Cytomorphologic characteristics of next-generation sequencing–positive bile duct brushing specimens

Abstract

Cytology of bile duct brushings (BDBs) is a specific, but insensitive, test for malignancy. Next-generation sequencing (NGS) of BDBs has recently been shown to improve sensitivity. We analyzed the cytologic features of NGS-positive (NGS+) and NGS-negative (NGS-) BDBs and correlated the morphology with the presence of mutations. A total of 96 BDBs were analyzed for 29 cytologic features by 2 pathologists who were unaware of the original diagnosis and NGS results. Clinicopathologic follow-up was used to determine the patient outcomes (ie, benign, low-grade neoplasm, malignant [carcinoma/high-grade dysplasia]). We analyzed 74 NGS+ BDBs from 66 patients and 22 NGS- BDBs from 22 patients. During follow-up, 58 of 66 NGS+ patients (88%) had malignancy compared with 0% of NGS- patients (P < 0.001). Fewer than 50% of the malignant cases had been interpreted as malignant on cytology; however, 100% had demonstrated mutations using NGS. Within the NGS+ cases, 53% showed late mutations (TP53, SMAD4, and CDKN2A) supportive of a high-risk stricture. Significant morphologic differences were seen in the background appearance, presence of single cells, architectural disarray, nucleomegaly, anisonucleosis, irregular nuclear borders, increased nuclear/cytoplasmic ratio, nuclear hyperchromasia, nucleoli, abnormal groups, clusters, and/or single cells, and overall impression. Naked nuclei, nucleomegaly, anisonucleosis, and coarse chromatin were more common in BDBs with late mutations than in those with KRAS/GNAS (Kirsten rat sarcoma viral oncogene homolog/guanine nucleotide binding protein, α-stimulating complex locus) mutations only. Cytology had a sensitivity of 16% and a specificity of 100% for malignancy. In contrast, NGS had a sensitivity of 100% and a specificity of 73%. Late mutations were 100% specific for malignancy compared with mutations in KRAS/GNAS only, of which 69% were malignant. We found significant overlap in the cytomorphologic features between neoplastic and non-neoplastic BDBs, and more than one half of cancer cases had been interpreted as "nonmalignant" on cytology. NGS showing late mutations was 100% specific for malignancy. Adding genetic testing to BDB cytology would be a valuable ancillary test for the detection of malignancy, and reflex testing should be considered.