Simple Detection of Telomere Fusions in Pancreatic Cancer, Intraductal Papillary Mucinous Neoplasm, and Pancreatic Cyst Fluid Tatsuo Hata, Marco Dal.

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Simple Detection of Telomere Fusions in Pancreatic Cancer, Intraductal Papillary Mucinous Neoplasm, and Pancreatic Cyst Fluid Tatsuo Hata, Marco Dal Molin, Anne McGregor-Das, Tae Jun Song, Christopher Wolfgang, James R. Eshleman, Ralph H. Hruban, Michael Goggins The Journal of Molecular Diagnostics Volume 20, Issue 1, Pages 46-55 (January 2018) DOI: 10.1016/j.jmoldx.2017.09.006 Copyright © 2018 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 1 Summary of the telomere fusion assay protocol. qPCR, real-time quantitative PCR. The Journal of Molecular Diagnostics 2018 20, 46-55DOI: (10.1016/j.jmoldx.2017.09.006) Copyright © 2018 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 2 Characteristics of telomere fusions. A: Classification of telomere fusions in pancreatic cancer cell lines and intraductal papillary mucinous neoplasm (IPMN) tissues. Telomere–telomere fusions contain telomere repeat sequences from both chromosomal ends (TTAGGG and its complement CCCTAA). Telomere–subtelomere fusions contain telomeric repeats from one chromosome fused with the subtelomeric region of another chromosome. Telomere–complex fusions contain short insertion between the subtelomere-telomere fusion junction. B: The length of telomeric DNA within fusion junctions. C: The number of TTAGGG repeats within fusions. HGD, high-grade dysplasia; IGD, intermediate-grade dysplasia. The Journal of Molecular Diagnostics 2018 20, 46-55DOI: (10.1016/j.jmoldx.2017.09.006) Copyright © 2018 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 3 Telomere fusion and telomere length in normal and intraductal papillary mucinous neoplasm (IPMN) tissues. Scatter plot graph with left y axis indicates telomere length. The longer horizontal bar represents the median value and shorter ones represent values of the 75th and 25th percentiles, respectively. Gray bar graph with right y axis indicates telomere fusion prevalence. There was insufficient DNA available from three low-grade dysplasia (LGD) cases for telomere length analysis. ∗∗∗P < 0.001, solid comparison bars represent the comparison of telomere lengths, and dashed comparison bars represent the comparison of telomere fusion prevalence. HGD, high-grade dysplasia; IGD, intermediate-grade dysplasia; T/S ratio, ratio of telomere repeat copy number to a single copy gene copy number. The Journal of Molecular Diagnostics 2018 20, 46-55DOI: (10.1016/j.jmoldx.2017.09.006) Copyright © 2018 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 4 Telomere fusions detected in intraductal papillary mucinous neoplasm (IPMN) cyst fluid samples. A: The prevalence of telomere fusions in IPMN cyst fluid samples according to their grade of dysplasia. B: Cyst fluid telomerase activity. The longer horizontal represents the median value and shorter ones represent values of the 75th and 25th percentiles, respectively. Red markers indicate fusion-positive samples. C: Telomerase activities in cyst fluid samples with or without telomere fusions. ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001. HGD, high-grade dysplasia; IGD, intermediate-grade dysplasia; INV, invasive cancer; LGD, low-grade dysplasia. The Journal of Molecular Diagnostics 2018 20, 46-55DOI: (10.1016/j.jmoldx.2017.09.006) Copyright © 2018 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Supplemental Figure S1 Examples of telomere fusion assay results. A: Amplification curves after nested real-time quantitative PCR (qPCR) classified by signal positive and negative. Green lines indicate the Ct value. B: Gel imaging after the electrophoresis of nested-qPCR products. Samples marked with blue circles are applied to third PCR. C: Gel imaging after the electrophoresis of third PCR. Sample in lane 30 (PL4 cell line) marked with red circles highlights the amplicon of similar size to that obtained after the nested-qPCR. D and E: DNA sequence in one of the subcloned AsPC-1 (D) and PL4 (E) fusion junctions. Rectangles indicate primer sequences, and telomeric repeats are underlined. M, DNA marker; N, nontemplate control. The Journal of Molecular Diagnostics 2018 20, 46-55DOI: (10.1016/j.jmoldx.2017.09.006) Copyright © 2018 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Supplemental Figure S3 Representative results of telomere fusion assays in pancreatic cancer xenograft samples. A: Amplification curves after nested real-time quantitative PCR (qPCR) classified by signal positive and negative. Green lines indicate the Ct value. B: Gel imaging after the electrophoresis of signal-positive nested-qPCR products. C: Gel imaging after the electrophoresis of signal-negative nested-qPCR products. Samples marked with blue circles are applied to third PCR. D: Gel imaging after the electrophoresis of third PCR. Samples marked with red circles highlight the amplicons of similar size to that obtained after the second-round nested-qPCR; these amplicons were subcloned and sequenced for validation. B, mouse whole blood DNA; M, DNA marker; N, nontemplate control; S, SU.86.86 cells; T, mouse tail DNA. The Journal of Molecular Diagnostics 2018 20, 46-55DOI: (10.1016/j.jmoldx.2017.09.006) Copyright © 2018 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Supplemental Figure S4 Kaplan-Meier curve of overall survival characterized by telomere fusion status in pancreatic cancer xenograft samples. Patients lost to follow-up were censored at their last visit. The Journal of Molecular Diagnostics 2018 20, 46-55DOI: (10.1016/j.jmoldx.2017.09.006) Copyright © 2018 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Supplemental Figure S5 Nested real-time quantitative PCR (qPCR) performance using various telomere fusion junction sequences as templates. A: Standard curve of serially diluted DNA for all tested clones. B and C: Amplification curve of serially diluted DNA (B) and fusion junction sequence (C) of PANC 215 clone 8. D and E: Amplification curve of serially diluted DNA (D) and fusion junction sequence (E) of PANC 215 clone 1. Rectangles indicate primer sequence, and telomeric repeats are underlined. The green lines indicate the Ct value (B and D). The Journal of Molecular Diagnostics 2018 20, 46-55DOI: (10.1016/j.jmoldx.2017.09.006) Copyright © 2018 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Supplemental Figure S6 Methodologic validation of telomere length measurement assay with the use of real-time quantitative PCR (qPCR) technique. A: Amplification curves using telomere telg/telc primers and serially diluted human genomic DNA. Green line indicates the Ct value. B: Dissociation curves of single product amplifications using telg/telc primers. C: Standard curves for telomeric repeat length and single copy reference (36B4). The Journal of Molecular Diagnostics 2018 20, 46-55DOI: (10.1016/j.jmoldx.2017.09.006) Copyright © 2018 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Supplemental Figure S7 Telomere lengths measured in 31 pancreatic cancer cell lines and HPDE and HPNE cells classified by their telomere fusion status. The longer horizontal bar represents the median value and shorter ones represent values of the 75th and 25th percentiles, respectively. The Journal of Molecular Diagnostics 2018 20, 46-55DOI: (10.1016/j.jmoldx.2017.09.006) Copyright © 2018 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Supplemental Figure S8 Representative hematoxylin and eosin images of intraductal papillary mucinous neoplasm frozen sections before and after laser capture microdissection. Scale bars: 400 μm. HGD, high-grade dysplasia; IGD, intermediate-grade dysplasia; LGD, low-grade dysplasia. The Journal of Molecular Diagnostics 2018 20, 46-55DOI: (10.1016/j.jmoldx.2017.09.006) Copyright © 2018 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Supplemental Figure S9 Representative examples of telomere fusion assay results. A: Nested real-time quantitative PCR (qPCR) amplification from fusion-positive and fusion-negative cases. Green lines indicate the Ct value. B: Gel electrophoresis images of nested-qPCR products. Samples marked with blue circles represent PCR-positive but qPCR negative telomere fusion amplicons. C–E: Gel electrophoresis images of third-round PCR to further evaluate telomere fusion amplicons. Examples of telomere fusion sequences from intraductal papillary mucinous neoplasms, including qPCR-positive (lane 3, D) and qPCR-negative (lane 22, E) fusions. Rectangles indicate primer sequences and underlined sequences are telomeric repeats. Lanes marked with red circles highlight the amplicons generated with the third-round PCR that are of similar size to that obtained after the second-round nested-qPCR. M, DNA marker; N, nontemplate control; S, SU.86.86 cell line as positive control. The Journal of Molecular Diagnostics 2018 20, 46-55DOI: (10.1016/j.jmoldx.2017.09.006) Copyright © 2018 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Supplemental Figure S10 Intra and interassay variation of telomere fusion assay. A: Ct values of four technical replicates measured by nested real-time quantitative PCR (qPCR) in the same assay, using five representative intraductal papillary mucinous neoplasm (IPMN) DNA samples and the SU.86.86 cell line. B: Amplification curves of five DNA samples corresponding to A. C: Interassay variation using five representative fusion-positive or fusion-negative DNA samples. Green lines indicate the Ct value (B and C). Green bars indicate mean values. CV; coefficient of variation. The Journal of Molecular Diagnostics 2018 20, 46-55DOI: (10.1016/j.jmoldx.2017.09.006) Copyright © 2018 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Supplemental Figure S11 Telomere lengths of intraductal papillary mucinous neoplasm (IPMN) stratified by histologic grade and telomere fusion status. A: Telomere length measurement and comparison by using paired of normal pancreas and IPMN. Median ratio of telomere repeat copy number to a single copy gene copy number is shown above the aligned dots. B: Mean Telomere lengths of fusion-positive and -negative IPMNs. The longer horizontal bar represents the median value and shorter ones represent values of the 75th and 25th percentiles, respectively. C: Relative telomere erosion between fusion-positive and -negative IPMN tissues. D: Relative telomere erosion across the three histologic grades of IPMN. ∗∗P < 0.01, ∗∗∗P < 0.001. HGD, high-grade dysplasia; IGD, intermediate-grade dysplasia; LGD, low-grade dysplasia. The Journal of Molecular Diagnostics 2018 20, 46-55DOI: (10.1016/j.jmoldx.2017.09.006) Copyright © 2018 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions