Table 1.
Patient group characteristics.
Fig 1.
Images showing representative regions for non-fibrotic and fibrotic areas within one lung biopsy.
(A) Low magnification image of H&E stained IPF lung tissue showing non-fibrotic (NF) and fibrotic (F) areas, and characteristic honeycombing (HC). (B) Enlarged boxed area of image A representing a fibrotic area in IPF lung biopsy. Characteristic fibroblast focus (FF) and alveolar type 2 (AT2) cell hyperplasia (black arrows) are highlighted. (C,D) Combined fluorescent images of AT2 and surrounding cells in non-fibrotic areas (C) and fibrotic areas (D) in IPF lung tissue. DNA in nucleus is displayed in blue (DAPI), pro-SPC in green and telomeres in red. Note the lower signal of AT2 cell (white arrows) telomere signal (red dots) in fibrotic areas (D) vs non-fibrotic (C) tissue, reflecting shorter telomeres in AT2 cells in fibrotic areas. Examples of pro-SPC negative surrounding cells are marked with an asterisk (*).
Fig 2.
Telomere length in sporadic IPF lungs.
(A) Representative example of telomere length per alveolar type 2 (AT2) cell for one sporadic IPF subject. Each dot represents one cell. Non-fibrotic AT2 FISH-TL is significantly higher compared to FISH-TL in fibrotic areas (p<0.0001). Bars represent medians and p-value is calculated using a Mann-Whitney test. (B, C, D) Median surrounding and AT2 cell FISH-TL in non-fibrotic and fibrotic areas for 16 sporadic IPF subjects. Each line connects, per subject, the FISH-TL of (B) AT2 cells in the non-fibrotic and fibrotic area or median FISH-TL differences between surrounding and AT2 cells in (C) non-fibrotic areas or (D) fibrotic areas. AT2 FISH-TL differences between non-fibrotic and fibrotic areas and between surrounding and AT2 cells were significant (2-tailed p<0.0006 and p<0.0001 respectively), which were indicated by asterisks (*** = p<0.001, **** = p<0.0001).
Table 2.
Median telomere length for non-fibrotic versus fibrotic areas per cohort.
Table 3.
Median telomere length for alveolar type 2 (AT2) cells versus surrounding cells per cohort.
Fig 3.
FISH telomere length in lungs of FIP-TERT and FIP-nonTERT subjects.
(A) Median FISH-TL in alveolar type 2 (AT2) cells in non-fibrotic and fibrotic areas of 9 FIP-TERT subjects. Telomeres are generally short and no significant difference between areas was observed (2-tailed, p = 0.36). (B, C) Median FISH-TL of same subjects as in figure A, showing differences between surrounding and AT2 cells in (B) non-fibrotic and (C) fibrotic areas. (D) Median FISH-TL in alveolar type 2 (AT2) cells in non-fibrotic and fibrotic areas of 10 FIP-nonTERT subjects. Non-fibrotic AT2 FISH-TL is significantly higher compared to FISH-TL in fibrotic areas (2-tailed, p = 0.02). (E, F) Median FISH-TL of same subjects as in figure D, showing differences between surrounding and AT2 cells in (E) non-fibrotic and (F) fibrotic areas. Asterisks indicate significant differences calculated by Wilcoxon matched-pairs signed rank analysis (* = p<0.05, ** = p<0.01).
Fig 4.
Correlation between FISH-TL and biopsy T/S in IPF subjects.
Correlation (Spearman) between FISH-TL measured in alveolar type 2 (AT2) cells and biopsy T/S measured by MMqPCR (n = 15) in non-fibrotic areas (black dots) and fibrotic areas (open squares). Significant correlations were established between biopsy T/S and AT2 cell FISH-TL in non-fibrotic (r2 = 0.53, p = 0.002) and fibrotic (r2 = 0.73, p<0.0001) areas.
Fig 5.
Correlation between FISH-TL and MMqPCR leukocyte TL (blood T/S) in FIP-TERT fibrotic areas.
Positive FIP-TERT correlation (Spearman) between alveolar type 2 (AT2) cell FISH-TL in fibrotic areas and MMqPCR blood T/S (n = 9, r2 = 0.67, p = 0.007).
Fig 6.
Correlations between MMqPCR measurements biopsy T/S and blood T/S in IPF.
No correlation between biopsy T/S and blood T/S was observed in sporadic IPF (n = 26), FIP-nonTERT (n = 12) and FIP-TERT (n = 10) subjects.
Fig 7.
(A) FISH-TL on lung biopsy; Kaplan-Meier curve of 15 IPF patients showing a median survival of 60 months for patients with AT2 cell FISH-TL in non-fibrotic areas above median TL (solid line) and a median survival of 26 months for patients with AT2 cell TL below median TL (dotted line). The difference in median survival is 34 months (p = 0.353, Mantel-Cox). (B) MMqPCR on lung biopsy; Kaplan-Meier curve of 34 IPF patients showing a median survival of 63 months for IPF patients with a lung biopsy MMqPCR T/S ratio above the median (solid line) and a median survival of 22 months for patients with a lung biopsy MMqPCR T/S ratio below the median T/S (dotted line). The significant difference in median survival is 41 months (p = 0.003, Mantel-Cox). Deceased n = 27, lung transplantation n = 4, still alive n = 3.