Belen Rubio-Viqueira

An in vivo platform for translational drug development in pancreatic cancer

Effective development of targeted anticancer agents includes the definition of the optimal biological dose and biomarkers of drug activity. Currently available preclinical models are not optimal to this end. We aimed at generating a model for translational drug development using pancreatic cancer as a prototype. Resected pancreatic cancers from 14 patients were xenografted and expanded in successive groups of nude mice to develop cohorts of tumor-bearing mice suitable for drug therapy in simulated early clinical trials. The xenografted tumors maintain their fundamental genotypic features despite serial passages and recapitulate the genetic heterogeneity of pancreatic cancer. The in vivo platform is useful for integrating drug screening with biomarker discovery. Passages of tumors in successive cohorts of mice do not change their susceptibility to anticancer agents and represent a perpetual live bank, facilitating the application of new technologies that will result in the creation of an integrated stable database of tumor-drug response data and biomarkers.

A Pilot Clinical Study of Treatment Guided by Personalized Tumorgrafts in Patients with Advanced Cancer

Patients with many advanced solid cancers have very poor prognosis, and improvements in life expectancy are measured only in months. We have recently reported the remarkable clinical outcome of a patient with advanced, gemcitabine-resistant, pancreatic cancer who was later treated with DNA-damaging agents, on the basis of the observation of significant activity of this class of drugs against a personalized tumorgraft generated from the patients surgically resected tumor. Here, we extend the approach to patients with other advanced cancers. Tumors resected from 14 patients with refractory advanced cancers were propagated in immunodeficient mice and treated with 63 drugs in 232 treatment regimens. An effective treatment regimen in the xenograft model was identified for 12 patients. One patient died before receiving treatment, and the remaining 11 patients received 17 prospectively guided treatments. Fifteen of these treatments resulted in durable partial remissions. In 2 subjects, no effective treatments were found. Overall, there was a remarkable correlation between drug activity in the model and clinical outcome, both in terms of resistance and sensitivity. The data support the use of the personalized tumorgraft model as a powerful investigational platform for therapeutic decision making and to efficiently guide cancer treatment in the clinic. Mol Cancer Ther; 10(8); 1311–6. ©2011 AACR.

A direct pancreatic cancer xenograft model as a platform for cancer stem cell therapeutic development

There is an enormous gap between the antiproliferative and in vivo antitumor efficacy of gemcitabine in cell line-based models and its clinical efficacy. This may be due to insensitiveness of the precursor, cancer stem cell (CSC) compartment to cytotoxic agents. The hedgehog pathway is associated with CSC signaling and control. We used a direct xenograft model of pancreatic cancer and a two-stage approach was used to test the hypotheses that targeting CSC could increase the efficacy of gemcitabine. Tumors from a gemcitabine-sensitive xenograft were treated with gemcitabine first, and randomized, after tumor regression to continuing treatment with gemcitabine, a hedgehog inhibitor alone or in combination with gemcitabine. We tested markers described as associated with CSC such as CD24, CD44, ALDH, nestin, and the hedgehog pathway. After induction with gemcitabine, treated tumor showed an enrichment in CSC markers such as ALDH and CD24. Subsequently, a release from gemcitabine prompted a repopulation of proliferating cells and a decrease in such markers to equilibrate from pretreatment levels. Combined treatment with gemcitabine and cyclopamine induced tumor regression and decrease in CSC markers and hedgehog signaling. Cytoplasmic CD24 and ALDH were inversely and strongly associated with growth and were expressed in a minority of cells that we propose constitute the CSC compartment. Hedgehog inhibitors as part of a dual compartment therapeutic approach were able to further reduce tumor growth and decreased both static and dynamic markers of CSC. Direct tumor xenografts are a valid platform to test multicompartment therapeutic approaches in pancreatic cancer. [Mol Cancer Ther 2009;8(2):310–4]

Tumor Engraftment in Nude Mice and Enrichment in Stroma- Related Gene Pathways Predict Poor Survival and Resistance to Gemcitabine in Patients with Pancreatic Cancer

Purpose: The goal of this study was to evaluate prospectively the engraftment rate, factors influencing engraftment, and predictability of clinical outcome of low-passage xenografts from patients with resectable pancreatic ductal adenocarcinoma (PDA) and to establish a bank of PDA xenografts. Experimental Design: Patients with resectable PDA scheduled for resection at the Johns Hopkins Hospital were eligible. Representative pieces of tumor were implanted in nude mice. The status of the SMAD4 gene and content of tumor-generating cells were determined by immunohistochemistry. Gene expression was carried out by using a U133 Plus 2.0 array. Patients were followed for progression and survival. Results: A total of 94 patients with PDA were resected, 69 tumors implanted in nude mice, and 42 (61%) engrafted. Engrafted carcinomas were more often SMAD4 mutant, and had a metastatic gene expression signature and worse prognosis. Tumors from patients resistant to gemcitabine were enriched in stroma-related gene pathways. Tumors sensitive to gemcitabine were enriched in cell cycle and pyrimidine gene pathways. The time to progression for patients who received treatment with gemcitabine for metastatic disease (n = 7) was double in patients with xenografts sensitive to gemcitabine. Conclusion: A successful xenograft was generated in 61% of patients attempted, generating a pool of 42 PDA xenografts with significant biological information and annotated clinical data. Patients with PDA and SMAD4 inactivation have a better engraftment rate. Engraftment is a poor prognosis factor, and engrafted tumors have a metastatic gene expression signature. Tumors from gemcitabine-resistant patients were enriched in stromal pathways. Clin Cancer Res; 17(17); 5793–800. ©2011 AACR.

Characterizing DNA methylation patterns in pancreatic cancer genome

We performed a global methylation profiling assay on 1505 CpG sites across 807 genes to characterize DNA methylation patterns in pancreatic cancer genome. We found 289 CpG sites that were differentially methylated in normal pancreas, pancreatic tumors and cancer cell lines. We identified 23 and 35 candidate genes that are regulated by hypermethylation and hypomethylation in pancreatic cancer, respectively. We also identified candidate methylation markers that alter the expression of genes critical to gemcitabine susceptibility in pancreatic cancer. These results indicate that aberrant DNA methylation is a frequent epigenetic event in pancreatic cancer; and by using global methylation profiling assay, it is possible to identify these markers for diagnostic and therapeutic purposes in this disease.

Epidermal growth factor receptor dynamics influences response to epidermal growth factor receptor targeted agents

Analysis of gene expression of cancer cell lines exposed to erlotinib, a small molecule inhibitor of the epidermal growth factor receptor (EGFR), showed a marked increase in EGFR mRNA in resistant cell lines but not in susceptible ones. Because cetuximab induces EGFR down-regulation, we explored the hypothesis that treatment with cetuximab would interfere with erlotinib-induced EGFR up-regulation and result in antitumor effects. Exposure of the resistant biliary tract cancer cell line HuCCT1 but not the susceptible A431 epidermoid cell line to erlotinib induced EGFR mRNA and protein expression. Combined treatment with cetuximab blunted the erlotinib-induced EGFR up-regulation and resulted in inhibition of cell proliferation and apoptosis in the HuCCT1 cells. Blockage of erlotinib-induced EGFR synthesis in HuCCT1 cells by small interfering RNA resulted in identical antitumor effects as cetuximab, providing mechanistic specificity. In mice xenografted with A431, HuCCT1, and the pancreatic cancer cell line Panc430, maximal growth arrest and decrease in Ki67 proliferation index were documented with combined therapy, and EGFR down-regulation was observed in cetuximab-treated tumors. These results may indicate that resistance to EGFR kinase inhibition may be, at least in part, mediated by a highly dynamic feedback loop consisting of up-regulation of the EGFR upon exposure to EGFR kinase inhibitors. Abrogation of this response by small interfering RNA-mediated EGFR mRNA down-regulation and/or by cetuximab-mediated protein clearance induced tumor arrest across several cancer models with different EGFR expression levels, suggesting that resistance and sensitivity are dynamic events where proportional decrease in the target rather than absolute content dictates outcome.

Coordinated epidermal growth factor receptor pathway gene overexpression predicts epidermal growth factor receptor inhibitor sensitivity in pancreatic cancer.

The epidermal growth factor receptor (EGFR) inhibitor erlotinib is approved for treatment of pancreatic cancer but the overall activity is minimal, and known predictive factors for EGFR inhibitor efficacy are infrequent in this disease. We tested the hypothesis that global activation of the EGFR pathway is predictive of EGFR inhibitor efficacy. Pancreatic cancer tumors directly xenografted at surgery were treated with the EGFR inhibitors erlotinib and cetuximab and analyzed for biological features. Two of 10 tumors were sensitive, and by global gene expression profiling with gene set enrichment analysis, the EGFR pathway was highly expressed in sensitive compared with resistant tumors. The core gene components driving EGFR pathway overexpression were pathway ligands and positive effectors. In a prospective validation, the EGFR pathway-based signature correctly predicted anti-EGFR treatment response in eight additional tumors and was not predictive of response to gemcitabine and CI1040 (a MEK inhibitor). Analysis of EGFR, KRAS, and PIK3CA mutations and gene amplification by fluorescence in situ hybridization and multiplex ligation-dependent probe amplification showed that none of these genetic abnormalities were neither predictive nor responsible for the EGFR pathway activation. Coordinated overexpression of the EGFR pathway predicts susceptibility to EGFR inhibitors in pancreatic cancer. These results suggest a phenomenon of pathway addiction and support the value of unbiased system biology approaches in drug development.

Inhibition of Ataxia Telangiectasia‐ and Rad3 ‐Related Function Abrogates the In Vitro and In Vivo Tumorigenicity of Human Colon Cancer Cells Through Depletion of the CD133+ Tumor‐Initiating Cell Fraction

The identification of novel approaches to specifically target the DNA‐damage checkpoint response in chemotherapy‐resistant cancer stem cells (CSC) of solid tumors has recently attracted great interest. We show here in colon cancer cell lines and primary colon cancer cells that inhibition of checkpoint‐modulating phosphoinositide 3‐kinase‐related (PIK) kinases preferentially depletes the chemoresistant and exclusively tumorigenic CD133+ cell fraction. We observed a time‐ and dose‐dependent disproportionally pronounced loss of CD133+ cells and the consecutive lack of in vitro and in vivo tumorigenicity of the remaining cells. Depletion of CD133+ cells was initiated through apoptosis of cycling CD133+ cells and further substantiated through subsequent recruitment of quiescent CD133+ cells into the cell cycle followed by their elimination. Models using specific PIK kinase inhibitors, somatic cell gene targeting, and RNA interference demonstrated that the observed detrimental effects of caffeine on CSC were attributable specifically to the inhibition of the PIK kinase ataxia telangiectasia‐ and Rad3‐related (ATR). Mechanistically, phosphorylation of CHK1 checkpoint homolog (S. pombe; CHK1) was significantly enhanced in CD133+ as compared with CD133− cells on treatment with DNA interstrand‐crosslinking (ICL) agents, indicating a preferential activation of the ATR/CHK1‐dependent DNA‐damage response in tumorigenic CD133+ cells. Consistently, the chemoresistance of CD133+ cells toward DNA ICL agents was overcome through inhibition of ATR/CHK1‐signaling. In conclusion, our study illustrates a novel target to eliminate the tumorigenic CD133+ cell population in colon cancer and provides another rationale for the development of specific ATR‐inhibitors. STEM CELLS 2011;29:418–429

A Comparison of EGFR Mutation Testing Methods in Lung Carcinoma: Direct Sequencing, Real-time PCR and Immunohistochemistry

The objective of this study is to compare two EGFR testing methodologies (a commercial real-time PCR kit and a specific EGFR mutant immunohistochemistry), with direct sequencing and to investigate the limit of detection (LOD) of both PCR-based methods. We identified EGFR mutations in 21 (16%) of the 136 tumours analyzed by direct sequencing. Interestingly, the Therascreen EGFR Mutation Test kit was able to characterize as wild-type one tumour that could not be analyzed by direct sequencing of the PCR product. We then compared the LOD of the kit and that of direct sequencing using the available mutant tumours. The kit was able to detect the presence of a mutation in a 1% dilution of the total DNA in nine of the 18 tumours (50%), which tested positive with the real-time quantitative PCR method. In all cases, EGFR mutation was identified at a dilution of 5%. Where the mutant DNA represented 30% of the total DNA, sequencing was able to detect mutations in 12 out of 19 cases (63%). Additional experiments with genetically defined standards (EGFR ΔE746-A750/+ and EGFR L858R/+) yielded similar results. Immunohistochemistry (IHC) staining with exon 19-specific antibody was seen in eight out of nine cases with E746-A750del detected by direct sequencing. Neither of the two tumours with complex deletions were positive. Of the five L858R-mutated tumours detected by the PCR methods, only two were positive for the exon 21-specific antibody. The specificity was 100% for both antibodies. The LOD of the real-time PCR method was lower than that of direct sequencing. The mutation specific IHC produced excellent specificity.

Integrated preclinical and clinical development of mTOR inhibitors in pancreatic cancer

Background:The purpose of this work was to determine the efficacy of inhibiting mammalian target of rapamycin (mTOR) in pancreatic cancer preclinical models and translate preclinical observations to the clinic.Methods:Temsirolimus (20 mg Kg−1 daily) was administered to freshly generated pancreatic cancer xenografts. Tumour growth inhibition was determined after 28 days. Xenografts were characterised at baseline by gene expression and comparative genomic hybridisation. Patients with advanced, gemcitabine-resistant pancreatic cancer were treated with sirolimus (5 mg daily). The primary end point was 6-month survival rate (6mSR). Correlative studies included immunohistochemistry assessment of pathway expression in baseline tumours, drug pharmacokinetics (PKs), response assessment by FDG-PET and pharmacodynamic effects in peripheral-blood mononuclear cells (PBMCs).Results:In all, 4 of 17 xenografts (23%) responded to treatment. Sensitive tumours were characterised by gene copy number variations and overexpression of genes leading to activation of the PI3K/Akt/mTOR pathway. Activation of p70S6K correlated with drug activity in the preclinical studies. Sirolimus was well tolerated in the clinic, showed predictable PKs, exerted pathway inhibition in post-treatment PBMCs and resulted in a 6mSR of 26%. No correlation, however, was found between activated p70S6K in tumour tissues and anti-tumour effects.Conclusion:Sirolimus activity in pancreatic cancer was marginal and not predicted by the selected biomarker.