儿童急性淋巴细胞白血病中p15基因缺失及转录异常的研究

目的：探讨ｐ１５基因缺失及表达异常在儿童急性淋巴细胞白血病（ＡＬＬ）发病中的作用。方法;采用Ｓｏｕｔｈｅｒｎ ｂｌｏｔ方法检测了５３例儿童ＡＬＬ骨髓样品中ｐ１５基因的缺失;用逆转录－聚合酶链反应（ＲＴ－ＰＣＲ０检测了２９例样品中ｐ１５ ｍＲＮＡ的转录。结果：全部被检样品中ｐ１５基因缺失１１例,缺失率为２０．８％,其中,９例Ｔ细胞型ＡＬＬ（Ｔ－ＡＬＬ）中５例显示有ｐ１５基因缺失,缺失率５５．６％;而     

小儿急性淋巴细胞白血病IgH及TCRγ基因重排研究

应用ＰＣＲ对３３例小儿急性淋巴细胞白血病（ＡＬＬ）进行免疫球蛋白重链（ＩｇＨ）及Ｔ细胞受体γ（ＴＣＲγ）基因重排研究,以探讨该２种重排在小儿ＡＬＬ的发生规律及与免疫分型的关系。结果显示：ＩｇＨ阳性１９例（５７．６％）,ＴＣＲγ阳性１０例（３０．３％）。有７例ＩｇＨ及ＴＣＲγ均呈阳性,其中５例来自Ｂ系或Ｔ系分化早期的淋巴细胞白血病,提示在儿童初发淋巴细胞白血病中,ＩｇＨ基因重排的发生率明显高于ＴＣＲγ基因重排,且在免疫表型为分化早期的Ｔ或Ｂ淋巴细胞白血病中,并存上述２种基因重排的可能性很大。     

小儿ALL细胞极限稀释液体微培养及微小残留病的检测

首次用ＣＦＵ－ＡＬＬ极限稀释液体微培养法，对１６例初诊，１４例ＣＲ期ＡＬＬ骨髓进行培养，并用８例正常，１１例再生骨髓作为ＣＦＵ－ＡＬＬ阴性对照。结果显示：初诊Ｂ系ＡＬＬ骨髓以重叠型集落伴ＣＤ（１０＋）细胞＞２０％为特点；初诊Ｔ－ＡＬＬ集落也属重叠型（ＣＤ（２＋）、ＣＤ（３＋）、ＣＤ（７＋））．而正常和再生骨髓虽形成集落，但为平展型或伴重叠型集落（ＣＤ（１０＋）＜２０％、以ＣＤ（１５＋）、ＣＤ（３３＋）细胞为主）。本文１４例ＣＲ期ＡＬＬ中，７例有重叠型集落（ＣＤ（１０＋）细胞＞２０％）；临床随访１年半，５例骨髓相继复发，１例诊断为睾丸白血病。     

子宫内膜癌中MTS1／p16基因缺失的研究

目的探讨ＭＴＳ１／ｐ１６基因缺失与子宫内膜癌发生发展的关系。方法采用聚合酶链式反应（ＰＣＲ）技术,扩增３２例子宫内膜癌组织的ＭＴＳ１／ｐ１６基因。结果３２例子宫内膜癌组织中,有６例出现ＭＴＳ１／ｐ１６基因缺失,缺失率为１８．８％;癌旁组织、正常子宫内膜组织和组织分级Ｇ１的癌组织未见ＭＴＳ１／ｐ１６基因缺失。结论ＭＴＳ１／ｐ１６基因缺失可能与子宫内膜癌病程进展有关,应对其进一步研究。     

乳腺癌MTS1/p16基因缺失及表达异常的初步研究

采用ＰＣＲ和免疫组化等方法对５１例乳腺肿瘤ＭＴＳ１／ｐ１６基因的缺失及表达异常情况进行了分析，结果显示，ＭＴＳ１／ｐ１６基因在乳腺癌中的阴性表达率为３２．６％（１５／４６），而其中６０％（９／１５）因纯合性缺失而无转录和表达产物，占总检测病例的１９．５％（９／４６）。ＭＴＳ１／ｐ１６基因缺失的病例多为淋巴结转移阳性，组织浸润程度高和分化程度低，提示ＭＴＳ１／ｐ１６基因的纯合性缺失与乳腺癌的发生发展及恶性程度密切相关。另外，在ｐ１６蛋白阴性表达的病例中除了基因的纯合性缺失造成的蛋白失表达外，还有４０％（６／１５）的病例有ＭＴＳ１／ｐ１６基因的扩增但没有表达产物，则可能是ＤＮＡ的异常甲基化，基因位移，点突变等其它基因变异作用的结果，进一步的研究正在进行中     

肺癌组织MTS1／p16基因缺失的研究

目的：研究ＭＴＳ１／ｐ１６基因在人肺癌组织中的变化。方法：应用Ｓｏｕｔｈｅｒｎ杂交分析了４６例肺癌组织标本,ＰＣＲ技术分析８９例肺癌组织标本。结果：Ｓｏｕｔｈｅｒｎ杂交检测ｐ１６基因的缺失率为１７．４％（８／１６）,ＰＣＲ检测ｐ１６基因缺失率为２５．８％（２３／８９）。结论：ＭＴＳ１／ｐ１６基因缺失在肺癌发生发展中可能起重要作用。     

PCR扩增TCRγ基因重排检测急性淋巴细胞白血病微量残留病的研究

应用多聚酶链反应（ＰＣＲ）技术检测急性淋巴细胞白血病（ＡＬＬ）骨髓白血病细胞ＴＣＲγ基因重排，敏感性达１０＾－５水平以上。２７例急性期ＡＬＬ有２１例检测到约４００ｂｐ的扩增产物，阳性检出率为７７．８％（２１／２７）；７例完全缓解（ＣＲ）的ＡＬＬ有２例检测阳性，其中１例于检测后１．５月复发，另１例随访６个月仍ＣＲ；而其余５例检测阴性者于检测后平均随访７．１月均持续ＣＲ。提示本法对ＡＬＬ ＴＣＲγ基因     

非小细胞肺癌中p16/MTS1基因失活及其意义

目的：研究抑癌基因ｐ１６ＭＴＳ１基因失活与肺癌发生发展的关系。方法：采用双重ＰＣＲ和免疫组化技术分析４８例原发性非小细胞肺癌中ｐ１６基因存在状态。结果：２５例ｐ１６蛋白表达阳性（５２．１％）,其中１４例ｐ１６基因第二外显子缺失。有淋巴结转移者和无淋巴结转移者的Ｐ１６蛋白表达阴性率有显著性差异。ｐ１６基因缺失率亦如此。肺鳞癌中ｐ１６蛋白表达阴性率显著高于腺癌。结论：ｐ１６基因存在状态与非小细胞肺癌的     

P16基因及其蛋白异常表达与肺腺癌病期及转移的关系

目的 探讨ｐ１６基因及其蛋白异常表达与肺腺癌病期及转移的关系。方法 肺癌手术标本采用免疫组化ＡＢＣ法分析ｐ１６蛋白的表达,其结果与ｐ１６基因第２外显子多聚酶链反应－单链构象多态性分析（ＰＣＲ－ＳＳＣＰ）结果进行对比。胸水中肺腺癌细胞采用ＰＣＲ－ＳＳＣＰ分析。结果（１）７４例肺腺癌和７６例肺鳞癌中ｐ１６蛋白阳性表达率分别率为４７．３％和４７．４％。二组间无显著差异（Ｐ〉０．０５）。但肺腺癌ｐ１６蛋白阴     

PCR扩增TCR_γ基因重排检测急性淋巴细胞白血病微量残留病的研究

应用多聚酶链反应（ＰＣＲ）技术检测急性淋巴细胞白血病（ＡＬＬ）骨髓白血病细胞ＴＣＲγ基因重排，敏感性达１０￣（－５）水平以上。２７例急性期ＡＬＬ有２１例检测到约４００ｂｐ的扩增产物，阳性检出率为７７．８％（２１／２７）；７例完全缓解（ＣＲ）的ＡＬＬ有２例检测阳性，其中１例于检测后１．５月复发，另１例随访６个月仍ＣＲ；而其余５例检测阴性者于检测后平均随访７．１月均持续ＣＲ。提示本法对ＡＬＬＴＣＲ＿γ基因重排的检测较具普遍性，可用于其微量残留病（ＭＲＤ）的检测，对ＡＬＬ预后的判断、复发的监测可能有重要意义。     

Prognostic significance of p16INK4a immunocytochemistry in adult ALL with standard risk karyotype.

The p16INK4a gene is frequently inactivated in acute lymphoblastic leukemia (ALL), by homozygous deletion. However, p16INK4a protein expression also varies widely in ALL blasts. We investigated the p16INK4a protein expression by immunocytochemistry (ICC) analysis in 76 cases adult ALL. We observed a great variation of the percentage of ICC-positive leukemic cells between samples even in which FISH analysis did not find p16INK4a gene deletion. All patients carrying a p16INK4a gene homozygous deletion were also negative by ICC. ALL with negative p16INK4a ICC were more frequently of T lineage, but no significant differences for white blood cell count, presence of bulky disease, karyotype, hemoglobin level, complete remission rate, overall and event-free survival (EFS) were found. However overall survival and EFS were significantly lower in patients negative by ICC, when analysis was performed in ALL with standard risk karyotype. We also analyzed sequentially at diagnosis and relapse nine cases and observed that one case lost p16INK4a expression between diagnosis and relapse, but that on the contrary three other samples showed increased expression at relapse. These findings suggest that p16INK4a ICC and deletion analysis provide distinct information about ALL cells and that the simple ICC method may be of prognostic value in standard risk adult ALL.     

INK4系列抑癌基因在白血病中纯合子缺失的实验研究

目的：探讨p16基因家族失活与白血病发生、发展及预后的关系。方法：PCR检测p16、p15、p18、p19基因在白血病中纯合子缺失。结果：p16、p15基因外显子1在AL组纯合子缺失率分别为22．37％、17．05％,在ALL组为45．95％、32．43％,在ANLL组均为5．88％;在CML的慢性期均为0。p16、p15基因外显子2在AL组纯合子缺失率分别为12．5％、5．68％;在ALL组为24．32％、10．81％;在ANLL组为3．92％、1．96％;在CML和对照组二者均无缺失。p18、p19基因外显子1在AL组纯合子缺失率分别为1．14％、0;在ALL组分别为2．70％、0;在ANLL和对照组均无缺失。结论：p16、p15基因纯合子缺失在AL的发生频率较高,ALL缺失率明显高于ANLL,复发一LLL组基因失活率最高。p16、p15基因纯合子缺失是AL,尤其是ALL的发病重要因素之一。p18、p19基因在AL组中几乎未见纯合子缺失。在ALL中,p16纯合子缺失率高于p15纯合子缺失率。两个基因的纯合子缺失常伴随存在。在ANLL中,p16、p15基因的纯合子缺失均少见。     

成人急性白血病p16基因的纯合缺失及甲基化研究

目的　观察成人急性白血病p16基因的纯合缺失及甲基化的情况。方法　用PCR法对成人急性白血病患者骨髓标本进行p16基因第 1、第 2外显子纯合缺失及甲基化检测。结果　 1) 71例成人急性白血病患者中p16基因纯合缺失 2例 ,均为B ALL ,AML及正常对照组p16基因未见纯合缺失 ;2 ) 2 8例ALL中 ,5例发生异常甲基化 ;43例ANLL中 ,有 7例发生了异常甲基化。结论　p16基因缺失、异常甲基化可能与白血病的发生及预后有关     

Deletion of the Ink4-locus (the p16ink4a, p14ARF and p15ink4b genes) predicts relapse in children with ALL treated according to the Nordic protocols NOPHO-86 and NOPHO-92.

Inactivation of the Ink4 gene locus locus on 9p comprising the tumour suppressor gene p16ink4a and its neighbours p14ARF and p15ink4b is common in childhood acute lymphoblastic leukaemia (ALL), but the prognostic significance is controversial. DNA from 230 patients was retrospectively analysed by Southern blotting, single strand conformation polymorphism (SSCP) and sequencing techniques. The results were correlated with clinical characteristics and outcome. One hundred and ninety-four fully analysed patients, similarly treated using the Nordic NOPHO-86 or the current NOPHO-92 protocols, were included in the outcome analysis. Deletions approached a minimally deleted region between the p16ink4a and p15ink4b genes, making the p14ARF gene the most commonly deleted coding sequence. Bi-allelic deletion was associated with high white blood cell count (WBC) (P < 0.001), T cell phenotype (P < 0.001) and mediastinal mass (P < 0.001). Patients with Ink4 locus bi-allelic deletions had an inferior pEFS (P < 0.01) and multivariate analysis indicated that bi-allelic deletion of the p16ink4a and the p14ARF genes was an independent prognostic risk factor (P < 0.05). Sub-group analysis revealed a pronounced impact of deletion status for high-risk patients, ie with high WBC. Deletion-status and clinical risk criteria (WBC) could thus be combined to further differentiate risk within the high-risk group. The analysis of the Ink4 locus adds independent prognostic information in childhood ALL treated by Nordic protocols and may help in selection of patients for alternative treatment.     

半巢式甲基化特异性PCR在急性淋巴细胞白血病患者p15基因甲基化检测中的应用

 【摘要】 目的 探讨半巢式甲基化特异性PCR（hn-MSP）的特性并了解p15基因甲基化和缺失在急性淋巴细胞白血病（ALL）发病中可能起到的作用。方法 运用hn-MSP方法和基因组硫化修饰PCR（BSP）后直接测序分析在25例初诊或复发不同阶段ALL患者以及部分恶性血液病细胞株中p15基因的甲基化和缺失状态,以10名健康者或非恶性血液病患者为对照组。以Molt-4细胞株为阳性对照,以对照组单个核细胞为阴性对照,分析hn-MSP方法的敏感性和特异性。结果　hn-MSP产物克隆测序结果与BSP结果一致,hn-MSP检测p15基因甲基化敏感性可达到1×10－5。hn-MSP检测,25例ALL患者p15基因甲基化发生率为68.0 ％（17例）;25例ALL患者中3例p15基因外显子1缺失,对照组p15基因无甲基化或缺失。结论　p15基因甲基化状态在ALL患者有较高的检出率,hn-MSP在分析p15基因甲基化状态上具有较高的特异性和敏感性。     

Aberrant expression of tumor suppressor genes and their association with chimeric oncogenes in pediatric acute lymphoblastic leukemia

Aberrant expression of tumor suppressor genes WT 1, RB 1, p53, homozygous deletion of p16 gene and their relationship with expression of oncogenes BCR-ABL, TEL-AML 1, MLL-AF 4, E2A-PBX 1, SIL-TAL 1 were determined in bone marrow samples of children with de novo B-lineage (n=170) and T-lineage (n=25) acute lymphoblastic leukemia (ALL). In contrast to expression of chimeric oncogenes alterations in p16, WT 1, RB 1 and p53 expression were T/B-lineage-unrestricted. Significant association between expression of MLL-AF 4 and WT 1, E2A-PBX 1 and p53; SIL-TAL 1 and homozygous deletion of p16 has been demonstrated.     

Hypermethylation of p16 and p15 genes and RB protein expression in acute leukemia

Both p16 and p15, encoded by genes located on chromosome 9p21, are inhibitors of cyclin-dependent kinases 4/6 (CDK4/6) and upstream regulators of RB function, and set up the RB/p16 tumor suppressive pathway, which is abrogated frequently in human neoplasms, either through inactivation of the RB or p16 tumor-suppressor protein, or alteration of the cyclin D1 or CDK4 oncoproteins. In hematological malignancies, deletion of p16/p15 locus has been shown to be highly specific to lymphoid malignancies, and more particularly to T-cell acute lymphoblastic leukemia (T-ALL). However, in the other subsets of ALL, deletions of p16 and p15 are relatively rare events. To investigate whether these genes are inactivated by methylation of the 5' CpG islands, we examined 35 leukemia cell lines and 29 childhood acute myeloid leukemia (AML) patients by Southern blot, polymerase chain reaction (PCR) and Western blot analyses. We found methylation of p16 in 12 (50%) of 24 ALL cell lines, 5 (50%) of 10 AML cell lines without homozygous deletion of p16, and 11 (38%) of 29 AML patients. Those leukemia cell lines subjected to p16 methylation were found to have lost p16 protein expression. The p15 gene was methylated in 10 (34%) of 29 ALL cell lines, 6 (60%) of 10 AML cell lines without homozygous deletion of p15, and 15 (52%) of 29 AML patients. These results revealed the frequent methylation of p16 and p15 genes in B-ALL and AML despite a low frequency of p16 and p15 deletions and mutations in these leukemias. In the study for expression of RB protein, we found no expression of RB in 4 of 16 leukemia cell lines. Inactivation of the p16 gene was found in all the cell lines with expression of RB. Neither amplification nor rearrangement of cyclin D1 gene was found in any cell lines. These results suggest that inactivation of p16 and p15 genes is one of the most common genetic events in acute leukemia, and plays an important role for the RB/p16 pathway in the pathogenesis of acute leukemia.     

食管鳞状细胞癌中p16基因变异的临床意义及地域性差异

目的 进一步了解Ｐ１６基因的变异与食管癌发生发展和生物学行为的关系及地域性差异。方法 采用免疫组化和聚合酶链反应技术,对来自林县高发区和浙江省的食管癌存档组织标本中Ｐ１６蛋白的表达及其因变异进行了检测,并对食管癌组织中ｐ１６基因变异表达的临床意义及地域性差异进行了回顾性比较分析。结果 ９２例食管癌组织中,４４例存在ｐ１６蛋白表达,２２例检测到ｐ１６基因的丢失,只有５例出现ＰＣＲ－ＳＳＣＰ的异常电泳