CORE--MINIMAL DISEASE DETECTION BY PCR

慢性粒细胞白血病（CML）是一种多能干细胞疾病， 这与一个异常的染色体有关，称为费城染色体， 染色体（Ph）。Ph融合了BCR和ABL基因的一部分， 产生的Bcr-Abl癌蛋白在血液和骨髓细胞中表达 CML患者。两种治疗模式在治疗上都非常有效， 诱导慢性期CML患者的长期缓解：骨髓 来自匹配供体的移植和干扰素-α治疗。 目前有几种测试用于监测CML的治疗，但只有 逆转录酶（RT）聚合酶链反应（PCR）方法， 足够敏感以检测微小残留疾病。RT-PCR是已知的 在总共约100个细胞中检测到少至一个表达BCR-ABL的细胞， 十万个细胞。然而，标准RT-PCR方法被广泛使用，因为没有 定量的竞争性定量PCR方法已经在 近年我们正在采用克罗斯和戈德曼开发的方法。 该方法包括添加已知水平的BCR-ABL连接序列， 含有一个插入片段以将其与天然连接序列区分开， 与未知样本进行比对并比较扩增物的强度的 通过分析连续的患者样品， 从一段时间内，以确定是否BCR-ABL-RNA的水平， 减少或增加。例如，一个模式的水平下降， BCR-ABL RNA将表明缓解加深。该补助金的目的是 是采用克罗斯和戈德曼的方法， 骨髓移植CML患者和接受以下治疗的CML患者： 干扰素α这种方法也将用于Ph+ ALL患者。的 将使用已知的Bcr-Abl阳性和阴性细胞建立方法 下一步的研究将在血液和骨髓中的细胞上进行， 强调微小残留病;并将通过以下方式评价效用： 对患者样本进行盲法研究。最后，核心实验室 将建立用于分析CML患者的血液和骨髓样本 通过竞争性PCR对P01研究范围内的样本进行检测。

Chronic myelogenous leukemia (CML) is a pluripotent stem cell disease, which is associated with an abnormal chromosome, termed the Philadelphia chromosome (Ph). The Ph fuses parts of the BCR and ABL genes, and the resulting Bcr-Abl oncoprotein is expressed in blood and bone marrow cells of CML patients. Two modes of therapy have been unusually effective at inducing long term remission in chronic phase CML patients: bone marrow transplantation from a matched donor and interferon-alpha treatment. Several tests are currently used to monitor treatment of CML but only the reverse transcriptase (RT) polymerase chain reaction (PCR) method is sufficiently sensitive to detect minimal residual disease. RT-PCR is known to detect as few as one cell expressing BCR-ABL in a total of about 100,000 cells. However, the standard RT-PCR method is widely used as not quantitative. Competitive quantitative PCR methods have been developed in recent years. We are adapting an approach developed by Cross and Goldman. This method involves adding known levels of BCR-ABL junction sequences, containing an insert to distinguish it from the native junction sequences, to an unknown sample and comparing the intensity of the amplimers. The reliability of the method is increased by analyzing serial patient samples from a period of time to determine whether the level of BCR-ABL-RNA is decreasing or increasing. For example, a pattern of decreasing level of BCR-ABL RNA would indicate a deepening remission. The aims of this grant are to adapt the Cross and Goldman method and use the test to monitor bone marrow transplant CML patients and CML patients being treated with interferon alpha. This approach will be also used in Ph+ ALL patients. The method will be established using known Bcr-Abl positive and negative cell lines; next studies will be done on cells from blood and marrow with emphasis on minimal residual disease; and the utility will be evaluated by performing a blind study with patient samples. Lastly, a core laboratory will be established for analysis blood and marrow samples of CML patient samples under study within the P01 grant by competitive PCR.