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Telomere Length Assessment in Human Archival Tumor Tissues By Quantitative PCR method (qTL) Hui Wang, Ning Fei Go, Zhu Pirot, and Katia Bassett Geron Corporation, Menlo Park, California Abstract Inter-assay variability and coefficient of variation were improved by using short amplicons Correlation between TRF and qTL in human archival breast tumor tissues Reproducibility of T/S ratios in normal and tumor archival breast tissues Quality of gDNA from archival tissue was improved by using a non-column-based FFPE DNA extraction kit Telomeres are essential for maintenance of genomic stability. Short telomeres have been associated with genomic instability, and initiation and progression of human cancers. To protect their telomeres, up to 90% of human cancers activate telomerase. Inhibition of telomerase by imetelstat, a specific and potent inhibitor, has resulted in telomere shortening, as well as tumor response in animal models. We propose that tumors with short telomeres may be more responsive to imetelstat than tumors with long telomeres. This hypothesis will be tested by measuring baseline telomere lengths in archival tumors of patients in imetelstat Phase II clinical trials, and correlating tumor telomere length with clinical outcome. In order to accomplish this, a fast, high-throughput qPCR-based method for telomere length measurement in genomic DNA extracted from Formalin Fixed Paraffin Embedded tissues (FFPE) sections was developed. Using this assay, telomere repetitive DNA sequences and single-copy gene control were successfully amplified. The assay required very little genomic DNA (1-10ng per PCR reaction). Thus, small tissue sections (20 mm 2 ) could be easily accommodated. Using this technique, we were able to perform the first comparison of relative telomere lengths in 22 human breast cancer samples and 8 human tumor cell lines in matched fresh-frozen and FFPE samples. Telomere lengths measured in FFPE samples by the qPCR method strongly correlated with the telomere lengths measured by TRF in matched fresh-frozen samples. Therefore, our method provides a useful tool for assessment of telomere length as a potential predictive biomarker of responsiveness to telomerase inhibition in clinical archival tumor tissue. Correlation between TRF and qTL in human FFPE tumor cell lines Summary Fig.3 (A) Telomere lengths of the eight cell lines were measured by the Telomere Restriction Fragment method (TRF). (B) Relative telomere lengths (T/S) of the eight cell lines were measured by the qTL method. (C) Reproducibility of telomere amplification was repeatedly (n=6) tested in eight human FFPE cell lines . Mean Ct values were inversely proportional to the telomere lengths as determined by the TRF (P<0.0001 in One-way ANOVA test). (D) Mean Ct values for the single copy gene were similar for all eight cell lines. Telomere length of a cell line had no impact on the Ct value for single copy gene (P=0.4809 in One-way ANOVA test). • We developed the first quantitative PCR assay for telomere length measurement (qTL) in formalin-fixed, paraffin-embedded tissues. • qPCR assay requires very little genomic DNA (0.1-10 ng per PCR reaction). Thus, very small tissue sections (~20 mm 2 ) from a single slide can be easily accommodated. • qPCR assay results correlate with the commonly used telomere repeat fragment southern blot (TRF) in matched non-formalin-fixed samples. • The assay enables us to measure telomere length in archived FFPE tumor samples and to correlate tumor telomere lengths with clinical outcome. Fig.1: Quality of genomic DNA from FFPE sections was improved by retaining small DNA fragments during DNA isolation process. ( A.) Genomic DNA from four human FFPE cell lines (A549, MDA-MB-231, CCRF-CEM and Ovcar-5) was isolated using three FFPE DNA extraction kits, manufactured by BioChain , QIAGEN, and TrimGene. Isolated genomic DNA was run on a 0.8% agarose gel. Small DNA fragments (<100bp) were only captured using the BioChain kit, not the QIAGEN and TrimGene kits. Column purification step was not required by the BioChain kit, but required by the other two kits, and could have caused the loss of small DNA fragments during DNA isolation process. ( B) and (C) Genomic DNA was repeatedly (n=5) isolated from four circled areas of mouse xenograft tumor sections using the BioChain kit. Mean DNA yields from the circled tissue areas were, 1.0, 2.2, 3.0, and 5.0 ng, and the coefficients of inter-assay variation were 15%, 8%, 19%, and 19%, respectively . (D). Linear regression coefficient between the area of the tissue section and the mean DNA yield was 0.9379. Fig.2 Telomeres and the single copy gene were amplified in triplicate from FFPE gDNA isolated from the A549 cell line. The primers were designed by Cawthon et. al (Nucleic Acids Res., 2002 and 2009), and the PCR conditions were modified to improve PCR efficiency. PCR products were resolved on a 15% PAGE gel. (A) Multiple sizes of PCR products were generated by the Tel1/Tel2 primers from telomeric DNA. ( B) A 79-bp, uniform PCR product was generated by the Telg/Telgc primers from telomeric DNA. (C) A 76-bp, uniform single copy gene (36B4) PCR product was generated by the 36Bu /36Bd primers. PCR efficiency (>95%) and inter-assay reproducibility of both telomere and single copy gene amplification were significantly improved by using short amplicons. Fig.4 Correlation of the average telomere lengths in human breast tumors measured by qTL and TRF was determined. Ten human breast tumors (matched fresh-frozen and FFPE sections) were purchased from Analytical Biological Services Inc (ABS). (A). Telomere lengths of ten human breast tumors were assessed by TRF in fresh-frozen tissues. (B). Relative telomere lengths were repeatedly (n=2) measured in ten human tumor FFPE sections by qTL. Fig.5 (A) The reproducibility of the qTL assay was measured in normal and tumor breast FFPE specimens. Ten paired normal and tumor breast samples were purchased from BioChain Institute. The intra-assay coefficient of variation was 6.12%. (B) The inter-assay reproducibility of T/S ratios was measured in three separate assays, which included DNA isolation and PCR reactions . Each DNA sample was tested in triplicate during each run. The inter-assay of coefficient of variation was 8.1%. The T/S ratios in three independent runs correlated significantly with each other (P< 0.0001 in 95% CI.)
