doi:10.1158/1078-0432.CCR-13-0788
PMCID: PMC3754450  PMID: 23780887
pancreatic cancer; protein phosphatase 2A; radiosensitization; homologous recombination repair
21.  Predictive Models for Regional Hepatic Function Based upon 99mTc-IDA SPECT and Local Radiation Dose for Physiological Adaptive RT 
Purpose
High dose radiation therapy (RT) for intrahepatic cancer is limited by the development of liver injury. This study investigated whether regional hepatic function assessed prior to and during the course of RT using 99mTc-labeled immindodiacetic acid (IDA) SPECT could predict regional liver function reserve after RT.
Methods and Materials
Fourteen patients treated with RT for intrahepatic cancers underwent dynamic 99mTc-IDA SPECT scans prior to RT, during, and one month after completion of RT. Indocyanine green (ICG) tests (a measure of overall liver function) were performed within 1 day of each scan. 3D volumetric hepatic extraction fraction (HEF) images of the liver were estimated by deconvolution analysis. After co-registration of the CT/SPECT and the treatment planning CT, HEF dose-response functions during and post-RT were generated. The volumetric mean of the HEFs in the whole liver was correlated with ICG clearance time. Three models, Dose, Priori and Adaptive models, were developed using multivariate linear regression to assess whether the regional HEFs measured before and during RT helped predict regional hepatic function post-RT.
Results
The mean of the volumetric liver HEFs was significantly correlated with ICG clearance half-life time (r = −0.80, p<0.0001), for all time points. Linear correlations between local doses and regional HEFs one month post-RT were significant in 12 patients. In the priori model, regional HEF post-RT was predicted by the planned dose and regional HEF assessed prior to RT (R=0.71, p<0.0001). In the adaptive model, regional HEF post-RT was predicted by regional HEF re-assessed during RT and the remaining planned local dose (R=0.83, p<0.0001).
Conclusions
99mTc-IDA SPECT obtained during RT could be used to assess regional hepatic function and helped predict post-RT regional liver function reserve. This could support individualized adaptive radiation treatment strategies to maximize tumor control and minimize the risk of liver damage.
doi:10.1016/j.ijrobp.2013.04.007
PMCID: PMC3710542  PMID: 23688813
99mTc-IDA SPECT; Hepatic extraction fraction; intrahepatic cancer; radiation-induced liver disease; radiation toxicity
22.  Low Dose Rate Radiosensitization of Hepatocellular Carcinoma In Vitro and in Patients1 
Translational Oncology  2014;7(4):472-478.
Transarterial radioembolization (TARE) with 90Y microspheres delivers low dose rate radiation (LDR) to intrahepatic tumors. In the current study, we examined clonogenic survival, DNA damage, and cell cycle distribution in hepatocellular carcinoma (HCC) cell lines treated with LDR in combination with varying doses and schedules of 5-fluorouracil (5-FU), gemcitabine, and sorafenib. Radiosensitization was seen with 1 to 3 μM 5-FU (enhancement ratio 2.2–13.9) and 30 to 100 nM gemcitabine (enhancement ratio 1.9–2.9) administered 24 hours before LDR (0.26 Gy/h to 4.2 Gy). Sorafenib radiosensitized only at high concentrations (3–10 μM) when administered after LDR. For a given radiation dose, greater enhancement was seen with LDR compared to standard dose rate therapy. Summarizing our clinical experience with low dose rate radiosensitization, 13 patients (5 with HCC, 8 with liver metastases) were treated a total of 16 times with TARE and concurrent gemcitabine. Six partial responses and one complete response were observed with a median time to local failure of 7.1 months for all patients and 9.9 months for patients with HCC. In summary, HCC is sensitized to LDR with clinically achievable concentrations of gemcitabine and 5-FU in vitro. Encouraging responses were seen in a small cohort of patients treated with TARE and concurrent gemcitabine. Future studies are needed to validate the safety and efficacy of this approach.
doi:10.1016/j.tranon.2014.05.006
PMCID: PMC4202782  PMID: 24956939
hepatocellular carcinoma; yttrium-90 microspheres; transarterial radioembolization; radiosensitization
23.  Opportunities and Challenges in the Era of Molecularly Targeted Agents and Radiation Therapy 
The first annual workshop for preclinical and clinical development of radiosensitizers took place at the National Cancer Institute on August 8–9, 2012. Radiotherapy is one of the most commonly applied and effective oncologic treatments for solid tumors. It is well recognized that improved clinical efficacy of radiotherapy would make a substantive impact in clinical practice and patient outcomes. Advances in genomic technologies and high-throughput drug discovery platforms have brought a revolution in cancer treatment by providing molecularly targeted agents for various cancers. Development of predictive biomarkers directed toward specific subsets of cancers has ushered in a new era of personalized therapeutics. The field of radiation oncology stands to gain substantial benefit from these advances given the concerted effort to integrate this progress into radiation therapy. This workshop brought together expert clinicians and scientists working in various disease sites to identify the exciting opportunities and expected challenges in the development of molecularly targeted agents in combination with radiation therapy.
doi:10.1093/jnci/djt055
PMCID: PMC3733308  PMID: 23503600
24.  Prognostic Significance of Carbohydrate Antigen 19-9 in Unresectable Locally Advanced Pancreatic Cancer Treated With Dose-Escalated Intensity Modulated Radiation Therapy and Concurrent Full-Dose Gemcitabine: Analysis of a Prospective Phase 1/2 Dose Escalation Study 
Purpose
Although established in the postresection setting, the prognostic value of carbohydrate antigen 19-9 (CA19-9) in unresectable locally advanced pancreatic cancer (LAPC) is less clear. We examined the prognostic utility of CA19-9 in patients with unresectable LAPC treated on a prospective trial of intensity modulated radiation therapy (IMRT) dose escalation with concurrent gemcitabine.
Methods and Materials
Forty-six patients with unresectable LAPC were treated at the University of Michigan on a phase 1/2 trial of IMRT dose escalation with concurrent gemcitabine. CA19-9 was obtained at baseline and during routine follow-up. Cox models were used to assess the effect of baseline factors on freedom from local progression (FFLP), distant progression (FFDP), progression-free survival (PFS), and overall survival (OS). Stepwise forward regression was used to build multivariate predictive models for each endpoint.
Results
Thirty-eight patients were eligible for the present analysis. On univariate analysis, baseline CA19-9 and age predicted OS, CA19-9 at baseline and 3 months predicted PFS, gross tumor volume (GTV) and black race predicted FFLP, and CA19-9 at 3 months predicted FFDP. On stepwise multivariate regression modeling, baseline CA19-9, age, and female sex predicted OS; baseline CA19-9 and female sex predicted both PFS and FFDP; and GTV predicted FFLP. Patients with baseline CA19-9 ≤90 U/mL had improved OS (median 23.0 vs 11.1 months, HR 2.88, P<.01) and PFS (14.4 vs 7.0 months, HR 3.61, P = .001). CA19-9 progression over 90 U/mL was prognostic for both OS (HR 3.65, P = .001) and PFS (HR 3.04, P = .001), and it was a stronger predictor of death than either local progression (HR 1.46, P = .42) or distant progression (HR 3.31, P = .004).
Conclusions
In patients with unresectable LAPC undergoing definitive chemoradiation therapy, baseline CA19-9 was independently prognostic even after established prognostic factors were controlled for, whereas CA19-9 progression strongly predicted disease progression and death. Future trials should stratify by baseline CA19-9 and incorporate CA19-9 progression as a criterion for progressive disease.
doi:10.1016/j.ijrobp.2012.11.020
PMCID: PMC3646633  PMID: 23265573
25.  Physiological Imaging-Defined Response-Driven Subvolumes of a Tumor1 
Summary
Dose painting of the physiological imaging-defined subvolumes of the tumors by intensity-modulated radiotherapy is hypothesized to lead to a better outcome than distributing a uniform dose within a target volume defined by anatomic imaging. We developed a general method to delineate the subvolumes of a tumor based upon multiple physiological imaging and tested their complementary roles for assessment of therapy response.
Purpose
To develop an image analysis framework to delineate the physiological imaging-defined subvolumes of a tumor in relating to treatment response and outcome.
Materials and Methods
Our proposed approach delineates the subvolumes of a tumor based upon its heterogeneous distributions of physiological imaging parameters. The method assigns each voxel a probabilistic membership function belonging to the physiological parameter classes defined in a sample of tumors, and then calculates the related subvolumes in each tumor. We applied our approach to regional cerebral blood volume (rCBV) and Gd-DTPA transfer constant (Ktrans) images of patients who had brain metastases and were treated by whole brain radiotherapy (WBRT). Forty-five lesions were included in the analysis. Changes in the rCBV (or Ktrans)-defined subvolumes of the tumors from pre RT to 2 weeks (2W) after the start of WBRT were evaluated for differentiation of responsive, stable and progressive tumors using Mann-Whitney U test. Performance of the newly developed metrics for predicting tumor response to WBRT was evaluated by Receiver Operating Characteristic (ROC) analysis.
Results
The percentage decrease in the high-CBV defined subvolumes of the tumors from pre-RT to 2W was significantly greater in the group of responsive tumors than the group of stable and progressive ones (p<0.007). The change in the high-CBV defined subvolumes of the tumors from pre-RT to 2W was a predictor for post-RT response significantly better than change in the gross tumor volume observed during the same time interval (p=0.012), suggesting the physiological change occurs prior to the volumetric change. Also, Ktrans did not add significant discriminatory information for assessing response with respect to rCBV.
Conclusion
The physiological imaging-defined subvolumes of the tumors delineated by our method could be a candidate for boost target, for which further development and evaluation is warranted.
doi:10.1016/j.ijrobp.2012.10.036
PMCID: PMC3638951  PMID: 23257692

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