Abstract 4532: A free and easy to use morphological biomarker to serve as an internal challenge in the study of biomarkers by systematic tissue microarray analysis
Authors: Dalton Leslie , Köhler Katharina , Poßögel Anne-Kathrin , Niendorf Axel 
Published: August 2018

Abstract
Background: Tissue microarrays (TMA) and digital microscopy (DM) are modern era innovations used here to assess a potential “free” internal control for TMA studies. Morphological nuclear grading can be performed on very few cells as is inherent with TMA samples. DM allows for ease in “shipment” of TMA slides for rapid central review by an experienced morphologist. The TMA used for this analysis are part of a larger initiative with a special focus on cases, whose conventional prognostic parameters (i.e. no special type histology [NST], estrogen receptor positive [ER+], Nottingham grade 2, nodal negative [N0]) indicate neither a good nor bad clinical course and thus constitute a so called gray zone (GZ).
Methods: We compared three grading systems based on the analysis of H&E-stained TMA. Assigned to 267 ER+, Nottingham grade 2 (GZ-ER+) tumors, and 97 triple negative (TN) tumors was a 3-9 score (nuclear grade sum score - NGSS) summed from 1-3 scores for each of nucleoli, size nuclei, and variation size/shape. The Helpap nucleolar (HN) component was analyzed separately as was a traditional Nottingham nuclear pleomorphism score (PS).
Results: A highly significant uneven distribution of NGSS was found between GZ-ER+ and TN tumors (p&lt;0.00001). 224 of 228 tumors (62% of all) NGSS &lt; 6 were GZ-ER+, while 52 of 54 tumors (14% of all) NGSS 8-9 were TN. Mid-range NGSS (6, 7) had 41 of 82 GZ-ER+ (22% of all were mid-range). There is a clear-cut inverse relationship between NGSS and ER+ vs. ER- cases: ER+ cases (n=267) have a distribution of 48, 129, 47, 21, 20, 2 and 0 for NGSS grades 3 through 9, whereas ER- cases (n=97) exhibit 0, 2, 2, 15, 26, 27 and 25 in these respective categories. Receiver operator curve analysis showed area under curve of 0.95 in the ability for the 7 category NGSS to predict TN cancer, 0.91 for PS and 0.84 for HN. HN and PS suffered from frequent intermediate grade assignment (respectively 40 and 45%). Interestingly HN had 46% grade 1 tumors of which only 9 of 167 were TN. 23% of tumors were assigned PS=1 and none were TN.
Conclusion: NGSS can easily be performed on the same tiny samples which are being used to study a novel biomarker. In the appropriate research setting, the challenge for a biomarker would be to offer significant improvement over what is evident by “old fashioned” morphology. Here NGSS highlighted the morphological contrast between poor prognosis TN cancer and more favorable prognosis GZ-ER+ tumors. This was even though GZ-ER+ tumors had originally been assigned intermediate Nottingham grade. Furthermore, the question has to be raised as to whether or not NGSS might serve as a way to adjust PS and thereby contribute to stratification within the gray zone. This would reclassify more tumors into low grade which would have important clinical implications.
Citation Format: Leslie Dalton, Katharina Köhler, Anne-Kathrin Poßögel, Axel Niendorf. A free and easy to use morphological biomarker to serve as an internal challenge in the study of biomarkers by systematic tissue microarray analysis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4532.