Analytical validation of a prognostic prostate cancer gene expression assay using formalin fixed paraffin embedded tissue.

BACKGROUND: There is a clear need for assays that can predict the risk of metastatic prostate cancer following curative procedures. Importantly these assays must be analytically robust in order to provide quality data for important clinical decisions. DNA microarray based gene expression assays measure several analytes simultaneously and can present specific challenges to analytical validation. This study describes the analytical validation of one such assay designed to predict metastatic recurrence in prostate cancer using primary formalin fixed paraffin embedded tumour material. METHODS: Accuracy was evaluated with a method comparison study between the assay development platform (Prostate Disease Specific Array) and an alternative platform (Xcel™ microarray) using 50 formalin-fixed, paraffin-embedded prostate cancer patient samples. An additional 70 samples were used to establish the assay reportable range. Determination of assay precision and sensitivity was performed on multiple technical replicates of three prostate cancer samples across multiple variables (operators, days, runs, reagent lots, and equipment) and RNA/cDNA inputs respectively using the appropriate linear mixed model. RESULTS: The overall agreement between the development and alternative platform was 94.7% (95% confidence interval, 86.9-98.5%). The reportable range was determined to be 0.150 to 1.107 for core needle biopsy samples and - 0.214 to 0.844 for radical prostatectomy samples. From the precision study, the standard deviations for assay repeatability and reproducibility were 0.032 and 0.040 respectively. The sensitivity study demonstrated that a total RNA input and cDNA input of 50 ng and 3.5 µg respectively was conservative. CONCLUSION: The Metastatic Assay was found to be highly reproducible and precise. In conclusion the studies demonstrated an acceptable analytical performance for the assay and support its potential use in the clinic.

Ex vivo metabolic fingerprinting identifies biomarkers predictive of prostate cancer recurrence following radical prostatectomy.

This corrects the article DOI: 10.1038/bjc.2017.346.

Ex vivo metabolic fingerprinting identifies biomarkers predictive of prostate cancer recurrence following radical prostatectomy.

BACKGROUND: Robust biomarkers that identify prostate cancer patients with high risk of recurrence will improve personalised cancer care. In this study, we investigated whether tissue metabolites detectable by high-resolution magic angle spinning magnetic resonance spectroscopy (HR-MAS MRS) were associated with recurrence following radical prostatectomy. METHODS: We performed a retrospective ex vivo study using HR-MAS MRS on tissue samples from 110 radical prostatectomy specimens obtained from three different Norwegian cohorts collected between 2002 and 2010. At the time of analysis, 50 patients had experienced prostate cancer recurrence. Associations between metabolites, clinicopathological variables, and recurrence-free survival were evaluated using Cox proportional hazards regression modelling, Kaplan-Meier survival analyses and concordance index (C-index). RESULTS: High intratumoural spermine and citrate concentrations were associated with longer recurrence-free survival, whereas high (total-choline+creatine)/spermine (tChoCre/Spm) and higher (total-choline+creatine)/citrate (tChoCre/Cit) ratios were associated with shorter time to recurrence. Spermine concentration and tChoCre/Spm were independently associated with recurrence in multivariate Cox proportional hazards modelling after adjusting for clinically relevant risk factors (C-index: 0.769; HR: 0.72; P=0.016 and C-index: 0.765; HR: 1.43; P=0.014, respectively). CONCLUSIONS: Spermine concentration and tChoCre/Spm ratio in prostatectomy specimens were independent prognostic markers of recurrence. These metabolites can be noninvasively measured in vivo and may thus offer predictive value to establish preoperative risk assessment nomograms.

Neuron navigator-2 and cyclin D2 are new candidate prognostic markers in uterine sarcoma.

The objective of this study was to validate the diagnostic and clinical role of four protein products of genes previously found to be differentially expressed in uterine low-grade endometrial stromal sarcoma (LG-ESS) compared to uterine leiomyosarcoma (LMS). Protein expression by immunohistochemistry of transgelin (TGLN), neuron navigator-2 (NAV2), fatty acid binding protein-3 (FABP3), and cyclin D2 (CCND2) was analyzed in 305 uterine sarcomas (231 LMS, 74 LG-ESS). Expression was analyzed for association with clinicopathologic parameters and survival. TGLN (p < 0.001), NAV2 (p < 0.001), and FABP3 (p = 0.005) were overexpressed in LMS compared to LG-ESS, whereas nuclear CCND2 (p < 0.001) was overexpressed in LG-ESS. NAV2 expression was associated with shorter overall survival in patients with LMS (p = 0.037), whereas nuclear CCND2 expression in LG-ESS was significantly related to longer survival (p = 0.012) in univariate analysis. Nuclear CCND2 expression was an independent prognosticator in Cox multivariate analysis (p = 0.023). In conclusion, TGLN, FABP3, NAV2, and nuclear CCND2 aid in differentiating LG-ESS from LMS. NAV2 and CCND2 are novel candidate prognostic markers in LMS and LG-ESS, respectively.

L1CAM as a prognostic marker in stage I endometrial cancer: a validation study.

BACKGROUND: L1 cell adhesion molecule (L1CAM) overexpression has been reported to be strongly associated with poor prognosis in early stage endometrial cancer (EC). We aimed at the validation of L1CAM as a marker of poor prognosis in an independent study population. METHODS: Patients with endometrioid EC FIGO stage I, were treated at Oslo University Hospital between 2005 and 2012. L1CAM expression was detected by immunohistochemistry with >10 % L1CAM staining defined as positive. Risks of relapse and death were estimated as hazard ratios (HRs) with 95 % confidence intervals (95 % CI). RESULTS: Of 450 patients, 388 (86 %) were evaluable for L1CAM expression and 35 (9 %) were L1CAM positive. After follow-up for a median time of 4.8 years (0.1-8.8), 33 (8 %) patients had recurred. 6/35 (17 %) L1CAM positive patients relapsed compared to 27/353 (8 %) L1CAM-negative patients. There were 7 (20 %) deaths in the L1CAM positive group, and 34 (10 %) in the negative group. In multivariate analysis, controlled for age and FIGO stage, L1CAM positivity was not significantly associated with the risk of relapse (HR 2.08, 95 % CI: 0.85-5.10, p = 0.11) or death of all-cause (HR 1.81, 95 % CI: 0.79-4.11, p = 0.16). In patients who were not treated with chemotherapy, L1CAM was significantly associated with risk of relapse (HR 2.9; 95 % CI: 1.08-7.56; p = 0.04). CONCLUSION: Our report confirms that L1CAM is associated with a more aggressive tumortype and more distant relapses. The overall recurrence rate in this population was low as were the absolute differences between L1CAM positive and negative patients. In this independent study sample, L1CAM failed to be a clinically relevant marker of poor prognosis in stage I endometrioid endometrial carcinoma.

PBX3 is a putative biomarker of aggressive prostate cancer.

There is a great need to identify new and better prognostic and predictive biomarkers to stratify prostate cancer patients for optimal treatment. The aims of this study were to characterize the expression profile of pre-B cell leukemia homeobox (PBX) transcription factors in prostate cancer with an emphasis on investigating whether PBX3 harbours any prognostic value. The expression profile of PBX3 and PBX1 in prostate tissue was determined by immunohistochemical and immunoblot analysis. Furthermore, the expression of PBX3 transcript variants was analyzed by RT-PCR, NanoString Technologies®, and by analyzing RNA sequence data. The potential of PBX3 to predict prognosis, either at mRNA or protein level, was studied in four independent cohorts. PBX3 was mainly expressed in the nucleus of normal prostate basal cells, while it showed cytosolic expression in prostatic intraepithelial neoplasia and cancer cells. We detected four PBX3 transcript variants in prostate tissue. Competing risk regression analysis revealed that high PBX3 expression was associated with slower progression to castration resistant prostate cancer (sub-hazard ratio (SHR) 0.18, 95% CI: 0.081-0.42, p values < 0.001). PBX3 expression had a high predictive accuracy (area under the curve (AUC) = 0.82) when combined with Gleason score and age. Patients undergoing radical prostatectomy, with high levels of PBX3 mRNA, had improved prostate cancer specific survival compared to patients expressing low levels (SHR 0.21, 95% CI: 0.46-0.93, p values < 0.001, and AUC = 0.75). Our findings strongly indicate that PBX3 has potential as a biomarker, both as part of a larger gene panel and as an immunohistochemical marker, for aggressive prostate cancer.

Low ß2-adrenergic receptor level may promote development of castration resistant prostate cancer and altered steroid metabolism.

The underlying mechanisms responsible for the development of castration-resistant prostate cancer (CRPC) in patients who have undergone androgen deprivation therapy are not fully understood. This is the first study to address whether ß2-adrenergic receptor (ADRB2)- mediated signaling may affect CRPC progression in vivo. By immunohistochemical analyses, we observed that low levels of ADRB2 is associated with a more rapid development of CRPC in a Norwegian patient cohort. To elucidate mechanisms by which ADRB2 may affect CRPC development, we stably transfected LNCaP cells with shRNAs to mimic low and high expression of ADRB2. Two UDP-glucuronosyltransferases, UGT2B15 and UGT2B17, involved in phase II metabolism of androgens, were strongly downregulated in two LNCaP shADRB2 cell lines. The low-ADRB2 LNCaP cell lines displayed lowered glucuronidation activities towards androgens than high-ADRB2 cells. Furthermore, increased levels of testosterone and enhanced androgen responsiveness were observed in LNCaP cells expressing low level of ADRB2. Interestingly, these cells grew faster than high-ADRB2 LNCaP cells, and sustained their low glucuronidation activity in castrated NOD/SCID mice. ADRB2 immunohistochemical staining intensity correlated with UGT2B15 staining intensity in independent TMA studies and with UGT2B17 in one TMA study. Similar to ADRB2, we show that low levels of UGT2B15 are associated with a more rapid CRPC progression. We propose a novel mechanism by which ADRB2 may affect the development of CRPC through downregulation of UGT2B15 and UGT2B17.

HUR mRNA expression in ovarian high-grade serous carcinoma effusions is associated with poor survival.

The objective of this study was to analyze the expression and clinical role of the RNA-binding molecule HuR in metastatic high-grade ovarian serous carcinoma (HGSC). HUR mRNA expression by reverse-transcription polymerase chain reaction was analyzed in 66 effusions from patients diagnosed with HGSC. Protein expression was analyzed in 262 HGSC effusions using immunohistochemistry. HUR mRNA was detected in all 66 effusions. HUR mRNA levels were unrelated to clinicopathological parameters. However, higher HUR mRNA levels were significantly related to poor overall survival in the entire cohort (P=.023), as well as in analysis limited to patients with prechemotherapy primary diagnosis specimens (P=.001) in univariate analysis. Cox multivariate analysis showed an independent prognostic role for HUR mRNA in the entire cohort (P=.033) and in patients with prechemotherapy primary diagnosis specimens (P=.002). HuR protein was detected in the nucleus and cytoplasm of tumor cells in 258 (98%) of 262 and 153 (58%) of 262 effusions, respectively. Higher HuR protein expression was associated with higher serum Cancer Antigen (CA) 125 levels at diagnosis (P=.01), but its presence at both cellular compartments was otherwise unrelated to clinicopathological parameters or survival. In conclusion, HuR is widely expressed in metastatic HGSC at both the mRNA and protein level. Higher HUR mRNA levels are associated with poor survival in metastatic HGSC, whereas protein expression has no prognostic value.

Synchronous resection of pancreatic serous cystadenocarcinoma and liver metastasis: First reported case and review of literature.

Cystic neoplasms account for approximately 10-20% of all pancreatic cysts and 1% of pancreatic cancers. Serous cystadenomas are considered benign tumors with almost no malignant potential, and thus the management is typically only observation with serial imaging. According to the current World Health Organization classification, cases with distant metastases are defined as serous cystadenocarcinomas. To date, only 17 such cases with concomitant synchronous or metachronous liver metastasis have been described in the literature, and eight of these reports described treatment of secondary liver lesions. This report describes the first case of synchronous resection of pancreatic serous cystadenocarcinoma and liver metastasis in a 56-year-old female patient. The patient is currently well after 30 months of follow-up with no tumor recurrence or new metastatic liver nodules based on magnetic resonance imaging.

MicroRNAs in Ovarian Cancer.

Ovarian cancer, consisting predominantly of ovarian carcinoma, is the eighth most common cancer in women and the most lethal gynecologic malignancy. Efforts focus on identifying biomarkers which may aid in early diagnosis and reduce mortality, as well as on characterizing therapeutic targets with the aim of circumventing chemoresistance and prolonging survival at advanced-stage disease. MicroRNAs (miRNAs) are small, non-coding RNAs that post-transcriptionally regulate gene expression, and have been found to play an important role in ovarian carcinoma. Recent research has identified multiple miRNAs involved in the biology and progression of the disease, and supports a role for miRNAs as potential biomarkers, predictive markers and prognostic factors. Many of the studies published to date nevertheless suffer from critical weaknesses which affect data quality and reproducibility, including the comparison of normal ovaries to tumor tissue without compensation for the highly discrepant target cell fraction in these two specimen types and the inclusion of carcinomas of different histotypes, non-epithelial tumors or tumors of non-specified histology. These shortcomings highlight the critical role of pathologists as part of the team in the setting of such research. This review summarizes current knowledge in this area and discusses the potential clinical relevance of miRNAs in ovarian carcinoma, with focus on studies of clinical specimens in which tissue selection has been deemed adequate.