Endocrinol Metab.  2021 Feb;36(1):123-133. 10.3803/EnM.2020.860.

Molecular Correlates and Nuclear Features of Encapsulated Follicular-Patterned Thyroid Neoplasms

• Jung CK
• Bychkov A
• Song DE
• Kim JH
• Zhu Y
• Liu Z
• Keelawat S
• Lai CR
• Hirokawa M
• Kameyama K
• Kakudo K

• KMID: 2513296
• DOI: http://doi.org/10.3803/EnM.2020.860

Abstract

Background
Assessing nuclear features is diagnostically challenging in the aspect of thyroid pathology. The aim of this study was to determine whether pathologists could distinguish BRAF-like and RAS-like nuclear features morphologically and identify morphological features to differentiate thyroid tumors with RAS-like mutations from encapsulated papillary thyroid carcinoma (PTC) with predominant follicular growth and BRAFV^600E mutation.
Methods
Representative whole slide images of 16 encapsulated thyroid tumors with predominant follicular growth were reviewed by 12 thyroid pathologists using a web browser-based image viewer. Total nuclear score was calculated from semi-quantitatively scored eight nuclear features. The molecular profile of RAS and BRAF genes was determined by Sanger sequencing.
Results
Total nuclear score ranging 0 to 24 could differentiate BRAF-like tumors from RAS-like tumors with a cut-off value of score 14. The interobserver agreement was the highest for the assessment of nuclear pseudoinclusions (NPIs) but the lowest for nuclear elongation and sickle-shaped nuclei. NPIs were found in tumors with BRAFV^600E mutation, but not in tumors with RAS-like mutations. Total nuclear scores were significantly higher for tumors with BRAFV^600E than for those with RAS-like mutations (P<0.001).
Conclusion
Our results suggest that NPIs and high nuclear scores have diagnostic utility as rule-in markers for differentiating PTC with BRAFV^600E mutation from benign or borderline follicular tumors with RAS-like mutations. Relaxation of rigid criteria for nuclear features resulted in an overdiagnosis of PTC. Immunostaining or molecular testing for BRAFV^600E mutation is a useful adjunct for cases with high nuclear scores to identify true PTC.

Keyword

Thyroid cancer, papillary; Proto-oncogene proteins B-raf; Genes, ras; Thyroid neoplasms; Mutation; Medical overuse; Pathologists; Observer variation

Figure

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  Fig. 1 Representative images of nuclear features of encapsulated follicular-patterned tumors. The right column shows high power views of the same tumor in the left column (H&E stain, left, ×10; right, ×1,000). (A) Case A01 with BRAFV600E shows fully developed nuclear features of papillary thyroid carcinoma, including nuclear pseudoinclusions (arrow). (B) In case A03 with NRASQ61R mutation, a low power view shows a mixed microfollicular and macrofollicular pattern. Microfollicular areas reveal nuclear enlargement, nuclear crowding/overlapping, nuclear elongation, irregular contour of nuclear membrane, nuclear grooves, and chromatin clearing. (C) Case A11 with NRASQ61R mutation shows small round dark and enlarged ovoid pale nuclei.

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  Fig. 2 (A, B) Distribution of total scores of eight histologic nuclear features assessed by 12 pathologists in 16 encapsulated follicular-patterned thyroid tumors. Total nuclear scores are significantly higher for cases with BRAFV600E than for those with RAS-like mutations.

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  Fig. 3 Heatmap illustrating variations of nuclear scores and molecular types among 12 pathologists (A–L) in 16 encapsulated follicular-patterned thyroid tumors. Each pathologist blinded to molecular data assessed whether tumors were BRAF-like (BL) or RAS-like (RL) based on their histologic features. The number in each cell means the sum of the grading score of eight nuclear histologic features. Values in the far-right column of the table mean the sum of the nuclear scores of 16 cases for each pathologist. All pathologists are listed in order according to their sum of nuclear scores. In the far-left column, three pathologists marked in blue had a tendency to give high scores and over-interpret histologic nuclear features whereas three pathologists marked in orange had a tendency to give low scores and underestimate degrees of nuclear changes.

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  Fig. 4 Association between the sum of total nuclear scores and correct answer rate for molecular type by six pathologists. Nuclear scores assessed by six pathologists were summed up to denote the sum of nuclear scores on the Y-axis on the left side of the graph. Of 16 encapsulated follicular-patterned thyroid tumors, two cases A16 and A01 had BRAFV600E mutation and the remaining 14 cases had RAS-like mutations. All six pathologists interpreted whether each tumor was BRAF-like or RAS-like based on its histologic features. They correctly answered molecular types for 10/16 cases (case A16, A01, A10, A14, A13, A11, A06, A05, A08, and A09).

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  Fig. 5 Heatmap illustrating the distribution of total score of each histologic feature assessed by six pathologists selected (D–I) for 16 encapsulated follicular-patterned thyroid tumors. The number in each cell means the sum of points grading with each histologic feature interpreted by six pathologists.

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  Fig. 6 Nuclear pseudoinclusions and nuclear vacuoles mimicking nuclear pseudoinclusions. (A) An intranuclear cytoplasmic pesudoinclusion found in case A01 contains a round eosinophilic intranuclear body well demarcated by a rim of dense nuclear membrane. The color and texture are the same as the surrounding cytoplasm. Mimics of nuclear pseudoinclusions in case 12 (B) and case 04 (C) showed pale chromatin and irregularly shaped empty-looking nuclear vacuoles that lacked color and texture of the cytoplasm (arrows). Nucleoli were found within the nuclear vacuoles (H&E stain, ×1,000).

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