■在之前的狒狒研究中,共鉴定了29个基因用于血液学的临床结果预测,急性,辐射,综合征(H-ARS)严重程度。其中,四个基因(FDXR,DDB2,POU2AF1,WNT3)似乎很有希望,并在五名白血病患者中得到了验证。在这项研究中,我们在大量接受全身照射的患者中寻求进一步的体内验证.
■在2-12Gy的分次(2Gy/天)全身照射(TBI)期间,从10名白血病患者中抽取外周血。RNA分离后,使用qRT-PCR对广泛用于生物剂量学和H-ARS预测的31个基因进行了基因表达(GE)评估。定制的低密度阵列(LDA)允许同时分析所有基因,96孔格式进一步检查了四个最有希望的基因。计算GE相对于预辐照的倍数变化(FC)。
■五名分别患有非霍奇金淋巴瘤(NHL)的急性淋巴细胞白血病(ALL)患者显示出足够的RNA量以及相应的淋巴细胞和中性粒细胞计数,以进行qRT-PCR,而急性髓系白血病(AML)和一名骨髓纤维化患者无法提供足够的RNA。一般来说,分离出1-2微克总RNA,而在暴露前和暴露样品中,RNA数量差异高达10倍(相关的抑制GE变化).来自31个基因,23在至少一个暴露前样品中表达。相对于曝光前,在辐照后48和72h,表达基因的数量可以减少一半。使用LDA,在人类样品中验证了13个基因。四个最有前途的基因(vid。sup.)要么不确定,要么太接近暴露前。然而,它们是用更灵敏的96孔格式测量的,除了无法检测到的WNT3.和以前的研究一样,在白血病患者中,GE对FDXR的调节(上调)相对于狒狒(下调)被再次确认.辐射诱导的DDB2(上调)和POU2AF1(下调)的GE变化在两个物种中的表现相似。因此,两个物种的23个基因中的16个显示出相同方向的GE变化,和在人体研究中上调的FDXR被重新验证。
■确定用于H-ARS严重程度预测的基因,以前在狒狒中检测到,在ALL中得到验证,但在AML患者中没有得到验证。与白血病类型有关的局限性,相关的减少的RNA量,抑制了GE的变化,和方法学上的挑战必须被视为对验证基因总数产生负面影响的因素。基于此,我们提出了额外的对照,包括血细胞计数和优选基于荧光的RNA数量测量,用于选择有希望的样品,并对基线拷贝数低的候选基因使用更灵敏的96孔格式.
UNASSIGNED: In a previous baboon-study, a total of 29 genes were identified for clinical outcome prediction of the hematologic, acute, radiation, syndrome (H-ARS) severity. Among them, four genes (FDXR, DDB2, POU2AF1, WNT3) appeared promising and were validated in five leukemia patients. Within this study, we sought further in-vivo validation in a larger number of whole-body irradiated patients.
UNASSIGNED: Peripheral blood was drawn from 10 leukemia patients before and up to 3 days during a fractionated (2 Gy/day) total-body irradiation (TBI) with 2-12Gy. After RNA-isolation, gene expression (GE) was evaluated on 31 genes widely used in biodosimetry and H-ARS prediction employing qRT-PCR. A customized low-density-array (LDA) allowed simultanously analyzing all genes, the 96-well format further examined the four most promising genes. Fold-changes (FC) in GE relative to pre-irradiation were calculated.
UNASSIGNED: Five patients suffering from acute-lymphoblastic-leukemia (ALL) respectively non-Hodgkin-lymphoma (NHL) revealed sufficient RNA-amounts and corresponding lymphocyte and neutrophile counts for running qRT-PCR, while acute-myeloid-leukemia (AML) and one myelofibrosis patient could not supply enough RNA. Generally, 1-2µg total RNA was isolated, whereas up to 10-fold differences in RNA-quantities (associated suppressed GE-changes) were identified among pre-exposure and exposure samples. From 31 genes, 23 were expressed in at least one of the pre-exposure samples. Relative to pre-exposure, the number of expressed genes could halve at 48 and 72h after irradiation. Using the LDA, 13 genes were validated in human samples. The four most promising genes (vid. sup.) were either undetermined or too close to pre-exposure. However, they were measured using the more sensitive 96-well format, except WNT3, which wasn´t detectable. As in previous studies, an opposite regulation in GE for FDXR in leukemia patients (up-regulated) relative to baboons (down-regulated) was reconfirmed. Radiation-induced GE-changes of DDB2 (up-regulated) and POU2AF1 (down-regulated) behaved similarly in both species. Hence, 16 out of 23 genes of two species showed GE-changes in the same direction, and up-regulated FDXR as in human studies were revalidated.
UNASSIGNED: Identified genes for H-ARS severity prediction, previously detected in baboons, were validated in ALL but not in AML patients. Limitations related to leukemia type, associated reduced RNA amounts, suppressed GE changes, and methodological challenges must be considered as factors negatively affecting the total number of validated genes. Based on that, we propose additional controls including blood cell counts and preferably fluorescence-based RNA quantity measurements for selecting promising samples and using a more sensitive 96-well format for candidate genes with low baseline copy numbers.