1. Evaluation of p16 expression in Gastrointestinal Stromal Tumors (GIST): A Tissue Microarray Study. V. Mambelli, F. Corini, A. D’Angelo, A. Braccischi, L. Diamanti, M. Del Vecchio, R. Taborro, R. Zamparese U.O. Anatomia Patologica Osp. C.G. Mazzoni Ascoli Piceno 1

2. Genetics alterations involving p16 gene have been shown to drive the patogenic development of GISTs, the most common mesenchymal tumour of the gastrointestinal tract. Losses on chromosome 14q and 22q are common in borderline tumors, while additional losses on chromosome 1p and 9p are overrepresented in malignant lesions. 2

3. w These losses often comprise 9p21 locus which contains the clusters of ARF/ INK4a gene. 9p21 locus 3

4. p16 is a tumor suppressor that inhibits cell cycling by arresting cell in G1 before entry into S phase. 4

5. Genetics alterations results in diminished p16 levels, related to proliferation and progression of cancer cells. 5 normal cell cancerous cell upregulation; downregulation

6. o  High throughput:  Uniform reaction conditions  Built-in controls  Economize use of reagents  Facilitates data recording and linking to clinical data Tissue Microarray Advantages Tissue MicroArrays (TMAs) are a relatively new innovation in pathology. TMAs represent an increasingly validated means of understanding the clinical impact of diagnostic-related, prognostic-related, and therapy-related markers.. 6

7. 7

8. The arrayed tissue cores are histologically guided samples from representative regions of standard formalin-fixed paraffin-embedded. 8

9. For TMA construction, tissue cylinders, 0.3 mm in diameter, were punched from the marked tumor regions of each donor tissue block. Tissue core punches were organized in four TMAs. 9

10. To overcome the problem of tissue variability, we replicated the TMAs by punching the donor tissue blocks at least three times. A B C A B C 10

11. To validate our TMA data, we compared the immunohistochemical staining results of p16 protein on whole tissue sections of 10 cases found completely concordance. 11 Full section core 1 core 2

12. The studied group consisted of 31 cases of GISTs (14 gastric tumors, 14 small and large bowel tumors, 1 peritoneal tumor, 1 retroperitoneal tumor and 1 duodenal tumor). We observed p16 protein expression in 19 of 31 GISTs (61.2%). x 20 x 40 x 20 x 40 12

13. Of 12 GISTs with loss of p16 expression, five GISTs (41.6%) were found to have worse prognosis (death for disease) x 20 x 40 13

14. negative A previuos study conducted on 284 cases of GISTs showed that a negative p16 expression is more likely to be associated with high-risk GISTs. positive A recent Norway study conducted on 434 cases of GISTs showed that a positive p16 phenotype is more likely to be associated with malignant GISTs 14

15. Our results Our results suggest that expression of p16 correlated significatly with favorable prognosis. However, we found strongly p16 expression in one GIST case with higher mitotic counts. Alternative changes in cell-cycle control could lead to a compensatory upregolation of p16. There was an excellent concordance of p16 evaluation between conventional sections and TMAs. This indicates that the TMA technology can be applied to high- throughput analysis of immunohistochemical expression studies. 15