Preanalytical variables and performance of diagnostic RNA-based gene expression analysis in breast cancer

Christopher Poremba, Jennifer Uhlendorff, Berit M Pfitzner, Guido Hennig, Kerstin Bohmann, Hans Bojar, Veit Krenn, Jan C Brase, Franziska Haufe, Manuela Averdick, Manfred Dietel, Ralf Kronenwett, Carsten Denkert, Christopher Poremba, Jennifer Uhlendorff, Berit M Pfitzner, Guido Hennig, Kerstin Bohmann, Hans Bojar, Veit Krenn, Jan C Brase, Franziska Haufe, Manuela Averdick, Manfred Dietel, Ralf Kronenwett, Carsten Denkert

Abstract

Prognostic multigene expression assays have become widely available to provide additional information to standard clinical parameters and to support clinicians in treatment decisions. In this study, we analyzed the impact of variations in tissue handling on the diagnostic EndoPredict test results. EndoPredict is a quantitative reverse transcription PCR assay conducted on RNA from formalin-fixed, paraffin-embedded (FFPE) tissue that predicts the likelihood of distant recurrence in patients with ER-positive/HER2-negative breast cancer. In this study, we performed a total of 138 EndoPredict assays to study the effects of preanalytical variables such as time to fixation, fixation time, tumor cell content, and section storage time on the EndoPredict test results. A time to fixation of up to 12 h and fixation of up to 5 days did not affect the results of the gene expression test. Paired samples of FFPE sections with tumor cell content ranging from 15 to 95 % and tumor-enriched samples showed a correlation coefficient of 0.97. Test results of tissue sections that have been stored for 12 months at +4 or +20 °C showed a correlation of 0.99 when compared to results of nonstored sections. In conclusion, preanalytical tissue handling is not a critical factor for diagnostic gene expression analysis with the EndoPredict assay. The test can therefore be easily integrated into the standard workflow of molecular pathology.

Figures

Fig. 1
Fig. 1
Scheme of preanaytical steps from tissue removal to archiving of the FFPE tumor material and related experiments that were performed in this study
Fig. 2
Fig. 2
EndoPredict test results achieved after different storage times and temperatures before fixation. The solid line represents the reference score which was derived from the mean EP score of the tissue sections that have been stored for 10 min at 20 °C before fixation. Dotted lines represent the reference score ±3× its standard deviation as determined by precision study [24]. Measurements were performed in duplicate (n = 2). Mean values are presented, with standard errors of the means indicated by bars
Fig. 3
Fig. 3
EndoPredict test results achieved after different fixation times. The solid line represents the reference score which was derived from the mean EP score of the tissue sections that have been fixed for 20 h in neutral buffered formalin. Dotted lines represent the reference score ±3× its standard deviation as determined by precision study [24]. Measurements were performed in duplicate (n = 2). Mean values are presented, with standard errors of the means indicated by bars
Fig. 4
Fig. 4
Correlation between the EP (a) and EPclin (b) scores of paired whole and tumor-enriched, manually microdissected tissue sections from ER+/HER2− breast cancer patients (n = 39). Pearson correlation coefficient of EP scores was 0.97 with a concordance of EndoPredict risk classification of 87 %. Pearson correlation coefficient of EPclin scores was 0.98 with a concordance of EndoPredict risk classification of 95 %. Black filled circles represent tissue samples with TCC ≥30 %, open circles tissue sections with TCC <30 %
Fig. 5
Fig. 5
Correlation of EP scores between stored and nonstored FFPE tissue sections. EP scores have been determined at the beginning of the study, after 12 months at +4 °C (a) or after 12 months at +20 °C (b), respectively. Pearson correlation coefficients were 0.99 and >0.99 with a concordance of EndoPredict risk classification of 100 %

References

    1. Filipits M, Rudas M, Jakesz R, Dubsky P, Fitzal F, Singer CF, Dietze O, Greil R, Jelen A, Sevelda P, Freibauer C, Muller V, Janicke F, Schmidt M, Kolbl H, Rody A, Kaufmann M, Schroth W, Brauch H, Schwab M, Fritz P, Weber KE, Feder IS, Hennig G, Kronenwett R, Gehrmann M, Gnant M. A new molecular predictor of distant recurrence in ER-positive, HER2-negative breast cancer adds independent information to conventional clinical risk factors Clin. Cancer Res. 2011;17:6012–6020.
    1. Paik S, Shak S, Tang G, Kim C, Baker J, Cronin M, Baehner FL, Walker MG, Watson D, Park T, Hiller W, Fisher ER, Wickerham DL, Bryant J, Wolmark N. A multigene assay to predict recurrence of tamoxifen-treated, node-negative breast cancer N. Engl J Med. 2004;351:2817–2826. doi: 10.1056/NEJMoa041588.
    1. Parker JS, Mullins M, Cheang MC, Leung S, Voduc D, Vickery T, Davies S, Fauron C, He X, Hu Z, Quackenbush JF, Stijleman IJ, Palazzo J, Marron JS, Nobel AB, Mardis E, Nielsen TO, Ellis MJ, Perou CM, Bernard PS. Supervised risk predictor of breast cancer based on intrinsic subtypes J. Clin Oncol. 2009;27:1160–1167. doi: 10.1200/JCO.2008.18.1370.
    1. van’t Veer LJ, Dai H, van de Vijver MJ, He YD, Hart AA, Mao M, Peterse HL, van der Kooy K, Marton MJ, Witteveen AT, Schreiber GJ, Kerkhoven RM, Roberts C, Linsley PS, Bernards R, Friend SH. Gene expression profiling predicts clinical outcome of breast cancer. Nature. 2002;415:530–536. doi: 10.1038/415530a.
    1. Specht K, Richter T, Muller U, Walch A, Werner M, Hofler H. Quantitative gene expression analysis in microdissected archival formalin-fixed and paraffin-embedded tumor tissue Am. J Pathol. 2001;158:419–429.
    1. Stanta G, Bonin S. RNA quantitative analysis from fixed and paraffin-embedded tissues: membrane hybridization and capillary electrophoresis. BioTechniques. 1998;24:271–276.
    1. Hatzis C, Sun H, Yao H, Hubbard RE, Meric-Bernstam F, Babiera GV, Wu Y, Pusztai L, Symmans WF. Effects of tissue handling on RNA integrity and microarray measurements from resected breast cancers J. Natl Cancer Inst. 2011;103:1871–1883. doi: 10.1093/jnci/djr438.
    1. Hewitt SM, Lewis FA, Cao Y, Conrad RC, Cronin M, Danenberg KD, Goralski TJ, Langmore JP, Raja RG, Williams PM, Palma JF, Warrington JA. Tissue handling and specimen preparation in surgical pathology: issues concerning the recovery of nucleic acids from formalin-fixed, paraffin-embedded tissue Arch. Pathol Lab Med. 2008;132:1929–1935.
    1. Elloumi F, Hu Z, Li Y, Parker JS, Gulley ML, Amos KD, Troester MA. Systematic bias in genomic classification due to contaminating non-neoplastic tissue in breast tumor samples. BMC Med Genomics. 2011;4:54. doi: 10.1186/1755-8794-4-54.
    1. Kotoula V, Kalogeras KT, Kouvatseas G, Televantou D, Kronenwett R, Wirtz RM, Fountzilas G. Sample parameters affecting the clinical relevance of RNA biomarkers in translational breast cancer research. Virchows Arch. 2013;462:141–154. doi: 10.1007/s00428-012-1357-1.
    1. Dubsky P, Filipits M, Jakesz R, Rudas M, Singer CF, Greil R, Dietze O, Luisser I, Klug E, Sedivy R, Bachner M, Mayr D, Schmidt M, Gehrmann MC, Petry C, Weber KE, Kronenwett R, Brase JC, Gnant M. EndoPredict improves the prognostic classification derived from common clinical guidelines in ER-positive, HER2-negative early breast cancer. Ann Oncol. 2013;24:640–647. doi: 10.1093/annonc/mds334.
    1. Simon RM, Paik S, Hayes DF. Use of archived specimens in evaluation of prognostic and predictive biomarkers J. Natl Cancer Inst. 2009;101:1446–1452. doi: 10.1093/jnci/djp335.
    1. Kronenwett R, Bohmann K, Prinzler J, Sinn BV, Haufe F, Roth C, Averdick M, Ropers T, Windbergs C, Brase JC, Weber KE, Fisch K, Muller BM, Schmidt M, Filipits M, Dubsky P, Petry C, Dietel M, Denkert C. Decentral gene expression analysis: analytical validation of the Endopredict genomic multianalyte breast cancer prognosis test. BMC Cancer. 2012;12:456. doi: 10.1186/1471-2407-12-456.
    1. Denkert C, Kronenwett R, Schlake W, Bohmann K, Penzel R, Weber KE, Hofler H, Lehmann U, Schirmacher P, Specht K, Rudas M, Kreipe HH, Schraml P, Schlake G, Bago-Horvath Z, Tiecke F, Varga Z, Moch H, Schmidt M, Prinzler J, Kerjaschki D, Sinn BV, Muller BM, Filipits M, Petry C, Dietel M. Decentral gene expression analysis for ER+/Her2- breast cancer: results of a proficiency testing program for the EndoPredict assay. Virchows Arch. 2012;460:251–259. doi: 10.1007/s00428-012-1204-4.
    1. Bohmann K, Hennig G, Rogel U, Poremba C, Mueller BM, Fritz P, Stoerkel S, Schaefer KL. RNA extraction from archival formalin-fixed paraffin-embedded tissue: a comparison of manual, semiautomated, and fully automated purification methods. Clin Chem. 2009;55:1719–1727. doi: 10.1373/clinchem.2008.122572.
    1. Hennig G, Gehrmann M, Stropp U, Brauch H, Fritz P, Eichelbaum M, Schwab M, Schroth W. Automated extraction of DNA and RNA from a single formalin-fixed paraffin-embedded tissue section for analysis of both single-nucleotide polymorphisms and mRNA expression Clin. Chem. 2010;56:1845–1853.
    1. Srinivasan M, Sedmak D, Jewell S. Effect of fixatives and tissue processing on the content and integrity of nucleic acids Am. J Pathol. 2002;161:1961–1971.
    1. Chung JY, Braunschweig T, Williams R, Guerrero N, Hoffmann KM, Kwon M, Song YK, Libutti SK, Hewitt SM. Factors in tissue handling and processing that impact RNA obtained from formalin-fixed, paraffin-embedded tissue J. Histochem Cytochem. 2008;56:1033–1042. doi: 10.1369/jhc.2008.951863.
    1. Godfrey TE, Kim SH, Chavira M, Ruff DW, Warren RS, Gray JW, Jensen RH. Quantitative mRNA expression analysis from formalin-fixed, paraffin-embedded tissues using 5″ nuclease quantitative reverse transcription-polymerase chain reaction. J Mole Diagn : JMD. 2000;2:84–91. doi: 10.1016/S1525-1578(10)60621-6.
    1. Abrahamsen HN, Steiniche T, Nexo E, Hamilton-Dutoit SJ, Sorensen BS. Towards quantitative mRNA analysis in paraffin-embedded tissues using real-time reverse transcriptase-polymerase chain reaction: a methodological study on lymph nodes from melanoma patients. J Mole Diagn : JMD. 2003;5:34–41. doi: 10.1016/S1525-1578(10)60449-7.
    1. De Cecco L, Musella V, Veneroni S, Cappelletti V, Bongarzone I, Callari M, Valeri B, Pierotti MA, Daidone MG. Impact of biospecimens handling on biomarker research in breast cancer. BMC Cancer. 2009;9:409. doi: 10.1186/1471-2407-9-409.
    1. Muller BM, Kronenwett R, Hennig G, Euting H, Weber K, Bohmann K, Weichert W, Altmann G, Roth C, Winzer KJ, Kristiansen G, Petry C, Dietel M, Denkert C. Quantitative determination of estrogen receptor, progesterone receptor, and HER2 mRNA in formalin-fixed paraffin-embedded tissue–a new option for predictive biomarker assessment in breast cancer Diagn. Mol Pathol. 2011;20:1–10. doi: 10.1097/PDM.0b013e3181e3630c.
    1. Macabeo-Ong M, Ginzinger DG, Dekker N, McMillan A, Regezi JA, Wong DT, Jordan RC. Effect of duration of fixation on quantitative reverse transcription polymerase chain reaction analyses Mod. Pathol. 2002;15:979–987.
    1. EndoPredict handbook, performance data (Sividon Diagnostics GmbH)
    1. Schobesberger M, Baltzer A, Oberli A, Kappeler A, Gugger M, Burger H, Jaggi R. Gene expression variation between distinct areas of breast cancer measured from paraffin-embedded tissue cores. BMC Cancer. 2008;8:343. doi: 10.1186/1471-2407-8-343.
    1. Tramm T, Hennig G, Kyndi M, Alsner J, Sorensen FB, Myhre S, Sorlie T, Overgaard J. Reliable PCR quantitation of estrogen, progesterone and ERBB2 receptor mRNA from formalin-fixed, paraffin-embedded tissue is independent of prior macro-dissection. Virchows Arch. 2013;463:775–786. doi: 10.1007/s00428-013-1486-1.
    1. Nielsen T, Wallden B, Schaper C, Ferree S, Liu S, Gao D, Barry G, Dowidar N, Maysuria M, Storhoff J. Analytical validation of the PAM50-based Prosigna Breast Cancer Prognostic Gene Signature Assay and nCounter Analysis System using formalin-fixed paraffin-embedded breast tumor specimens. BMC Cancer. 2014;14:177. doi: 10.1186/1471-2407-14-177.
    1. Cronin M, Pho M, Dutta D, Stephans JC, Shak S, Kiefer MC, Esteban JM, Baker JB. Measurement of gene expression in archival paraffin-embedded tissues: development and performance of a 92-gene reverse transcriptase-polymerase chain reaction assay Am. J Pathol. 2004;164:35–42.
    1. von Ahlfen S, Missel A, Bendrat K, Schlumpberger M. Determinants of RNA quality from FFPE samples. PloS One. 2007;2:e1261. doi: 10.1371/journal.pone.0001261.

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