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The extracellular matrix (ECM) is a complex meshwork of insoluble fibrillar proteins and signaling factors interacting together to provide architectural and instructional cues to the surrounding cells. Alterations in ECM organization or composition and excessive ECM deposition have been observed in diseases such as fibrosis, cardiovascular diseases, and cancer. We provide here optimized protocols to solubilize ECM proteins from normal or tumor tissues, digest the proteins into peptides, analyze ECM peptides by mass spectrometry, and interpret the mass spectrometric data. In addition, we present here two novel R-script-based web tools allowing rapid annotation and relative quantification of ECM proteins, peptides, and intensity/abundance in mass spectrometric data output files. We illustrate this protocol with ECMs obtained from two pairs of tissues, which differ in ECM content and cellularity: triple-negative breast cancer and adjacent mammary tissue, and omental metastasis from high-grade serous ovarian cancer and normal omentum. The complete proteomics data set generated in this study has been deposited to the public repository ProteomeXchange with the data set identifier: PXD005554.

Chimeric antigen receptor (CAR)-T cell therapies demonstrate the clinical potential of lymphocytes engineered with synthetic properties. However, CAR-T cells are ineffective in most solid tumors, partly due to inadequate activation of the infused lymphocytes at the site of malignancy. To selectively enhance anti-tumor efficacy without exacerbating off-target toxicities, CAR-T cells can be engineered to preferentially deliver immunostimulatory payloads in tumors. Here, we report a novel antigen-inducible promoter and single-vector sense-and-respond circuit for conditional payload expression in primary human T cells. In therapeutic T cell models, the novel NR4A-based promoter induced higher transgene expression than the conventional NFAT-based promoter under weakly immunogenic conditions, where payload expression is most needed. Minimal activity was detected from the inducible promoters in the absence of antigen and after withdrawal of stimulation. As a functional proof-of-concept, we used the NR4A-based promoter to express cytokines in an anti-mesothelin CAR-T model with suboptimal stimulation, and observed improved proliferation compared to T cells engineered with the conventional NFAT promoter or CAR alone. Our single-vector circuit achieves CAR-directed payload expression under weakly immunogenic conditions and could enable the next generation of cell therapies with enhanced anti-tumor efficacy.

Chromosome gains and losses are a frequent feature of human cancers. However, how these aberrations can outweigh the detrimental effects of aneuploidy remains unclear. An initial comparison of existing chromosomal instability (CIN) mouse models suggests that aneuploidy accumulates to low levels in these animals. We therefore developed a novel mouse model that enables unprecedented levels of chromosome missegregation in the adult animal. At the earliest stages of T-cell development, cells with random chromosome gains and/or losses are selected against, but CIN eventually results in the expansion of progenitors with clonal chromosomal imbalances. Clonal selection leads to the development of T-cell lymphomas with stereotypic karyotypes in which chromosome 15, containing the Myc oncogene, is gained with high prevalence. Expressing human MYC from chromosome 6 (MYCChr6) is sufficient to change the karyotype of these lymphomas to include universal chromosome 6 gains. Interestingly, while chromosome 15 is still gained in MYCChr6 tumors after genetic ablation of the endogenous Myc locus, this chromosome is not efficiently gained after deletion of one copy of Rad21, suggesting a synergistic effect of both MYC and RAD21 in driving chromosome 15 gains. Our results show that the initial detrimental effects of random missegregation are outbalanced by clonal selection, which is dictated by the chromosomal location and nature of certain genes and is sufficient to drive cancer with high prevalence.

Summary Myeloid suppressor cells promote tumor growth by a variety of mechanisms which are not fully characterized. We identified myeloid cells (MCs) expressing the latency-associated peptide (LAP) of TGF-β on their surface and LAPHi MCs that stimulate Foxp3+ Tregs while inhibiting effector T cell proliferation and function. Blocking TGF-β inhibits the tolerogenic ability of LAPHi MCs. Furthermore, adoptive transfer of LAPHi MCs promotes Treg accumulation and tumor growth in vivo. Conversely, anti-LAP antibody, which reduces LAPHi MCs, slows cancer progression. Single-cell RNA-Seq analysis on tumor-derived immune cells revealed LAPHi dominated cell subsets with distinct immunosuppressive signatures, including those with high levels of MHCII and PD-L1 genes. Analogous to mice, LAP is expressed on myeloid suppressor cells in humans, and these cells are increased in glioma patients. Thus, our results identify a previously unknown function by which LAPHi MCs promote tumor growth and offer therapeutic intervention to target these cells in cancer., Graphical abstract, Highlights • Several myeloid cell subsets express surface LAP • Myeloid cells that express surface LAP possess immunosuppressive properties • LAP expressing myeloid cells induce Tregs and inhibit effector T cell function • LAP expressing myeloid cells promote tumor growth, Immunology; Cancer

Many tumor types harbor specific chromosome gains or losses. In Ewing sarcoma (ES) chromosome 8 gain is extremely common, indicating that this trisomy drives tumorigenesis. However, in primary cells, whole chromosome gains and losses are universally detrimental. We hypothesize that gain of chromosome 8 suppresses cellular stresses associated with oncogenic transformation in ES. In this study, we induced expression of EWS-FLI1 fusion oncogene, which drives ES, in various primary human mesenchymal progenitor and fibroblast cells. We showed that expression of EWS-FLI1 results in a strong cell cycle-dependent replication stress in euploid mesenchymal progenitor cells and fibroblast cells. As a consequence, proliferation is impaired when EWS-FLI1 is expressed in these cells. However, primary fibroblast cells carrying an extra copy of chromosome 8 (trisomy 8) exhibit much less replication stress, and the impairment of proliferation was also not seen in these cells. The levels of replication-associated DNA damage are lower in the trisomy 8 cells than in the euploid cells in the presence of EWS-FLI1. Then, we applied weighted co-expression network analysis (WGCNA) to RNA sequencing data obtained from ES patient tumor tissues in combination of mouse synteny analysis. We identified multiple potential genes and regions on chromosome 8, which are beneficial to ES oncogenesis when gain of extra copies. Above all, we demonstrate that gain of chromosome 8 in Ewing sarcoma facilitates the primary mesenchymal-lineage cells to overcome certain oncogenic stresses and reduces DNA damage associated with tumorigenic processes, which is achieved by cooperative upregulation of multiple gene expression on chromosome 8. Citation Format: Xiaofeng A. Su, Duanduan Ma, James V. Parsons, John M. Replogle, James F. Amatruda, Charles A. Whittaker, Kimberly Stegmaier, Angelika Amon. Ewing sarcoma: A case study of clonal aneuploidy and DNA damage repair in pediatric cancer [abstract]. In: Proceedings of the AACR Special Conference on the Advances in Pediatric Cancer Research; 2019 Sep 17-20; Montreal, QC, Canada. Philadelphia (PA): AACR; Cancer Res 2020;80(14 Suppl):Abstract nr B52.

Inherited genetic variability contributes to susceptibility to cervical cancer. We investigated the association of single nucleotide polymorphisms (SNPs) in the human epidermal growth factor receptor (ERBB) family with cervical cancer.We used the transmission disequilibrium test (TDT) to look for excessive transmission of tag single nucleotide polymorphisms (tSNPs) in ERBB family members EGFR, ERBB2, ERBB3, and ERBB4 in a large sample of women with invasive and in situ cervical cancer and their biological parents (628 trios). The study used a discovery set of trios (244) analyzed by Illumina GoldenGate in which SNPs reaching a P.05 were re-tested by TaqMan in the combined set of 628. We also explored collaborative effects of different ERBB alleles.Based on single SNP TDT tests we identified 16 significant SNPs in the discover stage and six of 14 SNPs that could be assayed by TaqMan were significantly overtransmitted in women with cervical cancer in the combined replication set. Four SNPs were located in intron 1 of EGFR and two SNPs in intron 24 of ERBB4. The EGFR variants are located near multiple enhancers, silencers, and the previously identified functional common polymorphisms in intron 1.Our data provide evidence for the involvement of intron 1 EGFR variants and intron 24 ERBB4 variants in modulating risk for the development of in situ and invasive cervical cancer. These variants should be examined in additional populations and functional studies would be needed to confirm this hypothesis.

Fibronectin (FN) is a major component of the tumor microenvironment, but its role in promoting metastasis is incompletely understood. Here we show that FN gradients elicit directional movement of breast cancer cells, in vitro and in vivo. Haptotaxis on FN gradients requires direct interaction between α5β1 integrin and Mena, an actin regulator, and involves increases in focal complex signaling and tumor-cell-mediated extracellular matrix (ECM) remodeling. Compared to Mena, higher levels of the pro-metastatic MenaINV isoform associate with α5, which enables 3D haptotaxis of tumor cells towards the high FN concentrations typically present in perivascular space and in the periphery of breast tumor tissue. MenaINV and FN levels were correlated in two breast cancer cohorts, and high levels of MenaINV were significantly associated with increased tumor recurrence as well as decreased patient survival. Our results identify a novel tumor-cell-intrinsic mechanism that promotes metastasis through ECM remodeling and ECM guided directional migration., Ludwig Center for Molecular Oncology, Breast Cancer Research Program (U.S.) (post-doctoral fellowship, W81-XWH-12-1-0031), National Institutes of Health (U.S.) (NIH grant U54-CA112967), National Institutes of Health (U.S.) (NIH grant R01 CA142833), Howard Hughes Medical Institute, National Institutes of Health (U.S.) (NIH grant U01 CA143069), Kathy and Curt Marble Cancer Research Fund (Koch Institute Frontier Award), Prostate Cancer Foundation, David H. Koch Institute for Integrative Cancer Research at MIT (NCI core grant P30-CA14051)

To identify the causative gene in an autosomal dominant limb-girdle muscular dystrophy (LGMD) with skeletal muscle vacuoles.Exome sequencing was used to identify candidate mutations in the studied pedigree. Genome-wide linkage was then used to narrow the list of candidates to a single disease-associated mutation. Additional pedigrees with dominant or sporadic myopathy were screened for mutations in the same gene (DNAJB6) using exome sequencing. Skeletal muscle from affected patients was evaluated with histochemistry and immunohistochemical stains for dystrophy-related proteins, SMI-31, TDP43, and DNAJB6.Exome analysis in 3 affected individuals from a family with dominant LGMD and vacuolar pathology identified novel candidate mutations in 22 genes. Linkage analysis excluded all variants except a Phe93Leu mutation in the G/F domain of the DNAJB6 gene, which resides within the LGMD locus at 7q36. Analysis of exome sequencing data from other pedigrees with dominant myopathy identified a second G/F domain mutation (Pro96Arg) in DNAJB6. Affected muscle showed mild dystrophic changes, vacuoles, and abnormal aggregation of proteins, including TDP-43 and DNAJB6 itself.Mutations within the G/F domain of DNAJB6 are a novel cause of dominantly-inherited myopathy. DNAJB6 is a member of the HSP40/DNAJ family of molecular co-chaperones tasked with protecting client proteins from irreversible aggregation during protein synthesis or during times of cellular stress. The abnormal accumulation of several proteins in patient muscle, including DNAJB6 itself, suggest that DNAJB6 function is compromised by the identified G/F domain mutations.

Inherited genetic polymorphisms within immune response genes have been shown to associate with risk of invasive cervical cancer (ICC) and its immediate precursor, cervical intraepithelial neoplasia grade 3. Here, we used the transmission/disequilibrium test to detect disease-liability alleles and investigate haplotype transmission of KIR and HLA class I polymorphisms in a large family-based population of women with cervical cancer and their biological parents (359 trios). The effect of distinct human papillomavirus types was also explored. HLA-Cw group 1 (HLA-Cw alleles with asparagine at position 80), which serves as ligand for certain killer immunoglobulin-like receptors (KIR), was significantly overtransmitted in women with ICC (P = 0.04), and particularly in the subgroup of women infected with high risk HPV16 or 18 subtypes (P = 0.008). These data support the involvement of the HLA-C locus in modulating the risk of cervical neoplasia perhaps through its function as ligands for KIR, but functional studies are essential to confirm this hypothesis.

In some cancers, the oncogenic consequences of inactivating the retinoblastoma protein (Rb) appear to be mediated by unrestrained activity of the inhibitor of DNA binding protein Id2. The role of Id2 has not yet been investigated in the prototype cancer Rb-defective cancer, retinoblastoma itself. This study investigated whether loss of Id2 modified the effects of Rb inactivation in a mouse model of retinoblastoma.Id2 was analyzed in cultured cells using qPCR, Western blot, and colony formation assays. LH beta-Tag transgenic mice were crossed with Id2 heterozygotes to obtain mice with all three Id2 genotypes. Intraocular tumors were assessed for size, degree of differentiation, mitotic index, and tumor vascular density at 15 weeks of age.Retinoblastoma cell lines expressed low levels of Id2 mRNA and protein. Depletion of Id2 in Rb-inactivated cells increased clonogenic activity. Id2-deficient tumors in vivo were significantly larger, less differentiated, and more vascularized than Id2-wild-type tumors (P = 0.02, P = 0.01, P = 0.0001, respectively). There was a dosage effect for loss of each Id2 allele with respect to differentiation and vascular density.Id2 suppresses rather than promotes tumor progression in this mouse model of retinoblastoma. Id2 can act as either an oncogene or a tumor suppressor depending on context.

Host genetic variability modifies the risk of cervical cancer in women infected with oncogenic human papillomavirus (HPV). Studies have reported an association of the TP53 codon 72 arginine and cervical cancer, but the results are inconsistent. We examined the association of this single nucleotide polymorphism (SNP) in women with cervical cancer and cervical intraepithelial neoplasia grade 3, using a family-based association test. We further explored SNPs in two genes that regulate p53 stability: MDM2 (SNP309) and NQO1 (SNP609, SNP465). We also examined the relationship between host genotype and tumor HPV type. We genotyped 577 patients and their biological parents and/or siblings, using PCR-RFLP or Taqman assays. HPVs were typed by sequence-based methods. The transmission/disequilibrium test was used to detect disease-susceptibility alleles. The arginine peptide of TP53 codon 72 was overtransmitted in Caucasian families (P = 0.043), and the significance of this finding was enhanced in a subgroup of women infected with HPV16- and/or HPV18-related HPVs (P = 0.026). Allele C of NQO1 SNP609 was also overtransmitted in all cases (P = 0.026). We found no association between MDM2 SNP309 or NQO1 SNP465 and cervical cancer. Our results indicate that functional polymorphisms in TP53 codon 72 and NQO1 SNP609 associate with the risk of cervical cancer especially in women infected with type 16– and/or type 18–related HPVs. Cancer Epidemiol Biomarkers Prev; 19(3); 755–61

Glial cell line-derived neurotrophic factor (GDNF) shows potent and relatively specific protective effects on dopaminergic neurons. However the size of the GDNF protein (MW 32-42 kDa) precludes the clinical use of GDNF via parenteral administration. It would thus be useful to have a cell line that stably secretes GDNF with full biological activities. The present study shows that NIH 3T3 cells express a considerable amount of GDNF. After co-culturing with primary E14-E16 midbrain neurons, such cells protected primary rat midbrain TH-immunopositive neurons from degeneration and MPP+ toxicity. In order to enhance endogenous GDNF expression, NIH 3T3 cells were stably transfected with GDNF cDNA with the Kozak sequence. The clones with the highest GDNF expression level were selected. The protective effects of engineered cells increased as the GDNF expression level increased. These cell lines may merit clinical investigation. [Neural Res 2000; 22: 538-544]

Objective: To identify the gene responsible for 14q32-linked dominant spinal muscular atrophy with lower extremity predominance (SMA-LED, OMIM 158600). Methods: Target exon capture and next generation sequencing was used to analyze the 73 genes in the 14q32 linkage interval in 3 SMA-LED family members. Candidate gene sequencing in additional dominant SMA families used PCR and pooled target capture methods. Patient fibroblasts were biochemically analyzed. Results: Regional exome sequencing of all candidate genes in the 14q32 interval in the original SMA-LED family identified only one missense mutation that segregated with disease state—a mutation in the tail domain of DYNC1H1 (I584L). Sequencing of DYNC1H1 in 32 additional probands with lower extremity predominant SMA found 2 additional heterozygous tail domain mutations (K671E and Y970C), confirming that multiple different mutations in the same domain can cause a similar phenotype. Biochemical analysis of dynein purified from patient-derived fibroblasts demonstrated that the I584L mutation dominantly disrupted dynein complex stability and function. Conclusions: We demonstrate that mutations in the tail domain of the heavy chain of cytoplasmic dynein ( DYNC1H1 ) cause spinal muscular atrophy and provide experimental evidence that a human DYNC1H1 mutation disrupts dynein complex assembly and function. DYNC1H1 mutations were recently found in a family with Charcot-Marie-Tooth disease (type 2O) and in a child with mental retardation. Both of these phenotypes show partial overlap with the spinal muscular atrophy patients described here, indicating that dynein dysfunction is associated with a range of phenotypes in humans involving neuronal development and maintenance.

We investigated the association of metabolic syndrome (MetS) with a 500 k and a 50 k single-nucleotide polymorphism (SNP) gene chip in the Framingham Heart Study. We cross-sectionally evaluated the MetS longitudinal trends. Data analyzed were from the Offspring Cohort (four exams: first (n = 2,441), third (n = 2,185), fifth (n = 2,308), and seventh (n = 2,328)) and the Generation 3 Cohort (one exam: the first exam (n = 3,997)). The prevalence of MetS was determined using the National Cholesterol Education Program Adult Treatment Panel III diagnostic criteria, modified with a newly developed correction for medication use. The association test between an SNP and MetS was performed with a generalized estimating equations method under the additive genetic model. Multiple-testing corrections were also performed. The prevalence of MetS in the offspring cohort increased from one visit to the next, and reached the highest point by the seventh exam comparable with the prevalence reported for the general US population. The pattern of the MetS prevalence over time also reflected itself in the association tests, in which the highest significances were seen in the fifth and seventh exams. The association tests showed that SNPs within genes PRDM16, CETP, PTHB1, PAPPA, and FBN3, and also some SNPs not in genes were significant or close to significance at the genome-wide thresholds. These findings are important in terms of eventually identifying with the causal loci for MetS.

We previously mapped a nonrandom frequent loss of heterozygosity (LOH) region in cervical cancers to 1 Mb of 6p23. Here, we describe the identification of a novel cervical cancer susceptibility gene, CD83. The gene was identified by several complementary approaches, including a family-based association study, comparison of transcript expression in normal and cancerous tissue, and genomic sequencing of candidate. CD83 encodes an inducible glycoprotein in the immunoglobulin superfamily and is a marker for mature dendritic cells. The association study that includes 377 family trios showed that five single nucleotide polymorphisms (SNP) within 8 kb of its 3′-end showed significant allelic association that was strengthened in a subgroup of women with invasive cancers infected by high-risk human papillomavirus type 16 and 18 (rs9296925, P = 0.0193; rs853360, P = 0.0035; rs9230, P = 0.0011; rs9370729, P = 0.0012; rs750749, P = 0.0133). Investigation of CD83 uncovered three alternative transcripts in cervical tissue and cell lines, with variant 3 (lacking exons 3 and 4) being more frequent in cervical cancer than in normal cervical epithelium (P = 0.0181). Genomic sequencing on 36 paired normal and cervical tumors revealed several somatic mutations and novel SNPs in the promoter, exons, and introns of CD83. LOH was confirmed in >90% of cervical cancer specimens. Immunofluorescence colocalized CD83 protein to the Golgi apparatus and cell membrane of cervical cancer cell lines. None of seven nearby genes was differentially expressed in cervical cancer. The importance of CD83 in epithelial versus dendritic cells needs to be determined, as does its role in promoting cervical cancer. [Cancer Res 2007;67(23):11202–8]

Objective. To use microarray data to reveal regions of potential chromosomal loss or gain important in cervical intraepithelial neoplasia (CIN) and invasive cervical cancer by identifying mRNA expression biases in contiguous chromosomal regions. Methods. Data from three RNA expression microarray experiments were used: one primary experiment using cDNA arrays profiling gene expression in cervical epithelium from viral cytopathic effect to invasive cancer, one experiment using Affymetrix arrays profiling gene expression in invasive cancerous cervical epithelium, and one experiment using Affymetrix arrays profiling gene expression in CIN cervical biopsy specimens. Gene expression was aligned along chromosomes to reveal regions of significant chromosomal imbalance. Regions showing significant gain or loss and verified in more than one experiment are presented here. RT-PCR was performed to validate expression of one gene in a region. Results. Gain of 3q was detected from the CIN II (P = 0.018), CIN III (P = 0.005), and invasive cancer (P = 0.0002) cDNA arrays, and gain of 12q was detected from the CIN (P = 0.05) and invasive cancer (P = 0.05) Affymetrix arrays. Loss of 6p was detected from the CIN III (P = 0.004) cDNA arrays and invasive cancer (P = 0.05) Affymetrix arrays. Loss of 4q was detected from the invasive cancer (P = 0.04) cDNA arrays and invasive cancer (P=0.05) Affymetrix arrays. RAN, located in the region of gain on 12q24.3, was overexpressed in CIN and invasive cancer. Conclusions. Alignment of microarray expression data by chromosomes can be used to estimate regions of potential chromosomal aberration and identify differentially expressed genes important in the development of CIN and invasive cancer.

The retinoblastoma protein, Rb, suppresses tumorigenesis by inhibiting cell proliferation and promoting senescence and differentiation. Paradoxically, Rb also inhibits apoptosis, which would seem to oppose its tumor suppressor function. Further, most human cancer cells inactivate Rb by hyperphosphorylation and demonstrate increased proliferative capacity but not high levels of apoptosis. As a potential explanation for these findings, we show here that the tumor suppressor and antiapoptotic functions of Rb are regulated by distinct phosphorylation events. Phosphorylation of sites in the C terminus occurs efficiently every cell cycle and regulates proliferation. Phosphorylation of Ser567 is inefficient and does not occur during the normal cell cycle. However, high cyclin-dependent kinase activity promotes phosphorylation of Ser567 by inducing an intramolecular interaction that leads to release of E2F, degradation of Rb, and susceptibility to apoptosis. Thus, phosphorylation of Ser567 may limit excessive proliferation by triggering cell death under hyperproliferative conditions. These findings suggest that the antiproliferative and antiapoptotic activities of Rb may represent complementary functions that work in concert to maintain the proliferation rate of cells within certain limits. As a survival strategy, some cancer cells may exploit this dual role of Rb by phosphorylating sites that regulate tumor suppression but avoiding phosphorylation of Ser567 and consequent apoptotic stimulus.

Objective To determine whether transducible peptides that inhibit the oncoproteins HDM2 and Bcl-2 may selectively kill uveal melanoma and retinoblastoma cells. Methods Peptides were tested by viability assay, flow cytometry, TUNEL (terminal deoxynucleotidyl transferase–mediated fluorescein-dUTP nick-end labeling) assay, Western blot analysis, and reverse transcription–polymerase chain reaction in cultured eye tumor cells and normal cells. Preclinical studies were performed in a rabbit xenograft model of retinoblastoma. Main Outcome Measures Cell survival, apoptosis, gene expression, and tumor regression. Results The anti–Bcl-2 peptide induced apoptosis in tumor cells, but it also caused apoptosis in normal cells in culture and induced retinal damage after intravitreal injection. In contrast, the anti-HDM2 peptide induced rapid accumulation of p53, activation of apoptotic genes, preferential killing of tumor cells, and minimal retinal damage after intravitreal injection. The anti-HDM2 peptide also induced regression of human retinoblastoma cells in rabbit eyes. Conclusions Peptide transduction is a promising new approach to molecular eye cancer therapy. Inhibition of HDM2 can selectively activate p53 in transformed cells and may be an effective strategy for inducing apoptosis in eye cancer cells with minimal damage to normal ocular tissues. Clinical Relevance Molecular characteristics of uveal melanoma and retinoblastoma may be used to design novel therapeutic agents that have greater specificity and fewer adverse effects than current therapies.

Cervical cancer is the second-most common cancer among women worldwide (Parkin et al. 1999). Some studies address prognostic roles that different genes play after the development of cervical cancer. However, there is a lack of studies addressing polymorphisms in genes of normal tissues that influence susceptibility to cervical cancer prior to malignant transformation. Such studies could be pivotal in prevention and timely diagnosis. It is well-known that Human Papillomavirus (HPV) infection appears to be the primary causative factor for cervical cancer, but host factors must be crucial since only a fraction of HPV-infected women develop cervical cancer. HPV antigens are the ligands of HER family members, and the E5 protein may play a role in the early stage of HPV infection before transformation. Thus, we studied whether there are excessive parent-to-offspring transmissions of specific HER SNP variants in EGFR, ERBB2, ERBB3 and ERBB4, in cases of invasive cervical cancer (ICC) or cervical intraepithelial neoplasia grade 3 (CIN3). Based on single SNP TDT tests, we identified a significant association between ICC/CIN3 and rs11770506, which is located at intron1 of EGFR and in high linkage disequilibrium (LD) with multiple enhancers. Our study provides clues for the identification of vulnerable populations and will advance the development of early detection of ICC in appropriate populations. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1214. doi:10.1158/1538-7445.AM2011-1214