Aberrant expression of minichromosome maintenance protein-2 and Ki67 in laryngeal squamous epithelial lesions

Abstract

Histological classification of laryngeal epithelial lesions is highly subjective, and methods of cytological detection are not well developed. Improved determination of aberrant cell cycle entry may allow increased objectivity in histological assessment and enable the development of less invasive diagnostic cytology tests. Sections of normal larynx (n=10), laryngeal dysplasia (n=20) and laryngeal squamous cell carcinoma (SCC) (n=10) were classified according to the Ljubljana classification and stained for markers of cell cycle entry, minichromosome maintenance protein-2 (Mcm-2) and Ki67. Expression patterns were compared using double labelling confocal microscopy. There was a correlation between Mcm-2 and Ki67 labelling indices (rho=0.93; 95% CI [0.84, 0.97]) and both markers showed increased expression from normal epithelium to SCC (Mcm-2, P=0.001; Ki67, P=0.0002). Importantly, there was minimal expression of Mcm-2 or Ki67 in the most superficial layers of normal larynx and abnormal or atypical hyperplasia, in contrast to carcinoma in situ and SCC. Clusters of Mcm-2/5-positive cells were present in cytological preparations from SCC, but not from those showing atypical hyperplasia or inflammation in non-neoplastic tissue. Minichromosome maintenance protein-2 staining may increase the objectivity and reliability of histological grading of laryngeal epithelial lesions. Laryngeal brushings, combined with immuno-enhanced liquid-based cytology, could be useful, as a less invasive approach, to the detection of laryngeal malignant and premalignant lesions.

Figure 1

Immunohistochemical staining showing the distribution of Mcm-2 (middle panel) and Ki67 (right panel) in normal laryngeal squamous epithelium, abnormal hyperplasia, carcinoma in situ and dysplasia arising in squamous metaplasia affecting respiratory-type epithelium. The haematoxylin & eosin (H&E) appearances are given for comparison (left panels). All figures are at × 200 magnification. In carcinoma in situ, there is full-thickness staining for both markers, with clusters of immunopositive cells that appear to have sloughed away from the epithelial surface.

Figure 2

Box and whisker plots comparing Mcm-2 (A) and Ki67 (B) LIs in different compartments of normal laryngeal squamous epithelium and LD (atypical hyperplasia and carcinoma in situ combined). Control=normal larynx, bar=median value, box=upper to lower quartile ranges, whisker=2 × quartile range, asterisk and circle=extreme values.

Figure 3

(A): Correlation between Mcm-2 and Ki67 LIs for the entire epithelium in normal larynx (n=10), LD unaccompanied by invasive disease (n=10) and SCC (n=10) (ρ=0.93; 95% CI [0.84, 0.97]). (B) Comparison between Mcm-2 and Ki67 LIs for the entire epithelium in normal larynx (n=8), LD unaccompanied by invasive disease (n=10) and laryngeal SCC (n=10; median±IQR). Box=upper to lower quartile ranges; whisker=2 × quartile range; circles=extreme values.

Figure 4

Bar charts (median±IQR) comparing Mcm-2 LIs in different compartments of the laryngeal epithelium in normal larynx (n=8), atypical hyperplasia (n=10), carcinoma in situ (n=9). Substantial expression of Mcm-2 in the superficial epithelial third is only present in carcinoma in situ, although some increase in expression above normal is seen in atypical hyperplasia. Base=lower epithelial third, asterisk and circle=extreme values.

Figure 5

Double-labelling fluorescent confocal microscopy for Mcm-2 (green) and Ki67 (red) in carcinoma in situ. Many cells coexpress Mcm-2 and Ki67 (yellow), but there are also many cells showing Mcm-2-expression alone (green). Mitotic figures show chromatin exclusion of Mcm-2 (red staining) but the cytoplasm is positive for Mcm-2 (arrow). Some Mcm-2-expressing cells (green) are present in the superficial third whereas very few cells express Ki67 (yellow).

Figure 6

Normal squamous (A) and respiratory-type (B) epithelial cells are negative when stained with antibodies against Mcm-2/5. (C) A cluster of cells from a laryngeal SCC, which are strongly positive for Mcm-2/5 and were readily identified, even at low magnification.