Utility of Proliferation Markers Ki-67 and PHH3 in Predicting Malignancy in Basaloid Salivary Gland Neoplasms: A Study Using Digital Image Analysis of Cytology Cell Blocks

Abstract

Introduction: Basaloid salivary gland neoplasms (BSNs) are notoriously difficult to classify in fine needle aspiration (FNA) specimens due to the morphologic overlap of benign and malignant entities. Adenoid cystic carcinoma (AdCC) represents a particular diagnostic challenge, as it typically shows low-grade cytologic features despite its aggressive clinical behavior. We examined whether the proliferation markers Ki-67 and PHH3 could help predict malignancy in BSNs.

Methods: A retrospective search was conducted to identify FNA cases of BSNs that had adequate tumor cellularity in the cell block and a subsequent excision specimen. Ki-67 and PHH3 immunohistochemical stains were performed. Aperio (Leica Biosystems) was used to calculate the percentage of tumor cell nuclear expression. Proliferation scores and final histopathologic diagnoses were correlated using a two-sided p-value test.

Results: Ten benign and 14 malignant basaloid neoplasms were analyzed. Benign cases showed low mean percentages of tumor cell staining for Ki-67 (1.14%) and PHH3 (0.84%), while malignant cases showed significantly higher mean percentages, especially with Ki-67 (19% for low-grade malignancies and 25.5% for high-grade malignancies). The difference in proliferation marker scores between the benign and low-grade malignant cases showed statistical significance for both Ki-67 (p = 0.0041) and PHH3 (p = 0.00397). The difference between benign entities and AdCC was also statistically significant for Ki-67 (p = 0.0013) and PHH3 (p = 0.002).

Conclusion: Ki-67 and PHH3 analysis in cell block material may help predict malignancy in a cytologic specimen from a BSN, offering a valuable ancillary tool for cases with cytomorphologic ambiguity. In particular, the ability to suggest a sample is more likely to be AdCC rather than another morphologically similar low-grade BSN would be helpful for surgical planning.

Keywords: basaloid; cytopathology; digital image analysis; fine‐needle aspiration; immunohistochemistry; proliferation markers; salivary gland neoplasms.

FIGURE 1

Examples of regions excluded from digital analysis. (A) Non‐neoplastic salivary tissue (shown) in a cell block which also contained fragments of pleomorphic adenoma (Ki‐67, 20×). (B) Cell block from case of high grade myoepithelial carcinoma with extensive necrosis (Ki‐67, 20×). Note that the regions circled for analysis include the intact tumor cell fragments and exclude the dispersed tumor cells within the necrotic background. Ki‐67 proliferation index for this case was 45.83% (8196 total cells counted). [Color figure can be viewed at wileyonlinelibrary.com]

FIGURE 2

Digital analysis of case of basal cell adenoma. (A) Small tumor fragment in the cell block (H&E, 20×). (B) Tumor fragment circled prior to digital analysis with one tumor cell showing nuclear positivity of moderate intensity (Ki‐67, 20×) (C) Aperio algorithm identified the one positive (2+, indicated by orange color) tumor cell in this tumor fragment (Ki‐67, 20×). The total Ki‐67 index was 0.32%, with 934 total cells counted on the slide. PHH3 stain appeared similar. [Color figure can be viewed at wileyonlinelibrary.com]

FIGURE 3

Digital analysis of case of adenoid cystic carcinoma with tubular and cribriform patterns. (A) Tumor fragments in cell block (H&E, 20×). Same region stained with PHH3 (B) and Ki‐67 (C and D, pre‐ and post‐digital analysis, respectively). Proliferation indices were 1.39% with PHH3 (4541 total cells counted) and 27.14% with Ki‐67 (7162 total cells counted). [Color figure can be viewed at wileyonlinelibrary.com]

FIGURE 4

Digital analysis of case of adenoid cystic carcinoma with predominantly solid pattern. Direct smear (A, Diff‐Quik stain, 20×) and cell block (B, H&E, 20×) showing cellular basaloid neoplasm. Same tumor fragment in the cell block stained with PHH3 (C, D) and Ki‐67 (E, F), pre‐ and post‐digital analysis, respectively. Proliferation indices were 10.17% with PHH3 (13,836 total cells counted) and 29.93% with Ki‐67 (11,282 total cells counted). [Color figure can be viewed at wileyonlinelibrary.com]

FIGURE 5

Proliferation marker expression in benign vs. malignant basaloid neoplasms.

FIGURE 6

Proliferation marker expression in benign basaloid neoplasms compared to adenoid cystic carcinoma (excluding the one case with high‐grade transformation).