Unit of Clinical Pharmacokinetics, Hospital de Pediatría JP Garrahan, Buenos Aires, Argentina.
National Scientific and Technical Research Council, CONICET, Buenos Aires, Argentina.
Br J Ophthalmol. 2018 Mar;102(3):415-418. doi: 10.1136/bjophthalmol-2017-310574. Epub 2017 Oct 31.
Intravitreal injection of chemotherapy in retinoblastoma eyes with vitreous seeds may lead to a risk of extraocular tumour dissemination that has not been assessed so far.
To develop a sensitive and clinically feasible technique to assess for potential retinoblastoma cell reflux after intravitreal injection of melphalan.
Filter papers were cut in 6 mm diameter circles and sterilised before use. Eyes with retinoblastoma vitreous seeds (group D, International Classification) received weekly intravitreal melphalan injections (20 µg or 30 µg/dose) followed by cryotherapy as part of local treatment. Immediately after finishing the injection and cryotherapy, filter papers were placed on the injection site and on the cryoprobe tip to assess for the expression of the cone-rod homeobox gene (CRX) by real-time qPCR as a surrogate of retinoblastoma RNA. The assay was developed and validated to determine sensitivity, linearity, recovery, repeatability and reproducibility.
The assay for quantitation of CRX expression was linear in the range of 1 to 1000 cells. The lowest limit of detection was one retinoblastoma cell and allowed to recover 100% of the cell load in external supplementation. A total of 14 eyes received 22 cycles of intravitreal melphalan and were evaluated for potential extraocular tumour cell dissemination using the developed technique. None of the cycles were positive for CRX in samples from the scar or from the cryoprobe tip.
A sensitive and simple method of tumour cell assessment has been developed that can be used in the clinics to assess for potential extraocular dissemination after intravitreal injections to assure its performance.
眼内注射化疗药物治疗伴有玻璃体种植瘤的视网膜母细胞瘤可能会导致眼外肿瘤扩散的风险，目前尚未对此进行评估。
开发一种敏感且临床上可行的技术，以评估眼内注射氨甲蝶呤后潜在的视网膜母细胞瘤细胞反流。
滤纸片裁剪成 6mm 直径的圆形，使用前进行消毒。患有视网膜母细胞瘤玻璃体种植瘤（国际分类）的眼睛接受每周一次的眼内氨甲蝶呤注射（20µg 或 30µg/剂量），随后进行冷冻治疗作为局部治疗的一部分。在注射和冷冻治疗结束后，立即将滤纸片放在注射部位和冷冻探针尖端，通过实时 qPCR 检测 cone-rod homeobox 基因（CRX）的表达，作为视网膜母细胞瘤 RNA 的替代物。该检测方法已被开发和验证，以确定其灵敏度、线性、回收率、重复性和重现性。
CRX 表达定量检测的assay 在 1 到 1000 个细胞的范围内呈线性。检测下限为一个视网膜母细胞瘤细胞，允许在外源性补充中回收 100%的细胞负荷。共有 14 只眼睛接受了 22 个周期的眼内氨甲蝶呤治疗，并使用开发的技术评估了潜在的眼外肿瘤细胞扩散情况。在来自疤痕或冷冻探针尖端的样本中，没有一个周期的 CRX 呈阳性。
已经开发出一种敏感且简单的肿瘤细胞评估方法，可在临床上用于评估眼内注射后潜在的眼外扩散情况，以确保其性能。
