Selection of endogenous control genes for normalising gene expression data derived from formalin-fixed paraffin-embedded tumour tissue

From Springer Nature via Jisc Publications RouterHistory: received 2020-06-16, accepted 2020-09-28, registration 2020-10-01, online 2020-10-14, pub-electronic 2020-10-14, collection 2020-12Publication status: PublishedFunder: NIHRFunder: Mission Sector of the Egyptian Ministry of Higher Education and Scientific Research.Funder: CRUK; Grant(s): C147/A25254Abstract: Quantitative real time polymerase chain reaction (qPCR) data are normalised using endogenous control genes. We aimed to: (1) demonstrate a pathway to identify endogenous control genes for qPCR analysis of formalin-fixed paraffin-embedded (FFPE) tissue using bladder cancer as an exemplar; and (2) examine the influence of probe length and sample age on PCR amplification and co-expression of candidate genes on apparent expression stability. RNA was extracted from prospective and retrospective samples and subject to qPCR using TaqMan human endogenous control arrays or single tube assays. Gene stability ranking was assessed using coefficient of variation (CoV), GeNorm and NormFinder. Co-expressed genes were identified from The Cancer Genome Atlas (TCGA) using the on-line gene regression analysis tool GRACE. Cycle threshold (Ct) values were lower for prospective (19.49 ± 2.53) vs retrospective (23.8 ± 3.32) tissues (p < 0.001) and shorter vs longer probes. Co-expressed genes ranked as the most stable genes in the TCGA cohort by GeNorm when analysed together but ranked lower when analysed individually omitting co-expressed genes indicating bias. Stability values were < 1.5 for the 20 candidate genes in the prospective cohort. As they consistently ranked in the top ten by CoV, GeNorm and Normfinder, UBC, RPLP0, HMBS, GUSB, and TBP are the most suitable endogenous control genes for bladder cancer qPCR

Phosphatidylinositide 3-kinase (PI3K) and PI3K-related kinase (PIKK) activity contributes to radioresistance in thyroid carcinomas.

Anaplastic (ATC) and certain follicular thyroid-carcinomas (FTCs) are radioresistant. The Phosphatidylinositide 3-kinase (PI3K) pathway is commonly hyperactivated in thyroid-carcinomas. PI3K can modify the PI3K-related kinases (PIKKs) in response to radiation: How PIKKs interact with PI3K and contribute to radioresistance in thyroid-carcinomas is unknown. Further uncertainties exist in how these interactions function under the radioresistant hypoxic microenvironment. Under normoxia/anoxia, ATC (8505c) and FTC (FTC-133) cells were irradiated, with PI3K-inhibition (via GDC-0941 and PTEN-reconstitution into PTEN-null FTC-133s) and effects on PIKK-activation, DNA-damage, clonogenic-survival and cell cycle, assessed. FTC-xenografts were treated with 5 × 2 Gy, ± 50 mg/kg GDC-0941 (twice-daily; orally) for 14 days and PIKK-activation and tumour-growth assessed. PIKK-expression was additionally assessed in 12 human papillary thyroid-carcinomas, 13 FTCs and 12 ATCs. GDC-0941 inhibited radiation-induced activation of Ataxia-telangiectasia mutated (ATM), ATM-and Rad3-related (ATR) and DNA-dependent protein kinase catalytic subunit (DNA-PKcs). Inhibition of ATM and DNA-PKcs was PI3K-dependent, since activation was reduced in PTEN-reconstituted FTC-133s. Inhibition of PIKK-activation was greater under anoxia: Consequently, whilst DNA-damage was increased and prolonged under both normoxia and anoxia, PI3K-inhibition only reduced clonogenic-survival under anoxia. GDC-0941 abrogated radiation-induced cell cycle arrest, an effect most likely linked to the marked inhibition of ATR-activation. Importantly, GDC-0941 inhibited radiation-induced PIKK-activation in FTC-xenografts leading to a significant increase in time taken for tumours to triple in size: 26.5 ± 5 days (radiation-alone) versus 31.5 ± 5 days (dual-treatment). PIKKs were highly expressed across human thyroid-carcinoma classifications, with ATM scoring consistently lower. Interestingly, some loss of ATM and DNA-PKcs was observed. These data provide new insight into the mechanisms of hypoxia-associated radioresistance in thyroid-carcinoma

A miRNA signature predicts benefit from addition of hypoxia-modifying therapy to radiation treatment in invasive bladder cancer

From Springer Nature via Jisc Publications RouterHistory: received 2020-09-03, rev-recd 2021-02-11, accepted 2021-02-19, registration 2021-02-19, pub-electronic 2021-04-12, online 2021-04-12, pub-print 2021-07-06Publication status: PublishedFunder: U.S. Department of Defense (United States Department of Defense); doi: https://doi.org/10.13039/100000005; Grant(s): W81XWH-17-1-0543Abstract: Background: miRNAs are promising biomarkers in oncology as their small size makes them less susceptible to degradation than mRNA in FFPE tissue. We aimed to derive a hypoxia-associated miRNA signature for bladder cancer. Methods: Taqman miRNA array cards identified miRNA seed genes induced under hypoxia in bladder cancer cell lines. A signature was derived using feature selection methods in a TCGA BLCA training data set. miRNA expression data were generated for 190 tumours from the BCON Phase 3 trial and used for independent validation. Results: A 14-miRNA hypoxia signature was derived, which was prognostic for poorer overall survival in the TCGA BLCA cohort (n = 403, p = 0.001). Univariable analysis showed that the miRNA signature predicted an overall survival benefit from having carbogen–nicotinamide with radiotherapy (HR = 0.30, 95% CI 0.094–0.95, p = 0.030) and performed similarly to a 24-gene mRNA signature (HR = 0.47, 95% CI 0.24–0.92, p = 0.025). Combining the signatures improved performance (HR = 0.26, 95% CI 0.08–0.82, p = 0.014) with borderline significance for an interaction test (p = 0.065). The interaction test was significant for local relapse-free survival LRFS (p = 0.033). Conclusion: A 14-miRNA hypoxia signature can be used with an mRNA hypoxia signature to identify bladder cancer patients benefitting most from having carbogen and nicotinamide with radiotherapy

The hypoxia marker CAIX is prognostic in the UK phase III VorteX-Biobank cohort: an important resource for translational research in soft tissue sarcoma

BACKGROUND: Despite high metastasis rates, adjuvant/neoadjuvant systemic therapy for localised soft tissue sarcoma (STS) is not used routinely. Progress requires tailoring therapy to features of tumour biology, which need exploration in well-documented cohorts. Hypoxia has been linked to metastasis in STS and is targetable. This study evaluated hypoxia prognostic markers in the phase III adjuvant radiotherapy VorteX trial. METHODS: Formalin-fixed paraffin-embedded tumour biopsies, fresh tumour/normal tissue and blood were collected before radiotherapy. Immunohistochemistry for HIF-1α, CAIX and GLUT1 was performed on tissue microarrays and assessed by two scorers (one pathologist). Prognostic analysis of disease-free survival (DFS) used Kaplan-Meier and Cox regression. RESULTS: Biobank and outcome data were available for 203 out of 216 randomised patients. High CAIX expression was associated with worse DFS (hazard ratio 2.28, 95% confidence interval: 1.44-3.59, P<0.001). Hypoxia-inducible factor-1α and GLUT1 were not prognostic. Carbonic anhydrase IX remained prognostic in multivariable analysis. CONCLUSIONS: The VorteX-Biobank contains tissue with linked outcome data and is an important resource for research. This study confirms hypoxia is linked to poor prognosis in STS and suggests that CAIX may be the best known marker. However, overlap between single marker positivity was poor and future work will develop an STS hypoxia gene signature to account for tumour heterogeneity

Validation of a hypoxia related gene signature in multiple soft tissue sarcoma cohorts

Purpose: There is a need for adjuvant/neo-adjuvant treatment strategies to prevent metastatic relapse in soft tissue sarcoma (STS). Tumor hypoxia is associated with a high-risk of metastasis and is potentially targetable. This study aimed to derive and validate a hypoxia mRNA signature for STS for future biomarker-driven trials of hypoxia targeted therapy. // Materials and Methods: RNA sequencing was used to identify seed genes induced by hypoxia in seven STS cell lines. Primary tumors in a training cohort (French training) were clustered into two phenotypes by seed gene expression and a de novo hypoxia signature derived. Prognostic significance of the de novo signature was evaluated in the training and two independent validation (French validation and The Cancer Genome Atlas) cohorts. // Results: 37 genes were up-regulated by hypoxia in all seven cell lines, and a 24-gene signature was derived. The high-hypoxia phenotype defined by the signature was enriched for well-established hypoxia genes reported in the literature. The signature was prognostic in univariable analysis, and in multivariable analysis in the training (n = 183, HR 2.16, P = 0.0054) and two independent validation (n = 127, HR 3.06, P = 0.0019; n = 258, HR 2.05, P = 0.0098) cohorts. Combining information from the de novo hypoxia signature and a genome instability signature significantly improved prognostication. Transcriptomic analyses showed high-hypoxia tumors had more genome instability and lower immune scores. // Conclusions: A 24-gene STS-specific hypoxia signature may be useful for prognostication and identifying patients for hypoxia-targeted therapy in clinical trials

Expression of miR-210 in relation to other measures of hypoxia and prediction of benefit from hypoxia modification in patients with bladder cancer

The addition of hypoxia modifiers carbogen and nicotinamide (CON) to radiotherapy (RT) improved overall survival (OS) in bladder cancer patients in the BCON phase III clinical trial. We investigate whether expression of hsa-miR-210 in BCON patient samples reflects hypoxia and predicts benefit from hypoxia modification.In all, 183 T1-T4a bladder cancer samples were available for miR-210 analysis. A total of 86 received RT+CON and 97 received RT alone. TaqMan qPCR plates were used to assess miR-210 expression. Patients were classified as low (andlt;median expression) or high (⩾median) miR-210. Data on other hypoxia biomarkers were available for comparison.Patients with high miR-210 had a trend towards improved 5-year OS with RT+CON (53.2%) compared with RT alone (37.8%; hazard ratio (HR) 1.68, 95% CI 0.95-2.95, P=0.07). No benefit was seen with low miR-210 (HR 1.02, 95% CI 0.58-1.79, P=0.97). High miR-210 was significantly associated with high HIF-1α protein (P=0.001), CA9 protein (P=0.0004), Glut-1 protein (P=0.001), 26-gene hypoxia score (P=0.007), tumour necrosis (P=0.02) and concurrent pTis (P=0.03).High miR-210 may reflect hypoxia in bladder cancer. However, its ability to predict benefit from hypoxia modification does not improve upon other hypoxia markers. Investigation as part of a miRNA hypoxia signature may reveal the full potential of miR-210