肿瘤克隆起源之争与甲状腺乳头状癌研究
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摘要
甲状腺癌(thyroid carcinoma)是内分泌系统最常见的恶性肿瘤,约占人类新发恶性肿瘤的1%。近年来,甲状腺癌的发病率呈逐年上升的趋势,最新的Cancer Statistics,2009数据显示,美国甲状腺癌数以每年6.2%速度递增,首次超过卵巢癌发病率成为女性第七大常见恶性肿瘤。
甲状腺癌的发生发展过程中包含一系列高发的分子遗传学事件,如甲状腺滤泡状癌中的RAS基因突变、与甲状腺乳头状癌和部分甲状腺腺瘤相关的RET/PTC基因重排、以及在甲状腺未分化癌中常见的p53基因突变等。BRAF基因突变是近年来在甲状腺癌研究领域最突出的进展之一,其主要的突变方式是发生在15号外显子的BRAFV600E突变,在甲状腺乳头状癌中的平均发生率为44%。研究表明,BRAFV600E突变是甲状腺癌发生早期的分子遗传学事件,与肿瘤腺外浸润、淋巴结转移、TNM分期晚等都有显著的统计学关联,在临床上有很大的研究价值和运用前景。国内研究仅停留在较小样本的临床病理特征比较上,尚无大样本研究队列(超过200例)和长时间临床随访来研究BRAFV600E突变在中国人群中甲状腺癌的发生情况及预后价值。
甲状腺乳头状癌(Papillary thyroid carcinoma, PTC)是甲状腺癌最常见的病理类型,其临床特征之一是病灶多发,文献报道的发生率从18%-87%不等,多灶性甲状腺乳头状癌通常被认为与淋巴结转移、远处转移及初次治疗后局部复发率高有密切关联。双侧甲状腺乳头状癌(同时性或异时性),作为一种特殊类型的多灶癌,其克隆起源尚未被报道过,该类型的临床病理特征也未被重点研究过。
第一部分BRAF基因突变在我国甲状腺癌人群中的研究
目的：研究BRAF基因突变在我国甲状腺癌人群的发生情况,并进一步探索其在预测疾病复发和生存中的价值。
方法：建立我院十年甲状腺癌数据库(含甲状腺癌1006例),进行中位时间6年的电话随访和信访(最短2.5年,最长13.5年)。入组BRAF基因研究队列含甲状腺癌220例(乳头状癌208例、滤泡状癌8例、髓样癌1例、未分化癌3例),良性甲状腺病变46例。在比较并优化石蜡组织基因组DNA抽提方法的基础上,以直接测序法检测BRAF基因15号外显子的突变情况(BRAFV600E突变)。
结果：BRAFV600E突变集中发生在甲状腺乳头状癌中,突变率55.3%(115／208)；在甲状腺癌的其他病理类型及良性甲状腺病变组织中均未检测到该突变。统计学分析发现BRAFV600E突变与发病年龄密切相关(P=0.004)；长期随访结果显示该突变与疾病总生存率接近统计学差异(P=0.068)。
结论：BRAFV600E突变在我国甲状腺癌人群同样集中在甲状腺乳头状癌,且有相对较高的发生率；另外该突变可能与甲状腺乳头状癌的预后不良相关。
第二部分双侧甲状腺乳头状癌的克隆起源研究
目的：研究甲状腺乳头状癌同时性双侧病灶及异时性复发病灶之间的克隆起源。
方法：入组25对双侧甲状腺乳头状癌病灶(22对同时性的双侧病灶和3对异时性的复发病灶)以及15个转移淋巴结。以手工显微微切割肿瘤组织,从石蜡组织抽提基因组DNA；运用PCR和直接测序法检测成对病灶之间的BRAF基因状态；联合更为准确的X染色体失活分析技术判断肿瘤克隆起源。
结果：21对病例的62个病灶获得合格的DNA样品,18对病灶(18／21,85.7%)的BRAF基因状态完全一致,其中BRAF基因突变组12对、BRAF基因野生型组6对。18例女性病例中的11对病灶适合作X染色体失活分析,9对双侧病灶(9／11,81.1%)的X染色体失活形式相同。BRAF基因突变比较和X染色体失活分析在判断肿瘤克隆起源结果上一致性好(Chi-Square, two-sided P= 0.026; Spearman's rank correlation test, r= 0.671)。
结论：基于甲状腺癌发生相关的BRAF基因突变比较与胚胎时期就已确立的X染色体失活技术的联合,本研究首次证实甲状腺乳头状癌双侧病灶(同时性和异时性)系同一克隆起源,肿瘤腺内播散可能是双侧甲状腺癌发生的重要原因。这一发现为临床进一步认识甲状腺癌的生物学行为和合理治疗甲状腺癌(甲状腺全切)提供重要依据。
第三部分新型BRAF基因插入突变的初步研究
目的：在双侧甲状腺癌克隆起源研究中,我们通过测序筛查发现了一例极为罕见的插入型BRAF基因突变——BRAFV599Ins突变,该突变仅在一例意大利人群的甲状腺癌病人中被报道过。本部分研究拟初步探索该插入型突变可能获得的生物学功能。
方法：回顾性检测该患者所有病灶的BRAF基因突变情况,以TA克隆确认该突变类型。构建该罕见插入型BRAF基因突变(BRAFV599Ins突变)以及常见的BRAFV600E突变和BRAF基因野生型真核表达载体,转染HEK293细胞48小时后,检测BRAF蛋白及下游ERK信号通路的表达水平,流式细胞仪检测细胞周期变化,WST-8法(CCK-8)检测细胞增殖情况。
结果：TA克隆并测序验证BRAFV599Ins突变为1795位碱基前插入GTT三个碱基。瞬时转染293细胞后,BRAFV599Ins突变和BRAFV600E突变都明显激活下游ERK信号通路,细胞周期计数显示S期比例增高,细胞增殖实验也提示增殖活性增强。
结论：新型BRAF基因插入突变具有与BRAFV600E突变类似的功能,属于功能获得型突变。
Thyroid cancer is the most common endocrine neoplasm which accounts for roughly 1% of all new malignant disease. With the incidence increasing by 6.2% per year, thyroid cancer is currently the seventh most common malignancy diagnosed in women in U.S. population.
Previous studies have shown that thyroid cancer displays several highly prevalent genetic alterations, like RAS mutations in follicular thyroid carcinoma; rearrangements of the RET in papillary thyroid carcinoma and thyroid adenoma; and P53 mutations in anaplastic thyroid carcinoma. Among these alterations, mutations in BRAF gene are the most common. The point mutation in exon 15 of BRAF gene has been found to result in a valine to glutamine conversion at codon 600 (BRAFV600E mutation) with an average prevalence of 45% in papillary thyroid cancer. It has been reported that the BRAFV600E mutation is associated with one or more conventional high-risk clinicopathological characteristics of PTC, like lymph node metastasis, extrathyroidal invasion, and advanced disease stages. Until now, there is few study with a relative larger sample size to investigate the prevalence and possible prognostic value of BRAFV600E mutation in Chinese population.
Papillary thyroid carcinoma (PTC) is the most common histotype of thyroid cancer. PTC is often multifocal, with a reported frequency varying widely from 18 to 87 percent. Multifocality in PTC is associated with an increased risk of lymph node metastases and regional recurrence. However, as a specific subtype of multifocal PTCs, bilateral papillary thyroid carcinoma has not been widely investigated. In addition, in patients with local or distant recurrence after lobectomy, a tumor is found in more than 60% of the time in the resected contralateral lobe. The clonality of tumors appearing simultaneously in both lobes or recurring in the contralateral lobe remains unknown.
Part one. BRAF mutation in Chinese population of thyroid cancer
Purpose:To determine the incidence and define the possible role of BRAFV600E mutation in Chinese population of thyroid cancer.
Methods:We established a computer-based database comprising 1006 thyroid cancer patients and clinically followed them up with a mean time of six years (range from 2.5 years to 13.5 years). Two hundreds and twenty patients were enrolled from this database containing 208 papillary thyroid cancers,8 follicular thyroid cancers,1 medullary thyroid cancers and 3 anaplastic thyroid cancers. Also, a control group of 46 cases of benign thyroid tumors were studied. After compared the distinct methods of extracting genomic DNA from formalin-fixed paraffin-embedded tissues (FFPET), we screened the BRAF gene by DNA sequencing.
Results:One hundred and fifteen PTCs were positive for the BRAFV600E mutation (55.3%,115/208). We confirmed previous reports that BRAFv600E mutation did not occur in benign disorders. This mutation was associated with older age (P= 0.004) and poorer prognosis (close to statistic difference, P= 0.068).
Conclusions:BRAFV600E mutation was also common in Chinese population of thyroid cancer and might be an indicator of poorer prognosis.
Part two. Clonal analysis of bilateral papillary thyroid carcinomas
Purpose:To address the issue of clonal origin of tumors appearing simultaneously in both lobes or recurring in the contralateral lobe.
Methods:we examined twenty-five pairs of bilateral papillary thyroid carcinomas (synchronous or metachronous) and 15 matched metastatic lymph nodes. BRAF gene mutation analysis combined with X-chromosome inactivation was used to evaluate these tumors' clonal origins. Genomic DNA was prepared from paraffin-embedded tissues after microdissection.
Results:In total,62 tumors yielded DNA of adequate quality. Eighteen out of 21 informative cases (18/21,85.7%) showed concordant BRAF status in tumors from both thyroid lobes, be either BRAF mutation-positive (12 patients) or BRAF mutation-negative (6 patients). Metastatic lymph nodes in 12 patients (12/15,80%) had a complete concordance of BRAF state with their primaries. Of the 18 studied female patients, eleven were suitable for X-chromosome inactivation assay. Nine cases (9/11, 81.1%) showed the same pattern of inactivation in bilateral tumors. A close correlation was found between BRAF mutation and X-chromosome inactivation analysis.
Conclusions:our data provide evidence that bilateral, recurrent and metastatic papillary thyroid carcinomas often arise from a single clone, and that intrathyroidal metastasis may play an important role in the development of bilateral tumors, as well as in the recurrence of this malignancy.
Part three. Molecular characterization of novel BRAFV599Ins mutation
Purpose:In the study of Part two, we report a rare BRAF gene mutation in a bilateral PTC, namely a 1795GTT insertion, resulting in BRAFV599Ins, and here we describe its biochemical characterization.
Methods:We studied the MAPK cascade in HEK293 cells transiently transfected with the various BRAF constructs. Then MTT assay and flow cytometry were done to test the cell viability and the possible change of cell cycle.
Results:Phosphorylation of ERK was virtually low in vector-transfected cells, while the MAPK cascade was significantly more active in cells expressing BRAFV600E or BRAFV5991ns than in cells expressing BRAFWT. Cells transfected with BRAFV600E or BRAFV599Ins obviously promoted cell growth and had a higher rate of S phase. Conclusions:This study demonstrated that BRAFV599Ins mutation, as BRAFV600E, is a "gain of function" mutation
甲状腺癌的发生发展过程中包含一系列高发的分子遗传学事件,如甲状腺滤泡状癌中的RAS基因突变、与甲状腺乳头状癌和部分甲状腺腺瘤相关的RET/PTC基因重排、以及在甲状腺未分化癌中常见的p53基因突变等。BRAF基因突变是近年来在甲状腺癌研究领域最突出的进展之一,其主要的突变方式是发生在15号外显子的BRAFV600E突变,在甲状腺乳头状癌中的平均发生率为44%。研究表明,BRAFV600E突变是甲状腺癌发生早期的分子遗传学事件,与肿瘤腺外浸润、淋巴结转移、TNM分期晚等都有显著的统计学关联,在临床上有很大的研究价值和运用前景。国内研究仅停留在较小样本的临床病理特征比较上,尚无大样本研究队列(超过200例)和长时间临床随访来研究BRAFV600E突变在中国人群中甲状腺癌的发生情况及预后价值。
甲状腺乳头状癌(Papillary thyroid carcinoma, PTC)是甲状腺癌最常见的病理类型,其临床特征之一是病灶多发,文献报道的发生率从18%-87%不等,多灶性甲状腺乳头状癌通常被认为与淋巴结转移、远处转移及初次治疗后局部复发率高有密切关联。双侧甲状腺乳头状癌(同时性或异时性),作为一种特殊类型的多灶癌,其克隆起源尚未被报道过,该类型的临床病理特征也未被重点研究过。
第一部分BRAF基因突变在我国甲状腺癌人群中的研究
目的：研究BRAF基因突变在我国甲状腺癌人群的发生情况,并进一步探索其在预测疾病复发和生存中的价值。
方法：建立我院十年甲状腺癌数据库(含甲状腺癌1006例),进行中位时间6年的电话随访和信访(最短2.5年,最长13.5年)。入组BRAF基因研究队列含甲状腺癌220例(乳头状癌208例、滤泡状癌8例、髓样癌1例、未分化癌3例),良性甲状腺病变46例。在比较并优化石蜡组织基因组DNA抽提方法的基础上,以直接测序法检测BRAF基因15号外显子的突变情况(BRAFV600E突变)。
结果：BRAFV600E突变集中发生在甲状腺乳头状癌中,突变率55.3%(115／208)；在甲状腺癌的其他病理类型及良性甲状腺病变组织中均未检测到该突变。统计学分析发现BRAFV600E突变与发病年龄密切相关(P=0.004)；长期随访结果显示该突变与疾病总生存率接近统计学差异(P=0.068)。
结论：BRAFV600E突变在我国甲状腺癌人群同样集中在甲状腺乳头状癌,且有相对较高的发生率；另外该突变可能与甲状腺乳头状癌的预后不良相关。
第二部分双侧甲状腺乳头状癌的克隆起源研究
目的：研究甲状腺乳头状癌同时性双侧病灶及异时性复发病灶之间的克隆起源。
方法：入组25对双侧甲状腺乳头状癌病灶(22对同时性的双侧病灶和3对异时性的复发病灶)以及15个转移淋巴结。以手工显微微切割肿瘤组织,从石蜡组织抽提基因组DNA；运用PCR和直接测序法检测成对病灶之间的BRAF基因状态；联合更为准确的X染色体失活分析技术判断肿瘤克隆起源。
结果：21对病例的62个病灶获得合格的DNA样品,18对病灶(18／21,85.7%)的BRAF基因状态完全一致,其中BRAF基因突变组12对、BRAF基因野生型组6对。18例女性病例中的11对病灶适合作X染色体失活分析,9对双侧病灶(9／11,81.1%)的X染色体失活形式相同。BRAF基因突变比较和X染色体失活分析在判断肿瘤克隆起源结果上一致性好(Chi-Square, two-sided P= 0.026; Spearman's rank correlation test, r= 0.671)。
结论：基于甲状腺癌发生相关的BRAF基因突变比较与胚胎时期就已确立的X染色体失活技术的联合,本研究首次证实甲状腺乳头状癌双侧病灶(同时性和异时性)系同一克隆起源,肿瘤腺内播散可能是双侧甲状腺癌发生的重要原因。这一发现为临床进一步认识甲状腺癌的生物学行为和合理治疗甲状腺癌(甲状腺全切)提供重要依据。
第三部分新型BRAF基因插入突变的初步研究
目的：在双侧甲状腺癌克隆起源研究中,我们通过测序筛查发现了一例极为罕见的插入型BRAF基因突变——BRAFV599Ins突变,该突变仅在一例意大利人群的甲状腺癌病人中被报道过。本部分研究拟初步探索该插入型突变可能获得的生物学功能。
方法：回顾性检测该患者所有病灶的BRAF基因突变情况,以TA克隆确认该突变类型。构建该罕见插入型BRAF基因突变(BRAFV599Ins突变)以及常见的BRAFV600E突变和BRAF基因野生型真核表达载体,转染HEK293细胞48小时后,检测BRAF蛋白及下游ERK信号通路的表达水平,流式细胞仪检测细胞周期变化,WST-8法(CCK-8)检测细胞增殖情况。
结果：TA克隆并测序验证BRAFV599Ins突变为1795位碱基前插入GTT三个碱基。瞬时转染293细胞后,BRAFV599Ins突变和BRAFV600E突变都明显激活下游ERK信号通路,细胞周期计数显示S期比例增高,细胞增殖实验也提示增殖活性增强。
结论：新型BRAF基因插入突变具有与BRAFV600E突变类似的功能,属于功能获得型突变。
Thyroid cancer is the most common endocrine neoplasm which accounts for roughly 1% of all new malignant disease. With the incidence increasing by 6.2% per year, thyroid cancer is currently the seventh most common malignancy diagnosed in women in U.S. population.
Previous studies have shown that thyroid cancer displays several highly prevalent genetic alterations, like RAS mutations in follicular thyroid carcinoma; rearrangements of the RET in papillary thyroid carcinoma and thyroid adenoma; and P53 mutations in anaplastic thyroid carcinoma. Among these alterations, mutations in BRAF gene are the most common. The point mutation in exon 15 of BRAF gene has been found to result in a valine to glutamine conversion at codon 600 (BRAFV600E mutation) with an average prevalence of 45% in papillary thyroid cancer. It has been reported that the BRAFV600E mutation is associated with one or more conventional high-risk clinicopathological characteristics of PTC, like lymph node metastasis, extrathyroidal invasion, and advanced disease stages. Until now, there is few study with a relative larger sample size to investigate the prevalence and possible prognostic value of BRAFV600E mutation in Chinese population.
Papillary thyroid carcinoma (PTC) is the most common histotype of thyroid cancer. PTC is often multifocal, with a reported frequency varying widely from 18 to 87 percent. Multifocality in PTC is associated with an increased risk of lymph node metastases and regional recurrence. However, as a specific subtype of multifocal PTCs, bilateral papillary thyroid carcinoma has not been widely investigated. In addition, in patients with local or distant recurrence after lobectomy, a tumor is found in more than 60% of the time in the resected contralateral lobe. The clonality of tumors appearing simultaneously in both lobes or recurring in the contralateral lobe remains unknown.
Part one. BRAF mutation in Chinese population of thyroid cancer
Purpose:To determine the incidence and define the possible role of BRAFV600E mutation in Chinese population of thyroid cancer.
Methods:We established a computer-based database comprising 1006 thyroid cancer patients and clinically followed them up with a mean time of six years (range from 2.5 years to 13.5 years). Two hundreds and twenty patients were enrolled from this database containing 208 papillary thyroid cancers,8 follicular thyroid cancers,1 medullary thyroid cancers and 3 anaplastic thyroid cancers. Also, a control group of 46 cases of benign thyroid tumors were studied. After compared the distinct methods of extracting genomic DNA from formalin-fixed paraffin-embedded tissues (FFPET), we screened the BRAF gene by DNA sequencing.
Results:One hundred and fifteen PTCs were positive for the BRAFV600E mutation (55.3%,115/208). We confirmed previous reports that BRAFv600E mutation did not occur in benign disorders. This mutation was associated with older age (P= 0.004) and poorer prognosis (close to statistic difference, P= 0.068).
Conclusions:BRAFV600E mutation was also common in Chinese population of thyroid cancer and might be an indicator of poorer prognosis.
Part two. Clonal analysis of bilateral papillary thyroid carcinomas
Purpose:To address the issue of clonal origin of tumors appearing simultaneously in both lobes or recurring in the contralateral lobe.
Methods:we examined twenty-five pairs of bilateral papillary thyroid carcinomas (synchronous or metachronous) and 15 matched metastatic lymph nodes. BRAF gene mutation analysis combined with X-chromosome inactivation was used to evaluate these tumors' clonal origins. Genomic DNA was prepared from paraffin-embedded tissues after microdissection.
Results:In total,62 tumors yielded DNA of adequate quality. Eighteen out of 21 informative cases (18/21,85.7%) showed concordant BRAF status in tumors from both thyroid lobes, be either BRAF mutation-positive (12 patients) or BRAF mutation-negative (6 patients). Metastatic lymph nodes in 12 patients (12/15,80%) had a complete concordance of BRAF state with their primaries. Of the 18 studied female patients, eleven were suitable for X-chromosome inactivation assay. Nine cases (9/11, 81.1%) showed the same pattern of inactivation in bilateral tumors. A close correlation was found between BRAF mutation and X-chromosome inactivation analysis.
Conclusions:our data provide evidence that bilateral, recurrent and metastatic papillary thyroid carcinomas often arise from a single clone, and that intrathyroidal metastasis may play an important role in the development of bilateral tumors, as well as in the recurrence of this malignancy.
Part three. Molecular characterization of novel BRAFV599Ins mutation
Purpose:In the study of Part two, we report a rare BRAF gene mutation in a bilateral PTC, namely a 1795GTT insertion, resulting in BRAFV599Ins, and here we describe its biochemical characterization.
Methods:We studied the MAPK cascade in HEK293 cells transiently transfected with the various BRAF constructs. Then MTT assay and flow cytometry were done to test the cell viability and the possible change of cell cycle.
Results:Phosphorylation of ERK was virtually low in vector-transfected cells, while the MAPK cascade was significantly more active in cells expressing BRAFV600E or BRAFV5991ns than in cells expressing BRAFWT. Cells transfected with BRAFV600E or BRAFV599Ins obviously promoted cell growth and had a higher rate of S phase. Conclusions:This study demonstrated that BRAFV599Ins mutation, as BRAFV600E, is a "gain of function" mutation
引文
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