Endometrium kanserlerinde P53, PTEM ve mikrosatellitesinin hastalığın prognuzu üzerine etkisinin incelenmesi
Investigation of the effects of P53, pten and microsatellite on the prognosis of the disease in endometrial cancers
Yazar: MUTLU ÖĞÜT
Danışman: PROF. DR. TEVFİK GÜVENAL ; DR. ÖĞR. ÜYESİ AYÇA TAN
Manisa Celal Bayar Üniversitesi / Tıp Fakültesi / Kadın Hastalıkları ve Doğum Ana Bilim Dalı
Derece: Tıpta Uzmanlık, 2021
Konu: Kadın Hastalıkları ve Doğum = Obstetrics and Gynecology
Dizin: Endometrial neoplazmlar = Endometrial neoplasms ; Endometriyum = Endometrium ; Genler-P53 = Genes-P53 ; Neoplazmlar = Neoplasms ; Prognoz = Prognosis

Özet:
Endometrium kanserlerinde PTEN, p53 ve mikrosatellit instabilitesi' in hastalığın prognozu üzerindeki etkisinin incelenmesi
AMAÇ: Endometrium kanserinin yönetiminde son zamanlarda histo-patolojik faktörlere moleküler analizler eklenerek prognoz hakkında daha doğru bilgiler edinilmeye başlanmıştır. 2013'te Genom Atlas Pro-jesinde endometrium kanser 4 alt grupta toplanmıştır. Bunlar i) POLE ultra-mutant, (ii) MSI hipermutated, (iii) copy-number low ve (iv) copy-number high olarak sınıflandırılmıştır. Biz de bu çalışmada tümör doku-sunda PTEN, p53 ve mikrosatellit instabilitesi' inin endometrium kanse-rindeki bilinen prognostik faktörler ve sağ kalım arasındaki ilişkiyi incele-dik.

GEREÇ VE YÖNTEM: Ocak 2016- Aralık 2019 yılları arasında Ce-lal Bayar Üniversitesi Hafsa Sultan Hastanesi Kadın Hastalıkları ve Do-ğum Anabilim Dalı onkoloji polikliniğindeki endometrium kanseri olgula-rının retrospektif dosya taraması yapılarak çalışma için 50 hasta belir-lenmiştir. Bu hastalara ait formalinle fikse parafin bloklardan pozitif yüklü elektrostatik lamlara 4 mikronluk kesitler alınarak 58°C'lik etüvde 4 saat bekletilmiştir. Her olguya ait lam barkodları hazırlanan kesitlere yapıştı-rılarak tam otomatik immunohistokimyasal boyama cihazına (Bench-mark Ultra automated IHC/ISH slide staining system, Ventana Medical Systems) yerleştirilmiştir. İmmünohistokimyasal boyama için cihaz ile uyumlu Ultraview Universal DAB Detection Kit ve EZ prep, Reaction Buffer Concentrate solüsyonları, Endogenous Biotin Blocking Kit, He-matoksilen ve Blue Reagent kullanılmıştır. Tüm bloklara PTEN, p53 ve mikrosatellit instabilite markerları (MLH1, MSH2, MSH6 ve PMS2) ve ilgili immunohistokimyasal boyaları uygulanmıştır. Tüm değerlendirme-ler standart ışık mikroskobunda yapılmıştır. PTEN (sitoplazmik bo-yanma) ve p53 (nükleer boyanma) için tümör hücrelerindeki boyanma yüzdesi skorlanmıştır. Mikrosatellit markerler olan MLH1, MSH2, MSH6 ve PMS2 için tümör hücrelerinde nükleer boyanma olup olmaması

değerlendirilmiştir. Markerlardan biriyle hiç boyanma olmaması kayıp olarak kabul edilmiştir. Dört markerden hiçbirinde kayıp yoksa mikrosa-tellit stabil, birinde kayıp varsa düşük mikrosatellit instabil ve iki ve daha fazlasında kayıp varsa yüksek mikrosatellit instabil olarak kabul edilmiş-tir.

BULGULAR: Çalışmamızda histolojik tiplere göre değerlendirildi-ğinde 43 (%86) hastanın endometrioid tip, 3 (%6) hastanın seröz ve 4 (%8) hastanın mikst tip olduğu tespit edilmiştir. Endometrioid tip olan hastaların arasında ise; grade 1 olan 15 (%30) hasta iken grade 2 olan 23(%46) hasta ve grade 3 olan 10 (%20) hasta bulunmaktadır. Histopa-tolojik değerlendirmede olguların 27 (%54) hastada myometrial invaz-yon, 13(%26) hastada servikal tutulum olduğu belirlenmiştir. Çalışma-mızdaki 10 (%20) hastada ise pelvik lenf nodu ya da pelvik-paraaortik lenf nodu tutulumu olduğu tespit edilmiştir. Bu hastalar arasından 6 has-tada pelvik lenf nodu, 1 hasta paraaortik lenf nodu ve 3 hasta hem pelvik hem de paraaortik lenf nodu tutulumu mevcuttur. 2 (4%) hastanın batın sitolojisi ise malign özelliktedir ve 12 (%24) hastada ise lenfovasküler tutulum olduğu görülmüştür. Adjuvan tedaviler değerlendirildiğinde 31 (%62) hastada, olguya adjuvan tedavi verilmiştir. Bu hastalar arasında 20 hasta radyoterapi, 12 (%60) hasta external, 8 (%40) hasta brakiterapi almıştır. Bu olguların arasında 11 (%22) hastaya kemoterapi ve radyo-terapi uygulanmıştır. Çalışmamızda hastaların %54'ünün p53 ve %28'in PTEN ile boyandığı, %30'unun ise MSI kayıp olduğu saptanmıştır. PTEN ile boyanan hastaların %2'sinin güçlü boyanma, %8'sinin orta ve %18'sinin zayıf boyandığını; p53 ile boyanan hastaların ise %8'i güçlü, %10'nun orta ve %36'sının zayıf boyandığı görülmüştür. MSI kayıpla-rına bakıldığında ise %28'sinin MLH1PMS2 ve %2'sinin ise kaybı ol-duğu görülmüştür

SONUÇ: Endometrium kanserinde immunohistokimyasal değerlen-dirme sonucu PTEN ile hastalığın evresi, lenf nodu ve servikal alan tu-tulumu arasında anlamlı ilişki bulunmuştur. MSI ile hastalığın evresi ara-sında anlamlı ilişki bulunmuştur. P53'ün prognostik faktörler ile anlamlı ilişki saptanmamıştır. Ancak kötü prognostik belirteçlerinde diğer

çalışılan immünohistokimyasal belirteçlere göre daha yüksek oranda ol-duğu görülmüştür.. Çalışmamızda hastalığın nüks ve sağ kalım ara-sında anlamlı bir ilişki saptanmamıştır.

Anahtar sözcükler: endometrium kanseri, p53, PTEN, Mikrosatellit ins-tabilites

Abstract:
Investigation of the effect of PTEN, p53 and microsatellite instability in endometrial cancers on the prognosis of the disease

AİM: Endometrial cancer is the most commonly diagnosed cancer in the female genital system. Adjuvant treatments are applied after surgical treatment in the recurrence and survival of endometrial cancer. Adjuvant treatments are now determined based on certain prognostic factors. However, the most important marker in determining these prognostic factors is the subgroup of the endometrial cancer. Determining the subg-roups may vary according to the pathologist examining them. Hence, this subjectivity in the subgroup identification process changes the adju-vant therapy after surgery. For this reason, the Genome Atlas Project was carried out in 2013 and it was aimed that there were genetic chan-ges in many cancers and that the subgroup was made according to this classification. In this project, endometrium cancer has been divided into four subgroups. These subgroups can be listed as i) POLE ultra-mutant, (ii) MSI hypermutated, (iii) copy-number low ve (iv) copy-number high. Based on this classification, the investigation of new prognostic factors has been directed. Therefore, in our study, it was planned to investigate the effects of PTEN, p53 and microsatellite instability on the prognosis of endometrial cancer.

MATERIAL AND METHOD: 50 patients were selected for the study by retrospective file scanning of endometrial cancer cases in the onco-logy policlinic of Celal Bayar University Hafsa Sultan Hospital, Depart-ment of Obstetrics and Gynecology between January 2016 and Decem-ber 2019. Then, slides containing sufficient tumor from the hysterectomy preparations of the patients included in the study were determined. 4 micron sections of each case were taken from formalin-fixed paraffin blocks onto positively charged electrostatic slides and kept in an oven at 58°C for 4 hours. Slide barcodes of each case were affixed to the

prepared sections and placed in a fully automated immunohistochemi-cal staining device (Benchmark Ultra automated IHC / ISH slide staining system, Ventana Medical Systems). Ultraview Universal DAB Detection Kit and EZ prep, Reaction Buffer Concentrate solutions, Endogenous Biotin Blocking Kit, Hematoxylin and Blue Reagent were used for immu-nohistochemical staining. PTEN, p53 and microsatellite markers (MLH1, MSH2, MSH6 and PMS2) and related immunohistochemical stains were applied to all blocks. All evaluations were made under a standard light microscope. Percent staining in tumor cells was scored for PTEN (cytoplasmic staining) and p53 (nuclear staining). The presence or ab-sence of nuclear staining in tumor cells was evaluated for microsatellite markers MLH1, MSH2, MSH6 and PMS2. No staining with one of the markers was considered lost. If none of the four markers are missing, microsatellite is considered as stable. If one of the four markers is mis-sing, microsatellite is acknowledged as low microsatellite is unstable, and if two or more are missing the microsatellite is acknowledged as high microsatellite is considered unstable.

RESULTS: In our findings, it was determined that 43 (86%) of the patients were of the endometrioid type, 3 (6%) patients were of serous and 4 (8%) patients were of the mixed type according to histological ty-pes. Among the patients with endometrioid type, there were 15 (30%) patients with grade 1, 23 (46%) patients with grade 2, and 10 (20%) pa-tients with grade 3. Histopathological evaluation revealed 27 (54%) pa-tients with myometrial invasion and 13 (26%) patients with cervical in-volvement. In the study, 10 (20%) patients diagnosed with pelvic lymph node or pelvic-paraaortic lymph node involvement. Among these pati-ents, 6 patients had pelvic lymph node involvement, 1 patient had para-aortic lymph node involvement, and 3 patients had both pelvic and pa-raaortic lymph node involvement. The abdominal cytology of 2 (4%) pa-tients was malignant, and lymphovascular involvement was observed in 12 (24%) patients. When adjuvant treatments were evaluated, adjuvant treatment was given to 31 (62%) patients. Of these patients, 20 patients

received radiotherapy, 12 (60%) patients received external, 8 (40%) pa-tients received brachytherapy. Of these cases, 11 (22%) patients recei-ved chemotherapy and radiotherapy.In our study, 54% of patients were stained with p53 and 28% with PTEN, 30% of patients new MSI loss. 2% of the patients stained with PTEN had strong, 8% had moderate and 18% had weak staining. It was observed that 8% of the patients stained with p53 had a strong, 10% moderate, and 36% weak staining. When MSI losses were analyzed it was found that 28% of patients had MLH1PMS2 and 2% had MSH2MSH6 loss.

CONCLUSION: In immunohistochemical study of endometrium can-cer, a significant relationship was found between PTEN and disease stage, lymph node and cervical area involvement. A significant relations-hip was found between MSI and disease stage. No significant relations-hip was found between P53 and prognostic factors. However, it was fo-und to be higher in poor prognostic markers compared to other studied immunohistochemical markers. If our patients included in our study are more specifically selected from an advanced stage of the disease and then the study is carried out, different results can be obtained. In our study, no significant relationship was found between disease recurrence and survival. The reason for this result is that the size of our patient group is insufficient and the determined duration for the study is limited. If the number of patients is increased or the time span is extended, different results can be obtained.

Keywords: endometrium Cancer, p53, PTEN, Microsatellite Instability