Disertasi

Analisis Ekspresi mRNA Sel Punca Kanker (CD133, CD44, SOX2) dan AR pada Circulating Tumor Cells Pasien Metastasis Adenokarsinoma Asinar Prostat Terkait Respons Terapi Deprivasi Androgen = Analysis of mRNA Expression of Cancer Stem Cells (CD133, CD44, SOX2) and AR in Circulating Tumor Cells of Patients with Metastatic Prostatic Acinar Adenocarcinoma Associated with Androgen Deprivation Therapy Response.

Androgen deprivation therapy (ADT) merupakan salah satu pilihan terapi bagi pasien penyandang adenokarsinoma prostat stadium lanjut. Namun, dalam waktu singkat sel tumor akan membentuk resistensi terhadap ADT melalui jalur ARdependen dan AR-independen. Salah satu jalur AR-independen adalah reaktivasi gen penanda sel punca seperti CD133, CD44, dan SOX2. Salah satu proses penting metastasis adalah circulating tumor cells (CTC) yang diperkirakan memiliki ekspresi gen penanda sel punca. Tujuan penelitian ini adalah untuk mengetahui efek ADT terhadap ekspresi CD133, CD44, SOX2 dan AR pada CTC terkait pengaruh CTC dan gen-gen tersebut pada patogenesis resistensi. Penelitian ini adalah penelitian prospektif analitik komparatif before-and-after pada pasien dengan adenokarsinoma prostat yang telah bermetastasis. Pasien yang memenuhi kriteria inklusi diminta persetujuan untuk ikut serta dalam penelitian. Lima belas mililiter darah pasien diambil sebelum dan sesudah tiga bulan pasca-ADT. Darah disentrifugasi untuk memisahkan sel-sel mononuklear, lalu dipilih dengan metode magnetic activated cell sorting (MACS, Miltenyi Biotech) untuk mendapatkan selsel dengan ekspresi CD45(-) dan EpCam(+) dan dianggap sebagai CTC. Jumlah CTC dihitung dan dilakukan ekstraksi mRNA total serta dinilai ekspresi gen CD133, CD44 SOX2 dan AR. Biopsi prostat pasien diproses dan dilakukan pulasan imunohistokimia untuk menilai ekspresi protein CD133, CD44, SOX2 dan AR menggunakan H-score. Terdapat 35 subjek yang menyelesaikan follow up berusia 50–86 tahun. Terdapat penurunan kadar PSA serum yang bermakna pasca-ADT (p < 0,001). Pasien dengan penurunan PSA ≥ 90% dikelompokkan dalam respons baik (n = 27) dan < 90% dengan respons buruk (n = 8). Tidak terdapat perbedaan jumlah CTC sebelum dan sesudah terapi, maupun antara kedua kelompok respons. Terdapat peningkatan ekspresi mRNA AR (p < 0,001) pada CTC kedua kelompok respons sebelum dan sesudah terapi. Tidak terdapat perbedaan bermakna antara ekspresi H-score di tumor primer di antara kedua kelompok respons. Terdapat korelasi lemah sedang ekspresi CD44 di jaringan tumor primer dan ekspresi mRNA CD44 di CTC pada kelompok respons baik. Jumlah CTC yang tetap tinggi meskipun kadar PSA serum menurun secara signifikan dapat mengindikasikan kegagalan ADT dalam mengeradikasi CTC yang mengekspresikan penanda sel punca. Ekspresi AR yang juga dipengaruhi ekspresi penanda SPK mengindikasikan peran kunci dalam mekanisme resistensi terhadap ADT.
Kata kunci: Adenokarsinoma Prostat Metastatik, CD133, CD44, SOX2, AR, Circulating Tumor Cells, Sel Punca Kanker, Terapi Deprivasi Androgen.


Androgen deprivation therapy (ADT) is a treatment of choice for patients with advanced stage of prostatic adenocarcinoma. However, within short period of time, the tumor cells will develop resistance against ADT via AR-dependent and ARindependent pathway. One of AR-independent pathway is the reactivation of stem cell markers, namely CD133, CD44, and SOX2. An important part of metastatic process is the circulating tumor cells (CTC) that probably expressed stem cells markers. This research was aimed to elucidate the effect of ADT toward the expression of CD133, CD44, SOX2, and AR in CTC to understand the effect of CTC and the investigated genes in pathogenesis of drug resistance. This was a prospective, comparative analytic before-and-after study in patients with metastatic prostatic adenocarcinoma. Patients who fulfilled the inclusion criteria were recruited to be included in the research. Fifteen milliliter of the patients’ blood was drawn, before and after three months of ADT, and then was centrifuged to separate mononuclear cells and sorted through magnetic activated cell sorting (MACS, Miltenyi Biotech) to acquire CD45 (-) and EpCam(+) cells and considered as circulating tumor cells (CTC). The CTC were counted and total mRNA were extracted and evaluated for gene expression of CD133, CD44, SOX2 dan AR. The patients’ biopsy were stained immunohistologically for CD133, CD44, SOX2 dan AR protein expression and to determine H-score. Thirty-five subjects completed the follow up, aged 50–86 years. There were significant decline in serum PSA levels post-ADT (p < 0.001). Patient with PSA level declined ≥ 90% were considered as good response (n = 27) and < 90% were considered as poor response (n = 8). There was no significant difference in CTC number changes before and after therapy, nor between both groups. There was significant increase in AR mRNA expression (p < 0.001) in CTC in poor response groups. There was no significant difference of Hscore expression in the primary tumor between both response groups. There was only mild-to-moderate correlation of CD44 expression in the primary tumor and mRNA CD44 expression in CTC in good response group. The number of CTC remained high even though serum PSA was significantly declining may reflect ADT failure in eradicating CTC that express stem cell markers. Increased AR expression, which also influenced by CSC marker expression, may indicate its key role in mechanism to ADT resistance.
Keywords: Androgen deprivation therapy, cancer stem cells, CD133, CD44, SOX2, AR, circulating tumor cells, metastatic prostatic adenocarcinoma.