Z. Laura Tabatabai

Quantitative analysis of prostate metabolites using 1H HR-MAS spectroscopy

A method was developed to quantify prostate metabolite concentrations using 1H high‐resolution magic angle spinning (HR‐MAS) spectroscopy. T1 and T2 relaxation times (in milliseconds) were determined for the major prostate metabolites and an internal TSP standard, and used to optimize the acquisition and repetition times (TRs) at 11.7 T. At 1°C, polyamines (PAs; T1mean = 100 ± 13, T2mean = 30.8 ± 7.4) and citrate (Cit; T1mean = 237 ± 39, T2mean = 68.1 ± 8.2) demonstrated the shortest relaxation times, while taurine (Tau; T1mean = 636 ± 78, T2mean = 331 ± 71) and choline (Cho; T1mean = 608 ± 60, T2mean = 393 ± 81) demonstrated the longest relaxation times. Millimolal metabolite concentrations were calculated for 60 postsurgical tissues using metabolite and TSP peak areas, and the mass of tissue and TSP. Phosphocholine plus glycerophosphocholine (PC+GPC), total choline (tCho), lactate (Lac), and alanine (Ala) concentrations were higher in prostate cancer ([PC+GPC]mean = 9.34 ± 6.43, [tCho]mean = 13.8 ± 7.4, [Lac]mean = 69.8 ± 27.1, [Ala]mean = 12.6 ± 6.8) than in healthy glandular ([PC+GPC]mean = 3.55 ± 1.53, P < 0.01; [tCho]mean = 7.06 ± 2.36, P < 0.01; [Lac]mean = 46.5 ± 17.4, P < 0.01; [Ala]mean = 8.63 ± 4.91, P = 0.051) and healthy stromal tissues ([PC+GPC]mean = 4.34 ± 2.46, P < 0.01; [tCho]mean = 7.04 ± 3.10, P < 0.01; [Lac]mean = 45.1 ± 18.6, P < 0.01; [Ala]mean = 6.80 ± 2.95, P < 0.01), while Cit and PA concentrations were significantly higher in healthy glandular tissues ([Cit]mean = 43.1 ± 21.2, [PAs]mean = 18.5 ± 15.6) than in healthy stromal ([Cit]mean = 16.1 ± 5.6, P < 0.01; [PAs]mean = 3.15 ± 1.81, P < 0.01) and prostate cancer tissues ([Cit]mean = 19.6 ± 12.7, P < 0.01; [PAs]mean = 5.28 ± 5.44, P < 0.01). Serial spectra acquired over 12 hr indicated that the degradation of Cho‐containing metabolites was minimized by acquiring HR‐MAS data at 1°C compared to 20°C. Magn Reson Med, 2006.

Proton HR-MAS spectroscopy and quantitative pathologic analysis of MRI/3D-MRSI-targeted postsurgical prostate tissues.

Proton high‐resolution magic angle spinning (1H HR‐MAS) NMR spectroscopy and quantitative histopathology were performed on the same 54 MRI/3D‐MRSI‐targeted postsurgical prostate tissue samples. Presurgical MRI/3D‐MRSI targeted healthy and malignant prostate tissues with an accuracy of 81%. Even in the presence of substantial tissue heterogeneity, distinct 1H HR‐MAS spectral patterns were observed for different benign tissue types and prostate cancer. Specifically, healthy glandular tissue was discriminated from prostate cancer based on significantly higher levels of citrate (P = 0.04) and polyamines (P = 0.01), and lower (P = 0.02) levels of the choline‐containing compounds choline, phosphocholine (PC), and glycerophosphocholine (GPC). Predominantly stromal tissue lacked both citrate and polyamines, but demonstrated significantly (P = 0.01) lower levels of choline compounds than cancer. In addition, taurine, myo‐inositol, and scyllo‐inositol were all higher in prostate cancer vs. healthy glandular and stromal tissues. Among cancer samples, larger increases in choline, and decreases in citrate and polyamines (P = 0.05) were observed with more aggressive cancers, and a MIB‐1 labeling index correlated (r = 0.62, P = 0.01) with elevated choline. The elucidation of spectral patterns associated with mixtures of different prostate tissue types and cancer grades, and the inclusion of new metabolic markers for prostate cancer may significantly improve the clinical interpretation of in vivo prostate MRSI data. Magn Reson Med 50:944–954, 2003.

Evaluation of lactate and alanine as metabolic biomarkers of prostate cancer using 1H HR-MAS spectroscopy of biopsy tissues

The goal of this study was to investigate the use of lactate and alanine as metabolic biomarkers of prostate cancer using 1H high‐resolution magic angle spinning (HR‐MAS) spectroscopy of snap‐frozen transrectal ultrasound (TRUS)‐guided prostate biopsy tissues. A long‐echo‐time rotor‐synchronized Carr‐Purcell‐Meiboom‐Gill (CPMG) sequence including an electronic reference to access in vivo concentrations (ERETIC) standard was used to determine the concentrations of lactate and alanine in 82 benign and 16 malignant biopsies (mean 26.5% ± 17.2% of core). Low concentrations of lactate (0.61 ± 0.28 mmol/kg) and alanine (0.14 ± 0.06 mmol/kg) were observed in benign prostate biopsies, and there was no significant difference between benign predominantly glandular (N = 54) and stromal (N = 28) biopsies between patients with (N = 38) and without (N = 44) a positive clinical biopsy. In biopsies containing prostate cancer there was a highly significant (P < 0.0001) increase in lactate (1.59 ± 0.61 mmol/kg) and alanine (0.26 ± 0.07 mmol/kg), and minimal overlap with lactate concentrations in benign biopsies. This study demonstrates for the first time very low concentrations of lactate and alanine in benign prostate biopsy tissues. The significant increase in the concentration of both lactate and alanine in biopsy tissue containing as little as 5% cancer could be exploited in hyperpolarized 13C spectroscopic imaging (SI) studies of prostate cancer patients. Magn Reson Med 60:510–516, 2008.

Quantification of choline- and ethanolamine-containing metabolites in human prostate tissues using 1H HR-MAS total correlation spectroscopy.

A fast and quantitative 2D high‐resolution magic angle spinning (HR‐MAS) total correlation spectroscopy (TOCSY) experiment was developed to resolve and quantify the choline‐ and ethanolamine‐containing metabolites in human prostate tissues in ≈1 hr prior to pathologic analysis. At a 40‐ms mixing time, magnetization transfer efficiency constants were empirically determined in solution and used to calculate metabolite concentrations in tissue. Phosphocholine (PC) was observed in 11/15 (73%) cancer tissues but only 6/32 (19%) benign tissues. PC was significantly higher (0.39 ± 0.40 mmol/kg vs. 0.02 ± 0.07 mmol/kg, z = 3.5), while ethanolamine (Eth) was significantly lower in cancer versus benign prostate tissues (1.0 ± 0.8 mmol/kg vs. 2.3 ± 1.9 mmol/kg, z = 3.3). Glycerophosphocholine (GPC) (0.57 ± 0.87 mmol/kg vs. 0.29 ± 0.26 mmol/kg, z = 1.2), phosphoethanolamine (PE) (4.4 ± 2.2 mmol/kg vs. 3.4 ± 2.6 mmol/kg, z = 1.4), and glycerophosphoethanolamine (GPE) (0.54 ± 0.82 mmol/kg vs. 0.15 ± 0.15 mmol/kg, z = 1.8) were higher in cancer versus benign prostate tissues. The ratios of PC/GPC (3.5 ± 4.5 vs. 0.32 ± 1.4, z = 2.6), PC/PE (0.08 ± 0.08 vs. 0.01 ± 0.03, z = 3.5), PE/Eth (16 ± 22 vs. 2.2 ± 2.0, z = 2.4), and GPE/Eth (0.41 ± 0.51 vs. 0.06 ± 0.06, z = 2.6) were also significantly higher in cancer versus benign tissues. All samples were pathologically interpretable following HR‐MAS analysis; however, degradation experiments showed that PC, GPC, PE, and GPE decreased 7.7 ± 2.2%, while Cho+mI and Eth increased 18% in 1 hr at 1°C and a 2250 Hz spin rate. Magn Reson Med 60:33–40, 2008.

Ethnic group-related differences in CpG hypermethylation of the GSTP1 gene promoter among African-American, Caucasian and Asian patients with prostate cancer

The incidence and mortality of prostate cancer (PC) is approximately 2‐fold higher among African‐Americans as compared to Caucasians and very low in Asian. We hypothesize that inactivation of GSTP1 genes through CpG methylation plays a role in the pathogenesis of PC, and its ability to serve as a diagnostic marker that differs among ethnic groups. GSTP1 promoter hypermethylation and its correlation with clinico‐pathological findings were evaluated in 291 PC (Asian = 170; African‐American = 44; Caucasian = 77) and 172 benign prostate hypertrophy samples (BPH) (Asian = 96; African‐American = 38; Caucasian = 38) using methylation‐specific PCR. In PC cells, 5‐aza‐dC treatment increased expression of GSTP1 mRNA transcripts. The methylation of all CpG sites was found in 191 of 291 PC (65.6%), but only in 34 of 139 BPH (24.5%). The GSTP1 hypermethylation was significantly higher in PC as compared to BPH in each ethnic group (p < 0.0001). Logistic regression analysis (PC vs. BPH) showed that African‐Americans had a higher hazard ratio (HR) (13.361) compared to Caucasians (3.829) and Asian (8.603). Chi‐square analysis showed correlation of GSTP1 hypermethylation with pathological findings (pT categories and higher Gleason sum) in Asian PC (p < 0.0001) but not in African‐Americans and Caucasian PC. Our results suggest that GSTP1 hypermethylation is a sensitive biomarker in African‐Americans as compared to that in Caucasians or Asian, and that it strongly influences tumor progression in Asian PC. Ours is the first study investigating GSTP1 methylation differences in PC among African‐American, Caucasian and Asian.

MicroRNA-1280 Inhibits Invasion and Metastasis by Targeting ROCK1 in Bladder Cancer

MicroRNAs (miRNAs) are non-protein-coding sequences that can function as oncogenes or tumor suppressor genes. This study documents the tumor suppressor role of miR-1280 in bladder cancer. Quantitative real-time PCR and in situ hybridization analyses showed that miR-1280 is significantly down-regulated in bladder cancer cell lines and tumors compared to a non-malignant cell line or normal tissue samples. To decipher the functional significance of miR-1280 in bladder cancer, we ectopically over-expressed miR-1280 in bladder cancer cell lines. Over-expression of miR-1280 had antiproliferative effects and impaired colony formation of bladder cancer cell lines. FACS (fluorescence activated cell sorting) analysis revealed that re-expression of miR-1280 in bladder cancer cells induced G2-M cell cycle arrest and apoptosis. Our results demonstrate that miR-1280 inhibited migration and invasion of bladder cancer cell lines. miR-1280 also attenuated ROCK1 and RhoC protein expression. Luciferase reporter assays demonstrated that oncogene ROCK1 is a direct target of miR-1280 in bladder cancer. This study also indicates that miR-1280 may be of diagnostic and prognostic importance in bladder cancer. For instance, ROC analysis showed that miR-1280 expression can distinguish between malignant and normal bladder cancer cases and Kaplan-Meier analysis revealed that patients with miR-1280 high expression had higher overall survival compared to those with low miR-1280 expression. In conclusion, this is the first study to document that miR-1280 functions as a tumor suppressor by targeting oncogene ROCK1 to invasion/migration and metastasis. Various compounds are currently being used as ROCK1 inhibitors; therefore restoration of tumor suppressor miR-1280 might be therapeutically useful either alone or in combination with these compounds in the treatment of bladder cancer.

Wnt antagonist DICKKOPF-3 (Dkk-3) induces apoptosis in human renal cell carcinoma.

The Wnt signaling pathway is activated in most cancers while Wnt antagonist genes are inactivated. However, the functional significance and mechanisms of inactivation of Wnt antagonist Dkk‐3 gene in renal cell carcinoma (RCC) has not been reported. In this study, we examined potential epigenetic mechanisms regulating Dkk‐3 expression in RCC cells and whether Dkk‐3 expression affects cell growth and apoptosis. The expression of Dkk‐3 is regulated by histone modification rather than CpG island DNA methylation in renal cancer cells. Renal cancer cell proliferation was significantly inhibited and apoptosis was promoted in Dkk‐3 transfected renal cancer cells. Dkk‐3 did not inhibit the Wnt/beta‐catenin signaling pathway but induced apoptosis via the noncanonical JNK pathway in renal cancer cells. Expression of p21, MDM‐2, and Puma genes were increased after transfecting RCC cell lines with a Dkk‐3 expression plasmid. Overexpression of Dkk‐3 induced G0/G1 arrest together with an increase in p21 expression. Growth of stable Dkk‐3 transfected cells in nude mice was decreased compared to controls. Our data show for the first time that mRNA expression of Dkk‐3 is regulated by histone modification and that Dkk‐3 inhibits renal cancer growth through modulation of cell cycle and apoptotic pathways.

Pancreatic neuroendocrine tumors: Accurate grading with Ki-67 index on fine-needle aspiration specimens using the WHO 2010/ENETS criteria

The natural history of pancreatic neuroendocrine tumors (panNETs) is extremely variable. One of the most controversial problems in diagnosis is the accurate prediction of the clinical behavior of these tumors. PanNETs that behave aggressively with a malignant course may have bland cytologic features, while some tumors with previously described “malignant” features may behave in a benign or indolent fashion. Various classification schemes have been proposed for grading panNETs. The European Neuroendocrine Tumor Society (ENETS) and 2010 World Health Organization (WHO) classification schemes include counting the mitotic index and/or the Ki‐67 proliferation index for grading. The current study was undertaken to determine whether tumors sampled by endoscopic ultrasound‐guided fine‐needle aspiration (EUS‐FNA) can be accurately graded based on the Ki‐67 index when compared to surgical samples.

Metabolic, pathologic, and genetic analysis of prostate tissues: quantitative evaluation of histopathologic and mRNA integrity after HR‐MAS spectroscopy

The impact of high‐resolution magic angle spinning (HR‐MAS) spectroscopy on the histopathologic and mRNA integrity of human prostate tissues was evaluated. Forty prostate tissues were harvested at transrectal ultrasound (TRUS) guided biopsy (n = 20) or radical prostatectomy surgery (n = 20), snap‐frozen on dry ice, and stored at −80°C until use. Twenty‐one samples (n = 11 biopsy, n = 10 surgical) underwent HR‐MAS spectroscopy prior to histopathologic and cDNA microarray analysis, while 19 control samples (n = 9 biopsy, n = 10 surgical) underwent only histopathologic and microarray analysis. Frozen tissues were sectioned at 14‐µm intervals and placed on individual histopathology slides. Every 8th slide was stained with hematoxylin and eosin (H&E) and used to target areas of predominantly epithelial tissue on the remaining slides for mRNA integrity and cDNA microarray analysis. Histopathologic integrity was graded from 1 (best) to 5 (worst) by two ‘blinded’ pathologists. Histopathologic integrity scores were not significantly different for post‐surgical tissues (HR‐MAS vs controls); however, one pathologists scores were significantly lower for biopsy tissues following HR‐MAS while the other pathologists scores were not. mRNA integrity assays were performed using an Agilent 2100 Bioanalyzer and the electrophoretic traces were scored with an RNA integrity number (RIN) from 1 (degraded) to 10 (intact). RIN scores were not significantly different for surgical tissues, but were significantly lower for biopsy tissues following HR‐MAS spectroscopy. The isolated mRNA then underwent two rounds of amplification, conversion to cDNA, coupling to Cy3 and Cy5 dyes, microarray hybridization, imaging, and analysis. Significance analysis of microarrays (SAM) identified no significantly over‐ or under‐expressed genes, including 14 housekeeping genes, between HR‐MAS and control samples of surgical and biopsy tissues (5% false discovery rate). This study demonstrates that histopathologic and genetic microarray analysis can be successfully performed on prostate surgical and biopsy tissues following HR‐MAS analysis; however, biopsy tissues are more fragile than surgical tissues. Copyright

Postbrushing and fine-needle aspiration biopsy follow-up and treatment options for patients with pancreatobiliary lesions: the Papanicolaou Society of Cytopathology guidelines.

The Papanicolaou Society of Cytopathology (PSC) has developed a set of guidelines for pancreatobiliary cytology including indications for endoscopic ultrasound (EUS) guided fine-needle aspiration (FNA) biopsy, techniques of EUS-FNA, terminology and nomenclature for pancreatobiliary cytology, ancillary testing and post-procedure management. All documents are based on the expertise of the authors, a review of the literature and discussions of the draft document at several national and international meetings over an 18 month period and synthesis of online comments of the draft document on the PSC web site (www.papsociety.org). This document selectively presents the results of these discussions and focuses on the follow-up and treatment options for patients after procedures performed for obtaining cytology samples for the evaluation of biliary strictures and solid and cystic masses in the pancreas. These recommendations follow the six-tiered terminology and nomenclature scheme proposed by committee III.