Discrimination of grade 2 and 3 cervical intraepithelial neoplasia by means of analysis of water soluble proteins recovered from cervical biopsies

Abstract

BackgroundCervical intraepithelial neoplasia (CIN) grades 2 and 3 are usually grouped and treated in the same way as "high grade", in spite of their different risk to cancer progression and spontaneous regression rates. CIN2-3 is usually diagnosed in formaldehyde-fixed paraffin embedded (FFPE) punch biopsies. This procedure virtually eliminates the availability of water-soluble proteins which could have diagnostic and prognostic value.AimTo investigate whether a water-soluble protein-saving biopsy processing method followed by a proteomic analysis of supernatant samples using LC-MS/MS (LTQ Orbitrap) can be used to distinguish between CIN2 and CIN3.MethodsFresh cervical punch biopsies from 20 women were incubated in RPMI1640 medium for 24 hours at 4°C for protein extraction and subsequently subjected to standard FFPE processing. P16 and Ki67-supported histologic consensus review CIN grade (CIN2, n = 10, CIN3, n = 10) was assessed by independent gynecological pathologists. The biopsy supernatants were depleted of 7 high abundance proteins prior to uni-dimensional LC-MS/MS analysis for protein identifications.ResultsThe age of the patients ranged from 25-40 years (median 29.7), and mean protein concentration was 0.81 mg/ml (range 0.55 - 1.14). After application of multistep identification criteria, 114 proteins were identified, including proteins like vimentin, actin, transthyretin, apolipoprotein A-1, Heat Shock protein beta 1, vitamin D binding protein and different cytokeratins. The identified proteins are annotated to metabolic processes (36%), signal transduction (27%), cell cycle processes (15%) and trafficking/transport (9%). Using binary logistic regression, Cytokeratin 2 was found to have the strongest independent discriminatory power resulting in 90% overall correct classification.Conclusions114 proteins were identified in supernatants from fresh cervical biopsies and many differed between CIN2 and 3. Cytokeratin 2 is the strongest discriminator with 90% overall correct classifications.

Updating the Natural History of Human Papillomavirus and Anogenital Cancers

“…In an attempt to remove observer bias, Uleberg KE et al . [37] examined the protein expression from supernatants of fresh cervical biopsies and found that several proteins could differentiate CIN2 from CIN3 lesions. Specifically, Cytokeratin 2 was the strongest discriminator, with 90% correct classification.…”

Comparative Proteomics of Dying and Surviving Cancer Cells Improves the Identification of Drug Targets and Sheds Light on Cell Life/Death Decisions

“…For example, cytokeratins immunostaining is often used to detect metastatic tumor cells (47). In a study by Uleberg et al, cytokeratin 2 had the strongest independent discriminatory power among other proteins identified by LC-MS/MS in cervical biopsies and could correctly classify grade 2 and 3 cervical intraepithelial neoplasia in 90% of cases (48). The coexpression of vimentin and keratin intermediate filaments in human breast cancer cells has been shown to lead to phenotypic interconversion and increased invasiveness of these cells (49).…”

Strategies for non-invasive management of high-grade cervical intraepithelial neoplasia

“…Low grade CIN is characterized mainly by cytokines of the Th-1 profile, whereas high-grade CIN and cervical carcinoma are characterized by Th-2 profile cytokines, indicating a shift from cell-mediated to humoral immune response with increasing CIN grade. (37,38) Finally, the cellular immune response seems to be further deregulated by regulatory T-cells. Increased frequencies and suppressive activity of these cells has been found in patients with high-grade CIN and cervical carcinoma, indicating an active downregulation of the immune response in more progressive disease.…”

“…Both HPV antibodies and cytokine profiles, as markers of the host immune response, have been associated with disease grade in CIN, making them potential diagnostic biomarkers. (38,50) The evidence on their value in disease prognosis is limited, however. One author found a significantly higher regression rate of CIN in patients with a stronger Th-1 cytokine response.…”

“…A total of 19 women were included in our study. The mean age was 31 years (range [25][26][27][28][29][30][31][32][33][34][35][36][37][38][39][40][41]. The mean interval between the initial colposcopy and the follow-up colposcopy with LEEP was 115 days (91-154 days).…”