1、1,肿瘤的分子诊断 Molecular diagnosis of tumor,李江滨 副教授,2,第一节 肿瘤诊断的生物标志物染色体异常、基因异常、单核苷酸多态性、表观遗传异常、miRNA第二节 肿瘤的分子生物学检验技术第三节 肿瘤分子生物学检验的临床应用乳腺癌肿瘤分子诊断与个体化医疗,3,什么是肿瘤？ 肿瘤（Tumor） 是机体在各种致癌因素作用下，局部组织的某一个细胞在基因水平上失去对其生长的正常调控，导致其克隆性异常增生而形成的异常病变。 学界一般将肿瘤分为良性和恶性两大类。,4,Cancer vs Tumor,Tumors are sometimes cancerous but thi
2、s does not mean that tumors and cancers are synonyms (as most people think).,5,Abnormal cellular growth Tumors and cancers are different. A tumor develops when a lesion or lump病变或肿块is formed in your body due to abnormal cellular growth. In the case of cancer, this cellular growth is uncontrollable a
3、nd it spreads in the body.,6,Treatment Cancer Surgery, chemotherapy and radiotherapy. Tumor Removing a benign tumor is relatively easy through surgery, and the condition does not recur.,7,第一节 肿瘤诊断的生物标志物最早的肿瘤标志物：本-周蛋白，1846年 目前已发现一百多种肿瘤标志物 蛋白与核酸两大类 肿瘤相关染色体异常、基因异常、单核苷酸多态性、表观遗传异常、miRNA,8,一、肿瘤相关的染色体异常 多数
4、肿瘤细胞存在染色体异常。 1. 染色体数目异常 例：某个癌细胞的染色体共104条，包括许多异常的染色体,9,2. 染色体结构异常 包括易位、缺失、重复、环状染色体和双着丝粒染色体等。 例：Ph染色体（费城1号染色体） ，慢性粒细胞性白血病（CML） ，9号和22号染色体长臂易位 9号原癌基因abl和22号bcr基因组合成融合基因增高的酪氨酸激酶活性。Ph临床意义在于：95的CML都是Ph阳性，可以作为诊断的依据。有时Ph先于临床症状出现，故又可用于早期诊断。,10,二、肿瘤相关基因表达异常 原癌基因: sis、VEGF、EGFR、c-myc 抑癌基因: APC、BRCA、p53、Rb 细胞周期
5、调节基因: cyclins、CDKs、CKIs 细胞凋亡相关基因: Bcl-1、p53、bcr-abl 基因组稳定相关基因(DNA修复基因): APE1 肿瘤转移相关基因:nm23、WDNM、sis、p53 肿瘤血管生成相关基因: VEGF 、EGFR、p53,11,三、肿瘤相关单核苷酸多态性 What are single nucleotide polymorphisms (SNPs)? SNPs (pronounced “snips”), are the most common type of genetic variation among people. Each SNP represe
6、nts a difference in a single DNA building block, called a nucleotide. For example, a SNP may replace the nucleotide cytosine (C) with the nucleotide thymine (T) in a certain stretch of DNA.,12,13,SNPs occur normally throughout a persons DNA. They occur once in every 300 nucleotides on average, which
7、 means there are roughly 10 million SNPs in the human genome. They can act as biological markers, helping scientists locate genes that are associated with disease. When SNPs occur within a gene or in a regulatory region near a gene, they may play a more direct role in disease by affecting the genes
8、function.,14,Most SNPs have no effect on health. Researchers have found SNPs that may help predict an individuals response to certain drugs, susceptibility to environmental factors such as toxins, and risk of developing particular diseases. SNPs can also be used to track the inheritance of disease g
9、enes within families. Future studies will work to identify SNPs associated with complex diseases such as heart disease, diabetes, and cancer.,15,三、肿瘤相关单核苷酸多态性 SNP: Single nucleotide polymorphisms 1. SNP与肿瘤 肿瘤易感基因、肿瘤药物治疗相关基因 肿瘤个体化诊疗 2. SNP的研究思路 研究对象差异性 研究技术：PCR、芯片 研究难点：样本采集,16,四、肿瘤相关表观遗传异常 表观遗传： 表观遗传
10、（epigenetics）是指DNA序列不发生变化，但基因表达却发生了可遗传的改变。这种改变是细胞内除了遗传信息以外的其他可遗传物质发生的改变，且这种改变在发育和细胞增殖过程中能稳定传递。 几乎所有类型的人类肿瘤都存在表观遗传异常 抑癌基因的高甲基化和癌基因的去甲基化。 异常甲基化常发生在肿瘤细胞形成的早期。,17,五、肿瘤相关miRNA MicroRNA formation and function.flv,18,19,五、肿瘤相关miRNA 1. miRNA与肿瘤 miRNA与肿瘤发生、发展、诊断、治疗、预后 2. miRNA类肿瘤标志物的研究思路 研究对象：实体瘤、细胞、循环血、其他 研
11、究技术：定量PCR、western blot、芯片、免疫组化、细胞培养（增殖、迁移、浸润）等等,肿瘤相关长链非编码RNA,长链非编码RNA(long noncoding RNA, lncRNA) 长度在200-100000 nt之间的RNA分子 不编码蛋白lncRNA参与细胞内多种过程调控 种类、数量、功能都不明确 长链非编码RNA 在肿瘤发生发展中的位置 http:/ 肿瘤分子生物学检验技术,肿瘤分子诊断常用的检测标本 外周血细胞 病灶局部受损组织提取DNA或mRNA检测,22,肿瘤早期诊断标本的选择及处理 （1）样本选择体液、分泌物、排泄物作为早期分子诊断的检测样本，如：血、痰、大便等。
12、（2）样本处理：获取血浆DNA首先获取无血细胞血浆（Cell free plasma）：重复离心法：EDTA抗凝血4 1500rpm 20min 吸取上层血浆 3000rpm 10min。磁珠法和QIAamp DNA mini kit 在内的多种方法提取循环DNA。,23,循环DNA定量检测方法 早期检测方法：二苯胺法、溴化乙锭法、对流免疫电泳法、RNA-DNA杂交法 新的检测技术：放射免疫法，实时荧光定量PCR法等,24,新型检测标志物：血浆DNA 何谓血浆DNA (plasma DNA ) ？ 又称循环DNA ( circulating DNA ) ，是一种无细胞状态的细胞外DNA (ex
13、tra cellular DNA) ，由长度不等的单链或双链DNA及其混合物组成，主要以DNA-蛋白质混合物形式存在，但也存在部分游离DNA。,25,血浆DNA作为检测对象的优势 循环DNA来自外周血浆或血清，较组织标本易于获得，可实现无创检查。 DNA比RNA和蛋白质具有更高的稳定性，不宜降解，更适合作为检测的靶分子。 可以对其进行扩增，检测的敏感性高。 越来越多的证据表明，疾病（特别是肿瘤）患者的循环DNA存在量和质的改变。,26,Cell-free DNA resuscitated for tumor testing Nature Medicine 14, 914 - 915 (2008
14、) http:/ 以DNA为主要检测对象 1. 致病基因结构异常检测 2. 基因转录水平检测 3. 基因序列分析及表达检测,28,肿瘤分子诊断的常用方法,染色体数目异常 荧光原位杂交（Fluorescence in situ hybridization,FISH）,29,致病基因结构异常检测 （1） 斑点杂交（dot blot hybridization） （2）等位基因特异的寡核苷酸探针杂交（allele-specific oligonucleotide，ASO） （3）单链构象多态性（single strand conformation polymorphism, SSCP） （4）限制性
15、内切酶图谱（restriction map）分析 （5）限制性片段长度多态性（restriction fragment length polymorphism ,RFLP ） （6）DNA 分子杂交（Southern blot）,30,致病基因表达异常检测 （1） mRNA检测 定量PCR （2）蛋白检测 免疫组化：定性、半定量、定位 Western blot ：定量,31,第三节 肿瘤分子生物学检验的临床应用乳腺癌,32,一、 乳腺癌 女性发病率高，5%-10%为家族性。 90%家族性乳腺癌涉及BRCA1和BRCA2基因突变。 BRCA1突变可发生在所有细胞，若发生在精子或卵子则可以传给下一
16、代。 其他涉及基因：p53、PTEN、c-myc、端粒酶等。 论文导读: A strong candidate for the breast and ovarian卵巢 cancer susceptibility gene BRCA1 http:/www.sciencemag.org/cgi/rapidpdf/266/5182/66.pdf,33,BRCA1 BRCA1 (breast cancer 1, early onset早发) is a human tumor suppressor gene肿瘤抑制基因, which produces a protein, called breast
17、cancer type 1 susceptibility易感性 protein. It is found in the cells of breast and other tissue, where it helps repair damaged DNA, and destroy the cell when DNA cant be repaired. If BRCA1 itself is damaged, the damaged DNA can let the cell duplicate复制 without control, and turn into a cancer.,34,Mutati
18、ons and cancer riskCertain variations of the BRCA1 gene lead to an increased risk for breast cancer. Researchers have identified hundreds of mutations in the BRCA1 gene, many of which are associated with an increased risk of cancer. Women who have an abnormal BRCA1 or BRCA2 gene have up to an 60% ri
19、sk of developing breast cancer; increased risk of developing ovarian cancer is about 55% for women with BRCA1 mutations and about 25% for women with BRCA2 mutations.,35,These mutations can be changes in one or a small number of DNA base pairs. Those mutations can be identified with PCR and DNA seque
20、ncing. Other methods are proposed: Q-PCR定量 and Quantitative Multiplex PCR多重定量 of Shorts Fluorescents Fragments (QMPSF). New methods have been recently proposed: heteroduplex analysis异源双链分析(HDA) by multi-capillary electrophoresis多毛细管电泳or also oligonucleotides array based on comparative genomic hybrid
21、ization比较基因组杂交(array-CGH).,36,Researchers believe that the defective BRCA1 protein is unable to help fix mutations that occur in other genes. These defects accumulate and may allow cells to grow and divide uncontrollably to form a tumor.,37,女性排名第一的常见恶性肿瘤。 美国每8个人就有1个人一生中会得乳腺癌。美国患乳腺癌的女性占新发恶性肿瘤的30%，而其中
22、的大约10%的乳腺癌是遗传性的。 乳癌“株连”，一旦家里有人患此疾病，一级亲属（母亲、姐妹或孩子）的女性都应该做检查。,38,乳腺癌高危人群 有乳腺癌家族史、基因检测到BRAC1/2基因突变、曾有过胸部放疗史、乳腺活检为高危良性病变、乳腺密度高、初潮年龄早于12岁,以及绝经年龄推迟等的人群，一般患乳腺癌危险性会比较高。,39,40,安吉丽娜朱莉在2013年5月纽约时报上刊文“My Medical Choice”，讲述了决定手术的原因及经过，最后呼吁所有女性注意预防乳腺癌。 My doctors estimated that I had an 87 percent risk of breast
23、cancer and a 50 percent risk of ovarian cancer, although the risk is different in the case of each woman。 Only a fraction of breast cancers result from an inherited gene mutation. Those with a defect in BRCA1 have a 65 percent risk of getting it, on average。,41,Once I knew that this was my reality,
24、I decided to be proactive and to minimize the risk as much I could. I made a decision to have a preventive double mastectomy预防性的双乳切除手术 . I started with the breasts, as my risk of breast cancer is higher than my risk of ovarian cancer。,42,2015年3月纽约时报上刊文“diary of a surgery” I wanted other women at ris
25、k to know about the options. I promised to follow up with any information that could be useful, including about my next preventive surgery, the removal of my ovaries and fallopian tubes卵巢和输卵管. I had been planning this for some time. It is a less complex surgery than the mastectomy, but its effects a
26、re more severe. It puts a woman into forced menopause更年期.,43,Then two weeks ago I got a call from my doctor with blood-test results. “Your CA-125 is normal,” he said. I breathed a sigh of relief. That test measures the amount of the protein CA-125 in the blood, and is used to monitor ovarian cancer.
27、 I have it every year because of my family history. But that wasnt all. He went on. “There are a number of inflammatory markers that are elevated, and taken together they could be a sign of early cancer.” I took a pause. “CA-125 has a 50 to 75 percent chance of missing ovarian cancer at early stages
28、,” he said. He wanted me to see the surgeon immediately to check my ovaries.,44,The day of the results came. The PET/CT scan looked clear, and the tumor test was negative. I was full of happiness, although the radioactive tracer meant I couldnt hug my children. There was still a chance of early stag
29、e cancer, but that was minor compared with a full-blown tumor. To my relief, I still had the option of removing my ovaries and fallopian tubes and I chose to do it.,45,In my case, the Eastern and Western doctors I met agreed that surgery to remove my tubes and ovaries was the best option, because on
30、 top of the BRCA gene, three women in my family have died from cancer. My doctors indicated I should have preventive surgery about a decade before the earliest onset of cancer in my female relatives. My mothers ovarian cancer was diagnosed when she was 49. Im 39.,46,Last week, I had the procedure: a
31、 laparoscopic bilateral salpingo-oophorectomy腹腔镜双侧输卵管卵巢切除术. There was a small benign tumor on one ovary, but no signs of cancer in any of the tissues.在一侧卵巢有一个小的良性肿瘤，任何组织都没有癌症迹象。,47,I have a little clear patch透明贴 that contains bio-identical estrogen雌激素. A progesterone IUD黄体酮节育器 was inserted in my ute
32、rus子宫. It will help me maintain a hormonal balance, but more important it will help prevent uterine cancer子宫癌. I chose to keep my uterus because cancer in that location is not part of my family history.,48,Regardless of the hormone replacements Im taking, I am now in menopause更年期. I will not be able
33、 to have any more children, and I expect some physical changes. But I feel at ease with whatever will come, not because I am strong but because this is a part of life. It is nothing to be feared.,49,I feel deeply for women for whom this moment comes very early in life, before they have had their chi
34、ldren. Their situation is far harder than mine. I inquired and found out that there are options for women to remove their fallopian tubes but keep their ovaries, and so retain the ability to bear children and not go into menopause. I hope they can be aware of that.,50,预防性切乳术能保证今后百分百不患乳癌吗？ 国外研究表明，预防性
35、乳房乳腺切除手术，是目前对乳腺癌高危妇女最有效的预防措施。 预防性切乳术不能保证今后百分百不患乳癌 预防性双侧乳房切除术，分为两种。一种是乳房全切，即为整个乳房全部切掉。还有一种是保留乳头乳晕的皮下乳腺全切。 朱莉选择的是后者。癌变几率从手术前的87%，降到了5%。 切除输卵管保留卵巢还能怀孕吗？ 切除卵巢及输卵管还能怀孕吗？,51,肿瘤的分子筛查 通过特异性，敏感性的分子生物学技术手段，能够有效的筛查易患癌症体质的人群，对他们进行早期检测和治疗。 易感=患病？,52,临床检测实例： 华大基因 http:/birth.bgi- 金域检验 http:/ research program of a new plasma nucleic acid diagnostic marker of tumor2. How to carry out the detection of cancer susceptibility,
