CORE--HYPERMETAPHASE FISH AND PCR IN ACUTE MYELOGENOUS LEUKEMIA

荧光原位杂交（FISH）方法将进一步 开发和使用的一个主要程序， 周期性AML细胞频率的分辨率定量。AML 与下列染色体异常有关的疾病 将通过此程序进行研究- inv（16），t8;21，t15;17，+8， -7。该过程是基于我们在CML中的结果，其中我们有 再加上长期的有丝分裂停滞， 使用FISH探针的中期/载玻片（“超中期”） 它检测到了染色体重排， 恶性肿瘤（超中期/FISH或HMF）。在CML模型中， 表明癌细胞可以很容易地定量， 从病人的骨髓制备物中提取循环细胞。的 程序将允许检测<1%的癌症，电话和将 允许监测此类细胞频率的<4%变化。 在此将应用该方法来确定 AML患者在诊断时的疾病，并评估 治疗期间对AML患者的治疗有效性。的 来自+8和-7的数据将与间期FISH相关 由核心C生成的相同样本的数据。此外我们 将进行逆转录，然后进行PCR（RT/PCR）， 测定t15;17和t8;21中嵌合转录物的水平 AML与HMF数据以及间期FISH相关 以及在其上产生的标准G带细胞遗传学（CG）数据 最后，我们将完成开发并进行 对inv（16）样品进行基因组PCR，以评价细胞水平 含有与我们的HMF结果反应的嵌合基因 对同一样本进行RT/PCR、间期FISH和CG。 实验和相关性将评估与 由项目1和项目2确定的患者的进展（Estey博士 和Champlin分别），以确定效益和 每种技术在建立 患者对相关治疗的细胞遗传学反应。这些 程序将为我们提供新的见解， 残留病变的临床显著水平，并指导 进一步管理AML患者的个体恶性肿瘤。

A fluorescence in-situ hybridization (FISH) method will be further developed and utilized as a major procedure for the high resolution quantitation of the frequency of cycling AML cells. AML diseases associated with the following chromosomal abnormalities will be studied by this procedure - inv(16), t8;21, t15;17, +8, and -7. The procedure is based on our results in CML where we have coupled long term mitotic arrest to produce thousands of metaphases/slide ("hypermetaphase") with the use of a FISH probe that detects the chromosomal rearrangement associated with the malignancy (hypermetaphase/FISH or HMF). In the CML model we have demonstrated that cancer cells could be readily quantitated in cycling cells from patient bone marrow preparations. The procedures will allow detection of <1% cancer, calls and will permit monitoring of <4% changes in the frequency of such cells. The methodology will be applied here to identify severity of disease in AML patients at diagnosis and to evaluate the effectiveness of therapies on AML patients during treatment. The data from +8 and -7 will be correlated with the interphase FISH data on those same samples generated by Core C. In addition we will conduct reverse transcription followed by PCR (RT/PCR) to determine the level of chimeric transcript in t15;17 and t8;21 AMLs to correlate with the HMF data as well as the interphase FISH and standard G-band cytogenetics (CG) data generated on the same samples by Core Finally we will complete development and conduct genomic PCR on inv(16) samples to evaluate the level of cells containing the chimeric gene reactive to our HMF results as well as RT/PCR, interphase FISH and CG conducted on those same samples. The experiments and correlations will be evaluated with the progress of the patients determined by Projects 1 and 2 (Drs.Estey and Champlin respectively) to determine the benefits and shortcomings of each of the technologies in establishing the cytogenetic response of patients to therapies involved. These procedures will give us new insights into determining the clinically significant level of residual disease, and guide the further management of individual malignancies in AML patients.