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乳腺中心实验室 2012 级硕士 李满秀

乳腺中心实验室 2012 级硕士 李满秀. 研究背景. BRCA1 germline mutations 具有很高的乳腺癌及卵巢癌的患病风险，根据二次打击学说， BRCA1 野生型等位基因的失活被认为是肿瘤发生过程中的限速环节，而这种失活形式常常为 LOH 。. 正常细胞在急骤的 BRCA1 功能丧失下难以 生存， 如纯合的 BRCA1 突变可导致小鼠早期胚胎死亡. ？. loss of wild-type BRCA1 是否是 BRCA1-associated breast tumors 发生的始动环节， BRCA1 突变相关肿瘤究竟是如何演变的。.

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乳腺中心实验室 2012 级硕士 李满秀

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  1. 乳腺中心实验室 2012级硕士 李满秀

  2. 研究背景 BRCA1 germline mutations 具有很高的乳腺癌及卵巢癌的患病风险，根据二次打击学说，BRCA1野生型等位基因的失活被认为是肿瘤发生过程中的限速环节，而这种失活形式常常为LOH。

  3. 正常细胞在急骤的BRCA1功能丧失下难以生存，如纯合的BRCA1突变可导致小鼠早期胚胎死亡正常细胞在急骤的BRCA1功能丧失下难以生存，如纯合的BRCA1突变可导致小鼠早期胚胎死亡 ？ loss of wild-type BRCA1是否是BRCA1-associated breast tumors 发生的始动环节，BRCA1突变相关肿瘤究竟是如何演变的。 BRCA1突变携带者的正常细胞常呈现出异常的表型，如单倍剂量不足（haploinsufficiency） BRCA1相关联的乳腺癌常同时伴有其他基因的体系突变，如P53，PTEN

  4. 研究方法 • 55例BRCA1-associated和20例sporadic 乳腺癌 • 每个肿瘤记数100-200个细胞 • PTEN • P53 • BRCA1 LOH IHC immunofluorescence FISH

  5. 每个肿瘤计数100-200个细胞，记录3种基因的状态 (i.e., wild-type =wt and mutant =mut). 所有的肿瘤细胞均可归类为以下8种情况： PTENwtTP53wtBRCA1wt, PTENwtTP53wtBRCA1mut, PTENwtTP53mutBRCA1wt, PTENwtTP53mutBRCA1mut, PTENmutTP53wtBRCA1wt, PTENmutTP53wtBRCA1mut, PTENmutTP53mutBRCA1wt, PTENmutTP53mutBRCA1mut.

  6. 模型成立假设 (i) 浸润性肿瘤仍包含肿瘤演变过程中的各期细胞； (ii) 如果所有的肿瘤细胞均包含突变X，而只有少部分细胞含有Y突变，那么突变X必然发生在Y之前； (iii) 携带一种或多种改变的肿瘤细胞其增值与死亡率无明显区别； (iv) 通过计数单个或联合改变的细胞可以验证肿瘤可能的演变路径； (v) 通过分析少部分细胞可以提供肿瘤整体的信息.

  7. 同一患者的组织，具单一因子突变的所有细胞中，突变量最多的为first event； • the first event确定后，比较携带两种因子改变的细胞中，剩下两种因子突变的个数，较多者确定为the second； • 只要有具备三种突变的细胞存在，则剩余的因子为the third.

  8. Loss of PTEN was the first event in 28/55 tumors, followed by mutation in p53 or BRCA1 LOH with about equal probability; Mutation in p53 was the second most common first event in 17/55 tumors, almost followed by BRCA1 LOH. BRCA1 LOH was the least common first event in 10/55, only followed by p53mutation sometimes.

  9. loss of PTEN was the first event 的pathway 1改变的多为TNBC，TP53 or BRCA1 LOH为第一事件的pathway 2 肿瘤多为luminal。

  10. 在这些乳腺癌中，PTEN突变常见于ER阴性乳癌，而在luminal细胞中PTEN突变少见，因而考虑在luminal细胞中会不会存在PI3K的突变从而替代了PTEN的突变呢？在这些乳腺癌中，PTEN突变常见于ER阴性乳癌，而在luminal细胞中PTEN突变少见，因而考虑在luminal细胞中会不会存在PI3K的突变从而替代了PTEN的突变呢？ • 结果示：散发乳癌中，luminal型PI3K的突变率达6/10, 散发三阴中该比例为0/10. 而在BRCA1相关的55例患者中，检测到2例突变分别为（ER-,ER+).故推测BRCA1突变的luminal肿瘤发病途径与散发型不同。

  11. 瘤内异质性 The loss of wild-type BRCA1 may not be the first event in most BRCA1-associated breast tumors, and even in tumors that display apparent loss of the wild-type BRCA1 allele, not all tumor cells showed this change.

  12. 单倍定量不足 Loss of BRCA1在BRCA1-associated的乳腺癌中很少是 the first event ，乳腺上皮细胞中BRCA1单倍定量不足（haploinsufficient ）为BRCA1突变乳腺癌风险增加提供了很好的解释。

  13. Ki67, 增值标记物, PR-潜在的促分裂因子； • Immunofluorescence检测发现： significantly more Ki67+, PR+, and Ki67+ PR+ cells in contralateral normal breast tissue of BRCA1 mutation carriers diagnosed with breast cancer compared with that observed in controls.

  14. During the analysis of Ki67+ cells, we noticed occasional multipolar mitoses in normal breast tissues from BRCA1 mutation carriers, suggesting aberrant centrosome function. We found significantly higher (P ≤0.01) numbers of cells with more than 2 centrosomes in BRCA1 mutation carriers compared with controls

  15. 总结 • 1，The first event : loss of PTEN> P53 mutation> BRCA1 LOH; • 2,BRCA1 LOH的肿瘤中，仍有部分细胞保留有BRCA1蛋白功能； • 3，BRCA1突变携带者的正常乳腺具有细胞增值快，多级分化，中心粒增多等特点； • 4，这些皆提示BRCA1 单倍定量不足的促进细胞增殖及异常分裂，增加BRCA1突变者患癌风险。

  16. 谢谢！

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