Tesis

Biopsi Cairan Saliva sebagai Alternatif Non-invasif untuk Deteksi Mutasi Gen pada Karsinoma Sel Skuamosa Rongga Mulut: Studi Pendahuluan = Saliva-Based Liquid Biopsy As A Non-Invasive Alternative For Gene Mutation Detection In Oral Squamous Cell Carcinoma: A Preliminary Study.

Latar Belakang : Pembedahan diikuti dengan terapi adjuvan merupakan strategi tatalaksana pada KSSRM. Angka rekurensi lokal dan/atau regional yang tinggi pascaterapi definitif merupakan isu nyata yang jika dideteksi sejak dini dapat ditangani dengan pembedahan penyelamatan (salvage surgery). Ketiadaan protokol surveilans yang baku menyulitkan deteksi dini rekurensi, terutama karena baku emas berupa biopsi jaringan sulit dilakukan. Keberadaan ctDNA di saliva pasien KSSRM berpotensi mencerminkan status tumor sehingga bisa menjadi pengganti biopsi jaringan untuk mengkonfirmasi ada/tidaknya rekurensi. Tujuan : Sebagai studi pendahuluan, penelitian ini bertujuan mencari pola varian genetik pada jaringan KSSRM, protokol teknis untuk isolasi dan deteksi varian genetik dari saliva pasien KSSRM, serta kesesuaian varian genetik antara kedua spesimen tersebut. Metode : Penelitian ini merupakan penelitian potong-lintang yang mengumpulkan profil genetik dari KSSRM menggunakan next-generation sequencing terhadap jaringan tumor dan saliva. Proses ekstraksi DNA dan sekuensing jaringan tumor dilakukan di Litbang RS Kanker Dharmais menggunakan kit targeted gene sequencing TSO500. Proses ekstraksi DNA dan sekuensing spesimen saliva dilakukan di Departemen Kimia FKUI. Analisis sekunder dilakukan di Litbang RSKD dan Departemen Kimia FKUI menggunakan berbagai perangkat lunak dan database bioinformatika. Hasil : Isolasi DNA dengan konsentrasi dan kemurnian yang memuaskan dari spesimen saliva berhasil dilakukan pada 10 sampel. Tujuh sampel menjalani NGS jaringan tumor. Varian genetik dari berbagai tumor suppressor gene mendominasi temuan, yaitu gen TP53, ATR, FANCA, FANCE serta BRCA2. Ditemukan juga mutasi somatik berulang dari gen PRKDC, MDC1, NF1 serta NOTCH4. Terdapat perbedaan pola frekuensi varian genetik antara penelitian ini dengan pola frekuensi varian genetik dari TCGA, namun beberapa gen menunjukkan frekuensi varian genetik yang sesuai, yaitu TP53, CCND1, PIK3CA, PTEN serta FGFR1. Kesimpulan : Perbedaan profil genomik ini dapat menggambarkan perbedaan inheren KSSRM pada populasi Indonesia, namun dapat juga disebabkan perbedaan teknis seperti metode sekuensing serta protokol bioinformatika yang digunakan.
Kata kunci: kanker sel skuamosa rongga mulut; next-generation sequencing; saliva; varian genetik; ekstraksi; DNA


Background: Surgery combined with adjuvant therapy is the standard management for oral squamous cell carcinoma (OSCC). However, high rates of local and regional recurrence following definitive treatment remains a major issue. While early detection of recurrence is crucial for the success of salvage surgery, the lack of standardized surveillance protocols and the invasive nature of gold standard tissue biopsy hinders timely intervention. Salivary circulating tumor DNA (ctDNA) has emerged as a promising biomarker that may reflect tumor status and be a non-invasive alternative to tissue biopsy for monitoring recurrence. Objective: As a preliminary study, this study aimed to characterize genetic variants profile in OSCC tissue, to establish technical protocols for the extraction and detection of genetic variants from saliva, and compare the concordance of genetic variants between these two specimens. Methods: This was a cross-sectional study collecting genomic profiles from OSCC patients using Next-Generation Sequencing (NGS) on both tumor tissue and saliva samples. DNA extraction and sequencing of tumor tissues were conducted at the National Cancer Center Dharmais using TSO500 targeted gene sequencing panel. Saliva DNA extraction and sequencing were performed at the Department of Chemistry, Faculty of Medicine Universitas Indonesia FMUI. Tertiary bioinformatics analysis was conducted across both institutions utilizing various bioinformatics software and genomic databases. Results: DNA extraction from saliva was successfully performed on 10 samples, yielding satisfactory concentration and purity, while 7 samples underwent NGS for tumor tissue. Significant genomic variants were dominated by findings in several tumor suppressor genes, including TP53, ATR, FANCA, FANCE, and BRCA2. Recurrent somatic mutations were also identified in PRKDC, MDC1, NF1, and NOTCH4. While the overall frequency of genetic variants in this study diverged from the TCGA (The Cancer Genome Atlas) data, several genes showed similar rate of mutations, namely TP53, CCND1, PIK3CA, PTEN, and FGFR1. Conclusion: The observed differences in genomic profiles may reflect inherent characteristics of OSCC within the Indonesian population. However, these variations could also be attributed to technical factors, such as differences in sequencing methods and the bioinformatics protocols employed.
Keywords: oral squamous cell carcinoma; next-generation sequencing; saliva; genetic variants; extraction; DNA