Background The 2010 WHO classification recommends that pancreatic neuroendocrine tumors should be graded based on mitotic rate and Ki67 index, with grade 2 (G2) pancreatic neuroendocrine tumor (PanNET) defined as having a mitotic rate of 2–20 mitotic figures/10 high power fields (HPF) or a Ki67 index of 3–20%. Grade 3 (G3) pancreatic neuroendocrine carcinoma (NEC) is defined as having >20 mitotic figures/10 HPF or a Ki67 index of >20%. However, some PanNETs show discordance between the mitotic rate and Ki67 index, usually having a Ki67 index in the G3 range but a mitotic rate suggesting G2, prompting us to examine the clinical significance of the Ki67 index in a large series of clinically well characterized mitotic G2 PanNETs. Design Mitotic G2 well differentiated PanNETs, surgically resected at our institutions were reviewed. Of those, 19 cases had a Ki67 >20% and were selected as the study group of grade-discordant (mitotic count G2/Ki67 index G3) PanNETs. For comparison, 53 grade-concordant (both mitotic count and Ki67 index G2) PanNETs matched for presenting stage with the discordant group as well as 43 morphologically poorly differentiated (either small cell or large cell type) pancreatic NECs were also included. The percentage of Ki67 positive neoplastic cells was quantified by manual counting of at least 500 cells on printed photographic images of “hot spots”. Results The mean Ki67 index for grade-concordant and grade-discordant PanNETs and poorly differentiated NECs were 8.1% (range, 3–20), 40% (range, 24–80%) and 70% (range, 40–98), respectively. Overall, patients with grade-discordant PanNETs had significantly longer survival time compared to the patients with poorly differentiated NEC (median survival of 54.1 months vs 11 months and 5-year survival of 29.1% vs 16.1%; p=0.002). Also, the survival time of the patients with grade-discordant PanNETs was shorter than that of the patients with grade-concordant PanNETs (median survival of 67.8 months and 5-year survival of 62.4%); however, the difference was not statistically significant (p=0.2). Conclusion Our data support the notion that the mitotic rate and Ki67 index-based grades of PanNETs can be discordant, and when the Ki67 index indicates G3, the clinical outcome is slightly worse. More importantly, we demonstrate that well differentiated PanNETs that are G3 by Ki67 are significantly less aggressive than bona fide poorly differentiated NECs, suggesting that the current WHO G3 category is heterogeneous, contains two distinct neoplasms, and can be further separated into well differentiated PanNET with an elevated proliferation rate and poorly differentiated NEC.

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.

Triple Negative Breast Cancers (TNBC) is a heterogeneous disease at the molecular and clinical level with poor outcome. Molecular subclassification of TNBCs is essential for optimal use of current therapies and for development of new drugs. microRNAs (miRNA) are widely recognized as key players in cancer progression and drug resistance; investigation of their involvement in a TNBC cohort may reveal biomarkers for diagnosis and prognosis of TNBC. Here we stratified a large TNBC cohort into Core Basal (CB, EGFR and/or CK5, 6 positive) and five negative (5NP) if all markers are negative. We determined the complete miRNA expression profile and found a subset of miRNAs specifically deregulated in the two subclasses.We identified a 4-miRNA signature given by miR-155, miR-493, miR-30e and miR-27a expression levels, that allowed subdivision of TNBCs not only into CB and 5NP subgroups (sensitivity 0.75 and specificity 0.56; AUC=0.74) but also into high risk and low risk groups. We tested the diagnostic and prognostic performances of both the 5 IHC marker panel and the 4-miRNA expression signatures, which clearly identify worse outcome patients in the treated and untreated subcohorts. Both signatures have diagnostic and prognostic value, predicting outcomes of patient treatment with the two most commonly used chemotherapy regimens in TNBC: anthracycline or anthracycline plus taxanes. Further investigation of the patients' overall survival treated with these regimens show that regardless of IHC group subdivision, taxanes addition did not benefit patients, possibly due to miRNA driven taxanes resistance. TNBC subclassification based on the 5 IHC markers and on the miR-155, miR-493, miR-30e, miR-27a expression levels are powerful diagnostic tools. Treatment choice and new drug development should consider this new subtyping and miRNA expression signature in planning low toxicity, maximum efficacy therapies.

Although in this category repeat FNA is expected rather than excision, we suggest evaluation of all AUS/FLUS patients in multidisciplinary meetings to decide management and recommend follow-up of all patients with this diagnosis.

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