ObjectivePancreatic ductal adenocarcinoma (PDA) has among the highest stromal fractions of any cancer and this has complicated attempts at expression-based molecular classification. The goal of this work is to profile purified samples of human PDA epithelium and stroma and examine their respective contributions to gene expression in bulk PDA samples.DesignWe used laser capture microdissection (LCM) and RNA sequencing to profile the expression of 60 matched pairs of human PDA malignant epithelium and stroma samples. We then used these data to train a computational model that allowed us to infer tissue composition and generate virtual compartment-specific expression profiles from bulk gene expression cohorts.ResultsOur analysis found significant variation in the tissue composition of pancreatic tumours from different public cohorts. Computational removal of stromal gene expression resulted in the reclassification of some tumours, reconciling functional differences between different cohorts. Furthermore, we established a novel classification signature from a total of 110 purified human PDA stroma samples, finding two groups that differ in the extracellular matrix-associated and immune-associated processes. Lastly, a systematic evaluation of cross-compartment subtypes spanning four patient cohorts indicated partial dependence between epithelial and stromal molecular subtypes.ConclusionOur findings add clarity to the nature and number of molecular subtypes in PDA, expand our understanding of global transcriptional programmes in the stroma and harmonise the results of molecular subtyping efforts across independent cohorts.

Epidemiologic data support an inverse association between green tea intake and breast cancer risk and numerous experimental studies have demonstrated the anti-tumor effects of its main component, epigallocatechin gallate (EGCG). We conducted a phase IB dose escalation trial in women with a history of stage I-III hormone receptor-negative breast cancer of an oral green tea extract, Polyphenon E (Poly E) 400mg, 600mg, 800mg bid or matching placebo for 6 months. The primary endpoint was to determine the maximum tolerated dose (MTD), defined as the dose that causes 25% dose limiting toxicity (DLT, grade≥2). Assignment to dose level was based upon an adaptive design, the continual reassessment method. A mammogram and random core biopsy of the contralateral breast were obtained at baseline and 6 months and serial blood/urine collections every 2 months for biomarker analyses. Forty women were randomized: 10 to placebo, 30 to Poly E (16 at 400mg, 11 at 600mg, 3 at 800mg). There was 1 DLT at 400mg (grade 3 rectal bleeding), 3 DLTs at 600mg (grade 2 weight gain, grade 3 indigestion and insomnia), and 1 DLT at 800mg (grade 3 liver function abnormality). The DLT rate at 600mg was 27% (3/11). Pharmacologic levels of total urinary tea polyphenols were achieved with all three dose levels of Poly E. Using a novel phase I trial design, we determined the MTD for Poly E to be 600mg bid. This study highlights the importance of assessing toxicity for any chemopreventive agent being developed for chronic use in healthy individuals.

IntroductionPlacental-Cadherin (CDH3) is a cell adhesion molecule vital to cellular localization and tissue integrity. It is highly expressed in the placenta (PLC)and is overexpressed by many types of cancer. P-cadherin levels in colorectal cancer (CRC) remains poorly characterized. This study's purpose was to determine P-cadherin expression in CRC and normal tissues and to assess plasma CDH3 levels in order to determine the relationship, if any, between cancer stage, P-cadherin expression and plasma CDH3 levels.MethodsAn IRB approved plasma, tumor, and prospective data bank was utilized. CRC patients for whom tumor and normal colon tissue samples were available were enrolled. Tumor samples were OCT embedded and stored at -80C°. Total purified RNA was isolated from tissue samples and cDNA synthesized. CDH3 expression was analyzed by quantitative PCR (QPCR) using the SYBR Green platform. Tumor expression levels were determined and compared to levels in normal colonic tissue and PLC. Expression in 22 different normal tissues was also assessed by RT-PCR. Plasma CDH3 levels were determined via ELISA in patients for whom preoperative blood samples were available. Results: A total of 77 paired CRC and normal colon specimens (36 M/ 41 F, age 67.3±14.5) were assessed (82% colon, 18% rectal; Cancer Stage 2, 44; Stage 3, 33). All tested tumors had CDH3 expression levels over 0.1% of the PLC level and tumor to normal colon ratios greater than 1. CDH3 expression was noted in 14/22 normal organ tissues. There was a positive correlation between tumor CDH3 QPCR and preoperative CDH3 blood levels (n=57, P= 0.038). Expression levels were significantly higher in rectal vs. colon tumors (p=0.019). Conclusion: CDH3 expression was elevated in CRC tumors as compared to normal tissue. CDH3 was expressed by numerous normal organs and, thus, is not a viable vaccine target, however, the correlation between plasma and tumor CDH3 levels suggests CDH3 may have value as a diagnostic or prognostic marker.

process as reduction of ATP levels prevents nucleolar localization. In addition, p53 sub-nucleolar accumulation is abolished when cells are subjected to various types of genotoxic stress. Furthermore, we show that monoubiquitination of p53, which causes it to localize to the cytoplasm and nucleoplasm, does not prevent the association of p53 with the nucleolus after MG132 treatment. Importantly, we demonstrate that p53 nucleolar association occurs in lung and bladder carcinomas. Journal of Cell Science ATP-dependent p53 sub-nucleolar localizationThe nucleolus is the site of rRNA transcription, processing and ribosome assembly (Andersen et al., 2005; Carmo-Fonseca et al., 2000;Lam et al., 2005;Lamond and Sleeman, 2003;Leung et al., 2003;Olson and Dundr, 2005). As diagrammed in Fig. 1A the nucleolus consists of at least three morphologically distinct regions: the fibrillar center (FC), a dense fibrillar center (DFC) and a granular component (GC) (Scheer and Hock, 1999). Although, still not fully described, several studies indicate that the FC or the FC-DFC boundaries are the sites of pre-rRNA transcription (reviewed by Boisvert et al., 2007;Lamond and Sleeman, 2003). Ribosomal proteins and assembly factors are found in the GCs (Huang, 2002;Koberna et al., 2002;Lamond and Sleeman, 2003). FCs are particularly enriched in Pol I, topoisomerase I and the upstream binding factor (UBF, an activator of Pol I), whereas most processing molecules localize to the DFC and/or GC including nucleolin, nucleophosmin and fibrillarin [Casafont et al. (Casafont et al., 2007) and references therein].In a previous study we showed that p53 accumulation in the nucleolus, after proteasome inhibition, requires two regions within the carboxyl terminus of p53 (Karni-Schmidt et al., 2007). We show now that after proteasome inhibition, p53 can associate with a discrete sub-nucleolar component, the FC. We demonstrate that p53 association with the FC is ATP dependent and it is abrogated by genotoxic stress. Our results also suggest that monoubiquitination of p53 does not preclude its association with nucleoli. Perhaps most relevantly we have found that p53 associates with nucleoli in human bladder and lung carcinomas. Resultsp53 associates with a discrete sub-nucleolar compartment after MG132 treatment To extend our observations and those of others that the treatment of cells with a proteasome inhibitor causes nucleolar localization of p53 (Karni-Schmidt et al., 2007; Klibanov et al., 2001;Latonen et al., 2003;Pokrovskaja et al., 2001) we examined the precise location of ectopic p53 within the nucleolus. H1299 cells were transfected with a construct expressing wild-type p53 and after 24 hours, cells were treated with different concentrations of MG132 for various times or left untreated. The levels of transfected p53 either did not change or were modestly reduced by treatment with H1299 cells were transfected with wild-type p53 and after 24 hours were treated with different doses (5 μM, 10 μM and 20 μM for 6-10 hours and 1 μM, 5 μM and 10 μM f...

IntroductionIGF2BP3 (IMP3) is a mRNA binding protein that regulates IGF2 translation and function during embryogenesis. Because IGF2BP3 is undetectable in adult human tissues except the testis, and increased IGF2BP3 expression has been noted in several cancers, it is considered a cancer testis (CT) protein. IGF2BP3 mRNA expression in colorectal cancers (CRC) has not been well studied. This study's aim was to quantitatively assess IGF2BP3 mRNA expression in CRC and, thus, determine if IGF2BP3 has potential as a vaccine target.MethodData were collected prospectively from CRC patients in an IRB-approved tissue and data bank. Total RNA was isolated and purified from tumor and normal colonic tissue samples and cDNA synthesized. IGF2BP3 expression was analyzed by quantitative PCR (QPCR). Expression levels of IGF2BP3 in tumors and testis were determined and compared. Tumors with levels greater than 0.1% or more of the testis levels were considered positive. Analysis of IGF2BP3 protein expression by immunohistochemistry (IHC) in tumor and normal tissues was also performed.ResultsA total of 84 paired tumor and normal tissue specimens were assessed from patients with Stage 2 and 3 CRC; 43% of tumors had IGF2BP3 mRNA expression levels greater than 0.1 % of that of testis and were considered positive. The median tumor expression level was higher in women (p=0.042). No correlation was found between IGF2BP3 mRNA expression and tumor stage or lymph node involvement. IHC was carried out on paired tumor and normal tissue sections from 46 patients; IGF2BP3 staining was noted in 50% of the tumor sections and in 5% of the normal tissue sections.DiscussionIGF2BP3 mRNA was over expressed in 43% of the tumors whereas the protein was noted in 50% of samples. No correlation between mRNA expression and disease severity was noted. This protein holds promise as a vaccine target, however, a larger study that assesses a more diverse population of patients (Stage 1-4) as well as a study of preoperative serum samples for auto-antibodies to IGF2BP3 are needed to pursue this concept.

This study was conducted to determine the safety and efficacy of the green-tea derived Polyphenon E (Poly E) in patients with Barrett’s Esophagus (BE). Subjects were randomized to a 6-month, twice daily (BID) oral treatment of placebo or Poly E (200 mg, 400 mg, or 600 mg). Endoscopic evaluation, including biopsies, was performed before and after treatment. The primary objective was to demonstrate safety; secondary objectives investigated catechin accumulation and effects in clinical specimens. Of the 44 enrolled subjects, 11 received placebo, and 33 received Poly E. No dose-limiting toxicities were encountered, and a maximum tolerated dose (MTD) was not reached. The recommended phase 2 dose was 600 mg BID. The most common treatment-related adverse events (AEs) in Poly E-treated subjects were grade 1–2 nausea, grade 1 belching, and grade 1 LDH elevation. No treatment-related AEs were reported in placebo-treated subjects, aside from grade 1 laboratory abnormalities. Pill counts and subject diaries were not consistently collected, and compliance was difficult to determine. However, based on an intention-to-treat analysis there was a significant relationship between Poly E dose and esophageal EGCG level – mean changes (pmol/g) of 0.79 (placebo), 6.06 (200 mg), 35.67 (400 mg), and 34.95 (600 mg); p=0.005. There was a possible relationship between Poly E dose and urine PGE-M concentration. In conclusion, Poly E was well-tolerated, and treatment with Poly E (400 mg and 600 mg) but not Poly E (200 mg) or placebo resulted in clinically relevant and detectable EGCG accumulation in the target organ, esophageal mucosa.

Breast white adipose tissue inflammation (BWATi) is associated with obesity and higher breast cancer (BC) risk among non-Hispanic white women. Obesity is prevalent in Hispanic/Latina BC patients, and the occurrence of BWATi in this population is not well-characterized. The association between BWATi and body mass index (BMI) was evaluated in Hispanic/Latina BC patients who underwent mastectomy. BWATi was defined as the presence of crown-like structures of the breast (CLS-B), detected by CD68 immunohistochemistry in non-tumor breast tissue. BWATi severity was quantified as number of CLS-B/cm2. Adipocyte diameter was measured using hematoxylin and eosin (H&E) stained breast tissue sections. Preoperative BMI (within 1 week prior to mastectomy) was categorized as normal (18.5 to <25.0 kg/m2), overweight (25.0 to <30.0 kg/m2), class I obesity (30.0 to <35.0 kg/m2), and class II-III obesity (35.0 kg/m2 or above). Patient charts were abstracted to record clinicopathologic features and liver function tests <90 days before mastectomy. The study included 91 women (mean age 69 years; range 36–96 years). Prevalence of BWATi increased with BMI (24% in normal weight, 34% in overweight, 57% in class I obesity, and 65% in class II-III obesity; P for trend <0.01). Severe BWATi (>0.27 CLS-B/cm2) was associated with higher BMI (P for trend=0.046) and greater adipocyte diameter (P=0.04). Adjusting for BMI, neoadjuvant chemotherapy and elevated alanine aminotransferase were associated with severe BWATi, and current smoking was associated with mild BWATi (all P<0.05). BWATi was associated with higher BMI in Hispanic/Latina BC patients, consistent with previously described associations in other populations.

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