臺灣博碩士論文加值系統

造釉細胞瘤是顎骨最常見的齒源性上皮腫瘤，此腫瘤雖為良性，其特性卻相當具侵略性，容易復發，故目前的標準處置為大範圍切除，而導致大量組織缺損，影響口腔顏面的功能與美觀甚巨。然而最近幾年造釉細胞瘤之致病機轉有了突破性的發現，超過八成的造釉細胞瘤案例都有MAPK(FGFR-RAS-RAF) pathway的突變，其中BRAF(V600E)的突變占了六成，另外Sonic hedgehog pathway裡的SMO也被報導有一至三成的突變，由於在其他腫瘤已有使用MAPK pathway及Sonic hedgehog pathway相關的標靶藥物，且文獻中也有三個造釉細胞瘤使用MAPK標靶藥物的臨床案例，證明標靶藥物可能成為未來治療造釉細胞瘤的方法之一。
我們的研究致力於了解台灣造釉細胞瘤病患的MAPK(FGFR-RAS-RAF) pathway以及SMO之各種分子突變情形。我們共萃取了77個未脫鈣的造釉細胞瘤病理標本的DNA，針對FGFR2,KRAS,BRAF,SMO分子常見的基因突變區做DNA的定序，並針對最常見的BRAF(V600E)的偵測方法-定序及免疫組織化學染色，作進一步的分析。結果發現BRAF(V600E)的突變率高達78%，但沒有找到其他分子的突變。考量到在未來在臨床上標靶治療用藥前必須先檢測病人是否有BRAF(V600E)的突變，我們將BRAF的DNA定序結果當成標準來評估使用免疫組織化學染色偵測BRAF(V600E)的敏感度和特異度。結果發現敏感度為69%，特異度為33%。
由此實驗可了解到BRAF(V600E)的突變在台灣造釉細胞瘤病患的致病機轉當中扮演了重要角色。另外由於免疫組織化學染色的敏感度和特異度不佳，我們建議使用DNA定序來偵測造釉細胞瘤之BRAF(V600E)的突變。

Ameloblastoma is a benign neoplasm, and is the most prevalent amongst epithelial odontogenic neoplasm. Due to its locally aggressive behavior and the high risk of recurrence, surgical resection which results in facial deformity and significant morbidity is often needed in the treatment of ameloblastoma. However, in the past three years, several studies bring some insight into the molecular pathogenesis of ameloblastoma and the development of molecule-targeted therapy is foreseeable. Recent research have shown frequent MAPK (FGFR-RAS-RAF) pathway and SMO mutation in ameloblastomas, among which BRAF (V600E) mutation is the most common mutation. Because these MAPK pathway and SMO mutations have been identified in other neoplasms, the targeted therapy medications are available and have been used for treatment. Moreover, until now, there are already three case reports suggesting the efficacy of BRAF and MEK inhibitors in treating BRAF mutated ameloblastoma patients.
As the first step of using targeted therapy in the future, the aim of this study was to investigate the frequency of MAPK (FGFR-RAS-RAF) pathway and SMO mutations in various subtypes of ameloblastomas including follicular, plexiform and unicystic variants in Taiwan. Considering sequencing as gold standard to detect mutation, we also performed the BRAF (V600E) immunohistochemistry(IHC) with an antibody against BRAF (V600E)-mutated protein (clone VE1, Roche Molecular Diagnostics) that was proved well functioned in detecting other BRAF mutated neoplasms; then we compared the IHC results with BRAF (V600E) sequencing to evaluate the accuracy of using IHC in detecting BRAF mutation in ameloblastoma.
Seventy-seven non-decalcified ameloblastoma tissue sections were used for macro-dissection, DNA extraction and sequencing for examining the frequency of SMO FGFR2, KRAS, and BRAF mutations. BRAF (V600E) mutation status was further evaluated by BRAF (V600E) immunohistochemistry and the concordance between two methods was also examined. One dental follicle and one papillary thyroid carcinoma were used as negative and positive control, respectively.
In our results, sixty out of 77 ameloblastoma (78%) cases were BRAF (V600E) mutated. The mutation rate was 83.3% (25/30) for follicular type, 78% (15/19) for plexiform type, 76% (19/25) for unicystic type, 0/1 for desmoplastic type and 1/2 for granular cell type. No SMO, FGFR2 or KRAS mutation was identified in our cases. Notably, significant disconcordance between BRAF (V600E) sequencing and immunohistochemistry was observed in 48 ameloblastomas. The sensitivity and specificity of using BRAF (V600E) immunohistochemistry to detect the BRAF (V600E) mutation was 69% and 33%, respectively.
Our study showed that BRAF (V600E) mutation plays a predominant role in the pathogenesis of ameloblastoma in Taiwan. According to our results, we suggested using Sanger sequencing instead of IHC in detection of BRAF (V600E) mutation in ameloblastomas.

論文口試委員審定書i
誌謝ii
中文摘要iii
Abstractv
Literature Review
Part 1. Introduction 1
(i) Epidemiology of ameloblastoma1
(ii) Introduction of ameloblastoms1
(iii) Clinicoradiographic features of ameloblastoma4
(iv) Treatment of ameloblastoma5
Part 2. Pathogenesis of ameloblastoma9
(i) Genetic alterations in ameloblastoma9
(ii) Mitogen activated protein kinase(MAPK) pathway (FGFR2-RAS-BRAF) 12
(iii) Sonic hedgehog (Shh) signaling pathway15
(iv) Clinicopathologic associations of mutational profiles in ameloblastoma16
Part 3. New Treatment Options for ameloblastoma--Targeted therapy18
(i) Introduction of targeted therapy18
(ii) Potentially targeted molecules of ameloblastomas19
(iii) Clinical reports of targeted therapy for ameloblastoma patients24
Part 4. BRAF (V600E) immunohistochemistry27
(i) Introduction of BRAF protein27
(ii) BRAF (V600E) mutation28
(iii) Mutant-specific BRAF immunohistochemistry28
Materials and methods
Part 1. Patients and specimens31
Part 2. DNA isolation31
Part 3. Polymerase chain reaction (PCR)32
Part 4. Sanger sequencing33
Part 5. BRAF mutant-specific immunohistochemistry34
Part 6. Western blot to detect BRAF (V600E) in ameloblastoma cell lines35
Part 7. Assessment of clinical parameters36
Part 8. Statistical analysis37

Results
Part 1. Sanger sequencing results39
Part 2. BRAF mutant-specific immunohistochemistry in ameloblastomas40
Part 3. Comparison of BRAF (V600E) immunohistochemistry and sequencing40
Part 4. Western blot to detect BRAF (V600E) protein in ameloblastoma cell lines41
Part 5 Clinicopathological associations of BRAF genotypes42

Discussion44
Conclusion53
References54
Tables63
Figures68

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