Publications by authors named "David S Hsu"

40 Publications

Characterization of a castrate-resistant prostate cancer xenograft derived from a patient of West African ancestry.

Prostate Cancer Prostatic Dis 2021 Oct 13. Epub 2021 Oct 13.

Department of Medicine, Division of Medical Oncology, Duke University School of Medicine, Durham, NC, 27710, USA.

Background: Prostate cancer is a clinically and molecularly heterogeneous disease, with highest incidence and mortality among men of African ancestry. To date, prostate cancer patient-derived xenograft (PCPDX) models to study this disease have been difficult to establish because of limited specimen availability and poor uptake rates in immunodeficient mice. Ancestrally diverse PCPDXs are even more rare, and only six PCPDXs from self-identified African American patients from one institution were recently made available.

Methods: In the present study, we established a PCPDX from prostate cancer tissue from a patient of estimated 90% West African ancestry with metastatic castration resistant disease, and characterized this model's pathology, karyotype, hotspot mutations, copy number, gene fusions, gene expression, growth rate in normal and castrated mice, therapeutic response, and experimental metastasis.

Results: This PCPDX has a mutation in TP53 and loss of PTEN and RB1. We have documented a 100% take rate in mice after thawing the PCPDX tumor from frozen stock. The PCPDX is castrate- and docetaxel-resistant and cisplatin-sensitive, and has gene expression patterns associated with such drug responses. After tail vein injection, the PCPDX tumor cells can colonize the lungs of mice.

Conclusion: This PCPDX, along with others that are established and characterized, will be useful pre-clinically for studying the heterogeneity of prostate cancer biology and testing new therapeutics in models expected to be reflective of the clinical setting.
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http://dx.doi.org/10.1038/s41391-021-00460-yDOI Listing
October 2021

Circadian clock, carcinogenesis, chronochemotherapy connections.

J Biol Chem 2021 09 8;297(3):101068. Epub 2021 Aug 8.

Department of Biochemistry and Biophysics, University of North Carolina School of Medicine, Chapel Hill, North Carolina, USA. Electronic address:

The circadian clock controls the expression of nearly 50% of protein coding genes in mice and most likely in humans as well. Therefore, disruption of the circadian clock is presumed to have serious pathological effects including cancer. However, epidemiological studies on individuals with circadian disruption because of night shift or rotating shift work have produced contradictory data not conducive to scientific consensus as to whether circadian disruption increases the incidence of breast, ovarian, prostate, or colorectal cancers. Similarly, genetically engineered mice with clock disruption do not exhibit spontaneous or radiation-induced cancers at higher incidence than wild-type controls. Because many cellular functions including the cell cycle and cell division are, at least in part, controlled by the molecular clock components (CLOCK, BMAL1, CRYs, PERs), it has also been expected that appropriate timing of chemotherapy may increase the efficacy of chemotherapeutic drugs and ameliorate their side effect. However, empirical attempts at chronochemotherapy have not produced beneficial outcomes. Using mice without and with human tumor xenografts, sites of DNA damage and repair following treatment with the anticancer drug cisplatin have been mapped genome-wide at single nucleotide resolution and as a function of circadian time. The data indicate that mechanism-based studies such as these may provide information necessary for devising rational chronochemotherapy regimens.
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http://dx.doi.org/10.1016/j.jbc.2021.101068DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8403766PMC
September 2021

A Comparative Oncology Drug Discovery Pipeline to Identify and Validate New Treatments for Osteosarcoma.

Cancers (Basel) 2020 Nov 11;12(11). Epub 2020 Nov 11.

Department of Medicine, Duke University Medical Center, Durham, NC 27710, USA.

Background: Osteosarcoma is a rare but aggressive bone cancer that occurs primarily in children. Like other rare cancers, treatment advances for osteosarcoma have stagnated, with little improvement in survival for the past several decades. Developing new treatments has been hampered by extensive genomic heterogeneity and limited access to patient samples to study the biology of this complex disease.

Methods: To overcome these barriers, we combined the power of comparative oncology with patient-derived models of cancer and high-throughput chemical screens in a cross-species drug discovery pipeline.

Results: Coupling in vitro high-throughput drug screens on low-passage and established cell lines with in vivo validation in patient-derived xenografts we identify the proteasome and CRM1 nuclear export pathways as therapeutic sensitivities in osteosarcoma, with dual inhibition of these pathways inducing synergistic cytotoxicity.

Conclusions: These collective efforts provide an experimental framework and set of new tools for osteosarcoma and other rare cancers to identify and study new therapeutic vulnerabilities.
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http://dx.doi.org/10.3390/cancers12113335DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7696249PMC
November 2020

A Precision Medicine Drug Discovery Pipeline Identifies Combined CDK2 and 9 Inhibition as a Novel Therapeutic Strategy in Colorectal Cancer.

Mol Cancer Ther 2020 12 6;19(12):2516-2527. Epub 2020 Nov 6.

Department of Medicine, Division of Medical Oncology, Duke University Medical Center, Durham, North Carolina.

Colorectal cancer is the third most common cancer in the United States and responsible for over 50,000 deaths each year. Therapeutic options for advanced colorectal cancer are limited, and there remains an unmet clinical need to identify new treatments for this deadly disease. To address this need, we developed a precision medicine pipeline that integrates high-throughput chemical screens with matched patient-derived cell lines and patient-derived xenografts (PDX) to identify new treatments for colorectal cancer. High-throughput screens of 2,100 compounds were performed across six low-passage, patient-derived colorectal cancer cell lines. These screens identified the CDK inhibitor drug class among the most effective cytotoxic compounds across six colorectal cancer lines. Among this class, combined targeting of CDK1, 2, and 9 was the most effective, with ICs ranging from 110 nmol/L to 1.2 μmol/L. Knockdown of CDK9 in the presence of a CDK2 inhibitor (CVT-313) showed that CDK9 knockdown acted synergistically with CDK2 inhibition. Mechanistically, dual CDK2/9 inhibition induced significant G-M arrest and anaphase catastrophe. Combined CDK2/9 inhibition synergistically reduced PDX tumor growth. Our precision medicine pipeline provides a robust screening and validation platform to identify promising new cancer therapies. Application of this platform to colorectal cancer pinpointed CDK2/9 dual inhibition as a novel combinatorial therapy to treat colorectal cancer.
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http://dx.doi.org/10.1158/1535-7163.MCT-20-0454DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7718319PMC
December 2020

An Aptamer for Broad Cancer Targeting and Therapy.

Cancers (Basel) 2020 Oct 31;12(11). Epub 2020 Oct 31.

Department of Surgery, Duke Cancer Institute, Duke University School of Medicine, Durham, NC 27710, USA.

Recent advances in chemotherapy treatments are increasingly targeted therapies, with the drug conjugated to an antibody able to deliver it directly to the tumor. As high-affinity chemical ligands that are much smaller in size, aptamers are ideal for this type of drug targeting. Aptamer-highly toxic drug conjugates (ApTDCs) based on the E3 aptamer, selected on prostate cancer cells, target and inhibit prostate tumor growth in vivo. Here, we observe that E3 also broadly targets numerous other cancer types, apparently representing a universal aptamer for cancer targeting. Accordingly, ApTDCs formed by conjugation of E3 to the drugs monomethyl auristatin E (MMAE) or monomethyl auristatin F (MMAF) efficiently target and kill a range of different cancer cells. Notably, this targeting extends to both patient-derived explant (PDX) cancer cell lines and tumors, with the E3 MMAE and MMAF conjugates inhibiting PDX cell growth in vitro and with the E3 aptamer targeting PDX colorectal tumors in vivo.
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http://dx.doi.org/10.3390/cancers12113217DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7694147PMC
October 2020

Development of a precision medicine pipeline to identify personalized treatments for colorectal cancer.

BMC Cancer 2020 Jun 24;20(1):592. Epub 2020 Jun 24.

Department of Medicine, Division of Medical Oncology, Duke University Medical Center, 3008 Snyderman Building, 905 S. LaSalle St., Durham, NC, 27710, USA.

Background: Metastatic colorectal cancer (CRC) continues to be a major health problem, and current treatments are primarily for disease control and palliation of symptoms. In this study, we developed a precision medicine strategy to discover novel therapeutics for patients with CRC.

Methods: Six matched low-passage cell lines and patient-derived xenografts (PDX) were established from CRC patients undergoing resection of their cancer. High-throughput drug screens using a 119 FDA-approved oncology drug library were performed on these cell lines, which were then validated in vivo in matched PDXs. RNA-Seq analysis was then performed to identify predictors of response.

Results: Our study revealed marked differences in response to standard-of-care agents across patients and pinpointed druggable pathways to treat CRC. Among these pathways co-targeting of fibroblast growth factor receptor (FGFR), SRC, platelet derived growth factor receptor (PDGFR), or vascular endothelial growth factor receptor (VEGFR) signaling was found to be an effective strategy. Molecular analyses revealed potential predictors of response to these druggable pathways.

Conclusions: Our data suggests that the use of matched low-passage cell lines and PDXs is a promising strategy to identify new therapies and pathways to treat metastatic CRC.
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http://dx.doi.org/10.1186/s12885-020-07090-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7313200PMC
June 2020

Preclinical Testing of a Novel Niclosamide Stearate Prodrug Therapeutic (NSPT) Shows Efficacy Against Osteosarcoma.

Mol Cancer Ther 2020 07 5;19(7):1448-1461. Epub 2020 May 5.

Department of Orthopaedic Surgery, Duke University Medical Center, Durham, North Carolina.

Therapeutic advances for osteosarcoma have stagnated over the past several decades, leading to an unmet clinical need for patients. The purpose of this study was to develop a novel therapy for osteosarcoma by reformulating and validating niclosamide, an established anthelminthic agent, as a niclosamide stearate prodrug therapeutic (NSPT). We sought to improve the low and inefficient clinical bioavailability of oral dosing, especially for the relatively hydrophobic classes of anticancer drugs. Nanoparticles were fabricated by rapid solvent shifting and verified using dynamic light scattering and UV-vis spectrophotometry. NSPT efficacy was then studied for cell viability, cell proliferation, and intracellular signaling by Western blot analysis; pulmonary metastatic assay model; and pharmacokinetic and lung mouse metastatic model of osteosarcoma. NSPT formulation stabilizes niclosamide stearate against hydrolysis and delays enzymolysis; increases circulation with approximately 5 hours; reduces cell viability and cell proliferation in human and canine osteosarcoma cells at 0.2-2 μmol/L IC; inhibits recognized growth pathways and induces apoptosis at 20 μmol/L; eliminates metastatic lesions in the lung metastatic model; and when injected intravenously at 50 mg/kg weekly, it prevents metastatic spread in the lungs in a mouse model of osteosarcoma over 30 days. In conclusion, niclosamide was optimized for preclinical drug delivery as a unique prodrug nanoparticle injected intravenously at 50 mg/kg (1.9 mmol/L). This increased bioavailability of niclosamide in the blood stream prevented metastatic disease in the mouse. This chemotherapeutic strategy is now ready for canine trials, and if successful, will be targeted for human trials in patients with osteosarcoma.
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http://dx.doi.org/10.1158/1535-7163.MCT-19-0689DOI Listing
July 2020

Genome-wide single-nucleotide resolution of oxaliplatin-DNA adduct repair in drug-sensitive and -resistant colorectal cancer cell lines.

J Biol Chem 2020 05 16;295(22):7584-7594. Epub 2020 Apr 16.

Department of Biochemistry and Biophysics, University of North Carolina School of Medicine, Chapel Hill, North Carolina 27599-7260

Platinum-based chemotherapies, including oxaliplatin, are a mainstay in the management of solid tumors and induce cell death by forming intrastrand dinucleotide DNA adducts. Despite their common use, they are highly toxic, and approximately half of cancer patients have tumors that are either intrinsically resistant or develop resistance. Previous studies suggest that this resistance is mediated by variations in DNA repair levels or net drug influx. Here, we aimed to better define the roles of nucleotide excision repair and DNA damage in platinum chemotherapy resistance by profiling DNA damage and repair efficiency in seven oxaliplatin-sensitive and three oxaliplatin-resistant colorectal cancer cell lines. We assayed DNA repair indirectly as toxicity and directly measured bulky adduct formation and removal from the genome by slot blot and repair capacity in an excision assay, and used excision repair sequencing (XR-seq) to map repair events genome-wide at single-nucleotide resolution. Using this combinatorial approach and proxies for oxaliplatin-DNA damage, we observed no significant differences in repair efficiency that could explain the relative sensitivities and chemotherapy resistances of these cell lines. In contrast, the levels of oxaliplatin-induced DNA damage were significantly lower in the resistant cells, indicating that decreased damage formation, rather than increased damage repair, is a major determinant of oxaliplatin resistance in these cell lines. XR-seq-based analysis of gene expression revealed up-regulation of membrane transport pathways in the resistant cells, and these pathways may contribute to resistance. In conclusion, additional research is needed to characterize the factors mitigating cellular DNA damage formation by platinum compounds.
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http://dx.doi.org/10.1074/jbc.RA120.013347DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7261789PMC
May 2020

Single-Cell RNA Sequencing Identifies Yes-Associated Protein 1-Dependent Hepatic Mesothelial Progenitors in Fibrolamellar Carcinoma.

Am J Pathol 2020 01 24;190(1):93-107. Epub 2019 Oct 24.

Division of Gastroenterology, Department of Medicine, Duke University, Durham, North Carolina. Electronic address:

Fibrolamellar carcinoma (FLC) is characterized by in-frame fusion of DnaJ heat shock protein family (Hsp40) member B1 (DNAJB1) with protein kinase cAMP-activated catalytic subunit α (PRKACA) and by dense desmoplasia. Surgery is the only effective treatment because mechanisms supporting tumor survival are unknown. We used single-cell RNA sequencing to characterize a patient-derived FLC xenograft model and identify therapeutic targets. Human FLC cells segregated into four discrete clusters that all expressed the oncogene Yes-associated protein 1 (YAP1). The two communities most enriched with cells coexpressing FLC markers [CD68, A-kinase anchoring protein 12 (AKAP12), cytokeratin 7, epithelial cell adhesion molecule (EPCAM), and carbamoyl palmitate synthase-1] also had the most cells expressing YAP1 and its proproliferative target genes (AREG and CCND1), suggesting these were proliferative FLC cell clusters. The other two clusters were enriched with cells expressing profibrotic YAP1 target genes, ACTA2, ELN, and COL1A1, indicating these were fibrogenic FLC cells. All clusters expressed the YAP1 target gene and mesothelial progenitor marker mesothelin, and many mesothelin-positive cells coexpressed albumin. Trajectory analysis predicted that the four FLC communities were derived from a single cell type transitioning among phenotypic states. After establishing a novel FLC cell line that harbored the DNAJB1-PRKACA fusion, YAP1 was inhibited, which significantly reduced expression of known YAP1 target genes as well as cell growth and migration. Thus, both FLC epithelial and stromal cells appear to arise from DNAJB1-PRKACA fusion in a YAP1-dependent liver mesothelial progenitor, identifying YAP1 as a target for FLC therapy.
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http://dx.doi.org/10.1016/j.ajpath.2019.09.018DOI Listing
January 2020

Integrated chromatin and transcriptomic profiling of patient-derived colon cancer organoids identifies personalized drug targets to overcome oxaliplatin resistance.

Genes Dis 2021 Mar 29;8(2):203-214. Epub 2019 Oct 29.

Department of Biomedical Engineering, Pratt School of Engineering, Duke University, Durham, NC, 27708, USA.

Colorectal cancer is a leading cause of cancer deaths. Most colorectal cancer patients eventually develop chemoresistance to the current standard-of-care therapies. Here, we used patient-derived colorectal cancer organoids to demonstrate that resistant tumor cells undergo significant chromatin changes in response to oxaliplatin treatment. Integrated transcriptomic and chromatin accessibility analyses using ATAC-Seq and RNA-Seq identified a group of genes associated with significantly increased chromatin accessibility and upregulated gene expression. CRISPR/Cas9 silencing of fibroblast growth factor receptor 1 (FGFR1) and oxytocin receptor (OXTR) helped overcome oxaliplatin resistance. Similarly, treatment with oxaliplatin in combination with an FGFR1 inhibitor (PD166866) or an antagonist of OXTR (L-368,899) suppressed chemoresistant organoids. However, oxaliplatin treatment did not activate either FGFR1 or OXTR expression in another resistant organoid, suggesting that chromatin accessibility changes are patient-specific. The use of patient-derived cancer organoids in combination with transcriptomic and chromatin profiling may lead to precision treatments to overcome chemoresistance in colorectal cancer.
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http://dx.doi.org/10.1016/j.gendis.2019.10.012DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8099686PMC
March 2021

Dietary methionine influences therapy in mouse cancer models and alters human metabolism.

Nature 2019 08 31;572(7769):397-401. Epub 2019 Jul 31.

Department of Pharmacology and Cancer Biology, Duke University School of Medicine, Durham, NC, USA.

Nutrition exerts considerable effects on health, and dietary interventions are commonly used to treat diseases of metabolic aetiology. Although cancer has a substantial metabolic component, the principles that define whether nutrition may be used to influence outcomes of cancer are unclear. Nevertheless, it is established that targeting metabolic pathways with pharmacological agents or radiation can sometimes lead to controlled therapeutic outcomes. By contrast, whether specific dietary interventions can influence the metabolic pathways that are targeted in standard cancer therapies is not known. Here we show that dietary restriction of the essential amino acid methionine-the reduction of which has anti-ageing and anti-obesogenic properties-influences cancer outcome, through controlled and reproducible changes to one-carbon metabolism. This pathway metabolizes methionine and is the target of a variety of cancer interventions that involve chemotherapy and radiation. Methionine restriction produced therapeutic responses in two patient-derived xenograft models of chemotherapy-resistant RAS-driven colorectal cancer, and in a mouse model of autochthonous soft-tissue sarcoma driven by a G12D mutation in KRAS and knockout of p53 (Kras;Trp53) that is resistant to radiation. Metabolomics revealed that the therapeutic mechanisms operate via tumour-cell-autonomous effects on flux through one-carbon metabolism that affects redox and nucleotide metabolism-and thus interact with the antimetabolite or radiation intervention. In a controlled and tolerated feeding study in humans, methionine restriction resulted in effects on systemic metabolism that were similar to those obtained in mice. These findings provide evidence that a targeted dietary manipulation can specifically affect tumour-cell metabolism to mediate broad aspects of cancer outcome.
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http://dx.doi.org/10.1038/s41586-019-1437-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6951023PMC
August 2019

Organ-specific metastases obtained by culturing colorectal cancer cells on tissue-specific decellularized scaffolds.

Nat Biomed Eng 2018 06 23;2(6):443-452. Epub 2018 Apr 23.

Laboratory of Nano- and Translational Medicine, Department of Radiation Oncology, Lineberger Comprehensive Cancer Center, Carolina Center for Cancer Nanotechnology Excellence, Carolina Institute of Nanomedicine, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA.

Metastatic disease remains the primary cause of mortality in cancer patients. Yet the number of available in vitro models to study metastasis is limited by challenges in the recapitulation of the metastatic microenvironment in vitro, and by difficulties in maintaining colonized-tissue specificity in the expansion and maintenance of metastatic cells. Here, we show that decellularized scaffolds that retain tissue-specific extracellular-matrix components and bound signalling molecules enable, when seeded with colorectal cancer cells, the spontaneous formation of three-dimensional cell colonies that histologically, molecularly and phenotypically resemble in vivo metastases. Lung and liver metastases obtained by culturing colorectal cancer cells on, respectively, lung and liver decellularized scaffolds retained their tissue-specific tropism when injected in mice. We also found that the engineered metastases contained signet ring cells, which has not previously been observed ex vivo. A culture system with tissue-specific decellularized scaffolds represents a simple and powerful approach for the study of organ-specific cancer metastases.
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http://dx.doi.org/10.1038/s41551-018-0231-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6166886PMC
June 2018

Exercise inhibits tumor growth and central carbon metabolism in patient-derived xenograft models of colorectal cancer.

Cancer Metab 2018 15;6:14. Epub 2018 Nov 15.

1Division of Medical Oncology, Duke University Medical Center, 3008 Snyderman Building, 905 S. LaSalle St, Durham, NC 27710 USA.

Background: While self-reported exercise is associated with a reduction in the risk of recurrence in colorectal cancer, the molecular mechanisms underpinning this relationship are unknown. Furthermore, the effect of exercise on intratumoral metabolic processes has not been investigated in detail in human cancers. In our current study, we generated six colorectal patient patient-derived xenografts (CRC PDXs) models and treated each PDX to voluntary wheel running (exercise) for 6-8 weeks or no exposure to the wheel (control). A comprehensive metabolomics analysis was then performed on the PDXs to identify exercise induced changes in the tumor that were associated with slower growth.

Results: Tumor growth inhibition was observed in the voluntary wheel running group compared to the control group in three of the six models. A metabolomics analysis first revealed that central carbon metabolism was affected in each model irrespective of treatment. Interestingly, comparison of responsive and resistant models showed that levels of metabolites in nucleotide metabolism, known to be coupled to mitochondrial metabolism, were predictive of response. Furthermore, phosphocreatine levels which are linked to mitochondrial energy demands were associated with inhibition of tumor growth.

Conclusion: Altogether, this study provides evidence that changes to tumor cell mitochondrial metabolism may underlie in part the benefits of exercise.
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http://dx.doi.org/10.1186/s40170-018-0190-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6237036PMC
November 2018

Single-Cell Transcriptomics Reveals Heterogeneity and Drug Response of Human Colorectal Cancer Organoids.

Annu Int Conf IEEE Eng Med Biol Soc 2018 Jul;2018:2378-2381

Organoids are three-dimensional cell cultures that mimic organ functions and structures. The organoid model has been developed as a versatile in vitro platform for stem cell biology and diseases modeling. Tumor organoids are shown to share ~ 90% of genetic mutations with biopsies from same patients. However, it's not clear whether tumor organoids recapitulate the cellular heterogeneity observed in patient tumors. Here, we used single-cell RNA-Seq to investigate the transcriptomics of tumor organoids derived from human colorectal tumors, and applied machine learning methods to unbiasedly cluster subtypes in tumor organoids. Computational analysis reveals cancer heterogeneity sustained in tumor organoids, and the subtypes in organoids displayed high diversity. Furthermore, we treated the tumor organoids with a first-line cancer drug, Oxaliplatin, and investigated drug response in single-cell scale. Diversity of tumor cell populations in organoids were significantly perturbed by drug treatment. Single-cell analysis detected the depletion of chemosensitive subgroups and emergence of new drug tolerant subgroups after drug treatment. Our study suggests that the organoid model is capable of recapitulating clinical heterogeneity and its evolution in response to chemotherapy.
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http://dx.doi.org/10.1109/EMBC.2018.8512784DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6317967PMC
July 2018

Targeting HPV16 DNA using CRISPR/Cas inhibits anal cancer growth .

Future Virol 2018 Jul 12;13(7):475-482. Epub 2018 Jun 12.

Departments of Medicine & Molecular Genetics & Microbiology, Duke Cancer Institute, Duke University Medical Center, Durham, NC 27710, USA.

Aim: The goal of this study was to determine if a single AAV vector, encoding Cas9 and guide RNAs specific for the HPV16 E6 and E7 genes, could inhibit the growth of an HPV16-induced tumor .

Materials & Methods: We grew HPV16, patient-derived anal cancer explants in immunodeficient mice and then challenged these by injection of AAV-based vectors encoding Cas9 and control or HPV16-specific guide RNAs.

Results & Conclusion: We observed a significant and selective reduction in tumor growth when the HPV16 E6 and E7 genes were targeted using Cas9. These studies provide proof of principle for the hypothesis that CRISPR/Cas has the potential to be used to selectively treat HPV-induced tumors in humans.
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http://dx.doi.org/10.2217/fvl-2018-0010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6136077PMC
July 2018

Epidermal Growth Factor and Granulocyte Colony Stimulating Factor Signaling Are Synergistic for Hematopoietic Regeneration.

Stem Cells 2018 02 10;36(2):252-264. Epub 2017 Nov 10.

Division of Hematologic Malignancies and Cellular Therapy, Duke University, Durham, North Carolina, USA.

Hematopoietic regeneration following chemotherapy may be distinct from regeneration following radiation. While we have shown that epidermal growth factor (EGF) accelerates regeneration following radiation, its role following chemotherapy is currently unknown. We sought to identify EGF as a hematopoietic growth factor for chemotherapy-induced myelosuppression. Following 5-fluorouracil (5-FU), EGF accelerated hematopoietic stem cell regeneration and prolonged survival compared with saline-treated mice. To mitigate chemotherapy-induced injury to endothelial cells in vivo, we deleted Bax in VEcadherin cells (VEcadherinCre;Bax mice). Following 5-FU, VEcadherinCre;Bax mice displayed preserved hematopoietic stem/progenitor content compared with littermate controls. 5-FU and EGF treatment resulted in increased cellular proliferation, decreased apoptosis, and increased DNA double-strand break repair by non-homologous end-joining recombination compared with saline-treated control mice. When granulocyte colony stimulating factor (G-CSF) is given with EGF, this combination was synergistic for regeneration compared with either G-CSF or EGF alone. EGF increased G-CSF receptor (G-CSFR) expression following 5-FU. Conversely, G-CSF treatment increased both EGF receptor (EGFR) and phosphorylation of EGFR in hematopoietic stem/progenitor cells. In humans, the expression of EGFR is increased in patients with colorectal cancer treated with 5-FU compared with cancer patients not on 5-FU. Similarly, EGFR signaling is responsive to G-CSF in humans in vivo with both increased EGFR and phospho-EGFR in healthy human donors following G-CSF treatment compared with donors who did not receive G-CSF. These data identify EGF as a hematopoietic growth factor following myelosuppressive chemotherapy and that dual therapy with EGF and G-CSF may be an effective method to accelerate hematopoietic regeneration. Stem Cells 2018;36:252-264.
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http://dx.doi.org/10.1002/stem.2731DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5963258PMC
February 2018

Activation of the mTOR Pathway by Oxaliplatin in the Treatment of Colorectal Cancer Liver Metastasis.

PLoS One 2017 6;12(1):e0169439. Epub 2017 Jan 6.

Department of Medical Oncology, Duke University Medical Center, Durham, North Carolina, United States of America.

Background: Standard of care treatment for colorectal cancer liver metastasis consists of a cytotoxic chemotherapy in combination with a targeted agent. Clinical trials have guided the use of these combinatory therapies, but it remains unclear what the optimal combinations of cytotoxic chemotherapy with a targeted agent are.

Methods: Using a genomic based approach, gene expression profiling was obtained from tumor samples of patient with colorectal cancer liver metastasis who received an oxaliplatin based therapy. Early passaged colorectal cancer liver metastasis cell lines and patient derived xenografts of colorectal cancer liver metastasis were then treated with oxaliplatin and a mTOR inhibitor.

Results: Gene set enrichment analysis revealed that the mTOR pathway was activated in patients receiving oxaliplatin based therapy. Treatment of early passaged colorectal cancer lines and patient derived xenografts with oxaliplatin resulted in activation of the mTOR pathway. Combination therapy with oxaliplatin and a mTOR inhibitor resulted in a synergistic effect both in vitro and in vivo.

Conclusion: Our findings suggest a genomic based approach can be used to identify optimal combinations of cytotoxic chemotherapy with a targeted agent and that these observations can be validated both in vitro and in vivo using patient derived colorectal cancer cell lines and patient derived xenografts prior to clinical use.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0169439PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5218497PMC
August 2017

Development of a Novel c-MET-Based CTC Detection Platform.

Mol Cancer Res 2016 06 7;14(6):539-47. Epub 2016 Mar 7.

Division of Medical Oncology, Department of Medicine, Duke Cancer Institute, Duke University, Durham, North Carolina. Department of Pharmacology and Cancer Biology, Duke University, Durham, NC.

Unlabelled: Amplification of the MET oncogene is associated with poor prognosis, metastatic dissemination, and drug resistance in many malignancies. We developed a method to capture and characterize circulating tumor cells (CTC) expressing c-MET using a ferromagnetic antibody. Immunofluorescence was used to characterize cells for c-MET, DAPI, and pan-CK, excluding CD45(+) leukocytes. The assay was validated using appropriate cell line controls spiked into peripheral blood collected from healthy volunteers (HV). In addition, peripheral blood was analyzed from patients with metastatic gastric, pancreatic, colorectal, bladder, renal, or prostate cancers. CTCs captured by c-MET were enumerated, and DNA FISH for MET amplification was performed. The approach was highly sensitive (80%) for MET-amplified cells, sensitive (40%-80%) for c-MET-overexpressed cells, and specific (100%) for both c-MET-negative cells and in 20 HVs. Of 52 patients with metastatic carcinomas tested, c-MET CTCs were captured in replicate samples from 3 patients [gastric, colorectal, and renal cell carcinoma (RCC)] with 6% prevalence. CTC FISH demonstrated that MET amplification in both gastric and colorectal cancer patients and trisomy 7 with gain of MET gene copies in the RCC patient. The c-MET CTC assay is a rapid, noninvasive, sensitive, and specific method for detecting MET-amplified tumor cells. CTCs with MET amplification can be detected in patients with gastric, colorectal, and renal cancers.

Implications: This study developed a novel c-MET CTC assay for detecting c-MET CTCs in patients with MET amplification and warrants further investigation to determine its clinical applicability. Mol Cancer Res; 14(6); 539-47. ©2016 AACR.
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http://dx.doi.org/10.1158/1541-7786.MCR-16-0011DOI Listing
June 2016

Primary high-grade neuroendocrine carcinoma emerging from an adenomatous polyp in the setting of familial adenomatous polyposis.

BMJ Case Rep 2016 Feb 16;2016. Epub 2016 Feb 16.

Department of Pathology, Duke University Hospital, Durham, North Carolina, USA.

Familial adenomatous polyposis (FAP) is a rare inherited syndrome that is characterised by innumerable adenomas of the colon and rectum, a high risk of colorectal cancer and a variety of extracolonic manifestations. FAP presents as hundreds to thousands of colonic adenomas beginning in adolescence. The syndrome is associated with less than 1% of all colorectal cancer cases, but there is a nearly 100% lifetime risk of colorectal cancer in individuals with FAP. This case demonstrates a 60-year-old man with FAP who developed high-grade neuroendocrine carcinoma with glandular and squamous differentiation, and regional lymph node and liver metastases. Early diagnosis of FAP is of the utmost importance to start screening colonoscopies to assess disease burden, perform polypectomies and to make management decisions. Neuroendocrine carcinomas rarely occur in patients with FAP, and awareness of this association among general medical physicians and pathologists is essential for the diagnosis and care of these patients.
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http://dx.doi.org/10.1136/bcr-2015-214206DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5483537PMC
February 2016

Cystine Deprivation Triggers Programmed Necrosis in VHL-Deficient Renal Cell Carcinomas.

Cancer Res 2016 04 1;76(7):1892-903. Epub 2016 Feb 1.

Department of Molecular Genetics and Microbiology, Duke University, Durham, North Carolina. Center for Genomic and Computational Biology Duke University, Durham, North Carolina.

Oncogenic transformation may reprogram tumor metabolism and render cancer cells addicted to extracellular nutrients. Deprivation of these nutrients may therefore represent a therapeutic opportunity, but predicting which nutrients cancer cells become addicted remains difficult. Here, we performed a nutrigenetic screen to determine the phenotypes of isogenic pairs of clear cell renal cancer cells (ccRCC), with or without VHL, upon the deprivation of individual amino acids. We found that cystine deprivation triggered rapid programmed necrosis in VHL-deficient cell lines and primary ccRCC tumor cells, but not in VHL-restored counterparts. Blocking cystine uptake significantly delayed xenograft growth of ccRCC. Importantly, cystine deprivation triggered similar metabolic changes regardless of VHL status, suggesting that metabolic responses alone are not sufficient to explain the observed distinct fates of VHL-deficient and restored cells. Instead, we found that increased levels of TNFα associated with VHL loss forced VHL-deficient cells to rely on intact RIPK1 to inhibit apoptosis. However, the preexisting elevation in TNFα expression rendered VHL-deficient cells susceptible to necrosis triggered by cystine deprivation. We further determined that reciprocal amplification of the Src-p38 (MAPK14)-Noxa (PMAIP1) signaling and TNFα-RIP1/3 (RIPK1/RIPK3)-MLKL necrosis pathways potentiated cystine-deprived necrosis. Together, our findings reveal that cystine deprivation in VHL-deficient RCCs presents an attractive therapeutic opportunity that may bypass the apoptosis-evading mechanisms characteristic of drug-resistant tumor cells. Cancer Res; 76(7); 1892-903. ©2016 AACR.
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http://dx.doi.org/10.1158/0008-5472.CAN-15-2328DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4873412PMC
April 2016

Mismatch repair gone awry: Management of Lynch syndrome.

Crit Rev Oncol Hematol 2015 Mar 12;93(3):170-9. Epub 2014 Oct 12.

Duke Cancer Institute, Department of Medicine, Division of Medical Oncology, Duke University Medical Center, DUMC 3233, Durham, NC 27710, United States. Electronic address:

The hallmark of Lynch syndrome involves germline mutations of genes important in DNA mismatch repair. Affected family kindreds will have multiple associated malignancies, the most common of which is colorectal adenocarcinoma. Recently, evidence has shown that clinical diagnostic criteria provided by the Amsterdam Criteria and the Bethesda Guidelines must be linked with microsatellite instability testing to correctly diagnose Lynch syndrome. We present a case of metachronous colorectal adenocarcinomas in a patient less than 50 years of age, followed by a discussion of Lynch syndrome, with an emphasis on surveillance and prevention of malignancies.
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http://dx.doi.org/10.1016/j.critrevonc.2014.10.005DOI Listing
March 2015

Adjuvant chemotherapy for t1 node-positive colon cancers provides significant survival benefit.

Dis Colon Rectum 2014 Dec;57(12):1341-8

1Section of Colorectal Surgery, Department of Surgery, Duke University Medical Center, Durham, North Carolina 2Division of Medical Oncology, Department of Medicine, Duke University Medical Center, Durham, North Carolina.

Background: Contemporary treatment of node-positive (N+) colon cancer consists of adjuvant chemotherapy; however, randomized data supporting this practice were derived from lesions T2 or greater. Minimal data exist regarding the use and need for adjuvant chemotherapy in T1N+ disease.

Objective: The aim of this study was to determine treatment trends and the effects of adjuvant chemotherapy on T1N+ colon cancers by using the National Cancer Database.

Design: This was a retrospective study. Baseline demographics, tumor, and cancer treatment characteristics were compared. Groups were matched on the propensity to receive chemotherapy. Adjusted long-term survival stratified by chemotherapy use was compared by using the Kaplan-Meier method with the log-rank test. Predictors of not receiving chemotherapy were identified by using a multivariable logistic regression model.

Settings: Data were collected from the National Cancer Database, which collects cancer data from over 1500 cancer centers.

Patients: We identified patients from 1998 to 2006 with T1N+ disease, excluding those with metastatic disease or previous cancer. Patients were stratified based on whether or not they received chemotherapy.

Main Outcome Measures: The primary outcome measure of this study was long-term survival.

Results: Three thousand one hundred thirty-seven patients had T1N+ disease; 70.6% (n = 2216) received chemotherapy, and utilization significantly increased from 1998 to 2011 (p < 0.001). Unadjusted analysis revealed that patients treated with chemotherapy were statistically younger and healthier, and had shorter postoperative lengths of stay (all p < 0.001). Unadjusted 5-year survival was higher in patients receiving chemotherapy (87.9% vs 63.0% in patients with no chemotherapy; p < 0.001) and this persisted after propensity matching with (83.4% and 63.0% in patients with or without chemotherapy; p < 0.001). Only age (OR, 0.29; p < 0.001) predicted not receiving chemotherapy.

Limitations: Limitations include potential selection bias as well as the inability to compare disease-free survival/recurrence.

Conclusions: Adjuvant chemotherapy appears to significantly improve long-term survival in patients receiving chemotherapy in T1N+ disease. Thus, the use of chemotherapy in T1N+ disease is justified and provides a highly significant survival benefit.
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http://dx.doi.org/10.1097/DCR.0000000000000245DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4336178PMC
December 2014

The genomic landscape of mantle cell lymphoma is related to the epigenetically determined chromatin state of normal B cells.

Blood 2014 May 28;123(19):2988-96. Epub 2014 Mar 28.

Duke Institute for Genome Sciences and Policy, Duke University, Durham, NC;

In this study, we define the genetic landscape of mantle cell lymphoma (MCL) through exome sequencing of 56 cases of MCL. We identified recurrent mutations in ATM, CCND1, MLL2, and TP53. We further identified a number of novel genes recurrently mutated in patients with MCL including RB1, WHSC1, POT1, and SMARCA4. We noted that MCLs have a distinct mutational profile compared with lymphomas from other B-cell stages. The ENCODE project has defined the chromatin structure of many cell types. However, a similar characterization of primary human mature B cells has been lacking. We defined, for the first time, the chromatin structure of primary human naïve, germinal center, and memory B cells through chromatin immunoprecipitation and sequencing for H3K4me1, H3K4me3, H3Ac, H3K36me3, H3K27me3, and PolII. We found that somatic mutations that occur more frequently in either MCLs or Burkitt lymphomas were associated with open chromatin in their respective B cells of origin, naïve B cells, and germinal center B cells. Our work thus elucidates the landscape of gene-coding mutations in MCL and the critical interplay between epigenetic alterations associated with B-cell differentiation and the acquisition of somatic mutations in cancer.
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http://dx.doi.org/10.1182/blood-2013-07-517177DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4014841PMC
May 2014

Use of molecular biomarkers to inform adjuvant therapy for colon cancer.

Oncology (Williston Park) 2013 Aug;27(8):746-54

Institute for Genome Sciences & Policy, Duke University Medical Center, Durham, North Carolina 27710, USA.

The decision about who may derive benefit from adjuvant chemotherapy in colon cancer is often a difficult one for clinicians. While multiple trials have demonstrated that adjuvant chemotherapy reduces the risk of recurrence and improves overall survival in patients with stage III disease, the data supporting the use of adjuvant chemotherapy in patients with stage II disease are not as compelling. Because adjuvant therapy can have significant toxicity, tools to help clinicians determine who may derive a benefit from therapy are of the utmost importance. Recent advances in high throughput technologies have led to the identification of molecular biomarkers-including microsatellite instability (MSI), loss of heterozygosity (LOH), p53, Kirsten rat sarcoma viral oncogene homolog (KRAS), v-raf murine sarcoma viral oncogene homolog B1 (BRAF), thymidylate synthase (TS), and excision repair cross-complementation group 1 (ERCC1)--as well as various multigene assays that are being studied for their ability to offer both prognostic and predictive information to clinicians. Here we review the current knowledge about molecular biomarkers that may aid the clinician in offering personalized cancer therapy based on the genetic landscape of an individual patient's tumor.
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August 2013

Novel adenoviral vector induces T-cell responses despite anti-adenoviral neutralizing antibodies in colorectal cancer patients.

Cancer Immunol Immunother 2013 Aug 30;62(8):1293-301. Epub 2013 Apr 30.

Department of Medicine, Duke University Medical Center, Durham, NC 27710, USA.

First-generation, E1-deleted adenovirus subtype 5 (Ad5)-based vectors, although promising platforms for use as cancer vaccines, are impeded in activity by naturally occurring or induced Ad-specific neutralizing antibodies. Ad5-based vectors with deletions of the E1 and the E2b regions (Ad5 [E1-, E2b-]), the latter encoding the DNA polymerase and the pre-terminal protein, by virtue of diminished late phase viral protein expression, were hypothesized to avoid immunological clearance and induce more potent immune responses against the encoded tumor antigen transgene in Ad-immune hosts. Indeed, multiple homologous immunizations with Ad5 [E1-, E2b-]-CEA(6D), encoding the tumor antigen carcinoembryonic antigen (CEA), induced CEA-specific cell-mediated immune (CMI) responses with antitumor activity in mice despite the presence of preexisting or induced Ad5-neutralizing antibody. In the present phase I/II study, cohorts of patients with advanced colorectal cancer were immunized with escalating doses of Ad5 [E1-, E2b-]-CEA(6D). CEA-specific CMI responses were observed despite the presence of preexisting Ad5 immunity in a majority (61.3 %) of patients. Importantly, there was minimal toxicity, and overall patient survival (48 % at 12 months) was similar regardless of preexisting Ad5 neutralizing antibody titers. The results demonstrate that, in cancer patients, the novel Ad5 [E1-, E2b-] gene delivery platform generates significant CMI responses to the tumor antigen CEA in the setting of both naturally acquired and immunization-induced Ad5-specific immunity.
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http://dx.doi.org/10.1007/s00262-013-1400-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3732790PMC
August 2013

Histological and molecular evaluation of patient-derived colorectal cancer explants.

PLoS One 2012 4;7(6):e38422. Epub 2012 Jun 4.

Institute for Genome Sciences and Policy, Duke University, Durham, North Carolina, United States of America.

Mouse models have been developed to investigate colorectal cancer etiology and evaluate new anti-cancer therapies. While genetically engineered and carcinogen-induced mouse models have provided important information with regard to the mechanisms underlying the oncogenic process, tumor xenograft models remain the standard for the evaluation of new chemotherapy and targeted drug treatments for clinical use. However, it remains unclear to what extent explanted colorectal tumor tissues retain inherent pathological features over time. In this study, we have generated a panel of 27 patient-derived colorectal cancer explants (PDCCEs) by direct transplantation of human colorectal cancer tissues into NOD-SCID mice. Using this panel, we performed a comparison of histology, gene expression and mutation status between PDCCEs and the original human tissues from which they were derived. Our findings demonstrate that PDCCEs maintain key histological features, basic gene expression patterns and KRAS/BRAF mutation status through multiple passages. Altogether, these findings suggest that PDCCEs maintain similarity to the patient tumor from which they are derived and may have the potential to serve as a reliable preclinical model that can be incorporated into future strategies to optimize individual therapy for patients with colorectal cancer.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0038422PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3366969PMC
October 2012

Retraction in part: A genomic approach to identify molecular pathways associated with chemotherapy resistance.

Mol Cancer Ther 2012 May 29;11(5):1214-5. Epub 2012 Mar 29.

Duke Institute for Genome Sciences and Policy, Duke University and Division of Medical Oncology, Department of Medicine, Duke University, USA.

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http://dx.doi.org/10.1158/1535-7163.MCT-12-0210DOI Listing
May 2012

Characterization of an oxaliplatin sensitivity predictor in a preclinical murine model of colorectal cancer.

Mol Cancer Ther 2012 Jul 16;11(7):1500-1509. Epub 2012 Feb 16.

Division of Medical Oncology, Duke University, Durham, NC.

Despite advances in contemporary chemotherapeutic strategies, long-term survival still remains elusive for patients with metastatic colorectal cancer. A better understanding of the molecular markers of drug sensitivity to match therapy with patient is needed to improve clinical outcomes. In this study, we used in vitro drug sensitivity data from the NCI-60 cell lines together with their Affymetrix microarray data to develop a gene expression signature to predict sensitivity to oxaliplatin. To validate our oxaliplatin sensitivity signature, patient-derived colorectal cancer explants (PDCCE) were developed in nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice from resected human colorectal tumors. Analysis of gene expression profiles found similarities between the PDCCEs and their parental human tumors, suggesting their utility to study drug sensitivity in vivo. The oxaliplatin sensitivity signature was then validated in vivo with response data from 14 PDCCEs treated with oxaliplatin and was found to have an accuracy of 92.9% (sensitivity = 87.5%; specificity = 100%). Our findings suggest that PDCCEs can be a novel source to study drug sensitivity in colorectal cancer. Furthermore, genomic-based analysis has the potential to be incorporated into future strategies to optimize individual therapy for patients with metastatic colorectal cancer.
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http://dx.doi.org/10.1158/1535-7163.MCT-11-0937DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4521598PMC
July 2012

Gene expression profiling of peritoneal metastases from appendiceal and colon cancer demonstrates unique biologic signatures and predicts patient outcomes.

J Am Coll Surg 2012 Apr 17;214(4):599-606; discussion 606-7. Epub 2012 Feb 17.

Surgical Oncology Service, Department of General Surgery, Wake Forest University, Winston-Salem, NC 27157, USA.

Background: Treatment of peritoneal metastases from appendiceal and colon cancer with cytoreductive surgery and hyperthermic intraperitoneal chemotherapy (HIPEC) shows great promise. Although long-term disease-free survival is achieved in some cases with this procedure, many patients have recurrence. Oncologists have treated such recurrences of appendiceal cancer similarly to colorectal carcinoma, which has been largely ineffective. This study uses gene expression analysis of peritoneal metastases to better understand these neoplasms.

Study Design: From a prospectively maintained database and tissue bank, 41 snap frozen samples of peritoneal metastases (26 appendiceal, 15 colorectal) from patients undergoing HIPEC with complete cytoreduction and more than 3 years of follow-up underwent global gene expression analysis. Distinct phenotypes were identified using unsupervised hierarchical clustering based on differential gene expression. Survival curves restratified by genotype were generated.

Results: Three distinct phenotypes were found, 2 consisting of predominantly low grade appendiceal samples (10 of 13 in Cluster 1 and 15 of 20 in Cluster 2) and 1 consisting of predominantly colorectal samples (7 of 8 in Cluster 3). Cluster 1 consisted of patients with good prognosis and Clusters 2 and 3 consisted of patients with poor prognosis (p = 0.006). Signatures predicted survival of low- (Cluster 1) vs high-risk (Cluster 2) appendiceal (p = 0.04) and low-risk appendiceal (Cluster 1) vs colon primary (Cluster 3) (p = 0.0002).

Conclusions: This study represents the first use of gene expression profiling for appendiceal cancer, and demonstrates genomic signatures quite distinct from colorectal cancer, confirming their unique biology. Consequently, therapy for appendiceal lesions extrapolated from colonic cancer regimens may be unfounded. These phenotypes may predict outcomes guiding patient management.
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http://dx.doi.org/10.1016/j.jamcollsurg.2011.12.028DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3768122PMC
April 2012
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