Protocol detail

Relapsed AML Sample Preparation

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Tumor samples from 136 patients with relapsed AML from the St. Jude Children's Research Hospital tissue resource core facility were obtained with written informed consent using a protocol approved by the St. Jude Children's Research Hospital institutional review board (IRB). Studies were conducted in accordance with the International Ethical Guidelines for Biomedical Research Involving Human Subjects. These relapsed AML cases were part of multiple clinical studies, and detailed information of these patients and clinical trials are provided in Supplementary Table S1. CD3⁺ T cells were first depleted from all AML samples by magnetic beads (EasySep Human CD3 Positive Selection Kit II, 17851, StemCell Technologies). For samples with low-tumor purity (<60%), additional enrichment was performed by flow cytometry using a combination of mouse anti-human CD45 PerCP-Cy5.5 (eBioscience, #8045–9459–120, clone:2D1, RRID:AB_1907397) and mouse anti-human CD33 APC (eBioscience, #17–0338–42, clone: WM-53, RRID:AB_10667747), along with negative selection of T cells with mouse anti-human CD3 APC-Cy7 (BD Biosciences, #557832, clone:SK7, RRID:AB_396890). Matched germline DNA was obtained from purified T cells (n = 18), skin or bone marrow fibroblasts (n = 70), or remission samples (n = 3). Twenty-eight samples were sequenced through our clinical pipeline (n = 28; refs. 23, 25 (link)).

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Umeda M., Ma J., Huang B.J., Hagiwara K., Westover T., Abdelhamed S., Barajas J.M., Thomas ME I.I.I., Walsh M.P., Song G., Tian L., Liu Y., Chen X., Kolekar P., Tran Q., Foy S.G., Maciaszek J.L., Kleist A.B., Leonti A.R., Ju B., Easton J., Wu H., Valentine V., Valentine M.B., Liu Y.C., Ries R.E., Smith J.L., Parganas E., Iacobucci I., Hiltenbrand R., Miller J., Myers J.R., Rampersaud E., Rahbarinia D., Rusch M., Wu G., Inaba H., Wang Y.C., Alonzo T.A., Downing J.R., Mullighan C.G., Pounds S., Babu M.M., Zhang J., Rubnitz J.E., Meshinchi S., Ma X, & Klco J.M. (2022). Integrated Genomic Analysis Identifies UBTF Tandem Duplications as a Recurrent Lesion in Pediatric Acute Myeloid Leukemia. Blood Cancer Discovery, 3(3), 194-207.

Publication 2022

EasySep Human CD3 Positive Selection Kit II

Anti cd3 Bone marrow Clone Cy5 5 Fibroblasts Flow cytometry Germline Human Institutional review board Mouse Patients Skin Stemcell T cells Tissue Tumor

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Variable analysis

independent variables

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dependent variables

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control variables

CD3+ T cells were first depleted from all AML samples by magnetic beads
For samples with low-tumor purity (<60%), additional enrichment was performed by flow cytometry using a combination of mouse anti-human CD45 PerCP-Cy5.5 and mouse anti-human CD33 APC, along with negative selection of T cells with mouse anti-human CD3 APC-Cy7
Matched germline DNA was obtained from purified T cells (n = 18), skin or bone marrow fibroblasts (n = 70), or remission samples (n = 3)

positive controls

None explicitly mentioned

negative controls

None explicitly mentioned

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