Oral Cancer Genesis and Progression: DNA Near-Diploid Aneuploidization and Endoreduplication by High Resolution Flow Cytometry

Alessandra Donadini,Antonio Cavallero,Marco Scala,Sergio Gandolfo,Monica Pentenero,Alida Santelli,Davide Malacarne,Emanuela Di Nallo,Paola Mereu,Walter Giaretti,Roberto Navone,Mauro Truini,Massimo Maffei

doi:10.1155/2010/638014

Abstract

Oral potentially malignant lesions (OPMLs) with dysplasia and aneuploidy are thought to have a high risk of progression into oral squamous cell carcinomas (OSCCs). Non-dysplastic “oral distant fields” (ODFs), characterized by clinically normal appearing mucosa sited at a distance from co-existing OPMLs, and non-dysplastic OPMLs may also represent an early pre-cancerous state. ODFs, OPMLs without and with dysplasia and OSCCs were investigated by high resolution DNA content flow cytometry (FCM). ODFs and OPMLs without dysplasia were DNA aneuploid respectively in 7/82 (8.5%) and 25/109 (23%) cases. “True normal oral mucosa” and human lymphocytes from healthy donors were DNA diploid in all cases and were used as sex specific DNA diploid controls. Dysplastic OPMLs and OSCCs were DNA aneuploid in 12/26 (46%) and 12/13 (92%) cases. The DNA aneuploid sublines were characterized by the DNA Index (DI ≠ 1). Aneuploid sublines in ODFs and in non-dysplastic and dysplastic OPMLs were near-diploid (DI < 1.4) respectively in all, 2/3 and 1/3 of the cases. DNA aneuploid OSCCs, instead, were characterized prevalently by multiple aneuploid sublines (67%), which were commonly (57%) high-aneuploid (DI ≥ 1.4). DNA near-diploid aneuploid sublines in ODFs and OPMLs appear as early events of the oral carcinogenesis in agreement with the concept of field effect. Near-diploid aneuploidization is likely to reflect mechanisms of loss of symmetry in the chromosome mitotic division. High DNA aneuploid and multiple sublines in OPMLs with dysplasia and OSCCs suggest, instead, mechanisms of “endoreduplication” of diploid and near-diploid aneuploid cells and chromosomal loss. High resolution DNA FCM seems to enable the separation of subsequent progression steps of the oral carcinogenesis.

Highlights

Chromosomal instability (CIN) contributes together with gene mutations and epigenetic aberrations to cancer genesis and progression [11,20,25,28,47,51,53]
Several examples of DNA content high resolution flow cytometry (FCM) measurements using DAPI stained nuclei suspensions for 4 different oral lesions with the presence of DNA near-diploid aneuploid sublines are shown in Fig. 1a and b
These DI values could be clearly evaluated after measuring a mixed sample of lesion and control nuclei, which produced a relative increase of the DNA diploid control peak-height and a decrease of the DNA aneuploid lesion peak-height

Summary

Introduction

Chromosomal instability (CIN) contributes together with gene mutations and epigenetic aberrations to cancer genesis and progression [11,20,25,28,47,51,53]. A. Donadini et al / Oral cancer genesis and progression totic checkpoint, in particular, monitors microtubule attachment at kinetochores during mitosis and prevents cells with unaligned chromosomes from proceeding to anaphase by inhibiting the anaphase-promoting complex/cyclosome. Donadini et al / Oral cancer genesis and progression totic checkpoint, in particular, monitors microtubule attachment at kinetochores during mitosis and prevents cells with unaligned chromosomes from proceeding to anaphase by inhibiting the anaphase-promoting complex/cyclosome These CIN mechanisms can be leading to an imbalanced DNA content (DNA aneuploidy) in a cell and generate DNA aneuploid sublines, which may acquire a proliferative advantage with respect to the normal cells. This procedure has allowed to measure G0–G1 DNA diploid control nuclei with CV values commonly near 1% and to detect DNA at high resolution for neardiploid aneuploid sublines with slight DNA changes above/below the DNA diploid control, as for example a DNA increase in a OPML of 2.4%

Methods

Results

Conclusion