Clinical Impact of Proteomics in Surgical Pathology of Solid Tumors
Maria J. Merino
National Cancer Institute
Proteomics is an emerging area of research of the post-genomic era that deals with
the global analysis of gene expression using a combination of techniques to resolve (high resolution
two-dimensional polyacrylamide gel electrophoresis) (2-D PAGE), identify (peptide sequencing by Edman
degradation, mass spectrometry, Western immunoblotting, etc), quantify (scanners, phosphorimager, etc)
and characterize proteins, as well as store them in data bases. Each one of these technologies can be
applied independently, although their impact can be maximized when used in concert to the study of
complex biological problems. Proteomic analysis is capable of identifying known proteins and discovering
new ones, including those unique to different subtypes of a tumor. We therefore consider this an
excellent technique to study the differences in cellular protein expression between normal and
pathological tissues using 2D-gel electrophoresis (2D-EP).
A major factor limiting the application of proteomics has been the difficulty in obtaining pure
populations of cells. We have performed manual microdissection and protein extraction for proteomic
analysis from frozen sections performed in tissues, with the purpose of studying specific populations of
benign and or tumor cell. We have specifically applied the combined techniques of proteomics and
microdissection in the study of both benign and malignant thyroid conditions using human tissue
Figure 1 - Papillary Carcinoma (Click image to enlarge)
Approximately 300-500 polypeptides were detected in each specimen over the ranges of isoelectric
points (pI): 4.8-7.5 and molecular weights (Mr): 4.1-66.5 Kd. A general increase in the
level of protein expression was noted in malignant versus normal thyroid tissue specimens (Normal <
FA/Goiter < PTC << FTC).
Some of the proteins identified were expressed in a common fashion in both benign
and malignant thyroid growths. However, there were important differences among the groups of tissues
studied with regard to protein expression profiles. Importantly, several mitochondrial oxidation enzymes
were expressed in both benign and malignant tumors, reflecting the need for higher nutrient and
O2 metabolic demands in dividing cells. The level of expression of these proteins was
considerably higher in FTC.
Validation of these molecules can be done by standard immunohistochemical analysis in fixed tissues.
This is a crucial step for confirmation of potential markers before their clinical application in
Therefore, identification of new proteins by means of proteomics, may lead to the development of new
markers which will allow their utilization not only in clinical diagnosis but eventually, in the
development of new therapeutic modalities, and perhaps for monitoring therapeutic treatments.
Application of Proteomics in cytology specimens
Figure 3 - IHC Expression of proteins identified by proteomics (Click image to enlarge)
Preliminary studies have been performed with 12 cytology specimens from thyroid, prostate and kidney
(11 FNAC and 4 thinPrep). All specimens were ethanol-fixed, and stained with either Diff-Quik (4),
Papanicolaou (4), and unstained (4). Cytology smears were prepared from each case and manually
microdissected. A comparison of protein concentration and protein profiles between stains and type of
cytologic material was performed. Microdissected cells were directly lysed in lysis buffer, and protein
concentration was determined. First-dimensional electrophoresis was carried out on a Immobiline IPG
DryStrip system using 3-10 pH nonlinear gradient. Second-dimensional electrophoresis was carried out
using SDS-PAGE gels 1-mm thickness. Following electrophoresis, gels were fixed and stained using a
silver staining kit and scanned. Scanned images were analyzed and compared.
This preliminary study suggest that cytologic specimens can be used for proteomics since
they permitted the detection of approximately 500 distinct proteins as visualized by silver staining.
Cells from the ethanol-fixed and unstained cytologic slides gave the best results as compared with
Papanicolaou or Diff-Quik. Papanicolaou and DQ showed either weakly detectable or undetectable spots in
2D gel. ThinPrep specimens can not be used in proteomic analysis due to the very small amount of
- Merino MJ, Roberts DD, Bryant B, Panizo A: Differences of protein expression in benign and malignant microdissected thyroid tissues. Lab Invest 82: (1) 117A-118A JAN 2002.
- Panizo A, Roberts D, Al-Barazi H, Roberts D., Merino MJ: Utilization of cytology smears and manual micro-dissection for proteomic analysis. Lab Invest 81: (1) 59A-59A Jan 2001.
- Petricoin EE, Paweletz CP, Liotta LA. Related Articles, Links Abstract Clinical applications of proteomics: proteomic pattern diagnostics. J Mammary Gland Biol Neoplasia. 2002 Oct; 7(4): 433-40. Review.
- Wulfkuhle JD, Sgroi DC, Krutzsch H, McLean K, McGarvey K, Knowlton M, Chen S, Shu H, Sahin A, Kurek R, Wallwiener D, Merino MJ, Petricoin EF 3rd, Zhao Y, Steeg PS. Related Articles, Links Abstract Proteomics of human breast ductal carcinoma in situ. Cancer Res. 2002 Nov 15; 62(22): 6740-9.
- Wu W, Hu W, Kavanagh JJ. Related Articles, Links Abstract Proteomics in cancer research. Int J Gynecol Cancer. 2002 Sep-Oct; 12(5): 409-23. Review.
- Clarke W, Zhang Z, Chan DW. Related Articles, Links Abstract The application of clinical proteomics to cancer and other diseases. Clin Chem Lab Med. 2003 Dec; 41(12): 1562-70.
- Petricoin EF, Liotta LA. Related Articles, Links No abstract Clinical proteomics: application at the bedside. Contrib Nephrol. 2004; 141: 93-103.
- Torres-Cabala CA, Bibbo M, Roberts DD, et al. Identification of new proteins in the spectrum of thyroid lesions and their application as possible tumor markers in cytology. Modern Pathol 16 (1): 374 JAN 2003