Xinchun Zhou

Identification of Plasma Lipid Biomarkers for Prostate Cancer by Lipidomics and Bioinformatics

Background Lipids have critical functions in cellular energy storage, structure and signaling. Many individual lipid molecules have been associated with the evolution of prostate cancer; however, none of them has been approved to be used as a biomarker. The aim of this study is to identify lipid molecules from hundreds plasma apparent lipid species as biomarkers for diagnosis of prostate cancer. Methodology/Principal Findings Using lipidomics, lipid profiling of 390 individual apparent lipid species was performed on 141 plasma samples from 105 patients with prostate cancer and 36 male controls. High throughput data generated from lipidomics were analyzed using bioinformatic and statistical methods. From 390 apparent lipid species, 35 species were demonstrated to have potential in differentiation of prostate cancer. Within the 35 species, 12 were identified as individual plasma lipid biomarkers for diagnosis of prostate cancer with a sensitivity above 80%, specificity above 50% and accuracy above 80%. Using top 15 of 35 potential biomarkers together increased predictive power dramatically in diagnosis of prostate cancer with a sensitivity of 93.6%, specificity of 90.1% and accuracy of 97.3%. Principal component analysis (PCA) and hierarchical clustering analysis (HCA) demonstrated that patient and control populations were visually separated by identified lipid biomarkers. RandomForest and 10-fold cross validation analyses demonstrated that the identified lipid biomarkers were able to predict unknown populations accurately, and this was not influenced by patients age and race. Three out of 13 lipid classes, phosphatidylethanolamine (PE), ether-linked phosphatidylethanolamine (ePE) and ether-linked phosphatidylcholine (ePC) could be considered as biomarkers in diagnosis of prostate cancer. Conclusions/Significance Using lipidomics and bioinformatic and statistical methods, we have identified a few out of hundreds plasma apparent lipid molecular species as biomarkers for diagnosis of prostate cancer with a high sensitivity, specificity and accuracy.

The expression level of lysophosphatidylcholine acyltransferase 1 (LPCAT1) correlates to the progression of prostate cancer

BACKGROUND Lysophosphatidylcholine acyltransferase 1 (LPCAT1), the enzyme catalyzing the reaction in remodeling of phosphatidylcholine (PC) has been reported to express in prostate. However, its diagnostic and prognostic values remain unclear. METHODS Immunohistochemistry (IHC) for LPCAT1 was performed on the tissue microarray (TMA) slides containing 251 samples from 148 patients with various prostatic disorders. The association of expression level of LPCAT1 with the progression of prostate cancer was analyzed. RESULTS LPCAT1 IHC mean score was the highest in metastatic prostate cancer (8.00±1.28), which was significantly higher than that in primary prostate cancer (4.63±3.00, p=9.73E-07), in high grade prostatic intraepithelial neoplasia (HGPIN, 2.72±2.47, p=1.02E-12), and in benign prostate (2.68, p=6.17E-12). The mean score in primary prostate cancer was significantly higher than that in HGPIN (p=4.09E-04) and in benign prostate (p=2.74E-04). There was no significant difference in the mean score between HGPIN and benign prostate (p=0.951). LPCAT1 IHC score also correlated to the tumor grade and stage of prostate cancer. Patients who underwent prostatectomy for prostate cancer and developed biochemical recurrence or clinical metastasis had higher LPCAT1 IHC score than those who underwent prostatectomy for prostate cancer and did not develop biochemical recurrence and clinical metastasis. The association of LPCAT1 with the progression of prostate cancer was independent of patient race and age, PSA level and positivity of surgical resection margins. CONCLUSIONS LPCAT1 correlates with the progression of prostate cancer and could be a new biomarker in diagnosis, prognosis and studying the pathogenesis of prostate cancer.

A retrospective study on pathologic features and racial disparities in prostate cancer.

We reviewed more than 3,000 pathology reports on prostate cancer-related surgical specimens and analyzed racial disparities in histological and clinical features at the time of initial biopsy, diagnosis of prostate cancer, and prostatectomy, as well as in characteristics of tumor evolution between African American and Caucasian patients. As compared to Caucasians, African American patients had younger age, higher cancer detection rate, higher Gleason score of prostate cancer, and more bilateral involvement of the prostate. African Americans also had larger prostates, greater volume of tumor, and more positive margins. The diagnosis of HGPIN or ASAP in prostate biopsies and African American race conferred an increased risk of diagnosis of prostate cancer. The interval between prior noncancerous biopsy and the subsequent biopsy with diagnosis of prostate cancer was shorter in men with HGPIN, with ASAP, or of African American race.

Systemic distribution, subcellular localization and differential expression of sphingosine-1-phosphate receptors in benign and malignant human tissues.

AIMS Five sphingosine-1-phosphate receptors (S1PR): S1PR1, S1PR2, S1PR3, S1PR4 and S1PR5 (S1PR1-5) have been shown to be involved in the proliferation and progression of various cancers. However, none of the S1PRs have been systemically investigated. In this study, we performed immunohistochemistry (IHC) for S1PR1-S1PR5 on different tissues, in order to simultaneously determine the systemic distribution, subcellular localization and expression level of all five S1PRs. METHODS We constructed tissue microarrays (TMAs) from 384 formalin-fixed paraffin-embedded (FFPE) blocks containing 183 benign and 201 malignant tissues from 34 human organs/systems. Then we performed IHC for all five S1PRs simultaneously on these TMA slides. The distribution, subcellular localization and expression of each S1PR were determined for each tissue. The data in benign and malignant tissues from the same organ/tissue were then compared using the Students t-test. In order to reconfirm the subcellular localization of each S1PR as determined by IHC, immunocytochemistry (ICC) was performed on several malignant cell lines. RESULTS We found that all five S1PRs are widely distributed in multiple human organs/systems. All S1PRs are expressed in both the cytoplasm and nucleus, except S1PR3, whose IHC signals are only seen in the nucleus. Interestingly, the S1PRs are rarely expressed on cellular membranes. Each S1PR is unique in its organ distribution, subcellular localization and expression level in benign and malignant tissues. Among the five S1PRs, S1PR5 has the highest expression level (in either the nucleus or cytoplasm), with S1PR1, 3, 2 and 4 following in descending order. Strong nuclear expression was seen for S1PR1, S1PR3 and S1PR5, whereas S1PR2 and S1PR4 show only weak staining. Four organs/tissues (adrenal gland, liver, brain and colon) show significant differences in IHC scores for the multiple S1PRs (nuclear and/or cytoplasmic), nine (stomach, lymphoid tissues, lung, ovary, cervix, pancreas, skin, soft tissues and uterus) show differences for only one S1PR (cytoplasmic or nuclear), and twenty three organs/tissues show no significant difference in IHC scores for any S1PR (cytoplasmic or nuclear) between benign and malignant changes. CONCLUSION This is the first study to evaluate the expression level of all S1PRs in benign and malignant tissues from multiple human organs. This study provides data regarding the systemic distribution, subcellular localization and differences in expression of all five S1PRs in benign and malignant changes for each organ/tissue.

Expression of allograft inflammatory factor-1 (AIF-1) in acute cellular rejection of cardiac allografts

BACKGROUND To date, there are no good biomarkers for diagnosing and predicting the progression of cardiac allograft rejection. Previous studies have shown a correlation of allograft inflammatory factor-1 (AIF-1) to human cardiac allograft rejection; however these studies were limited in sample size and did not address whether AIF-1 could be used as a biomarker in diagnosing and predicting the progression of cardiac allograft rejection. METHODS Using immunohistochemistry, AIF-1 expression was determined in cardiomyocytes (CMCs), mononuclear cells (MNCs) and Quilty lesions in 192 allograft endomyocardial biopsies and 37 heart specimens from nontransplant patients with diverse heart diseases. RESULTS AIF-1 was found in both cardiac allografts and hearts with other cardiac diseases. In cardiac allografts, expression levels of AIF-1 in both CMCs and MNCs directly correlated with the severity of cardiac cellular rejection. AIF-1 expression was also elevated in Quilty B lesions, but not in Quilty A lesions. The rejection grade in subsequent biopsies increased in biopsies that had low-grade rejection with high CMC AIF-1 scores or Quilty B lesions. CONCLUSION AIF-1 was expressed in both CMCs and MNCs in hearts with various adverse conditions including but not limited to heart transplantation. In cardiac transplantation, AIF-1 was associated with the severity of cardiac allograft rejection and Quilty B lesions, which could predict subsequent increases in rejection grade. Thus, AIF-1 shows promise that it can be a potential biomarker for cardiac allograft rejection.

Cardiac allograft rejection correlates with increased expressions of Toll-like receptors 2 and 4 and allograft inflammatory factor 1.

BACKGROUND Evidence suggests that injury-induced activation of the recipients innate immune response determines the outcome of allograft transplantation. The mechanism responsible for the induction of such innate immune response is not clear yet. We hypothesized that in cardiac transplantation settings, the initial myocardial ischemia and postischemia graft reperfusion may release allograft inflammatory factor (AIF) 1, causing Toll-like receptor (TLR)-mediated activation of macrophages and dendritic cells, leading to the production of cytokines and the activation of adaptive alloimmunity. Therefore, our goal was to validate the presence of these biomarkers in the peripheral blood and biopsy specimens of patients presenting allograft rejection. METHODS We studied 90 peripheral blood and 30 endomyocardial biopsy specimens from patients who had undergone cardiac transplantation. Specimens were tested by quantitative reverse-transcription polymerase chain reaction to determine TLR-2 and -4 and AIF-1 expression levels, correlating with clinical rejection grades. The group differences for mRNA transcript levels between the rejection grades were determined by 1-way analysis of variance. The level of significance was set at P < .05 for comparison between the groups. RESULTS The mean ± SEM level of TLR-2 mRNA expression was increased 1.7-fold in monocytes (P < .05) and 4.2-fold in biopsy samples from groups with grade 3A compared with grade 1A or grade 0 rejection (P < .0001). AIF-1 expression was increased 2.4-fold in monocytes (P < .05) and 4.2-fold in biopsy samples comparing grade 3A versus 1A rejections. The TLR-4 mRNA expression was also increased in the group with 3A rejections; however, the difference was only significant in biopsy specimens (P < .0001). CONCLUSIONS Our data demonstrated that expression profiles of AIF-1 and TLR-2 correlated with biopsy-proven allograft rejection in both peripheral blood and local tissue, suggesting their potential as diagnostic biomarkers for early detection of allograft rejection.

Differential association of cytochrome P450 3A4 genotypes with onsets of breast tumors in African American versus Caucasian patients.

Introduction Malignant breast tumors are often hormone-dependent, and to this end, both estrogen and progesterone receptors are good prognostic markers for evaluation of the outcomes after therapy. In addition, HER-2/neu, whose expression is increasingly being associated with poor prognosis of breast cancer, has predictive potential after immunotherapy. Cytochrome P450 3A4 is highly involved in the metabolism of steroids. Thus, we investigated the impact of CYP 3A4 gene variants in association with clinical outcomes in African American (AFAM) versus Caucasian (CAU) patients with breast cancer diagnosis. Methods Patients who had undergone biopsy procedures for diagnosis or for partial or radical mastectomy were recruited. The CYP 3A4 genotypes (A or G) were detected using polymerase chain reaction amplification and primers designed for a single nucleotide polymorphism. The messenger RNA (mRNA) transcripts were screened by reverse transcription-polymerase chain reaction. Clinical data including tumor staging, pathology grades, and family history were evaluated. Results Frequency of the CYP 3A4-G (mutated variant) was significantly increased in AFAM patients as compared with controls (P < 0.001). No statistically significant difference was observed between the genotypes comparing the benign versus ductal carcinomas in situ (DCIS) or infiltrating ductal carcinomas (IDCAs). In AFAM patients, GG alleles were increased in IDCA with stage III tumors, and in CAU patients, the AA alleles were increased with stage III tumors. The mRNA expression was reduced in patients with IDCA versus DCIS or benign tumors (benign vs IDCA, P < 0.0009; DCIS vs IDCA, P < 0.005), as well in HER-2/neu-positive tumors versus samples negative for receptors (P < 0.0024). Conclusions Genotype association was affected by race. Expression levels of total CYP 3A4 mRNA were inversely correlated with clinical diagnosis. This may suggest mRNA testing as an additional tool that accelerates improvement in the diagnosis of the onsets of breast cancer.

Abstract 4973: The role of cholesteryl esters in progression and racial disparity of prostate cancer

Background Prostate cancer (PCa) is featured by uncertainty of future outcomes at the time of diagnosis, development of castration resistant prostate cancer (CRPC), and racial disparity in morbidity and mortality, which is especially prominent between African American (AA) and Caucasian American (CA). This study is aimed to investigate whether accumulation of cholesteryl esters (CE) in PCa is a key biological process that determines progression potential, and serves as a common mechanism underlying these features. Methods The methods used in this study include: 1) ESI/MS-MS on 47 fresh-frozen prostatic tissues for global lipid profiling; 2) Real-time PCR on 16 fresh-frozen prostatic tissues for the expression level of genes related to the pathogenesis of prostate cancer and metabolism of cholesteryl esters; and 3) immunohistochemistry on 165 formalin-fixed and paraffin embedded prostatic tissues for the expression level of ACAT1 and LAL. Results We found that 1) prostatic concentration of total lipids was significantly higher in PCa than benign prostatic tissues (BPT), with a PCa to BPT (P/B) ratio of 2.3, p = 0.013; 2) when total lipids were stratified into groups of neutral lipids and polar lipids, only neutral lipids are significantly higher in PCa than BPT (P/B ratio: 3.1, p = 0.02); 3) after neutral lipids were further stratified into class of triglycerides, diglycerides, free fatty acids (FFA) and CEs, total CEs are most sensitive in differentiation of PCa from BPT (P/B ratio: 5.8, p = 0.025); 4) 17 out of 31 prostatic individual CE species are significantly higher in PCa than in BPT all the studied populations; and 5) intriguingly, 14 of these 17 prostatic CE individual species remain significantly different between PCa and BPT in AA population, whereas only 3 of them remain significantly different between PCa and BPT in the CA population. The real-time PCR results indicated that the expression levels of genes Pten, LIPA, and ABCA1 are obviously lower in high grade than in low grade PCa (not obviously for ACAT1). The expression levels of proteins ACAT1 and LAL (LIPA gene product) are reversely correlated with the progression of PCa: in prostate cancer, ACAT1 is highly expressed, but LAL is not expressed. In contrary, in benign ACAT1 is not expressed, but LAL is highly expressed. Such a reverse correlation is especially prominent in AA than CA population. Conclusion and Significance Accumulation of cholesterol esters correlates with the progression and racial disparity of PCa. Down regulation of LIPA gene and its product LAL could play major role in CE accumulation. Thus prostatic CE and its individual species could serve as racially specific biomarkers in diagnosis and in differentiation of indolent from aggressive cases of PCa, and LAL could be potential therapeutic targets for treatment of advanced prostate cancer, including castration resistant prostate cancer. Citation Format: Xinchun Zhou, Timera Brown, Janice Lage, Jinghe Mao. The role of cholesteryl esters in progression and racial disparity of 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 4973.

Abstract 5003: Differential expression of lysophosphatidic acid receptors (LPARs) between benign and malignant tissues in humans

Background: Lysophosphatidic acid receptors (LPARs) are important to normal cellular functions and implicated in the pathogenesis and survival of various cancers recently. Previous studies were mostly focused on one LPAR using experiments on animal models and cell cultures. This study was executed to investigate systemic distribution of LPAR1, LPAR2, and LPAR3 in human organs/tissues and to compare the expression levels of these receptors between benign and malignant changes in major human organs/tissues. Method: Tissue Microarrays TMAs) were constructed with 384 formalin-fixed and paraffin-embedded benign and malignant specimens from 33 different human organs/tissues. Immunohistochemistry (IHC) for each LPAR was performed on the TMA slides. In addition to observing the subcellular localization of each LPAR, the expression level of each LPAR at each subcellular localization was recorded as final IHC score, calculated by IHC intensity score (0 to3) multiplied with IHC area score (0 to 3). A 2-sample t test was used to statistical analysis. Results: All three LPARs were widely distributed throughout human organs/tissues. All LPARs were expressed higher in nuclei than in cytoplasm. Nuclear expressed LPARs were all higher in overall malignant changes than in overall benign changes, which is especially true for nuclear expressed LPAR1 (LPAR1N): the IHC score for LPAR1N was 6.38 in overall malignant changes and 5.84 in overall benign changes (p = 0.016). The expression level of LPAR1 in nuclei or in cytoplasm is significantly (p 0.05) different between benign and malignant changes in eight organs/tissues. The number of organs/tissues with such difference was five for LPAR2 and six for LPAR3. Hepatocellular carcinoma (HCC) had significantly higher expression level of LPAR1N (p = 0.000000001), LPAR2N (p = 0.00002) and LPAR3N (p = 0.00001) as compared with benign hepatic changes. The malignancies showed obviously alterations in expression level of two LPARs in three organs/tissues and one LPAR in eight organs/tissues. The malignancies in the rest organs/tissues, including breast and prostate showed no obvious difference between benign and malignant changes. Conclusions: This study is first to investigate systemic distribution of the LPAR1, LPAR2 and LPAR3 in benign and malignant changes throughout human organs/tissues. The results indicate that each LPAR has a unique expression pattern in a spectrum of organs/tissues. Differentially expressed LPARs between benign and malignant changes in different organs/tissues will help to understand the involvement of LPARs in the pathogenesis of cancer, and to find therapeutic targets in treatment of cancer. Citation Format: Andrew Bingham, Jinghe Mao, Chunyi Wang, Janice Lage, Xinchun Zhou. Differential expression of lysophosphatidic acid receptors (LPARs) between benign and malignant tissues in humans. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5003. doi:10.1158/1538-7445.AM2015-5003

Abstract 3011: Differential expressions of lysophospholipid receptors (LPRs) in benign and malignant tissues from various human system/organs

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Background: Lysophospholipid receptors (LPRs), including Lysophosphatidic acid (LPA) receptors and sphingosine 1-phosphate (S1P) receptors, are a group of G protein-coupled receptors. LPRs are involved in a wide range of biological processes, such as embryogenesis, systemic development, immune cell trafficking, and inflammatory reactions. The emerging evidence has suggested that the activities of LPRs may be associated with pathogenesis and metastasis of several human cancers. In this study, the expression levels of several LPRs were determined by immunohistochemistry (IHC) in various benign and malignant tissues from various human system/organs, in order to correlate the expression levels of these LPRs with different malignancies. Methods: IHC for EDG1 (S1P receptor 1, S1P1, C- and N-terminals), EDG8 (S1P receptor 5, S1P5), EDG2 (LPA receptor 1, LPA1) and EDG4 (LPA receptor 2, LPA2) was performed on tissue microarray slides containing 384 formalin-fixed paraffin-embedded benign and malignant tissues from 33 human system/organs. The expression level of each LPR was reported as a final IHC score calculated as staining extent score (0-3) multiplied by intensity score (0-3) with a maximal score of 9. Student t-test was employed to analyze the differences in IHC score of each studied LPR between benign and malignant tissues in overall and in individual system/organ. Results: The IHC signals for all studied LPRs were seen in both cytoplasm and nuclei of most tissues. In the level of overall benign and malignant tissues, the expression pattern was similar for all studied LPRs: the cytoplasmic IHC signals were decreased, but the nuclear IHC signals were increased in malignant tissues as compared with benign tissues. Among them, the expression level of cytoplasmic S1P1 (C-terminal) was significantly decreased, and expression levels of nuclear S1P1 (N-terminal) and LPA1 were significantly increased in overall malignant tissues as compared with overall benign tissues. At the organ level, a significant increase in nuclear expression was observed in liver malignancies for all studied LPRs, whereas a decrease in nuclear expression was observed in lung malignancies for all studied LPRs. In colon malignancies, the expression levels of S1P1 (both N- and C-terminals) were significantly increased, but the expression level of LPA1 was significantly increased as compared with benign colon tissues. Conclusion: The constitutive expression of cytoplasmic LPRs may be essential for physiological processes in all tissues. However alterations in nuclear expression of studied LPRs may differentially correlate with the tumorogenesis in different system/organs. Citation Format: Chunyi Wang, Samantha Redfield, Jinghe Mao, Yinyuan Mo, Xinchun Zhou. Differential expressions of lysophospholipid receptors (LPRs) in benign and malignant tissues from various human system/organs. [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 3011. doi:10.1158/1538-7445.AM2014-3011