Kristina Schwamborn (Presenter)
Institute of Pathology, TU Munich
Authorship: Kristina Schwamborn (1)
(1) Institute of Pathology, TU Munich, Munich, Germany
Reliable markers for diagnostic purposes or markers that correlate with prognosis and therapeutic response are needed in Pathology day to day practice. Since matrix assisted laser desorption ionization (MALDI) imaging mass spectrometry (IMS) goes far beyond microscopy it is ideal for this endeavor. MALDI IMS can generate molecular maps of tissue sections that can elucidate the underlying biochemistry or provide information on how therapeutics or toxins influence the function or misfunction of an organ. Thus, it has the potential to overcome limitations of other approaches in the identification and routine diagnostic measurement of new marker molecules/profiles.
In Pathology day to day practice, reliable markers for diagnostic purposes or markers that correlate with disease severity as well as prognosis and therapeutic response are needed. MALDI IMS can measure hundreds of molecules at the cellular level with direct correlation to histological features. It generates molecular maps of tissue sections that can elucidate the underlying biochemistry. Thus, it has the potential to overcome limitations of other approaches in the identification and routine diagnostic measurement of new marker molecules/profiles. Prostate cancer (PCa) is the most common cancer in men in the United States. Currently, three major clinical challenges remain: overdiagnosis due to prostate specific antigen screening, the inability to distinguish between indolent and aggressive tumors, and drug resistance. One key to improve PCa diagnosis and therapy is the better understanding of its molecular basis. Tissue microarrays (TMA) were analyzed by MALDI Imaging Mass Spectrometry (IMS) to identify disease related markers by comparing benign prostatic hyperplasia (BPH), prostate cancer, hormone refractory prostate cancer (HRPCa), and prostate cancer metastases.
Four different TMAs comprising formalin fixed and paraffin embedded (FFPE) tissue cores from BPH (n=115), PCa (n=535), HRPCa (n=57), and metastases (n=46) were subjected to on-tissue tryptic digestion. Briefly, sections were mounted onto conductive glass slides and underwent paraffin removal (xylene and ethanol washes) as well as antigen retrieval (tris-EDTA buffer). On-tissue digestion was achieved by spotting trypsin onto the tissue in an array pattern using a Portrait 630 reagent multi-spotter. Following digestion, CHCA (10 mg/ml in 1:1 ACN/ 0.5% TFA) was spotted directly onto the array of tryptic spots. Samples were analyzed utilizing an UltrafleXtreme MALDI-TOF/TOF mass spectrometer. Additionally, MS/MS measurements of selected peptides were acquired. Data analysis was performed by using the ClinProTools 2.2 and FlexImaging 2.1 software.
On-tissue tryptic digestion of prostate tissue revealed on average hundreds of tryptic peptides in the mass range from m/z 600-3000. Supervised classification analysis comparing PCa and BPH resulted in a classifier composed of 25 peptides that could predict the class of cancer and benign samples achieving a sensitivity and specificity of 94.8% and 81.1%, respectively. Similarly, utilizing a different classifier, 94.5% of all hormone naïve and 70.4% of all hormone refractory PCa samples could be classified correctly. In total 73 peptides from 34 different proteins could be identified. For example, fatty acid binding protein, epidermal, glutathione S-transferase P and heat shock protein beta-1 were differentially expressed between prostate cancer and benign hyperplasia. Peptides significantly differentially expressed between primary PCa and metastases (p-value < 0.0001) were among others collagen alpha-1(I) chain, heterogeneous nuclear ribonucleoproteins A2/B1, histone H4 and H3, desmin, myosin, and tropomyosin. Immunhistochemical validation analyses utilizing an independent validation cohort with 158 PCa samples and 118 BPH could verify biglycan as significantly differentially expressed between PCa and BPH.
Conclusions & Discussion
In providing clinically relevant information that could be validated using immunohistochemistry, this study shows the potential of MALDI IMS in Pathology. Additionally, it could provide insight into the discovery of novel biomarkers for prostate cancer that can be used in day to day Pathology practice.
References & Acknowledgements:
IP Royalty: no
|Planning to mention or discuss specific products or technology of the company(ies) listed above:||