Immunohistochemical estimation of cell cycle phase in laryngeal neoplasia

Abstract

We previously developed an immunohistochemical method for estimating cell cycle state and phase in tissue samples, including biopsies that are too small for flow cytometry. We have used our technique to examine whether primary abnormalities of the cell cycle exist in laryngeal neoplasia. Antibodies against the markers of cell cycle entry, minichromosome maintenance protein-2 (Mcm-2) and Ki67, and putative markers of cell cycle phase, cyclin D1 (G1-phase), cyclin A (S-phase), cyclin B1 (G2-phase) and phosphohistone H3 (Mitosis) were applied to paraffin-embedded sections of normal larynx (n ¼ 8), laryngeal dysplasia (n ¼ 10) and laryngeal squamous cell carcinoma (n ¼ 10). Cells expressing each marker were determined as a percentage of total cells, termed the labelling index (LI), and as a percentage of Mcm-2-positive cells, termed the labelling fraction (LF). The frequency of coexpression of each putative phase marker was investigated by confocal microscopy. There was a correlation between Mcm-2 and Ki67 LIs (r ¼ 0.93) but Mcm-2 LIs were consistently higher. All cells expressing a phase marker coexpressed Mcm-2, whereas Ki67 was not expressed in a proportion of these cells. The putative phase markers showed little coexpression. Labelling index values increased on progression from normal larynx through laryngeal dysplasia to squamous cell carcinoma for Mcm-2 (P ¼ 0.001), Ki67 (P ¼ 0.0002), cyclin D1 (P ¼ 0.015), cyclin A (P ¼ 0.0001) and cyclin B1 (P ¼ 0.0004). There was no evidence of an increase in the LF for any phase marker. Immunohistochemistry can be used to estimate cell cycle state and phase in laryngeal biopsies. Our data argues against primary cell cycle phase abnormalities in laryngeal neoplasia.

Correlation between levels of expression of minichromosome maintenance proteins, Ki-67 proliferation antigen and metallothionein I/II in laryngeal squamous cell cancer

“…They found that MCM2 protein is expressed in the same cells as cyclins A and D1, and also in some cells expressing cyclin B1 and phosphorylated histone H3. This fact suggests that MCM2 proteins are present during all phases of cell cycle, but their strongest expression is observed in phase G1 and S (51). MT-I/II, similarly to Ki-67, are associated with cell proliferation process.…”

“…However, there is still a lack of analysis of MCM protein expression and the prognostic significance in LsCC. The importance of MCM2 protein as a prognostic marker in LSCC was described in two studies (27,51), whereas the significance of MCM3 and MCM7 in LSCC has not been reported so far. Expression of MCM7 was in turn evaluated in other cancers of head and neck area, e.g.…”

Expression of Minichromosome Maintenance MCM6 Protein in Meningiomas is Strongly Correlated With Histologic Grade and Clinical Outcome

“…32 Labeling index with anti-MCM antibodies is correlated with Ki-67 but is significantly higher. 9,46 Indeed, in contrast with Ki-67, MCM proteins are expressed in early G1 and are later downregulated when the cells adopt a terminally differentiated phenotype. 14,31 MCMs were shown to be useful markers of proliferation 5,7,[20][21][22]30 In the field of neuropathology, few studies have analyzed MCM3 and MCM7 in astrocytoma 15,16,49 and MCM2 in oligodendroglioma.…”

OPN/CD44v6 overexpression in laryngeal dysplasia and correlation with clinical outcome

“…The overexpression of the EGF receptor is also an early event in carcinogenesis (Rubin Grandis et al, 1998). Amplification and overexpression of the CCND1 gene, encoding cyclin D1 is a common late event in HNSCC formation (Michalides et al, 1995;Izzo et al, 1998;Chatrath et al, 2006). Our findings suggest that the upregulation of the OPN/CD44v6 axis is an additional early event during the progression of laryngeal dysplasia.…”