Jeffrey R Shuster1, Raymond S Lance, Dean A Troyer. 1. Depts, Of Pathology and Microbiology and Molecular Cell Biology, Eastern Virginia Medical School, Norfolk, VA, USA. troyerda@evms.edu.
Abstract
BACKGROUND: Histopathology is the standard method for cancer diagnosis and grading to assess aggressiveness in clinical biopsies. Molecular biomarkers have also been described that are associated with cancer aggressiveness, however, the portion of tissue analyzed is often processed in a manner that is destructive to the tissue. We present here a new method for performing analysis of small molecule biomarkers and histology in exactly the same biopsy tissue. METHODS: Prostate needle biopsies were taken from surgical prostatectomy specimens and first fixed, each in a separate vial, in 2.5 ml of 80% methanol:water. The biopsies were fixed for 24 hrs at room temperature and then removed and post-processed using a non-formalin-based fixative (UMFIX), embedded, and analyzed by hematoxylin and eosin (H&E) and by immunohistochemical (IHC) staining. The retained alcohol pre-fixative was analyzed for small molecule biomarkers by mass spectrometry. RESULTS: H&E analysis was successful following the pre-fixation in 80% methanol. The presence or absence of tumor could be readily determined for all 96 biopsies analyzed. A subset of biopsy sections was analyzed by IHC, and cancerous and non-cancerous regions could be readily visualized by PIN4 staining. To demonstrate the suitability for analysis of small molecule biomarkers, 28 of the alcohol extracts were analyzed using a mass spectrometry-based metabolomics platform. All extracts tested yielded successful metabolite profiles. 260 named biochemical compounds were detected in the alcohol extracts. A comparison of the relative levels of compounds in cancer containing vs. non-cancer containing biopsies showed differences for 83 of the compounds. A comparison of the results with prior published reports showed good agreement between the current method and prior reported biomarker discovery methods that involve tissue destructive methods. CONCLUSIONS: The Molecular Preservation by Extraction and Fixation (mPREF) method allows for the analysis of small molecule biomarkers from exactly the same tissue that is processed for histopathology.
BACKGROUND: Histopathology is the standard method for cancer diagnosis and grading to assess aggressiveness in clinical biopsies. Molecular biomarkers have also been described that are associated with cancer aggressiveness, however, the portion of tissue analyzed is often processed in a manner that is destructive to the tissue. We present here a new method for performing analysis of small molecule biomarkers and histology in exactly the same biopsy tissue. METHODS: Prostate needle biopsies were taken from surgical prostatectomy specimens and first fixed, each in a separate vial, in 2.5 ml of 80% methanol:water. The biopsies were fixed for 24 hrs at room temperature and then removed and post-processed using a non-formalin-based fixative (UMFIX), embedded, and analyzed by hematoxylin and eosin (H&E) and by immunohistochemical (IHC) staining. The retained alcohol pre-fixative was analyzed for small molecule biomarkers by mass spectrometry. RESULTS:H&E analysis was successful following the pre-fixation in 80% methanol. The presence or absence of tumor could be readily determined for all 96 biopsies analyzed. A subset of biopsy sections was analyzed by IHC, and cancerous and non-cancerous regions could be readily visualized by PIN4 staining. To demonstrate the suitability for analysis of small molecule biomarkers, 28 of the alcohol extracts were analyzed using a mass spectrometry-based metabolomics platform. All extracts tested yielded successful metabolite profiles. 260 named biochemical compounds were detected in the alcohol extracts. A comparison of the relative levels of compounds in cancer containing vs. non-cancer containing biopsies showed differences for 83 of the compounds. A comparison of the results with prior published reports showed good agreement between the current method and prior reported biomarker discovery methods that involve tissue destructive methods. CONCLUSIONS: The Molecular Preservation by Extraction and Fixation (mPREF) method allows for the analysis of small molecule biomarkers from exactly the same tissue that is processed for histopathology.
Authors: John W Gillespie; Carolyn J M Best; Verena E Bichsel; Kristina A Cole; Susan F Greenhut; Stephen M Hewitt; Mamoun Ahram; Yvonne B Gathright; Maria J Merino; Robert L Strausberg; Jonathan I Epstein; Stanley R Hamilton; Gallya Gannot; Galina V Baibakova; Valerie S Calvert; Michael J Flaig; Rodrigo F Chuaqui; Judi C Herring; John Pfeifer; Emmanuel F Petricoin; W Marston Linehan; Paul H Duray; G Steven Bova; Michael R Emmert-Buck Journal: Am J Pathol Date: 2002-02 Impact factor: 4.307
Authors: Martine J Piccart-Gebhart; Marion Procter; Brian Leyland-Jones; Aron Goldhirsch; Michael Untch; Ian Smith; Luca Gianni; Jose Baselga; Richard Bell; Christian Jackisch; David Cameron; Mitch Dowsett; Carlos H Barrios; Günther Steger; Chiun-Shen Huang; Michael Andersson; Moshe Inbar; Mikhail Lichinitser; István Láng; Ulrike Nitz; Hiroji Iwata; Christoph Thomssen; Caroline Lohrisch; Thomas M Suter; Josef Rüschoff; Tamás Suto; Victoria Greatorex; Carol Ward; Carolyn Straehle; Eleanor McFadden; M Stella Dolci; Richard D Gelber Journal: N Engl J Med Date: 2005-10-20 Impact factor: 91.245
Authors: Melissa K Tuck; Daniel W Chan; David Chia; Andrew K Godwin; William E Grizzle; Karl E Krueger; William Rom; Martin Sanda; Lynn Sorbara; Sanford Stass; Wendy Wang; Dean E Brenner Journal: J Proteome Res Date: 2009-01 Impact factor: 4.466
Authors: Arun Sreekumar; Laila M Poisson; Thekkelnaycke M Rajendiran; Amjad P Khan; Qi Cao; Jindan Yu; Bharathi Laxman; Rohit Mehra; Robert J Lonigro; Yong Li; Mukesh K Nyati; Aarif Ahsan; Shanker Kalyana-Sundaram; Bo Han; Xuhong Cao; Jaeman Byun; Gilbert S Omenn; Debashis Ghosh; Subramaniam Pennathur; Danny C Alexander; Alvin Berger; Jeffrey R Shuster; John T Wei; Sooryanarayana Varambally; Christopher Beecher; Arul M Chinnaiyan Journal: Nature Date: 2009-02-12 Impact factor: 49.962
Authors: Michael F Berger; Michael S Lawrence; Francesca Demichelis; Yotam Drier; Kristian Cibulskis; Andrey Y Sivachenko; Andrea Sboner; Raquel Esgueva; Dorothee Pflueger; Carrie Sougnez; Robert Onofrio; Scott L Carter; Kyung Park; Lukas Habegger; Lauren Ambrogio; Timothy Fennell; Melissa Parkin; Gordon Saksena; Douglas Voet; Alex H Ramos; Trevor J Pugh; Jane Wilkinson; Sheila Fisher; Wendy Winckler; Scott Mahan; Kristin Ardlie; Jennifer Baldwin; Jonathan W Simons; Naoki Kitabayashi; Theresa Y MacDonald; Philip W Kantoff; Lynda Chin; Stacey B Gabriel; Mark B Gerstein; Todd R Golub; Matthew Meyerson; Ashutosh Tewari; Eric S Lander; Gad Getz; Mark A Rubin; Levi A Garraway Journal: Nature Date: 2011-02-10 Impact factor: 49.962
Authors: P Eschwège; S Moutereau; S Droupy; R Douard; J-L Gala; G Benoit; M Conti; P Manivet; S Loric Journal: Br J Cancer Date: 2009-02-24 Impact factor: 7.640
Authors: Tzu-Fang Lou; Deepa Sethuraman; Patrick Dospoy; Pallevi Srivastva; Hyun Seok Kim; Joongsoo Kim; Xiaotu Ma; Pei-Hsuan Chen; Kenneth E Huffman; Robin E Frink; Jill E Larsen; Cheryl Lewis; Sang-Won Um; Duk-Hwan Kim; Jung-Mo Ahn; Ralph J DeBerardinis; Michael A White; John D Minna; Hyuntae Yoo Journal: Cancer Prev Res (Phila) Date: 2015-10-28
Authors: Rachel S Kelly; Matthew G Vander Heiden; Edward Giovannucci; Lorelei A Mucci Journal: Cancer Epidemiol Biomarkers Prev Date: 2016-04-06 Impact factor: 4.254
Authors: Meredith V Brown; Jonathan E McDunn; Philip R Gunst; Elizabeth M Smith; Michael V Milburn; Dean A Troyer; Kay A Lawton Journal: Genome Med Date: 2012-04-30 Impact factor: 11.117
Authors: Dong Lin; Susan L Ettinger; Sifeng Qu; Hui Xue; Noushin Nabavi; Stephen Yiu Chuen Choi; Robert H Bell; Fan Mo; Anne M Haegert; Peter W Gout; Neil Fleshner; Martin E Gleave; Michael Pollak; Colin C Collins; Yuzhuo Wang Journal: Oncotarget Date: 2017-04-18
Authors: Annika Kurreck; Lindsey A Vandergrift; Taylor L Fuss; Piet Habbel; Nathalie Y R Agar; Leo L Cheng Journal: Prostate Cancer Prostatic Dis Date: 2017-12-05 Impact factor: 5.554
Authors: Maria K Andersen; Sebastian Krossa; Therese S Høiem; Rebecca Buchholz; Britt S R Claes; Benjamin Balluff; Shane R Ellis; Elin Richardsen; Helena Bertilsson; Ron M A Heeren; Tone F Bathen; Uwe Karst; Guro F Giskeødegård; May-Britt Tessem Journal: Anal Chem Date: 2020-01-28 Impact factor: 6.986