Linking prostate cancer recurrence to the spermine pathway: a retrospective study using High Resolution Magic Angle Spinning 1H Magnetic Resonance Spectroscopy at 14T and Quantitative Polymerase Chain Reaction

Abstract

In this study, we attempted to link prostate cancer recurrence to the spermine pathway. To establish and explore this link we combined a quantitative method of radiology, namely HRMAS 1H MRS, with a quantitative method of molecular biology, namely qPCR. Results of the study indicate that qPCR enzymatic profiles differ significantly between recurrent and non-recurrent cases. Looking ahead, these enzymatic profiles, possibly in addition to or combination with MRS metabolomic profiles, may be developed into clinical tools predicting the likelihood of disease relapse after radical surgery. To date clinicians have virtually no tools at hand to predict the likelihood of recurrence. With the present study, we recognize the need for such predictive tools. The study has been designed as a retrospective analysis of two cohorts of male patients (n = 16) having undergone radical prostatectomy. The average follow-up period during which the non-recurrent cohort had gone without detectable serum PSA levels was 55.8 months. The average time to the first detectable rise of serum PSA > 0.2 ng/ml after prostatectomy in the recurrent cohort was 19.9 months. Patients in the recurrent cohort were matched to patients in the non-recurrent cohort based on age, Gleason Score and clinical staging. Biopsy tissue recovered for histopathological assessment at the time of prostatectomy served as the original specimens for the study. Specimens were subjected to histopathological assessment, HRMAS 1H MRS at 14.1 T and qPCR. Using qPCR, we chose to quantify the relative mRNA concentration of the genes coding for what can be assumed to be the most influential enzymatic players of the polyamine pathway: the two rate limiting anabolic enzymes of the polyamine pathway, ornithine decarboxylase (ODC) and S-adenosyl-methionine decarboxylase (Ado-MetDC); the rate-limiting catabolic enzyme, acetyl-CoA spermidine/spermine N1-acetyl-transferase (SSAT); and the ODC antizyme, ornithine decarboxylase antizyme (OAZ). We found that all of the four enzymes are expressed to a greater degree in the prostates of recur-rent patients. The differences were statistically significant for the expression of AdoMetDC in epithelial cells (p = 0.0134) and the log-transformed expression of OAZ in epithelial cells (p = 0.0252). When comparing the non-recurrent to the recurrent group, relative enzyme expression was 44.30% and 56.54% for AdoMetDC and OAZ, respectively. In addition, we found that the anabolic enzymes ODC and AdoMetDC appear to be co-regulated across both cohorts (R2 = 0.5864 and R2 = 0.6945 for epithelial and stroma tissue, respectively). Similarly, the catabolic enzymes SSAT and OAZ correlated (R2 = 0.6024 and R2 = 0.6890 for epithelial and stroma tis-sue, respectively). We also detected an inverse correlation of the expression of anabolic and ca-tabolic enzymes relative to the general expression level of the pathway. Combining the qPCR data with HRMAS 1H MRS data, we found that singular correlations between individual enzymes and specific MRS spectral regions were, if detectable at all, weak and statistically insignificant. Using a combined model integrating several enzymes delivered more convincing results, with a top fitting correlation of R2 = 0.823. We conclude that metabolomic changes appear to be best explained by complex trans-pathway changes rather than singular changes of individual enzyme expression levels. In sum, there is solid evidence that (a) enzymatic profiles differ between recurrent and non-recurrent cases, and (b) differences in NMR spectra between recurrent and non-recurrent cases are linked to the enzymes of the spermine pathway. The present study may be a step towards a better understanding of prostate cancer in general and BCR in particular. With further development still to come, enzymatic profiles, in addition to or in combination with HRMAS 1H MRS metabolomic profiles, may be developed into clinical tools. As clinical tools, the profiles may be applied to help (a) better judge the benefits of radical surgery, a procedure that often comes with drastic side effects, (b) weigh its benefits against alterna-tive treatment options, and (c) adjust post-surgery monitoring regimes according to whether re-currence is probable or not. While we recognize the limitations of the present study, it demonstrates the potential of qPCR-based enzymatic profiles and of HRMAS 1H MRS-based metabolomic profiles. And while this study is experimental and explorative in nature, it may encourage further research.

HINTERGRUND: Die Studie untersucht den postulierten Zusammenhang zwischen Spermin-Stoffwechsel und Prostatakarzinom. Sie versucht, diesen für die Vorhersage von Rezidiven nutzbar zu machen. Dazu kombiniert sie Histopathologie, High Resolution Magic Angle Spinning 1H- Magnetresonanzspektroskopie (HRMAS 1H MRS) und quantitative Polymerase- Kettenreaktion (qPCR). METHODIK: Die Studie betrachtet retrospektiv eine Kohorte männlicher Patienten mit Prostatakarzinom (n = 16) im Zeitraum (19,9 Monate) zwischen radikaler Prostatektomie und biochemischem Rezidiv (PSA > 0,2 ng/ml). Die Kontrollkohorte setzt sich aus randomisiert ausgewählten und per Matching (Alter, Gleason-Score, klinisches Stadium) zugewiesenen Patienten mit Prostatakarzinom zusammen, bei denen im Beobachtungszeitraum (55,8 Monate nach Prostatektomie) kein PSA-Rezidiv nachweisbar war. Untersuchungsmaterial sind zum Zeitpunkt der Prostatektomie gewonnene Biopsat-Serien. Es erfolgte (1) eine histopathologische Bewertung, (2) die Aufzeichnung und quantitative Auswertung von Magnetresonanzspektren per HRMAS 1H MRS (14 Tesla), (3) die mRNA-Quantifizierung der Genexpression von Schrittmacherenzymen im Polyaminstoffwechsel per qPCR. ERGEBNISSE: Die Expression der für AdoMetDC (p = 0,0134) und OAZ (p = 0,0252) kodierenden Gene unterscheidet sich in der Rezidiv-Kohorte signifikant von derjenigen in der Kontrollkohorte. Die anabolen Enzyme ODC und AdoMetDC korrelieren dabei miteinander, sowohl in Epithel (R2 = 0,5864) als auch Stroma (R2 = 0,6945). Gleiches gilt für die katabolen Enzyme SSAT (R2 = 0,6024) und OAZ (R2 = 0,6890). In multivariaten Analysen korrelieren diese Genexpressionsmuster mit der Intensität bestimmter Regionen des NMR-Spektrums der Proben. Während für individuelle Enzyme kein signifikanter Vorhersagewert nachgewiesen werden kann, erreichen kombinierte lineare Modelle (Methode der kleinsten Fehlerquadrate) in den betrachteten Kohorten Korrelationskoeffizienten von bis zu R2 = 0,823. DISKUSSION: Die Studie legt nahe, dass sich die Genexpressionsmuster von Rezidiv-Patienten bereits bei Prostatektomie signifikant von denjenigen der Patienten ohne späteres Rezidiv unterscheiden. Unterschiede der Genexpressionsmuster korrelieren zudem mit Unterschieden im Magnetresonanzspektrum der Gewebe. Perspektivisch könnte das helfen, die Rezidivwahrscheinlichkeit von Prostatakarzinomen besser einzuschätzen und das klinische Monitoring fallgemäß anzupassen.