We report here a new method for the extraction of molecular biomarkers from human tissue that is non-destructive to the tissue and is compatible with downstream workflow including histopathology and immunohistochemical analysis. The method involves the simple immersion of the tissue in 80% methanol (aqueous) which both extracts the small molecules and fixes the tissue. The analysis of the tissue extracts has the potential to provide for identification of molecular biomarkers associated with any biological process in a wide array of different tissue types. The method is compatible with small tissue specimens such as are obtained by needle biopsies and therefore has potential utility for clinical practice.
Histology is the gold standard for classifying disease and assigning prognostic grade, particularly in oncology. The direct examination of the architecture of intact tissue sections by pathologists remains powerful in classifying disease, however the data generated is usually qualitative, or semi-quantitative and the methods for preparing histological sections can be limiting for some downstream analyses. Studies of precision in histology have shown that scoring is generally consistent, however, there is not always full agreement across different laboratories [43–46]. Analysis of molecular biomarkers provides for the possibility of quantitative measurements to augment histology in the characterization of disease. Innovations intended to improve biomarker detection must take into account several factors. Biomarkers originating from tissues have often proven difficult to discover in blood because of the dilutive factor. For example, the most frequently used biomarker for the early detection of prostate cancer, PSA, is not routinely seen in proteomics discovery studies in blood from prostate cancer patients because of its low levels. To increase the probability of biomarker discovery, the closer in proximity one can get to the diseased tissue, the more concentrated will be disease specific proteins, peptides, nucleic acids, and metabolites. The mPREF method described in this study addresses many of the issues for the discovery of biomarkers and their translation to clinical practice. It is compatible with the long established diagnostic and prognostic histology-based methods, and IHC and FISH can still be performed on the paraffin embedded tissue. The small molecule biomarkers appear to reflect meaningful biological differences amongst tumors, and assays for these classes of molecules can be developed that are cost effective, high throughput, and highly accurate.
The use of alcohols as tissue fixatives is not new to histology and has been recently described as an alternative to formaldehyde-based fixatives [8–10]. We have demonstrated that alcohol fixation of prostate biopsies using the mPREF method produces tissue histology slides that can be readily read for the presence or absence of cancer (Figure 2). In addition, IHC with the PIN4 cocktail stain demonstrated good resolution of basal cells and basal keratin in normal prostate glands. In prostatic adenocarcinoma, basal cells and basal keratin were absent while racemase was detected in the cytoplasm. Other IHC stains will need to be tested empirically to determine their compatibility with the mPREF method. It is recognized that any change of fixation procedure has the potential to change the appearance of the tissues and that a larger number of tissues will need to be examined before general utility can be validated. The drivers for any change in procedures are related to the strength of the utility of any new biomarkers for the classification of disease. It was not the purpose of this study to extensively validate the histology and staining characteristics of alcohol vs. formalin as a fixative for prostate biopsies. We recognize that most pathologists are trained using formalin fixed tissues, however, pathologists must also read cytology, hematopathology smears and cryosections, which are frequently fixed in aqueous alcohol. We anticipate the formation of a study group of interested pathologists to share slides and more formally evaluate this method's acceptability for diagnosis and grading of disease.
The primary goal of this work was to describe a new method for the analysis of molecular biomarkers from human tissue that allows for histopathology to be performed in the same exact tissue. In addition, although the numbers of samples analyzed by mass spectrometry was limited in number, the mPREF method produced data that is consistent with published reports for candidate biomarkers for prostate cancer. Metabolite candidate biomarkers previously identified using a tissue grinding method and reported to increase in the progression of prostate cancer  also showed increased levels with the mPREF method when comparing cancer vs. non-cancer. The levels of these candidate biomarkers were also increased in more aggressive non-organ-confined (T3) disease, vs. the less aggressive organ-confined (T2) disease (Figure 4A). Other compounds also previously reported to be increased in prostate cancer [28, 30, 39–41], were also observed to be increased in cancer containing biopsies using the mPREF method (Figure 4B). The relatively small numbers of prostate biopsy extracts analyzed for metabolite profiles in this study precluded an in depth statistical analysis, however, further experiments are planned to investigate these results in more detail.
In this study, 82 compounds were observed to increase in cancer vs. non-cancer biopsy extracts. Eighteen of the 20 common amino acids and a number of long chain fatty acids and phospholipids were increased. Analysis of the amino acids showed that the increase in the levels of these compounds is not directly related to the percent of tumor in the biopsy (Figure 3). It is possible that the metabolite levels have a degree of a zone effect showing an increase in amount in areas close to histopathologically confirmed tumor. The compounds with the highest fold increases observed (4-6 fold) were cysteine, dihomo-linoleate, docosapentaenoate, N-acetylaspartate, N-acetylglucosamine, uracil, xanthine, and 1-stearoyl-glycerophosphoinositol. It is difficult to know which of the compounds are increased as a result of a general higher metabolic rate in more rapidly dividing cells, and which, if any, compounds represent changes in metabolism specifically associated with the oncogenic process. With regard to the potential utility of these types of changes, it has been demonstrated that the expression of cell proliferation-related genes has prognostic value for cancer disease progression , and it will be of interest to determine in later experiments which of the metabolites may have similar utilities.
The polyamines have been well studied as biomarkers in oncology [30, 41, 42]. In cancers of non-prostatic origin, polyamines are increased, yet, as a differentiated function in the prostate, the polyamines decrease in prostate cancer as the tissue becomes de-differentiated. In this study, the polyamines were observed to be the lowest levels from the highest grade cancer (T3) containing biopsies (Figure 4C). This is consistent with polyamines decreasing in prostate cancer, however, the polyamines were higher in pT2 cancer containing biopsies as compared with non-cancer containing biopsies.
With the notable exception of genomic DNA-based analysis, the discovery and performance of molecular biomarkers is highly dependent upon clinical sampling, processing, and storage [48–50]. In this study, we describe the mPREF method for prostate needle biopsy specimens obtained from fresh prostate tissues following prostatectomy. The future translation of this work to clinically relevant studies will be to include biopsies obtained in vivo using TRUS guided trans-rectal biopsy procedures. The use of image guided small core needle biopsies is likely to continue to increase while excisional biopsies decrease in part because the latter are more invasive, can cause more scarring and deformation, and require use of the operating room, the most expensive location in a hospital . The use of core needle biopsies for molecular testing such as mutational analysis to identify therapeutic targets provides a new rationale both for initial diagnosis and for repeat core needle biopsies to monitor therapy. Examples include EGFR mutation analysis in lung cancer  and HER-2 in breast cancer . The mPREF method described in this study should be widely applicable to a variety of tissues. The approach described has no inherent limitations for tissue type or disease state. While providing a method for quantitative analysis of small molecules, it is also compatible with histology, mutational analysis, RNA expression, immunohistochemical analysis, and other in situ methods.