Effect of Tumor Heterogeneity on the Assessment of Ki67 Labeling Index in Well-differentiated Neuroendocrine Tumors Metastatic to the Liver

The Ki67 labeling index is known to correlate with survival in patients with neuroendocrine tumors (NETs). A grading scheme recently endorsed by the World Health Organization for gastroenteropancreatic NETs classifies well-differentiated NETs into 2 categories based on the Ki67 labeling index: low (G1) and intermediate grades (G2). Tumor heterogeneity is a common finding in many tumors including NETs. Metastatic NETs to the liver are often diagnosed by radiographically guided needle core biopsy from which the Ki67 index is determined, which randomly samples the lesion without being targeted to regions that may show a higher proliferative rate. Whether the Ki67 index obtained from this type of limited material represents the whole tumor has been questioned. Forty-five surgically resected liver metastases of well-differentiated NETs were retrieved. A 9 core (3 core-triplets) tissue microarray (TMA) was constructed from the paraffin blocks of each tumor, each triplet considered to represent a virtual biopsy. Immunohistochemical staining for Ki67 was performed on TMA and whole slides, and the Ki67 labeling indices were determined by digital image analysis. Correlation of the Ki67 index with patient survival was analyzed. Forty-seven percent of cases showed intratumoral heterogeneity in Ki67 index that translated into discrepant grades among subsections on the whole slide. A similar trend was recapitulated on the virtual biopsies, although to a lesser degree. When the definitive grade of the tumor was based on the highest Ki67 index identified on the whole slide, the virtual biopsies perfectly predicted G1 cases (100%), but were much less accurate for G2 cases (47.8% with 3 biopsies and 34.8% with single biopsy). Accordingly, the predictive value for G1 on the virtual biopsies was low (64.7% and 59.5% for 3 and 1 biopsy, respectively), but was perfect for G2 (100%). By Kaplan-Meier survival analysis, there was a statistically significant difference between G1 and G2 in terms of overall survival, disease-free survival, and progression-free survival when graded on either whole-slide subsections or virtual biopsies. On the whole slides, the highest Ki67 grade showed a better correlation with overall survival than the mean Ki67 grade. In summary, by image analysis, we found that about half of the NETs metastatic to the liver show intratumoral heterogeneity resulting in discrepant Ki67 grade. In most cases, in particular G1, the virtual biopsy is representative of the whole slide, but for G2 the representation is <50%. Nevertheless, grades based on virtual biopsy had statistically significant prognostic values on patient survival, and there is no clear difference between the 3 and single virtual biopsy. Ki67 staining of core biopsies usually provides an adequately reliable method of proliferation assessment for prognosis of metastatic NETs to the liver, although the choice of treatment may be affected by intratumoral grade heterogeneity.