Aqeel Ahmed

Phase IB Randomized, Double-Blinded, Placebo-Controlled, Dose Escalation Study of Polyphenon E in Women with Hormone Receptor–Negative Breast Cancer

Epidemiologic data support an inverse association between green tea intake and breast cancer risk, and numerous experimental studies have shown the antitumor effects of its main component, epigallocatechin gallate (EGCG). We conducted a phase IB dose escalation trial in women with a history of stage I to III hormone receptor–negative breast cancer of an oral green tea extract, polyphenon E (Poly E) 400, 600, 800 twice daily 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 ≥II). 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 400 mg, 11 at 600 mg, 3 at 800 mg). There was one DLT at 400 mg (grade III rectal bleeding), three DLTs at 600 mg (grade II weight gain, grade III indigestion and insomnia), and one DLT at 800 mg (grade III liver function abnormality). The DLT rate at 600 mg was 27% (3 of 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 600 mg twice daily. This study highlights the importance of assessing toxicity for any chemopreventive agent being developed for chronic use in healthy individuals. Cancer Prev Res; 5(9); 1144–54. ©2012 AACR.

p53 is localized to a sub-nucleolar compartment after proteasomal inhibition in an energy-dependent manner

The tumor suppressor p53 is activated in response to many forms of cellular stress leading to cell cycle arrest, senescence or apoptosis. Appropriate sub-cellular localization is essential for modulating p53 function. We recently showed that p53 localizes to the nucleolus after proteasome inhibition with MG132 and this localization requires sequences within its carboxyl terminus. In the present study, we found that after treatment with MG132, p53 associates with a discrete sub-nucleolar component, the fibrillar center (FC), a region mainly enriched with RNA polymerase I. Moreover, we now demonstrate that this localization is an energy-dependent 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.

CpG ODN, Toll Like Receptor (TLR)-9 Agonist, Inhibits Metastatic Colon Adenocarcinoma in a Murine Hepatic Tumor Model

BACKGROUND Colorectal liver metastases (mets) are often refractory to conventional therapies. CpG oligodeoxynucleotide 1826 (CpG), a Toll like receptor (TLR)-9 agonist, inhibits murine tumor growth by augmenting Th1 immunity. The impact of CpG on metastatic colon tumors is unknown. The purpose of this study was to determine the effect of CpG on the growth of hepatic colon cancer mets. METHODS Two studies with separate control groups were performed using 40 Balb/C mice (study A, CpG 50 μg/dose; study B, 100 μg/dose; n = 9-11/subgroup). Tumors were induced via portal vein injection of 2 × 10(4) CT26 colon tumor cells. After surgery, the mice were randomized; test groups were given 14 daily intraperitoneal (i.p.) CpG injections (50 or 100 μg/dose) while the control group received i.p. saline. On d 21 mice were sacrificed, the livers and spleens excised and weighed and the mets counted (reported as median ± 95% confidence interval [CI]) and histologically assessed. RESULTS The CpG mice had significantly fewer hepatic mets/mouse (study A, median two nodules, 95% CI, 0-3; study B, 0 nodules, 95% CI 0-0) than the control mice (study A, 6 nodules, 95% CI, 3-9, P = 0.002; Study B, 6 nodules, 95% CI, 3-9, P < 0.001). In study B, there were no mets in 9/11 CpG mice (versus 2/10 for CpG 50 μg and 0/19 for control mice). The mean liver/spleen weights of the CpG mice in both studies were significantly greater than in control mice. Histologically, high mitotic rates were noted in control mets while fewer tumor cells and histiocytic and lymphocytic infiltrates were found in CpG livers. CONCLUSIONS CpG inhibited liver tumor growth in this model (100 μg/dose more than 50 μg/dose). CpG was associated with increased liver and spleen weights not related to tumor burden. Increased lymphocytic and histiocytic infiltrates were noted in CpG-treated tumor nodules.

The Cancer Testis Antigens CABYR a/b and CABY C Are Expressed in a Subset of Colorectal Cancers and Hold Promise as Targets for Specific Immunotherapy

Introduction: Human CABYR is a testis specific polymorphic calcium binding tyrosine phosphorylation regulative protein isolated from spermatozoa. This protein is localized to the sperm flagellum in association with the fibrous sheath; it can bind calcium after being phosphorylated during sperm capacitation. Six transcript variants of CABYR encoding five protein isoforms have been identified. Normally, CABYR is expressed in the human germ line but not in adult human tissues, thus, it is considered a cancer testis (CT) protein. A high frequency of expression in tumor tissues and restricted expression in normal tissues make CT proteins attractive cancer vaccine targets. Increased expression of CABYR a/b and c isoforms in lung cancers and isoforms c and d in brain tumors have been reported. The expression patterns of CABYR genes in colorectal cancer (CRC) are not well characterized. The aim of this study is to evaluate the expression of CABYR a/b and c in CRC tumors and to determine if they hold promise as vaccine targets. Method: Consenting CRC patients (pts) who underwent elective resection for whom tumor samples were available were included in the study. Demographic, clinical, and pathologic data were collected prospectively. Tissues were OCT embedded and stored at -80°C until analysis. Total purified RNA was isolated from tissue samples and cDNA synthesized. CABYR a/b and c expression was analyzed by quantitative PCR (QPCR) using the SYBR Green platform. Tumor and normal tissue expression levels were determined and compared as were tumor and testis CABYR expression levels. Tumors with expression levels 0.1% or more than the testis were considered positive. Expression of CABYR a/b and c in a set of normal human tissues was analyzed by RT-PCR. Results: A total of 47 paired CRC and normal tissue specimens (18 M/ 29 F, age 65+/-16.8) were studied (85% colon,15% rectal). Stage breakdown was as follows: Stage 2, 26; Stage 3, 19; and Stage 4, 2 pts. The percent of pts with a relative expression ratio of malignant to normal tissues (MN ratio) over 1 was 70% for CABYR a/b and 72% for CABYR-c. The percent with both a MN ratio over 1 and expression levels over 0.1% of testis was 23.4% for CABYR-a/b and 25.5% for CABYR-c. Except for very low expression of CABYR a/b in the brain, CABYR a/b or c was not expressed in normal tissues. Conclusion: RT-PCR analysis confirmed the frequent overexpression of CABYR a/b and c in most CRC tumors compared to adjacent normal tissues. In 23-26% of tumors expression was more than 0.1% of the expression level of testis. A larger and more diverse group of tumors (Stage 1-4) needs to be assessed to determine if CABYR expression correlates with T, N, or final tumor stage. Evaluation of blood for anti-CABYR antibodies is also needed. These results support CABYR as a potential therapeutic vaccine candidate in CRC.

Transcriptional deconvolution reveals consistent functional subtypes of pancreatic cancer epithelium and stroma

Bulk tumor tissues comprise intermixed populations of neoplastic cells and multiple stromal cell lineages. We used laser capture microdissection and RNA sequencing to disentangle the transcriptional programs active in the malignant epithelium and stroma of pancreatic ductal adenocarcinoma (PDA). This led to the development of a new algorithm (ADVOCATE) that accurately predicts the compartment fractions of bulk tumor samples and can computationally purify bulk gene expression data from PDA. We also present novel stromal subtypes, derived from 110 microdissected PDA stroma samples, that were enriched in extracellular matrix– and immune–associated processes. Finally, we applied ADVOCATE to systematically evaluate cross–compartment subtypes spanning four patient cohorts, revealing consistent functional classes and survival associations despite substantial compositional differences.

Perioperative Polyphenon E, a Green Tea Extract, Does Not Affect the Wound Complication Rate in Mice After Sham Laparotomy yet Has an Inhibitory Effect on Wound Healing

Introduction. Major surgery is associated with physiologic alterations that may promote tumor growth, and catechins in green tea may inhibit tumor growth. This study’s aim was to assess the impact of a green tea extract on laparotomy wound healing in mice. Methods. Mice were randomized to daily oral catechins solution (n = 25) or placebo (n = 20), underwent sham laparotomy after 10 days, and were sacrificed on postoperative day 7 or 21. The peak force and total energy required to rupture the abdominal wall wound, wound collagen content, and histology were assessed. Results. There were no wound complications in either group, and mean peak wound rupture forces and collagen concentration were similar. Mean energy was lower and more fibroblast proliferation was found in the treatment group on postoperative day 21. Conclusions. These results suggest that catechins has only mild clinically significant adverse effect on wound healing, and its perioperative use warrants further study.

Su2020 The Tumor Associated Antigen P-Cadherin (CDH3) in Colorectal Cancer Holds Promise as a Prognostic Marker Rather Than as Specific Immunotherapy Target

Introduction: Variability of percentage excess weight loss (%EWL) in LAGB patients can be influenced by many preoperative factors, such as gender, race/ethnicity, and age. We hypothesize that race/ethnicity plays an important predictor in the post-operative weight loss. Methods: A retrospective analysis of 428 patients using electronic medical record was performed to assess differential %EWL for patients across a period of three years post-band implantation, with an average of 1.53 years for all groups and no significant differences between ethnic groups. Average initial BMI is 42 ± 5. ANOVA was used to analyze data and P<0.05 considered significant. Results: Percent excess weight loss (EWL) are reported for the following racial groups. Asians lost the most, followed by Caucasians, the Hispanics and finally African Americans. The Caucasian group lost 66.33% ± 2.4% %EWL (N=209); the Asian group lost 88.6% ± 7.7 %EWL (N=23); the Hispanic group (50.2% ± 2.3%; P= 1.5E-05, N=123), and African American group lost 44.4% ± 3.3%; P=9.96E-06, (N=73). Furthermore, the average number of adjustments was 6 for the Caucasian group, 5 for the Asian group, and 4 for the Hispanic and African American groups. Conclusion: Our findings suggest that weight loss outcome for LAGB may be related to a patients race/ethnicity. Patients in the Caucasian group have significantly more adjustments than any other group. Excess weight loss is correlated with race and number of adjustments as well as willingness to diet and exercise. Cultural differences in types of food and exercise is also important in weight loss outcome.

W1756 Expression of the Cancer Testis Antigens PLAC1 and MAGE3A in Colon Tumors As Potential Targets for Specific Immunotheraphy for a Subset of CRC Patients

Introduction: Genes that encode Cancer testis (CT) antigens are normally expressed only in the human germline. High frequencies of expression in various tumors and restricted expression in normal tissues make the CT antigens attractive cancer vaccine targets. PLAC1, MAGE3A and GAGE are genes with high expression in placenta/or testis and different cancer types with relatively restricted expression in normal tissues. The aim of this study was to evaluate PLAC1, MAGE3A and GAGE as vaccine targets in colorectal cancers. Method: Consenting CRC patients who underwent elective resection for whom tumor samples and preoperative blood samples were available comprise the study population. Basic demographic and clinical as well as short term outcome data was prospectively collected. Tissues were paraffin embedded and also stored at -80οC. Total purified RNA was isolated and cDNA synthesized. Comparative quantitative PCR (QPCR) was performed using the SYBR Green platform. Comparative quantitative analysis was performed based on delta-delta Ct method using GAPDH as internal control. Expression levels in tumors were compared to expression levels in Testis (MAGE3 and GAGE ) or levels in Placenta (PLAC1) and samples found to express 0.1% or more of the testis or placenta levels were considered positive for the expression of these genes. Results: 35 tumors and paired normal tissue samples were studied (83% colon, 17% rectal). Stage breakdown was as follows: stage 2, 18; stage 3, 16; and stage 4, 1 patient. Relative QPCR values of 35 malignant and paired normal samples for MAGEA3 and QPCR values of 31 pairs for PLAC1 and GAGE were available for analysis. Relative expression ratio of malignant to normal tissues over one(1) was higher for PLAC1 (77%) and moderate for GAGE (35.5%) and MAGEA3 (31%). However, both expression ratio over one (1) and expression levels above 0.1% of the levels in testis or placenta were noted for MAGE3A in 26%, PLAC1 (19%) but not for GAGE1. Conclusion: In a subset of tumors, the relative expression of MAGE3A and PLAC1 was considered above the expression in paired normal colon tissues and had more than 0.1% of the levels in testis and placenta. Serological evaluation and immunohistochemistry studies are needed to further evaluate the tumor subsets presenting MAGEA3 and PLAC1 expression. These results suggest that MAGEA3 and PLAC1 might be useful as vaccine targets for a subset of CRC patients and further studies are warranted.