Background: Gene expression profiling is a highly sensitive technique which is used for profiling tumor samples for medical prognosis. RNA quality and degradation influence the analysis results of gene expression profiles. The impact of this influence on the profiles and its medical impact is not fully understood. As patient samples are very valuable for clinical studies, it is necessary to establish criteria for the RNA quality to be able to use these samples in later analysis.Methods: To investigate the effects of RNA integrity on gene expression profiling, whole genome expression arrays were used. We used tumor biopsies from patients diagnosed with locally advanced rectal cancer. To simulate degradation, the isolated total RNA of all patients was subjected to heat-induced degradation in a time-dependent manner. Expression profiling was then performed and data were analyzed bioinformatically to assess the differences.Results: The differences introduced by RNA degradation were largely outweighed by the biological differences between the patients. Only a relatively small number of probes (275 out of 41,000) show a significant effect due to degradation. The genes that show the strongest effect due to RNA degradation were, especially, those with short mRNAs and probe positions near the 5' end.Conclusions: Degraded RNA from tumor samples (RIN > 5) can still be used to perform gene expression analysis. A much higher biological variance between patients is observed compared to the effect that is imposed by degradation of RNA. Nevertheless there are genes, very short ones and those with the probe binding side close to the 5' end that should be excluded from gene expression analysis when working with degraded RNA. These results are limited to the Agilent 44 k microarray platform and should be carefully interpreted when transferring to other settings.

Background: Patients with locally advanced rectal cancer are treated with preoperative chemoradiotherapy followed by surgical resection. Despite similar clinical parameters (uT2-3, uN+) and standard therapy, patients’ prognoses differ widely. A possible prediction of prognosis through microRNAs as biomarkers out of treatment-naïve biopsies would allow individualized therapy options. Methods: Microarray analysis of 45 microdissected preoperative biopsies from patients with rectal cancer was performed to identify potential microRNAs to predict overall survival, disease-free survival, cancer-specific survival, distant-metastasis-free survival, tumor regression grade, or nodal stage. Quantitative real-time polymerase chain reaction (qPCR) was performed on an independent set of 147 rectal cancer patients to validate relevant miRNAs. Results: In the microarray screen, 14 microRNAs were significantly correlated to overall survival. Five microRNAs were included from previous work. Finally, 19 miRNAs were evaluated by qPCR. miR-515-5p, miR-573, miR-579 and miR-802 demonstrated significant correlation with overall survival and cancer-specific survival (p < 0.05). miR-573 was also significantly correlated with the tumor regression grade after preoperative chemoradiotherapy. miR-133b showed a significant correlation with distant-metastasis-free survival. miR-146b expression levels showed a significant correlation with nodal stage. Conclusion: Specific microRNAs can be used as biomarkers to predict prognosis of patients with rectal cancer and possibly stratify patients’ therapy if validated in a prospective study.

Neoadjuvant chemoradiation (nCRT) is an established procedure in stage union internationale contre le cancer (UICC) II/III rectal carcinomas. Around 53% of the tumours present with good tumor regression after nCRT, and 8%–15% are complete responders. Reliable selection markers would allow the identification of poor or non-responders prior to therapy. Tumor biopsies were harvested from 20 patients with rectal carcinomas, and stored in liquid nitrogen prior to therapy after obtaining patients’ informed consent (Erlangen-No.3784). Patients received standardized nCRT with 5-Fluoruracil (nCRT I) or 5-Fluoruracil ± Oxaliplatin (nCRT II) according to the CAO/ARO/AIO-04 protocol. After surgery, regression grading (Dworak) of the tumors was performed during histopathological examination of the specimens. Tumors were classified as poor (Dworak 1 + 2) or good (Dworak 3 + 4) responders. Laser capture microdissection (LCM) for tumor enrichment was performed on preoperative biopsies. Differences in expressed proteins between poor and good responders to nCRT I and II were identified by proteomic analysis (Isotope Coded Protein Label, ICPL™) and selected markers were validated by immunohistochemistry. Tumors of 10 patients were classified as histopathologically poor (Dworak 1 or 2) and the other 10 tumor samples as histopathologically good (Dworak 3 or 4) responders to nCRT after surgery. Sufficient material in good quality was harvested for ICPL analysis by LCM from all biopsies. We identified 140 differentially regulated proteins regarding the selection criteria and the response to nCRT. Fourteen of these proteins were synchronously up-regulated at least 1.5-fold after nCRT I or nCRT II (e.g., FLNB, TKT, PKM2, SERINB1, IGHG2). Thirty-five proteins showed a complete reciprocal regulation (up or down) after nCRT I or nCRT II and the rest was regulated either according to nCRT I or II. The protein expression of regulated proteins such as PLEC1, TKT, HADHA and TAGLN was validated successfully by immunohistochemistry. ICPL is a valid method to identify differentially expressed proteins in rectal carcinoma tissue between poor vs. good responders to nCRT. The identified protein markers may act as selection criteria for nCRT in the future, but our preliminary findings must be reproduced and validated in a prospective cohort.

Since the response to chemoradiotherapy in patients with locally advanced rectal cancer is heterogeneous, valid biomarkers are needed to monitor tumor response. Circulating microRNAs are promising candidates, however analyses of circulating microRNAs in rectal cancer are still rare. 111 patients with rectal cancer and 46 age-matched normal controls were enrolled. The expression levels of 30 microRNAs were analyzed in 17 pre-treatment patients’ plasma samples. Differentially regulated microRNAs were validated in 94 independent patients. For 52 of the 94 patients a paired comparison between pre-treatment and post-treatment samples was performed. miR-17, miR-18b, miR-20a, miR-31, and miR-193a_3p, were significantly downregulated in pre-treatment plasma samples of patients with rectal cancer (p < 0.05). miR-29c, miR-30c, and miR-195 showed a trend of differential regulation. After validation, miR-31 and miR-30c were significantly deregulated by a decrease of expression. In 52 patients expression analyses of the 8 microRNAs in matched pre-treatment and post-treatment samples showed a significant decrease for all microRNAs (p < 0.05) after treatment. Expression levels of miR-31 and miR-30c could serve as valid biomarkers if validated in a prospective study. Plasma microRNA expression levels do not necessarily represent miRNA expression levels in tumor tissue. Also, expression levels of microRNAs change during multimodal therapy.

Zusammenfassung: Hintergrund Nach Diagnose eines papillären Schilddrüsenkarzinoms (PTC) mit einer Primärtumorgröße von ≥ 10 mm und < 10 mm wird, vor dem Hintergrund eines möglichen bilateralen Tumorbefalls, das Konzept der weiteren chirurgischen Therapie nach wie vor kontrovers diskutiert. Ziel dieser Studie war es, die Inzidenz sowie prädiktive Faktoren für das Vorhandensein eines bilateralen papillären Schilddrüsenkarzinoms hinsichtlich der Auswahl einer optimalen chirurgischen Therapie zu identifizieren. Material und Methoden Anhand einer retrospektiven Datenbank wurden bei 123 Patienten mit einem PTC, die entweder primär eine totale Thyreoidektomie oder eine Komplettierungsoperation nach Hemithyreoidektomie erhielten, die Parameter Tumorgröße, histopathologische Charakteristika, Multifokalität sowie Lymphknotenmetastasierung mit dem Vorhandensein eines bilateralen Tumorbefalls für die Primärtumorgrößen ≥ 10 mm und < 10 mm sowie ≥ 7 mm und < 7 mm korreliert. Ergebnisse Im gesamten Patientenkollektiv zeigte sich in 26 Fällen ein bilaterales PTC. Dieses war signifikant häufiger, wenn die Primärtumorgröße ≥ 10 mm betrug (77 %). Im Vergleich dazu boten Patienten mit einer Primärtumorgröße von < 10 mm nur in 23 % der Fälle ein bilaterales PTC (p = 0,004). Die Multifokalität des Primärtumors erwies sich hierbei als positiver prädiktiver Faktor für ein bilaterales PTC (p = 5,022e-18). Bei bilateralem Nachweis eines PTCs zeigte sich ein Trend für das Auftreten von metachronen Lymphknotenmetastasen (p = 0,0691). Darüber hinaus konnten in den Analysen bezogen auf eine Primärtumorgröße von ≥ 7 mm und < 7 mm die oben genannten Ergebnisse reproduziert werden. Schlussfolgerung Die vorliegende retrospektive Datenanalyse zeigt, dass eine Primärtumorgröße von ≥ 10 mm mit einem signifikant häufigeren bilateralen Tumorbefall korreliert. Multifokalität erwies sich als positiver prädiktiver Faktor für das Vorhandensein eines bilateralen PTCs. Bei Nachweis eines multifokalen PTCs, auch bei Primärtumorgrößen < 10 mm, sollte demnach eine primäre totale Thyreoidektomie bzw. eine Komplettierungsoperation als chirurgisches Therapieverfahren erwogen werden. Bei Multifokalität und nicht erfolgter Komplettierungsoperation ist eine engmaschige Nachsorge notwendig, um frühzeitig einen bilateralen Tumorbefall oder Lymphknotenmetastasen detektieren zu können.