Telomere length (TCR) differences between age groups.

<p>Telomere length (TCR) differences between age groups.</p

Assessment of Telomere Length in Archived Formalin-Fixed, Paraffinized Human Tissue Is Confounded by Chronological Age and Storage Duration

<div><p>Telomeres shorten with physiological aging but undergo substantial restoration during cancer immortalization. Increasingly, cancer studies utilize the archive of formalin-fixed, paraffin-embedded (FFPE) tissues in diagnostic pathology departments. Conceptually, such studies would be confounded by physiological telomere attrition and loss of DNA integrity from prolonged tissue storage. Our study aimed to investigate these two confounding factors. 145 FFPE tissues of surgically-resected, non-diseased appendixes were retrieved from our pathology archive, from years 2008 to 2014. Cases from 2013 to 2014 were categorized by patient chronological age (0–20 years, 21–40 years, 41–60 years, > 60 years). Telomere lengths of age categories were depicted by telomere/chromosome 2 centromere intensity ratio (TCR) revealed by quantitative fluorescence <i>in situ</i> hybridization. Material from individuals aged 0–20 years from years 2013/2014, 2011/2012, 2009/2010, and 2008 were compared for storage effect. Telomere integrity was assessed by telomere fluorescence intensity (TFI). Epithelial TCRs (mean ± SD) for the respective age groups were 4.84 ± 2.08, 3.64 ± 1.21, 2.03 ± 0.37, and 1.93 ± 0.45, whereas corresponding stromal TCRs were 5.16 ± 2.55, 3.84 ± 1.36, 2.49 ± 1.20, and 2.93 ± 1.24. A trend of inverse correlation with age in both epithelial and stromal tissues is supported by r = -0.69, p < 0.001 and r = -0.42, p < 0.001 respectively. Epithelial TFIs (mean ± SD) of years 2013/2014, 2011/2012, 2009/2010 and 2008 were 852.60 ± 432.46, 353.04 ± 127.12, 209.24 ± 55.57 and 429.22 ± 188.75 respectively. Generally, TFIs reduced with storage duration (r = -0.42, p < 0.001). Our findings agree that age-related telomere attrition occurs in normal somatic tissues, and suggest that an age-based reference can be established for telomere studies on FFPE tissues. We also showed that FFPE tissues archived beyond 2 years are suboptimal for telomere analysis.</p></div

Representative fluorescence images of telomere Q-FISH from various patient age groups.

<p>Q-FISH performed with PNA FITC-labelled telomere probe (green) and Cy3-labelled CEN2 probe (red), and nuclei counterstained with DAPI (blue) (original magnification: ×1000). (A) Strong telomere signals (green) are evident in the normal appendix epithelial cells of a 7-week-old infant (age group: 0–20 years). (B) Bright telomere signals are observed in the normal appendix epithelial cells of a 22-year-old individual (age group: 21–40 years), but are slightly reduced in comparison with A. (C) Moderately bright telomere signals in the normal appendix epithelial cells of a 43-year-old individual (age group: 41–60 years), but are reduced compared to younger individuals. (D) Diminished telomere signals in the normal appendix epithelial cells of a 64-year-old individual (age group: > 60 years), compared to those of the younger individuals.</p

Fluorescence image of telomere Q-FISH denoting spermatocytes from normal testicular tissue of a 63-year-old individual used as experimental positive control.

<p>Q-FISH performed with PNA FITC-labelled telomere probe (green) and Cy3-labelled CEN2 probe (red), and nuclei counterstained with DAPI (blue) (original magnification: ×1000). The telomere signals (green) were very strong in the germ cells.</p

Fluorescence images of telomere Q-FISH from 0 to 20-year-old individuals after various storage durations.

<p>There are reduced telomere and CEN2 signals in the normal appendix epithelial cells from years (A) 2011/2012, (B) 2009/2010, and (C) 2008 compared to those from the most recent years (2013/2014) as depicted in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0161720#pone.0161720.g002" target="_blank">Fig 2A</a>.</p

Mean telomere length (TFI) measurements in appendiceal epithelial cells according to storage duration.

<p>Mean telomere length (TFI) measurements in appendiceal epithelial cells according to storage duration.</p

Number of samples with quantifiable CEN2 signals for each surgery year category.

<p>Number of samples with quantifiable CEN2 signals for each surgery year category.</p

Scatter plots showing the inverse relationship between telomere length and chronological age.

<p>(A) Epithelial telomere length decreases with increasing chronological age. (B) Mucosal stromal telomere length decreases with increasing chronological age. </p

Survival probabilities and Hazard Ratios for all cause mortality by pN classification and LNR.

*<p>Model A is adjusted for: age, radiotherapy, surgery type, grade and tumor size and pN stage and stratified by ER Status. Model B is adjusted for: age, radiotherapy, surgery type, grade and tumor size and LNR and stratified by ER Status. Both models were internally validated using bootstrap resampling.</p

Patient, tumor characteristics and treatment along with the unadjusted Hazard Ratio for all cause mortality.

*<p>indicates valid proportions have been calculated (i.e., not considering unknown).</p