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Who really needs a cervical biopsy?
that, with the ThinPrep test, clinicians are instructed to place cells in a vial of preservative immediately after collection. Then, the samples are adhered to slides and stained for cytological analysis. “One of the things we had to test first was whether we could handle these samples,” says Karger. Pilot tests on HeLa and SiHa cells treated with the preservative and a commonly used combination of stains suggested that these chemicals would not interfere with the analysis of cervical specimens. Next, they studied patient samples. The cells in question were adhered to slides, stained, and categorized. Of
the two sets of cells with SDS and ran the proteins on 1D gels. The gels were Each year, in the U.S. alone, ~60 million cut into three sections, and the proteins women have cervical samples taken were in-gel digested. Peptides were for routine microscopic analysis, or a separated by reversed-phase LC and Pap test, during annual gynecological analyzed by MS and MS/MS with an exams. As many as 3 million of these ion trap/FT instrument. In total, 1011 women have abnormal test results, says proteins were identified. Of those, >150 Barry Karger at Northeastern Univerproteins were up-regulated ≥3-fold in sity. Typically, the next step for these HSIL samples compared with NILM patients is to have a biopsy performed samples, as determined by label-free to determine whether they have a lequantitation methods. sion that could lead to cervical cancer. The researchers selected ~50 of the In most cases, the biopsy results are up-regulated HSIL proteins, and a negative. Although these women are subset was cross-checked by immunorelieved to be cancer-free, an invasive, cytochemistry on an independent set of costly procedure is needclinical samples. Only a ed to get this news. subset could be exam(a) (b) To reduce the number ined with this method, of unnecessary biopsies because antibodies performed every year, were not available for Karger, Shiaw-Lin Wu, all of the candidates. In David Hanlon, and addition, some antibodcolleagues at Northies were finicky and eastern University and required a lot of optimiCytyc Corp. searched for zation. Currently, the protein biomarker canresearchers are studydidates that could distining all 50 proteins in guish high-grade cervical much smaller samples Now you see it; now you don’t. (a) An arrow points to a cluster of HSIL cells on cells that are likely to (~3000–6000 cells) with a ThinPrep slide. (b) Laser-capture microdissection specifically removed the HSIL progress to a cancerous selected reaction monicells for proteomic analysis. state from those that are toring (known as SRM), likely to remain benign. a specific and sensitive In this issue of JPR (pp 4256–4268), these samples, a group of nine that MS method. Karger says the final sethey report a proteomics workflow for were classified as having high-grade lected biomarkers will be validated by analyzing cells that already have been squamous intraepithelial lesions immunocytochemistry in larger cliniprocessed and adhered to a microscope (HSILs) and another group of nine that cal studies in the future. slide for the ThinPrep test (developed were classified as negative for intraepiIn this study, collaboration was esby Cytyc), which is the most widely thelial lesions or malignancy (NILM; sential, says Karger. “To really do this used version of the Pap test. Ultimately, normal) were selected for further study. type of study, you need people like the researchers plan to develop a speCells were removed from the slides with me who are analytical chemists, but cific and sensitive immunostaining test laser-capture microdissection. For each you also need clinicians,” he says. The to identify high-grade cervical cells. specimen, ~12,000 cells were analyzed. clinicians of the group were experts at Although a DNA test recently has Karger points out that working with tispreparing and classifying clinical cerbeen developed to detect the presence sue is a trade-off: biomarkers are likely vical samples. The analytical chemists of the human papillomavirus (HPV) in to be more concentrated in diseased were experts at conducting proteomics patients, it doesn’t indicate whether the tissue than in plasma, but tissue speciexperiments and analyzing the data. “If infection will lead to cervical cancer. mens often are available only in small you don’t have the correct samples, you Many women have HPV infections but quantities. “What we’re illustrating in might as well not do anything,” says never develop cancer. Thus, a need still this paper is that you can successfully Karger. “You’ve got to have the right diexists for better screening techniques, take a limited number of cells and reagnosis, and you have to work together. says Karger. ally do something with it,” he says. That’s why this project has been sucOne challenge in performing proteo To compare the proteomes of HSIL cessful.” mics experiments on cervical cells is and NILM cells, the investigators lysed —Katie Cottingham
4104 Journal of Proteome Research • Vol. 6, No. 11, 2007
© 2007 American Chemical Society
