AEG-1/MTDH/LYRIC was initially identified as a HIV-1-inducible gene in primary human fetal astrocytes. Astrocyte Elevated Gene-1 (AEG-1) is ubiquitously overexpressed in all or most cancers and plays a regulatory role in diverse and multiple processes of carcinogenesis. AEG1 is a multi-functional regulator of normal and abnormal physiology; it contributes to broad-spectrum resistance to various chemotherapeutics; and it was recently proposed as the first potential ‘pan-cancer’ gene However, despite its critical relevance to carcinogenesis, until now genetic polymorphisms for this important gene have not been studied for association with cancer susceptibility. For example, though studies have reported AEG-1 gene expression in animal models and tumor tissues, no studies have been reported in Caucasians on the AEG-1 gene polymorphisms in large samples to establish an association with cancers. We report a PCR-RFLP screening protocol for AEG-1 gene polymorphism which is cost effective, simple and reproducible. This study also shows the strong association of AEG-1 gene with prostate cancer risk and metastasis. DNA and RNA was isolated from 232 normal healthy age matched male Caucasians and 129 prostate cancer patients recruited in the study with an informed consent. AEG-1 gene polymorphism (rs2438211) was screened using PCR-RFLP method. Patients were compared with normal healthy controls and also within patient group comparisons were made between primary and metastasis cases. Genotype analysis was done using IBM SPSS 22 software and gene expression analysis was done using RT2 profiler PCR Array Data analysis version 3.5 (SABIOSCIENCES). A strong association of AEG-1 gene polymorphism with prostate cancer (p<0.0001) was observed. A high frequency of the mutant allele was observed in patients (27%) as compared to normal healthy controls (7.9%).This study also highlights the possible effect of AEG-1 interaction with other genes (p53, BCCIP) contributing to tumorigenesis. Citation Format: Radhika Gade Andavolu, Catherine Stafford, Patrick Herling, Isabella Bianco, Jean-Luc Cardenas, Henry Go, Svetlana Rubakovic, Murthy V. Andavolu. Astrocyte elevated gene-1(AEG-1)as a potential diagnostic/prognostic marker for prostate cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4775.

Background: Our previous study on the gene expression profiling in FFPE prostate cancer tissue using Oligo GEArray® (440 oligonucleotide probes) reported 139 differentially expressed genes in malignant vs. normal tissue (AACR San Diego, 2008). To validate the performance of these low density Oligo GEArray® microarray, and for reliable and reproducible data in FFPE malignant and normal prostate tissue we subjected the processed samples for immunohistochemistry and Real-Time™ PCR assays. In this paper we evaluate our study with the most significant (p<0.0001) lactotransferrin gene (LTF, also referred to as the lactoferrin gene, LF) because, of its low or no signals observed in malignant FFPE prostate tissue as compared to the surrounding normal tissue. Lactoferrin (LF) is a multifunctional protein with major role in regulation of iron homeostasis, host defense against a broad range of microbial infections, anti-inflammatory activity, regulation of cellular growth and differentiation and protection against cancer development and metastasis. Materials and Methods: Formalin-Fixed Paraffin-Embedded (FFPE) tissue sections were made at the Anatomic Pathology laboratory located at Eisenhower medical center. RNA isolations and purifications were done in these archived samples and 24 Oligo GEArray® were used for screening & identification of differential gene expressions in malignant and normal tissues as described earlier (AACR 2008). Two validation assays were done 1). To study the presence of LF antigen in different regions of the prostate, sections of the malignant and normal prostate tissues were stained with an antibody against LF (a polyclonal rabbit antibody to lactoferrin (Abcam, Cambridge, MA, USA). 2). The most reliable SYBR® Green-based quantitative real-time PCR assay for gene expression analysis was also done in Stratagene MX3000P instrument using the standard protocol (SABiosciences) given with the commercially designed LTF Primer sets (Accession #:NM_002343). Results: Immunohistochemistry: Staining patterns of LF was evaluated in normal and malignant tissue sections. In general, most of the benign glands show patchy positive staining mostly luminal and with some cytoplasmic staining. Few benign glands with hyperplastic pattern stained negative. No signal was detected in malignant tissue in general; and all cancers are negative with few exceptions. When positive, it is usually focal and only luminal (not cytoplasmic). Real-Time.™ qPCR assay: Low expression levels correspond to high Ct values (in malignant) and high expression levels correspond to low Ct values (normal) were observed. Raw Ct values did show distinct patterns but not as clear as seen in the arrays. The overall comparisons confirmed the results with a significance of p<0.002 suggesting low expression of LTF gene in malignant tissue. Conclusions: Immunostaining experiments and the Real-Time™ qPCR assay confirmed the significant over expressions of LTF in normal tissue as compared to no or low expression in the malignant tissue. In conclusion, Oligo GEArray® can be a good source for initial screening to identify molecular signatures of the known class in FFPE tissues. With the selected panel of molecular gene signatures, a Real-Time™ qPCR array can be custom designed for evaluation of patients' prognosis, treatment and to improve clinical diagnostics with the discovery of new markers. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C168.

Background: Genetic alterations can contribute to the malignant transformation and progression of prostate cancer. We have previously shown a strong association between specific leptin gene alleles, elevated serum leptin level, and body mass index in subjects with prostate and breast cancer. These studies showing association with leptin suggest a strong link with obesity as an increased risk factor. Studies have shown that adipose tissue TNFa and leptin, which are both overexpressed in obesity, induces the production of acute phase reactants through their autocrine or endocrine role. When GSTP1 is inactivated, prostate cells appear to become more vulnerable to somatic alterations upon chronic exposure to genome-damaging stresses such as oxidants and electrophiles that are contributed by environment and lifestyle. Studies also show that the methylation status of glutathione S-transferase p1 (GSTP1) could individually distinguish prostate cancer from benign prostatic hyperplasia. Based on prior studies, it is possible that multi gene analysis can provide better diagnostic or prognostic values for prostate cancer. Methods: In this study, we screened leptin gene markers, TNFa, GSTP1 and BCCIP(TOK-1A) in 126 prostate cancer and normal healthy age & sex matched controls (Prostate cases: Age range 45-94, Mean age 73.3 Control males: Age range 65-98, Mean age 73.5). Polymerase chain reaction was used to amplify Leptin, TNF-308 and GSTP1 genes and the products are analyzed using 3130 Genetic analyzer and PCR-RFLP methods. Statistical analysis was performed using the SPSS/PC statistical program (version 10.0 for Windows; SPSS, Inc.,Chicago, IL). Relationships between two groups and genotype variables were assessed by Pearson's correlation coefficient. Conclusions: Prostate cancer is a complex, heterogeneous disease and there a variety of gene-based biomarkers that have been associated with prostate cancer. Our results show strong association of leptin (p<0.006), GSTP1 (p<0.023) with prostate cancer. Individually TNF and BCCIP showed a modest association but TNF/BCCIP gene interaction had a significant effect on prostate cancer risk and disease progression suggesting a multigene model for prostate cancer screening. Citation Format: Radhika G. Andavolu, Jean-Luc Cardenas, Ross Shore, Zach Rubin, James MacMurray, Krishna Kanth Chiravuri, Murthy VS Andavolu, Svetlana Rubakovic. Association of genetic variants with prostate cancer risk. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1932. doi:10.1158/1538-7445.AM2013-1932

BACKGROUND: Human acid phosphatases are normally found in low concentrations, but pronounced changes in their synthesis occur in particular diseases where unusually high or low enzyme expression is seen as part of the pathophysiological process,suggesting the clinical relevance of ACP as a disease marker. Acid Phosphatase 1 (ACP1) is considered to play a key role as a regulator of signaling pathways in receptor-stimulated immune cells and is known to be widely expressed and endowed with metabolic and proliferative activity. We hypothesized ACP1 could serve as a predictive marker in identifying cancer patients at an earlier stage, especially when disease is treatable. Acid phosphatase enzymes is generally correlated with the 3 co-dominant alleles A, B & C. This is the first study screening two ACP1 gene polymorphisms and ACP gene expressions in different cancer types. In this study, we exploit a novel connection of enzymatic activity in correlations to these two ACP1 polymorphisms and with more specific quantification of acid phosphatase in tumor tissues in primary and metastatic cancer patients. METHODS: All study subjects were non-Hispanic Caucasians. The study sample was comprised of 661 cancer patients (221 breast; 121 prostate;137 malignant melanoma;196 other cancers) and 827 healthy controls. DNA & RNA was isolated from all cancer patients & controls using standard procedure.PCR-RFLP method was used. ACP1 alleles (i.e., ACP1*A, ACP1*B, and ACP1*C) were determined with 2 of the selected SNPs: rs11553742 (C>T) is a synonymous polymorphism located in the codon 44 (exon 3) and rs7576247 (A/G) encodes an amino acid change in the codon 105 (exon 6) from arginine, present in ACP1*A allele, to glutamine in ACP1*B and *C alleles. Genotype and haplotype analysis was done using PASW 18 (SPSS IBM). qPCR is performed in tumor tissues and comparisons were made among cancer types and also in correlation with the haplotype based enzyme activity. RESULTS: A very significant association of ACP haplotypes AA & AB with cancers is observed (p<0.00001). Further analysis of these haplotypes in cancer types controlling for age and sex showed significant association mainly with Melanoma (p<0.0001); prostate ca (p<0.002); breast ca (p<0.013) and all other cancers (p< 0.002). Primary cases showed low activity while the metastatic cases showed high activity (> 2fold). CONCLUSIONS:The enzymatic activity from gene combination correlates with the gene expression data. The primary cancer cases showed a low expression. Our data also shows an over expression of ACP1(>2 fold) in metastatic cases as compared to the primary cases. This data supports the previous report on alkaline phosphatases (APs) catalyse and facilitate important physiological changes within cells and their expression is altered in many disease processes. It is clearly evident from this study that the ACP1 gene can be a critical marker in screening patients who are deemed low to high risk for cancer and metastasis. Citation Format: Radhika G. Andavolu, Jean-Luc Cardenas, Svetlana Rubakovic, Sina J. Torabi, Patrick Herling, James MacMurray, Murthy VS Andavolu. Genetic variations in acid phosphatase 1 (ACP 1) gene and cancer risk. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1858. doi:10.1158/1538-7445.AM2014-1858

Cyclin D1 (CCND1) G870A polymorphism has been known to be a risk factor in multiple cancers. Cyclin D1 is a cell cycle regulator frequently mutated, amplified and overexpressed in a variety of tumors. Activation and over expression of CCND1 have been found in variety of tumors and alterations in CCND1 are thought to be involved in carcinogensis. CCND1 polymorphism A870G interferes with splicing and studies showed DNA damage in cells from subjects with ‘A’ allele to bypass G1/S check point of the cell cycle control mechanism more easily than damage in cells with G allele (wild type). A single base pair polymorphism (exon 4, G870A) in the CCND1 gene affects gene splicing. This identification and validation of contributing genetic factors such as single-nucleotide polymorphisms (SNPs) represents a critical step in the advance toward personalized medicine. Several gene expression correlation studies have been done but to our knowledge studies have not been reported on SNP’s of ERBB2 gene, ESR gene and CCND1 combined effect correlating with high risk. In this study, we examined the relationship between ERBB2 (codon 655), Estrogen receptor (ER Intron1 T<C) and cyclin D1 (Exon 4) polymorphic additive effect in cancers more specific to breast cancer. Genomic DNA was extracted from whole blood samples collected on study. The SNPs were tested by the PCR-restriction fragment length polymorphism (PCR-RFLP) method. Our results show individuals carrying CCND1 ‘A allele in combination with variant ERBB2/BsmAI uncut allele are significantly high (P<0.004) in cancer patients (N=232) as compared to controls. (N=222). Similarly individuals carrying CCND1 Variant ‘A ‘ allele in combination with ESR ‘C’ Allele showed high frequency with p<0.009. Both these combinations showed a strong and significant association with cancer but individually, ERBB2 variant did not show any significant association, ESR gene showed a borderline association and CCND1 showed strong association independent of these two gene polymorphisms. Earlier few studies have shown no association of ERBB2 mutation in agreement with the previous reports. In conclusion our data suggests that in complex diseases a single gene may not contribute to show its effect but in combination may show a profound effect in evaluating the risk. Citation Format: Radhika Gade Andavolu, Olivia Rodriguez, Anastasia Al Rubakovic, Catherine Stafford, Murthy V. Andavolu. Combined effect of multiple gene polymorphisms on cancer risk stratification [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3267. doi:10.1158/1538-7445.AM2017-3267