Ki67 index is now an essential part of classification of pancreatic neuroendocrine tumors. However, its adaptation into daily practice has been fraught with challenges related to counting methodology. In this study, three reviewers used four counting methodologies to calculate Ki67 index in 68 well-differentiated pancreatic neuroendocrine tumors: (1) ‘eye-ball’ estimation, which has been advocated as reliable and is widely used; (2) automated counting by image analyzer; (3) manual eye-counting (eye under a microscope without a grid); and (4) manual count of camera-captured/printed image. Pearson’s correlation (R) was used to measure pair-wise correlation among three reviewers using all four methodologies. Average level of agreement was calculated using mean of R values. The results showed that: (1) ‘eye-balling’ was least expensive and fastest (average time <1 min) but had poor reliability and reproducibility. (2) Automated count was the most expensive and least practical with major impact on turnaround time (limited by machine and personnel accessibility), and, more importantly, had inaccuracies in overcounting unwanted material. (3) Manual eye count had no additional cost, averaged 6 min, but proved impractical and poorly reproducible. (4) Camera-captured/printed image was most reliable, had highest reproducibility, but took longer than ‘eye-balling’. In conclusion, based on its comparatively low cost/benefit ratio and reproducibility, camera-captured/printed image appears to be the most practical for calculating Ki67 index. Although automated counting is generally advertised as the gold standard for index calculation, in this study it was not as accurate or cost-effective as camera-captured/printed image and was highly operator-dependent. ‘Eye-balling’ produces highly inaccurate and unreliable results, and is not recommended for routine use.

The diagnosis of low-grade adrenal cortical carcinoma (ACC) confined to the adrenal gland can be challenging. Although there are diagnostic and prognostic molecular tests for ACC, they remain largely unutilized. We examined the diagnostic and prognostic value of altered reticulin framework and the immunoprofile of biomarkers including IGF-2, proteins involved in cell proliferation and mitotic spindle regulation (Ki67, p53, BUB1B, HURP, NEK2), DNA damage repair (PBK, γ-H2AX), telomere regulation (DAX, ATRX), wnt-signaling pathway (beta-catenin) and PI3K signaling pathway (PTEN, phospho-mTOR) in a tissue microarray of 50 adenomas and 43 carcinomas that were characterized for angioinvasion as defined by strict criteria, Weiss score, and mitotic rate-based tumor grade. IGF-2 and proteins involved in cell proliferation and mitotic spindle regulation (Ki67, p53, BUB1B, HURP, NEK2), DNA damage proteins (PBK, γ-H2AX), regulators of telomeres (DAXX, ATRX), and beta-catenin revealed characteristic expression profiles enabling the distinction of carcinomas from adenomas. Not all biomarkers were informative in all carcinomas. IGF-2 was the most useful biomarker of malignancy irrespective of tumor grade and cytomorphologic features, as juxtanuclear Golgi-pattern IGF-2 reactivity optimized for high specificity was identified in up to 80% of carcinomas and in no adenomas. Loss rather than qualitative alterations of the reticulin framework yielded statistical difference between carcinoma and adenoma. Angioinvasion defined as tumor cells invading through a vessel wall and intravascular tumor cells admixed with thrombus proved to be the best prognostic parameter, predicting adverse outcome in the entire cohort as well as within low-grade ACCs. Low mitotic tumor grade, Weiss score, global loss of DAXX expression, and high phospho-mTOR expression correlated with disease-free survival, but Weiss score and biomarkers failed to predict adverse outcome in low-grade disease. Our results underscore the importance of careful morphologic assessment coupled with ancillary diagnostic and prognostic biomarkers of ACC.

von Hippel-Lindau (VHL) disease is a hereditary autosomal dominant disorder associated with deletions or mutations in the VHL tumor suppressor gene. Characteristically, up to 60 % of neuroendocrine tumors (NETs) associated with VHL disease display a spectrum of clear cell morphology including multivacuolated lipid-rich cell change. Unlike neurofibromatosis type 1 and multiple endocrine neoplasia type 1 syndromes, ampullary NETs have not been described in association with VHL disease. In this report, we discuss the features of an incidental ampullary clear cell NET occurring in a patient with pancreatic VHL disease including multiple pancreatic NETs. The ampullary lesion consisted of epithelial cells resembling lipoblasts or signet ring cells. In our case, all NETs showing clear cell change were positive for inhibin. While the underlying mechanism of this finding is largely unknown, it is of note that positivity for inhibin has not been observed in clear cell NETs associated with multiple endocrine neoplasia type 1 syndrome. Our case proves that NETs can develop in the ampullary region in patients with VHL; clear cell change can occur in these lesions and can mimic signet ring cell carcinoma. This issue is of clinical significance especially in small biopsy samples; thus, positivity for keratin alone should not be taken as evidence of an adenocarcinoma. Moreover, demonstration of inhibin expression in a NET with clear cell change along with other clinical stigmata should alert the diagnostician to the possibility of VHL disease. However, further larger series examining inhibin expression in both syndrome-related and sporadic clear cell NETs are needed to confirm our findings.

In this study, we compared the diagnostic value of TROP-2 expression in distinguishing between benign and malignant thyroid lesions to those of HBME-1, CK19 and galectin-3. We selected 102 cases from our archive including 20 normal thyroid tissues, 23 follicular nodular diseases, 17 follicular adenomas, 20 follicular variant papillary carcinomas and 22 classical variant papillary carcinomas. Tissue microarrays constructed from these cases were immunohistochemically analyzed with HBME-1, CK19, galectin-3 and TROP-2. Respectively 73.8%, 83.3%, 69% and 50% of all papillary carcinomas were positive with HBME-1, CK19, galectin-3 and TROP-2. CK19 was positive respectively by 100%, 43.5% and 35.3% in cases of normal thyroid, follicular nodular diseases and follicular adenoma, while the other markers were negative. In distinguishing benign and malignant lesions, which constitutes this study, HBME-1, CK19, galectin-3 and TROP-2 were statistically significant (p < 0.001). In distinguishing cases of follicular variant papillary carcinoma from follicular nodular diseases and follicular adenoma, HBME-1 and galectin-3 were statistically significant (p < 0.001). Consequently, in this study, we found that all immunohistochemical markers were effective in distinguishing benign and malignant thyroid lesions. In determining malignancy, HBME-1 had the highest diagnostic accuracy, while CK19 was the most sensitive marker. The sensitivity increased when the markers were used together.

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