Association of p16 Loss and EZH2 Overexpression with Clinicopathological Characteristics and Prognosis in Gastrointestinal Stromal Tumors

Öz

Abstract Aim: Gastrointestinal stromal tumors (GISTs) exhibit marked heterogeneous clinical behavior that is not fully captured by conventional risk classification systems. This study aimed to evaluate the prognostic significance of loss of p16 expression and EZH2 overexpression and their relationship with clinicopathological parameters in GIST. Methods: In this retrospective study, 113 GIST cases diagnosed at a single institution between 2016 and 2026 were evaluated. Expression of p16 and EZH2 was evaluated immunohistochemically and correlated with clinicopathological parameters and overall survival using Kaplan-Meier and Cox regression analyses. Results: p16 loss was detected in 34 cases (30.1%) and EZH2 overexpression in 12 cases (10.6%). Both markers were significantly correlated with high mitotic activity, tumor necrosis, metastasis, multifocality, a high AFIP risk category, and elevated Ki-67 proliferation index (p<0.001 for all). Patients with EZH2 overexpression and those with p16 loss had significantly shorter overall survival in Kaplan–Meier analysis (log-rank p=0.001 and p<0.001, respectively). EZH2 overexpression was significantly more frequent in p16-loss cases (26.5% vs 3.8%, p=0.001). In multivariate analysis, loss of p16 expression (HR=2.159, p=0.040) and AFIP risk category (HR=1.661, p<0.001) remained independent prognostic factors, whereas EZH2 overexpression did not remain independent prognostic factor (HR=1.001, p=0.999). Conclusions: Loss of p16 expression is an independent adverse prognostic factor in GIST. Although EZH2 overexpression is associated with agressive clinicopathological features and shorter overall survival, its prognostic effect is not independent of established pathological risk factors. The significant association between EZH2 overexpression and p16 loss supports the potential involvement of the EZH2/PRC2-CDKN2A epigenetic pathway in GIST progression and warrants further molecular investigation. Keywords: EZH2; Gastrointestinal stromal tumor; Immunohistochemistry; p16; Prognosis

Anahtar Kelimeler

• EZH2
• Gastrointestinal stromal tumor
• Immunohistochemistry
• p16
• Prognosis

Destekleyen Kurum

This research received no external funding.

Etik Beyan

This study was approved by the Çukurova University Faculty of Medicine Non-Interventional Clinical Research Ethics Committee (Approval No: [69], Date: [10/07/2026]). The study was conducted in accordance with the Declaration of Helsinki. Due to the retrospective nature of the study, the requirement for informed consent was waived by the Ethics Committee.

Gastrointestinal Stromal Tümörlerde p16 Ekspresyon Kaybı ve EZH2 Aşırı Ekspresyonunun Prognostik Önemi ve Klinikopatolojik Parametrelerle İlişkisi

Öz

Özet Amaç: Gastrointestinal stromal tümörler (GİST), geleneksel risk sınıflandırma sistemleriyle tam olarak açıklanamayan belirgin klinik heterojenlik gösteren tümörlerdir. Bu çalışmada, GİST'lerde p16 ekspresyon kaybı ve EZH2 aşırı ekspresyonunun prognostik önemi ile bu belirteçlerin klinikopatolojik parametrelerle ilişkilerinin değerlendirilmesi amaçlandı. Yöntem: Bu retrospektif çalışmada, tek bir merkezde 2016–2026 yılları arasında tanı alan 113 GİST olgusu değerlendirildi. p16 ve EZH2 ekspresyonları immünohistokimyasal olarak değerlendirildi ve klinikopatolojik parametreler ile genel sağkalım arasındaki ilişkileri Kaplan–Meier ve Cox regresyon analizleri kullanılarak incelendi. Bulgular: p16 ekspresyon kaybı 34 (%30,1) olguda, EZH2 aşırı ekspresyonu ise 12 (%10,6) olguda saptandı. Her iki belirteç de yüksek mitotik aktivite, tümör nekrozu, metastaz, multifokalite, yüksek AFIP risk kategorisi ve yüksek Ki-67 proliferasyon indeksi ile anlamlı olarak ilişkili bulundu (tümü için p<0,001). Kaplan–Meier analizinde, EZH2 aşırı ekspresyonu gösteren ve p16 ekspresyon kaybı bulunan olgularda genel sağkalımın anlamlı olarak daha kısa olduğu belirlendi (sırasıyla log-rank p=0,001 ve p<0,001). EZH2 aşırı ekspresyonu, p16 ekspresyon kaybı bulunan olgularda,p16 ekspresyonuna sahip olgulara kıyasla anlamlı derecede daha sık izlendi (%26,5'e karşı %3,8; p=0,001). Çok değişkenli analizde, p16 ekspresyon kaybı (HR=2,159; p=0,040) ve AFIP risk kategorisi (HR=1,661; p<0,001) bağımsız prognostik faktörler olarak belirlenirken, EZH2 aşırı ekspresyonunun bağımsız prognostik önemini korumadığı görüldü (HR=1,001; p=0,999). Sonuç: p16 ekspresyon kaybı, GİST'lerde bağımsız bir olumsuz prognostik faktördür. EZH2 aşırı ekspresyonu agresif klinikopatolojik özellikler ve daha kısa genel sağkalım ile ilişkili olmakla birlikte, yerleşik patolojik risk faktörlerinden bağımsız prognostik değer göstermemektedir. EZH2 aşırı ekspresyonu ile p16 ekspresyon kaybı arasında saptanan anlamlı ilişki, EZH2/PRC2–CDKN2A epigenetik yolunun GİST progresyonundaki olası rolünü desteklemekte olup, bu ilişkinin moleküler düzeyde daha ileri çalışmalarla araştırılmasını gerekli kılmaktadır. Anahtar Kelimeler: EZH2; Gastrointestinal stromal tümör; İmmünohistokimya; p16; Prognoz.

Anahtar Kelimeler

• EZH2
• Gastrointestinal stromal tümör
• immünohistokimya
• p16
• prognoz

Destekleyen Kurum

Bu çalışma için herhangi bir finansal destek alınmamıştır.

Etik Beyan

Bu çalışma, Çukurova Üniversitesi Tıp Fakültesi Girişimsel Olmayan Klinik Araştırmalar Etik Kurulu tarafından onaylanmıştır (Karar No: 69, Tarih: 10/07/2026). Çalışma Helsinki Bildirgesi ilkelerine uygun olarak yürütülmüştür. Çalışmanın retrospektif olması nedeniyle bilgilendirilmiş onam gerekliliği Etik Kurul tarafından kaldırılmıştır.

Kaynakça

1. WHO Classification of Tumours Editorial Board. Digestive system tumours. In WHO Classification of Tumours Series, 5th ed.; International Agency for Research on Cancer: Lyon, France, 2019; Vol. 1, pp. 1–435.
2. Joensuu H, Kindblom LG. Gastrointestinal stromal tumors–a review. Acta Orthop Scand Suppl. 2004;75(311):62-71. https://doi.org/10.1080/00016470410001708340
3. Miettinen M, Lasota J. Gastrointestinal stromal tumors: pathology and prognosis at different sites. Semin Diagn Pathol. 2006;23(2):70-83. doi: https://doi.org/10.1053/j.semdp.2006.09.001
4. Hirota S, Isozaki K, Moriyama Y, Hashimoto K, Nishida T, Ishiguro S, et al. Gain-of-function mutations of c-kit in human gastrointestinal stromal tumors. Science. 1998;279(5350):577-80. https://doi.org/10.1126/science.279.5350.577
5. Emile JF, Brahimi S, Coindre JM, Bringuier PP, Monges G, Samb P, et al. Frequencies of KIT and PDGFRA mutations in the MolecGIST prospective population-based study differ from those of advanced GISTs. Med Oncol. 2012;29(3):1765-72. doi: https://doi.org/10.1007/s12032-011-0074-y
6. Calabuig-Fariñas S, López-Guerrero JA, Llombart-Bosch A. The GIST paradigm: how to establish diagnostic and prognostic criteria. Arkhiv Patol. 2011;73(4):13-21.
7. Adil MT, Sagar J, Das P, Jain V. Gastrointestinal stromal tumours: a review on genetics, pathology, risk stratification, clinical characteristics, investigation, and treatment. EMJ Oncol. 2019;7(1):62-70.
8. Rossi S, Gasparotto D, Miceli R, Toffolatti L, Gallina G, Scaramel E, et al. KIT, PDGFRA, and BRAF mutational spectrum impacts on the natural history of imatinib-naive localized GIST: a population-based study. Am J Surg Pathol. 2015;39(7):922-30. https://doi.org/10.1097/PAS.0000000000000418

9. Fletcher CD, Berman JJ, Corless C, Gorstein F, Lasota J, Longley BJ, et al. Diagnosis of gastrointestinal stromal tumors: a consensus approach. Hum Pathol. 2002;33(5):459-65. https://doi.org/10.1053/hupa.2002.123545
10. Miettinen M, Lasota J. Gastrointestinal stromal tumors: review on morphology, molecular pathology, prognosis, and differential diagnosis. Arch Pathol Lab Med. 2006;130(10):1466-78. https://doi.org/10.5858/2006-130-1466-GSTROM
11. Joensuu H, Rutkowski P, Nishida T, Steigen SE, Brabec P, Plank L, et al. KIT and PDGFRA mutations and the risk of GI stromal tumor recurrence. J Clin Oncol. 2015;33(6):634-42. https://doi.org/10.1200/JCO.2014.57.4970
12. Demetri GD, von Mehren M, Blanke CD, Van den Abbeele AD, Eisenberg B, Roberts PJ, et al. Efficacy and safety of imatinib mesylate in advanced gastrointestinal stromal tumors. N Engl J Med. 2002;347(7):472-80. https://doi.org/10.1056/NEJMoa020461
13. Serrano M, Hannon GJ, Beach D. A new regulatory motif in cell-cycle control causing specific inhibition of cyclin D/CDK4. Nature. 1993;366(6456):704-7. https://doi.org/10.1038/366704a0
14. Ricci R, Arena V, Castri F, Martini M, Maggiano N, Murazio M, et al. Role of p16/INK4a in gastrointestinal stromal tumor progression. Am J Clin Pathol. 2004;122(1):35-43. https://doi.org/10.1309/MJ4X-N2M5-7HNC-8X5H
15. Haller F, Löbke C, Ruschhaupt M, Cameron S, Füzesi L, Sültmann H, et al. Loss of 9p leads to p16INK4A down-regulation and enables RB/E2F1-dependent cell cycle promotion in gastrointestinal stromal tumours (GISTs). J Pathol. 2008;215(3):253-62. https://doi.org/10.1002/path.2352
16. Haller F, Agaimy A, Cameron S, Beyer M, Gunawan B, Happel N, et al. Expression of p16INK4A in gastrointestinal stromal tumours (GISTs): two different forms exist that independently correlate with poor prognosis. Histopathology. 2010;56(3):305-18. https://doi.org/10.1111/j.1365-2559.2010.03478.x
17. Zong L, Chen P, Jiang J, Wang H, Wang L. Correlation between p16 expression and malignant risk of gastrointestinal stromal tumor: evidence from nine studies. Hepatogastroenterology. 2012;59(117):1458-1463. https://doi.org/10.5754/hge11473
18. Sooryanarayana V, Dhanasekaran SM, Zhou M, Barette S, Sanda MG, Ghosh D, et al. The polycomb group protein EZH2 is involved in progression of prostate cancer. Nature. 2002;419(6907):624-9. https://doi.org/10.1038/nature01075
19. Cao R, Wang L, Wang H, Xia L, Erdjument-Bromage H, Tempst P, et al. Role of histone H3 lysine 27 methylation in Polycomb-group silencing. Science. 2002;298(5595):1039-43. https://doi.org/10.1126/science.1076997
20. Simon JA, Kingston RE. Mechanisms of polycomb gene silencing: knowns and unknowns. Nat Rev Mol Cell Biol. 2009;10(10):697-708. https://doi.org/10.1038/nrm2763
21. Cao W, Ribeiro RO, Liu D, Saintigny P, Xia R, Xue Y, et al. EZH2 promotes malignant behaviors via cell cycle dysregulation and its mRNA level associates with prognosis of patient with non-small cell lung cancer. PLoS One. 2012;7(12):e52984. https://doi.org/10.1371/journal.pone.0052984
22. Bai J, Chen J, Ma M, Cai M, Xu F, Wang G, et al. Inhibiting enhancer of zeste homolog 2 promotes cellular senescence in gastric cancer cells SGC-7901 by activation of p21 and p16. DNA Cell Biol. 2014;33(6):337-44. https://doi.org/10.1089/dna.2014.2340
23. Lu F, Xu H, Wang Q, Li M, Meng J, Kuang Y. Inhibition of enhancer of zeste homolog 2 increases the expression of p16 and suppresses the proliferation and migration of ovarian carcinoma cells in vitro and in vivo. Oncol Lett. 2018;15(3):3233-39. https://doi.org/10.3892/ol.2017.7712
24. Jiang T, Wang Y, Zhou F, Gao G, Ren S, & Zhou C. Prognostic value of high EZH2 expression in patients with different types of cancer: a systematic review with meta-analysis. Oncotarget, 2015;7(4), 4584. https://doi.org/10.18632/oncotarget.6612
25. Schneider-Stock R, Boltze C, Lasota J, Miettinen M, Peters B, Pross M, Roessner A, Günther T. High prognostic value of p16INK4 alterations in gastrointestinal stromal tumors. J Clin Oncol. 2003;21(9):1688-97. https://doi.org/10.1200/JCO.2003.08.101
26. Huang HY, Huang WW, Lin CN, Eng HL, Li SH, Li CF, Lu D, Yu SC, Hsiung CY. Immunohistochemical expression of p16INK4A, Ki-67, and Mcm2 proteins in gastrointestinal stromal tumors: prognostic implications and correlations with risk stratification of NIH consensus criteria. Ann Surg Oncol. 2006;13(12):1633-44. https://doi.org/10.1245/s10434-006-9188-4
27. Sioulas AD, Vasilatou D, Pappa V, Dimitriadis G, Triantafyllou K. Epigenetics in gastrointestinal stromal tumors: clinical implications and potential therapeutic perspectives. Dig Dis Sci. 2013;58(11):3094-102. https://doi.org/10.1007/s10620-013-2785-8
28. Miao Z, Wu F, Wei H, Luo Z, Wu K, Zhang j. Enhancer of zeste homolog 2-mediated paired box 8 methylation promotes gastrointestinal stromal tumor progression through Wnt4 downregulation. Cancer Gene Ther. 2021;28(10):1162-74. https://doi.org/10.1038/s41417-020-00266-5
29. Ji Y, Xu X, Long C, Wang J, Ding L, Zheng Z, et al. SMYD2 aggravates gastrointestinal stromal tumor via upregulation of EZH2 and downregulation of TET1. Cell Death Discov. 2022;8(1):278. https://doi.org/10.1038/s41420-022-01038-w
30. Meseure D, Vacher S, Alsibai KD, Nicolas A, Chemlali W, Caly M, et al. Expression of ANRIL-polycomb complexes-CDKN2A/B/ARF genes in breast tumors: identification of a two-gene (EZH2/CBX7) signature with independent prognostic value. Mol Cancer Res. 2016;14(7):623-33. https://doi.org/10.1158/1541-7786.MCR-15-0418
31. Mitomi H, Fukui N, Kishimoto I, Tanabe S, Kikuchi S, Saito T, et al. Role for p16(INK4a) in progression of gastrointestinal stromal tumors of the stomach: alteration of p16(INK4a) network members. Hum Pathol. 2011;42(10):1505-13. https://doi.org/10.1016/j.humpath.2011.01.005
32. Yakovtsova II, Miroshnichenko YM, Chertenko TM. Immunohistochemical features of gastrointestinal stromal tumors and the role of expression of p16ink4A, Ki-67, VEGF and MMP-9 in their behavior. Patologìa. 2021;18(2):136-41. https://doi.org/10.14739/2310-1237.2021.2.233425
33. Zaki MM, Enan ET, Hany H. Immunohistochemical expression of p16, p53, BCL2, and cyclin D1 in gastrointestinal stromal tumors: correlation with clinicopathological parameters. Cureus. 2025;17(10):e93731. https://doi.org/10.7759/cureus.93731
34. Gounder M, Schöffski P, Jones RL, Agulnik M, Cote GM, Villalobos VM, et al. Tazemetostat in advanced epithelioid sarcoma with loss of INI1/SMARCB1: an international, open-label, phase 2 basket study. Lancet Oncol. 2020;21(11):1423-32. https://doi.org/10.1016/S1470-2045(20)30451-4