Intratumoral heterogeneity of microRNA expression in rectal cancer

Introduction: An increasing number of studies have investigated microRNAs (miRNAs) as potential markers of diagnosis, treatment and prognosis. So far, agreement between studies has been minimal, which may in part be explained by intratumoral heterogeneity of miRNA expression. The aim of the present study was to assess the heterogeneity of a panel of selected miRNAs in rectal cancer, using two different technical approaches. Materials and Methods: The expression of the investigated miRNAs was analysed by real-time quantitative polymerase chain reaction (RT-qPCR) and in situ hybridization (ISH) in tumour specimens from 27 patients with T3-4 rectal cancer. From each tumour, tissue from three different luminal localisations was examined. Inter- and intra-patient variability was assessed by calculating intraclass correlation coefficients (ICCs). Correlations between RT-qPCR and ISH were evaluated using Spearman's correlation. Results: ICCsingle (one sample from each patient) was higher than 50% for miRNA-21 and miRNA-31. For miRNA-125b, miRNA-145, and miRNA-630, ICCsingle was lower than 50%. The ICCmean (mean of three samples from each patient) was higher than 50% for miRNA-21(RT-qPCR and ISH), miRNA-125b (RT-qPCR and ISH), miRNA-145 (ISH), miRNA-630 (RT-qPCR), and miRNA-31 (RT-qPCR). For miRNA-145 (RT-qPCR) and miRNA-630 (ISH), ICCmean was lower than 50%. Spearman correlation coefficients, comparing results obtained by RT-qPCR and ISH, respectively, ranged from 0.084 to 0.325 for the mean value from each patient, and from -0.085 to 0.515 in the section including the deepest part of the tumour. Conclusion: Intratumoral heterogeneity may influence the measurement of miRNA expression and consequently the number of samples needed for representative estimates. Our findings with two different methods suggest that one sample is sufficient for adequate assessment of miRNA-21 and miRNA-31, whereas more samples would improve the assessment of miRNA-125b, miRNA-145, and miRNA-630. Interestingly, we found a poor correlation between the expression estimates obtained by RT-qPCR and ISH, respectively

Intraclass Correlation Coefficient (ICC) for one sample from each tumour (ICC_single) and for three different samples from each tumour (ICC_mean) as obtained by RT-qPCR and ISH.

<p>Intraclass Correlation Coefficient (ICC) for one sample from each tumour (ICC_single) and for three different samples from each tumour (ICC_mean) as obtained by RT-qPCR and ISH.</p

Inter- and intra-patient variation and correlation between RT-qPCR and ISH measurements for miRNA-21, miRNA-125b, miRNA-145, and miRNA-630.

<p>All data were normalised to be plotted on a common scale: see the main text for details. Each vertical line represents the range of the three measures obtained in the individual patient with different colours to represent RT-qPCR and ISH measurements, respectively. For each miRNA, patients were sorted according to their mean RT-qPCR measurement for plotting.</p

<i>In situ</i> hybridization (ISH) for miRNA-21, miRNA-125b, miRNA-145, and miRNA-630: Classifiers, relative area fractions and localisation of ISH signal.

<p><i>In situ</i> hybridization (ISH) for miRNA-21, miRNA-125b, miRNA-145, and miRNA-630: Classifiers, relative area fractions and localisation of ISH signal.</p

<i>In situ</i> hybridization (ISH) visualizing the expression of miRNA-21, miRNA-125b, miRNA-145, and miRNA-630 in rectal cancer tissue.

<p>The ISH signal is corresponding to the blue colour in the left row. St: stroma; Ca: epithelial tumor cells. <b>(A)</b> Expression of miRNA-21 mainly present in the stroma adjacent to the epithelial tumour cells. <b>(B)</b> Pixel classifier corresponding to image A. <b>(C)</b> Expression of miRNA-125b with strong ISH-signal present in an enteric neuron (arrows) and weaker ISH signal representing different stromal cells. <b>(D)</b> Pixel classifier corresponding to image C. <b>(E)</b> Expression of miRNA-145 mainly present in smooth muscle cells (SMC) and myofibroblastic cells. <b>(F)</b> Pixel classifier corresponding to image E. <b>(G)</b> Expression of miRNA-630 scattered in epithelial tumour cells and adjacent stromal cells (arrows indicate examples of ISH-positive cells in the stroma). <b>(H)</b> Pixel classifier corresponding to image G.</p

Spearman Correlation Coefficients comparing RT-qPCR and ISH for average measures for individual patients and for a single sample from each patient (the one of the three sections including the deepest part of the tumour).

<p>Spearman Correlation Coefficients comparing RT-qPCR and ISH for average measures for individual patients and for a single sample from each patient (the one of the three sections including the deepest part of the tumour).</p

Late Relapses in Stage I Testicular Cancer Patients on Surveillance

Background: Comprehensive data on late relapse (LR) and very LR (VLR) in patients with clinical stage I (CS-1) testicular cancer followed on surveillance are missing. These data are essential for planning optimal follow-up. Objective: Assess incidence and outcome of LR (>2 yr) and VLR (>5 yr) in a large cohort of CS-1 surveillance patients, and examine differences in the clinical characteristics of patients with early relapse (ER), LR, and VLR. Design, setting, and participants: CS-1 surveillance patients diagnosed between 1984 and 2007 were identified from the retrospective Danish Testicular Cancer (DaTeCa) database. Outcome measurements and statistical analysis: We estimated survival and relapse probabilities and compared the results using log-rank tests and Cox regression analyses. We compared differences in patient characteristics by using χ2, Fisher exact, and Mann-Whitney tests. Results and limitations: Our study included 3366 (2000 seminoma and 1366 nonseminoma) patients. Median follow-up was 15 yr. Five-year conditional risk of LR was 5.0% and 2.1% for seminoma and nonseminoma patients, respectively. There were no significant differences in disease-specific or overall survival when comparing the LR(VLR) and ER patients by log-rank, but Cox regression adjusted for age showed a significant effect of time to relapse on survival for seminoma patients. Apart from significantly more ER nonseminoma patients with elevated human chorionic gonadotropin at relapse, there were no significant differences in patient characteristics at orchiectomy or relapse. Limitations include retrospective design and exclusion of patients who had been offered adjuvant therapy. Conclusions: The risk of VLR is minimal, and the patients carry a good prognosis. Patient characteristics of CS-1 surveillance patients with LR(VLR) do not differ significantly from patients with ER. Patient summary: We compared stage I testicular cancer surveillance patients with early relapse (ER) versus late relapse (LR; >2 yr). LR patients as a group did no worse than ER patients, although increased time to relapse was negatively associated with survival for seminoma patients

Heterogeneous FDG-guided dose-escalation for locally advanced NSCLC (the NARLAL2 trial): Design and early dosimetric results of a randomized, multi-centre phase-III study

Background and purpose: Local recurrence is frequent in locally advanced NSCLC and is primarily located in FDG-avid parts of tumour and lymph nodes. Aiming at improving local control without increasing toxicity, we designed a multi-centre phase-III trial delivering inhomogeneous dose-escalation driven by FDG-avid volumes, while respecting normal tissue constraints and requiring no increase in mean lung dose. Dose-escalation driven by FDG-avid volumes, delivering mean doses of 95 Gy (tumour) and 74 Gy (lymph nodes), was pursued and compared to standard 66 Gy/33 F plans. Material and methods: Dose plans for the first thirty patients enroled were analysed. Standard and escalated plans were created for all patients, blinded to randomization, and compared for each patient in terms of the ability to escalate while protecting normal tissue. Results: The median dose-escalation in FDG-avid areas was 93.9 Gy (tumour) and 73.0 Gy (lymph nodes). Escalation drove the GTV and CTV to mean doses for the tumour of 87.5 Gy (GTV-T) and 81.3 Gy (CTV-T) in median. No significant differences in mean dose to lung and heart between standard and escalated were found, but small volumes of e.g. the bronchi received doses between 66 and 74 Gy due to escalation. Conclusions: FDG-driven inhomogeneous dose-escalation achieves large increment in tumour and lymph node dose, while delivering similar doses to normal tissue as homogenous standard plans