Droplet Digital PCR for Oncogenic KMT2A Fusion Detection
2023; Elsevier BV; Volume: 25; Issue: 12 Linguagem: Inglês
10.1016/j.jmoldx.2023.09.006
ISSN1943-7811
AutoresAndrew L. Young, Hannah C. Davis, Grant A. Challen,
Tópico(s)Cancer Genomics and Diagnostics
ResumoAcute myeloid leukemia (AML) is an aggressive blood cancer diagnosed in approximately 120,000 individuals worldwide each year. During treatment for AML, detecting residual disease is essential for prognostication and treatment decision-making. Currently, methods for detecting residual AML are limited to identifying approximately 1:100 to 1:1000 leukemic cells (morphology and DNA sequencing) or are difficult to implement (flow cytometry). AML arising after chemotherapy or radiation exposure is termed therapy-related AML (t-AML) and is exceptionally aggressive and treatment resistant. t-AML is often driven by oncogenic fusions that result from prior treatments that introduce double-strand DNA breaks. The most common t-AML–associated translocations affect KMT2A. There are at least 80 known KMT2A fusion partners, but approximately 80% of fusions involve only five partners—AF9, AF6, AF4, ELL, and ENL. We present a novel droplet digital PCR assay targeting the most common KMT2A-rearrangements to enable detection of rare AML cells harboring these fusions. This assay was benchmarked in cell lines and patient samples harboring oncogenic KMT2A fusions and demonstrated a limit of detection of approximately 1:1,000,000 cells. Future application of this assay could improve disease detection and treatment decision-making for patients with t-AML with KMT2A fusions and premalignant oncogenic fusion detection in at-risk individuals after chemotherapy exposure. Acute myeloid leukemia (AML) is an aggressive blood cancer diagnosed in approximately 120,000 individuals worldwide each year. During treatment for AML, detecting residual disease is essential for prognostication and treatment decision-making. Currently, methods for detecting residual AML are limited to identifying approximately 1:100 to 1:1000 leukemic cells (morphology and DNA sequencing) or are difficult to implement (flow cytometry). AML arising after chemotherapy or radiation exposure is termed therapy-related AML (t-AML) and is exceptionally aggressive and treatment resistant. t-AML is often driven by oncogenic fusions that result from prior treatments that introduce double-strand DNA breaks. The most common t-AML–associated translocations affect KMT2A. There are at least 80 known KMT2A fusion partners, but approximately 80% of fusions involve only five partners—AF9, AF6, AF4, ELL, and ENL. We present a novel droplet digital PCR assay targeting the most common KMT2A-rearrangements to enable detection of rare AML cells harboring these fusions. This assay was benchmarked in cell lines and patient samples harboring oncogenic KMT2A fusions and demonstrated a limit of detection of approximately 1:1,000,000 cells. Future application of this assay could improve disease detection and treatment decision-making for patients with t-AML with KMT2A fusions and premalignant oncogenic fusion detection in at-risk individuals after chemotherapy exposure. Acute myeloid leukemia (AML) is an aggressive blood cancer driven by a diverse but finite set of oncogenic drivers.1Ley T.J. Miller C. Ding L. Raphael B.J. Mungall A.J. et al.Cancer Genome Atlas Research NetworkGenomic and epigenomic landscapes of adult de novo acute myeloid leukemia.N Engl J Med. 2013; 368: 2059-2074Crossref PubMed Scopus (3605) Google Scholar,2Papaemmanuil E. Gerstung M. Bullinger L. Gaidzik V.I. Paschka P. Roberts N.D. Potter N.E. Heuser M. Thol F. Bolli N. Gundem G. Van Loo P. Martincorena I. Ganly P. Mudie L. McLaren S. O'Meara S. Raine K. Jones D.R. Teague J.W. Butler A.P. Greaves M.F. Ganser A. Dohner K. Schlenk R.F. Dohner H. Campbell P.J. Genomic classification and prognosis in acute myeloid leukemia.N Engl J Med. 2016; 374: 2209-2221Crossref PubMed Scopus (2655) Google Scholar Detecting persistent leukemic cells after treatment is essential for subsequent treatment decision-making and long-term prognostication. Currently, the methods for detecting measurable residual disease (MRD) after treatment for AML include bone marrow morphology, multiparameter flow cytometry, and DNA sequencing.3Schuurhuis G.J. Heuser M. Freeman S. Bene M.C. Buccisano F. Cloos J. Grimwade D. Haferlach T. Hills R.K. Hourigan C.S. Jorgensen J.L. Kern W. Lacombe F. Maurillo L. Preudhomme C. van der Reijden B.A. Thiede C. Venditti A. Vyas P. Wood B.L. Walter R.B. Dohner K. Roboz G.J. Ossenkoppele G.J. Minimal/measurable residual disease in AML: a consensus document from the European LeukemiaNet MRD Working Party.Blood. 2018; 131: 1275-1291Crossref PubMed Scopus (684) Google Scholar Morphologic assessment only detects leukemic cells at a 5% limit of detection. Multiparameter flow cytometry has a more sensitive limit of detection at 0.01% to 0.001% but is challenging to implement and interpret and is not standardized among laboratories. DNA sequencing approaches can identify leukemic cells by their somatic mutation profile but are expensive assays to implement and can be confounded by clonal hematopoiesis in nonleukemic blood cells.4Jongen-Lavrencic M. Grob T. Hanekamp D. Kavelaars F.G. Al Hinai A. Zeilemaker A. Erpelinck-Verschueren C.A.J. Gradowska P.L. Meijer R. Cloos J. Biemond B.J. Graux C. van Marwijk Kooy M. Manz M.G. Pabst T. Passweg J.R. Havelange V. Ossenkoppele G.J. Sanders M.A. Schuurhuis G.J. Lowenberg B. Valk P.J.M. Molecular minimal residual disease in acute myeloid leukemia.N Engl J Med. 2018; 378: 1189-1199Crossref PubMed Scopus (499) Google Scholar For patients with AML with oncogenic fusions driving their disease, the fusion itself is a molecular marker than can be leveraged for sensitive MRD detection. Already, oncogenic fusions are used for disease monitoring in hematologic malignant tumors, such as chronic myeloid leukemia (CML), which is driven by the BCR-ABL1 fusion.5Nowell P. Hungerford D. A minute chromosome in human granulocytic leukemia.Science. 1960; 132: 1497Google Scholar Quantitative real-time RT-PCR (RT-qPCR) sensitively identifies BCR-ABL1 fusions by using primers that span the fusion.6Mensink E. van de Locht A. Schattenberg A. Linders E. Schaap N. Geurts van Kessel A. De Witte T. Quantitation of minimal residual disease in Philadelphia chromosome positive chronic myeloid leukaemia patients using real-time quantitative RT-PCR.Br J Haematol. 1998; 102: 768-774Crossref PubMed Scopus (217) Google Scholar Response to therapy is assessed by log-order decreases in transcript abundance measured by RT-qPCR with a 10−3 BCR-ABL1 abundance classified as a major molecular response and 10−4.5 to 10−5 BCR-ABL1 abundance marking a deep molecular response and the limit of detection for most available assays.7Berman E. How I treat chronic-phase chronic myelogenous leukemia.Blood. 2022; 139: 3138-3147Crossref PubMed Scopus (15) Google Scholar Because tyrosine kinase inhibitors have become more effective, individuals who clear their CML (molecular response 10−4.5 to 10−5) can discontinue therapy, with approximately half of these individuals remaining disease free in the long term.8Imagawa J. Tanaka H. Okada M. Nakamae H. Hino M. Murai K. Ishida Y. Kumagai T. Sato S. Ohashi K. Sakamaki H. Wakita H. Uoshima N. Nakagawa Y. Minami Y. Ogasawara M. Takeoka T. Akasaka H. Utsumi T. Uike N. Sato T. Ando S. Usuki K. Morita S. Sakamoto J. Kimura S. Group D.T. Discontinuation of dasatinib in patients with chronic myeloid leukaemia who have maintained deep molecular response for longer than 1 year (DADI trial): a multicentre phase 2 trial.Lancet Haematol. 2015; 2: e528-e535Abstract Full Text Full Text PDF PubMed Scopus (248) Google Scholar,9Mahon F.X. Rea D. Guilhot J. Guilhot F. Huguet F. Nicolini F. Legros L. Charbonnier A. Guerci A. Varet B. Etienne G. Reiffers J. Rousselot P. Intergroupe Français des Leucémies Myéloïdes ChroniquesDiscontinuation of imatinib in patients with chronic myeloid leukaemia who have maintained complete molecular remission for at least 2 years: the prospective, multicentre Stop Imatinib (STIM) trial.Lancet Oncol. 2010; 11: 1029-1035Abstract Full Text Full Text PDF PubMed Scopus (1254) Google Scholar Although BCR-ABL1 is almost universally associated with CML, there is no similar singular oncogenic fusion found in AML. The most common translocations associated with de novo AML, including RUNX1-RUNX1T1, CBFB-MYH11, and PML-RARA, can be detected with qPCR-based assays.10van Dongen J.J. Macintyre E.A. Gabert J.A. Delabesse E. Rossi V. Saglio G. Gottardi E. Rambaldi A. Dotti G. Griesinger F. Parreira A. Gameiro P. Diaz M.G. Malec M. Langerak A.W. San Miguel J.F. Biondi A. Standardized RT-PCR analysis of fusion gene transcripts from chromosome aberrations in acute leukemia for detection of minimal residual disease. Report of the BIOMED-1 concerted action: investigation of minimal residual disease in acute leukemia.Leukemia. 1999; 13: 1901-1928Crossref PubMed Scopus (1032) Google Scholar,11Hourigan C.S. Gale R.P. Gormley N.J. Ossenkoppele G.J. Walter R.B. Measurable residual disease testing in acute myeloid leukaemia.Leukemia. 2017; 31: 1482-1490Crossref PubMed Scopus (174) Google Scholar Droplet digital PCR (ddPCR) improves on qPCR by partitioning individual DNA molecules into microfluidic droplets, enabling absolute quantification of nucleic acids in a sample.12Hindson B.J. Ness K.D. Masquelier D.A. Belgrader P. Heredia N.J. Makarewicz A.J. et al.High-throughput droplet digital PCR system for absolute quantitation of DNA copy number.Anal Chem. 2011; 83: 8604-8610Crossref PubMed Scopus (1909) Google Scholar The improvements of ddPCR over qPCR are ease of assay implementation, improved lower limit of detection, high specificity, and absolute quantification (compared with relative quantification with a standard curve in qPCR). ddPCR has already demonstrated utility for detecting BCR-ABL1 fusions associated with CML and PML-RARA fusions associated with acute promyelocytic leukemia.13Jiang X.W. Chen S.Z. Zhu X.Y. Xu X.X. Liu Y. Development and validation of a droplet digital PCR assay for the evaluation of PML-RARalpha fusion transcripts in acute promyelocytic leukemia.Mol Cell Probes. 2020; 53101617Crossref Scopus (4) Google Scholar, 14Jennings L.J. George D. Czech J. Yu M. Joseph L. Detection and quantification of BCR-ABL1 fusion transcripts by droplet digital PCR.J Mol Diagn. 2014; 16: 174-179Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar, 15Shelton D.N. Bhagavatula P. Sepulveda N. Beppu L. Gandhi S. Qin D. Hauenstein S. Radich J. Performance characteristics of the first food and drug administration (FDA)-cleared digital droplet PCR (ddPCR) assay for BCR::ABL1 monitoring in chronic myelogenous leukemia.PLoS One. 2022; 17e0265278Crossref Scopus (8) Google Scholar However, these techniques are difficult to implement when the gene fusion involves many different partners and is not detectable with a single assay. Therapy-related AML (t-AML) is a unique subpopulation of AML that arises after chemotherapy or radiation exposure usually used to treat an antecedent solid tumor or lymphoma. Prior work has demonstrated that cytotoxic therapy can select for preexisting premalignant hematopoietic stem and progenitor cells that lead to t-AML.16Wong T.N. Ramsingh G. Young A.L. Miller C.A. Touma W. Welch J.S. Lamprecht T.L. Shen D. Hundal J. Fulton R.S. Heath S. Baty J.D. Klco J.M. Ding L. Mardis E.R. Westervelt P. DiPersio J.F. Walter M.J. Graubert T.A. Ley T.J. Druley T. Link D.C. Wilson R.K. Role of TP53 mutations in the origin and evolution of therapy-related acute myeloid leukaemia.Nature. 2015; 518: 552-555Crossref PubMed Scopus (599) Google Scholar,17Young A.L. Wong T.N. Hughes A.E. Heath S.E. Ley T.J. Link D.C. Druley T.E. Quantifying ultra-rare pre-leukemic clones via targeted error-corrected sequencing.Leukemia. 2015; 29: 1608-1611Crossref PubMed Scopus (66) Google Scholar In other cases, the therapy itself creates the oncogenic initiating event. Topoisomerase II (TOP2) inhibitors are chemotherapeutics uniquely associated with oncogenic fusions that involve the KMT2A gene.18McNerney M.E. Godley L.A. Le Beau M.M. Therapy-related myeloid neoplasms: when genetics and environment collide.Nat Rev Cancer. 2017; 17: 513-527Crossref PubMed Scopus (223) Google Scholar KMT2A fusions are potent drivers of leukemia. Mouse models show that introduction of these fusions into healthy bone marrow progenitor cells can drive an aggressive AML.19Krivtsov A.V. Twomey D. Feng Z. Stubbs M.C. Wang Y. Faber J. Levine J.E. Wang J. Hahn W.C. Gilliland D.G. Golub T.R. Armstrong S.A. Transformation from committed progenitor to leukaemia stem cell initiated by MLL-AF9.Nature. 2006; 442: 818-822Crossref PubMed Scopus (1195) Google Scholar Moreover, in pediatric de novo leukemia, KMT2A fusions often arise without any cooperating mutations.20Andersson A.K. Ma J. Wang J. Chen X. Gedman A.L. Dang J. et al.The landscape of somatic mutations in infant MLL-rearranged acute lymphoblastic leukemias.Nat Genet. 2015; 47: 330-337Crossref PubMed Scopus (360) Google Scholar In patients with cancer who receive high doses of TOP2 inhibitor therapy, t-AML is a devastating complication that is difficult to treat and typically fatal. The ability to detect preleukemic KMT2A fusions during therapy or after treatment could identify patients at high risk of t-AML who may benefit from early intervention. In contrast to most oncogenic fusions, there are at least 80 known KMT2A fusion partners. However, approximately 80% of KMT2A fusions involve only five partners—AF9, AF6, AF4, ELL, and ENL.21Winters A.C. Bernt K.M. MLL-rearranged leukemias-an update on science and clinical approaches.Front Pediatr. 2017; 5: 4Crossref PubMed Scopus (262) Google Scholar Given the varied fusion partners, it is difficult to detect these fusions by qPCR. This manuscript presents a novel ddPCR assay enabling the detection of the five most common KMT2A fusions, accounting for the vast majority of oncogenic KMT2A fusions found in patients with t-AML. The assay was benchmarked using cell lines and primary patient samples with KMT2A fusions. Together, this assay is an inexpensive, rapid, sensitive, and specific platform for KMT2A fusion detection that could improve MRD detection for patients with AML with KMT2A fusions and enable screening for patients at risk for developing t-AML after receiving TOP2 inhibitor therapy. Human cell lines known to harbor KMT2A fusions were used to design, benchmark, and validate the ddPCR assay. Cell lines used were THP-1 (KMT2A-AF9), MOLM-13 (KMT2A-AF9), MV4-11 (KMT2A-AF4), OCI-AML2 (KMT2A-AF6), and KOPN8 (KMT2A-ENL). A sample from a patient with t-AML harboring the KMT2A-ELL fusion was used to design and test the KMT2A-ELL reagents. Cell lines without KMT2A fusions were used as controls, including K562, HEL, Kasumi, Jurkat, and OCI-AML3. For each ddPCR experiment, cell lines harboring a KMT2A fusion and cell lines without a KMT2A fusion were used as controls. THP-1 and HEL cells were grown in RPMI 1640 medium (ATCC, Manassas, VA), 10% heat-inactivated fetal bovine serum (HI-FBS; Gibco, Billings, MT), and 1% penicillin-streptomycin (P/S; Gibco) with 0.05 mmol/L B-mercaptoethanol (Sigma-Aldrich, St. Louis, MO) added to the THP-1 media. MOLM-13, Jurkat, and KOPN8 cells were grown in RPMI 1640 medium (Gibco), 10% HI-FBS, and 1% P/S. Kasumi cells were grown in RPMI 1640 medium (Gibco), 20% HI-FBS, and 1% P/S. The MV-4 to 11 cells were grown in IMDM (Gibco), 10% HI-FBS, and 1% P/S. OCI-AML2 and OCI-AML3 cells were grown in Mem Alpha (Gibco), 20% HI-FBS, and 1% P/S. Cryopreserved human samples from patients with t-AML were banked at Washington University in St. Louis, MO. All patients with t-AML provided written informed consent for tissue repository and genomic sequencing in accordance with protocol 201011766 approved by the Washington University in St. Louis Institutional Review Board. Deidentified control patient peripheral blood or bone marrow samples were obtained according to a protocol approved by the Washington University Human Studies Committee. RNA was extracted from cell lines and patient samples using the RNeasy Plus Mini Kit (Qiagen, Hilden, Germany). Up to 1 × 106 cells were processed per experiment. RNA was extracted per manufacturer recommendations without modification. The RNeasy spin columns were eluted with 25 to 50 μL of RNase-free water. The RNA concentration was quantified using Qubit Fluorometric Quantification (Thermo Fisher Scientific, Waltham, MA). cDNA was synthesized using SuperScript IV VILO (Thermo Fisher Scientific). Synthesized cDNA molecules were stored at −20 °C. The genomic locations for the translocation in each cell line harboring a KMT2A fusion were identified from the Cancer Dependency Map (https://depmap.org/portal, last accessed December 17, 2021) and used to design primer sequences to span the fusions (Figure 1A, Supplemental Figure S1, and Table 1). Primers and probes were designed using Primer3Plus (https://www.primer3plus.com).22Untergasser A. Nijveen H. Rao X. Bisseling T. Geurts R. Leunissen J.A. Primer3Plus, an enhanced web interface to Primer3.Nucleic Acids Res. 2007; 35: W71-W74Crossref PubMed Scopus (2005) Google Scholar Multiple exonic primers were designed for each translocation partner. Fusion-specific cDNA amplicons generated from cell lines harboring a KMT2A fusion were Sanger sequenced and mapped to the hg38 reference genome using BLAT23Kent W.J. BLAT--the BLAST-like alignment tool.Genome Res. 2002; 12: 656-664Crossref PubMed Scopus (6424) Google Scholar to verify the fidelity of the primer pairs (Supplemental Figure S1). PrimeTime fluorescent probes (Integrated DNA Technologies, Coralville, IA) were designed to anneal within the fusion-specific primers to add sensitivity and specificity to the assay (Figure 1A and Supplemental Figure S1 and Table 1). For each fusion, one fluorescein (FAM)–labeled probe was designed to anneal to KMT2A upstream of the fusion, and one hexachlorofluorescein (HEX) –labeled probe was designed to anneal to the fusion partner downstream of the fusion. A control primer-probe pair was designed to tag the wild-type KMT2A cDNA with FAM- and HEX-labeled probes annealing in KMT2A exons.Table 1Primer and Probe Sequences for KMT2A Fusion DetectionPrimer-probe labelNucleotide sequence and modificationsKMT2A_e7_fwd∗Primers moved forward for the droplet digital PCR assay.5′-CCACTCCAGCTTCCAGGAAG-3′KMT2A_e9_fwd∗Primers moved forward for the droplet digital PCR assay.5′-CAGCACTCTCTCCAATGGCA-3′KMT2A_e9_rev∗Primers moved forward for the droplet digital PCR assay.5′-TGCCATTGGAGAGAGTGCTG-3′KMT2A_e11_rev∗Primers moved forward for the droplet digital PCR assay.5′-TTTGCAACGACGACAACACC-3′AF4_AFF1_e5_rev∗Primers moved forward for the droplet digital PCR assay.5′-AACTTGGATGGCTCAGCTGT-3′AF4_AFF1_e10_rev5′-TTCTGACTCTGCACTGCTGG-3′AF9_MLLT3_e6_rev∗Primers moved forward for the droplet digital PCR assay.5′-CTTGTTGCCTGGTCTGGGAT-3′AF9_MLLT3_e8_rev5′-TGCTTTCACTATCGCTGCCA-3′AF6_AFDN_e2_rev∗Primers moved forward for the droplet digital PCR assay.5′-GTCGAAATTTCTCCGCGAGC-3′AF6_AFDN_e2_rev25′-GGAGAGGACAGCATTCGCAT-3′ENL_MLLT1_e2_rev5′-CAGTCGTGAGTGAACCCCTC-3′ENL_MLLT1_e4_rev5′-GGTCGTAGGTGAAGCAGACC-3′ENL_MLLT1_e7_rev∗Primers moved forward for the droplet digital PCR assay.5′-GTTCTGGGATGGCTCGAAGT-3′ELL_ELL_e3_rev∗Primers moved forward for the droplet digital PCR assay.5′-GGCCGATGTTGGAGAGGTAG-3′ELL_ELL_e4_rev5′-CAGTCCAGGTGAACTTCCCC-3′KMT2A_e7_FAMprobe5′-/56-FAM/AGCAGGTCT/ZEN/CCCAGCCAGCA/3IABkFQ/-3′AF4_AFF1_HEXprobe5′-/5HEX/ACCCATTCA/ZEN/TGGCCGCCTCCTTTG/3IABkFQ/-3′AF9_MLLT3_HEXprobe5′-/5HEX/CCTGCCAGC/ZEN/TCCAGCTCCAG/3IABkFQ/-3′AF6_AFDN_HEXprobe5′-/5HEX/TCGGGTCTC/ZEN/TAGTACTGCCACCACTC/3IABkFQ/-3′KMT2A_e9_HEXprobe5′-/5HEX/ACCACCTCC/ZEN/GGTCAATAAGCAGGA/3IABkFQ/-3′ENL_MLLT1_HEXprobe5′-/5HEX/AGTCTGAGC/ZEN/TGGAGTCTGAG/3IABkFQ/-3′KMT2A_e9_FAMprobe5′-/56-FAM/CAGCAGATG/ZEN/GAGTCCACAGGATCAGAG/3IABkFQ/-3′ELL_e3_HEXprobe5′-/5HEX/AACGTCCGC/ZEN/GCCTCTGCG/3IABkFQ/-3′All primers tested listed above.fwd, forward; rev, reverse.∗ Primers moved forward for the droplet digital PCR assay. Open table in a new tab All primers tested listed above. fwd, forward; rev, reverse. ddPCR experiments were conducted on the QX200 Droplet Digital PCR System (Bio-Rad Laboratories, Hercules, CA). For each ddPCR reaction, the input cDNA was diluted to ensure that <330 ng of cDNA was input per reaction. Each ddPCR reaction was composed of 10 μL of 2× ddPCR Supermix for Probes no dUTP, 1000 nmol/L fusion-specific primers, 250 nmol/L fusion-specific probes, cDNA (maximum, 330 ng), and RNase/DNase-free water to 20 μL total. Droplets were generated on the QX200 Droplet Generator (Bio-Rad) per manufacturer instructions. Droplet PCR amplification occurred using the following thermocycler conditions: 94 °C for 10 seconds, 40 cycles of 94 °C for 30 seconds, 60 °C for 1 minute, 98 °C for 10 minutes, and 4 °C hold. Amplification was followed by imaging on the QX200 Droplet Reader (Bio-Rad) and analyzed using the QuantaSoft Analysis Pro software package version 7 (Bio-Rad). Multiple negative (cell lines without known KMT2A fusions) and positive (cell lines with known KMT2A fusions) controls were used per experiment. For each sample analyzed for KMT2A fusions, a separate aliquot was analyzed using primers and probes that targeted the wild-type KMT2A locus spanning exon 7 to 9 to provide an estimate of wild-type transcript abundance (Table 1). To enable simultaneous detection for all five KMT2A fusions targeted by the assay, a pooled ddPCR assay was designed and benchmarked. In a single reaction mixture, forward primers and probes for KMT2A exon 7 and KMT2A exon 9 were combined with reverse primers and probes for AF9 exon 6, AF4 exon 5, AF6 exon 2, ENL exon 7, and ELL exon 3 (Table 2). The pooled primer-probe mixture was 10× concentrated, such that 2 μL of the pooled primer-probe mixture added to a 20-μL ddPCR reaction would yield a final concentration of 1000 nmol/L for each primer and 250 nmol/L for each probe. Droplet generation, thermocycler conditions, droplet imaging, and droplet analysis were performed as described above.Table 2Pooled Primers and Probes for 10× Concentrated Master Mix (Final Volume of 200 μL)ReagentVolume, μLKMT2A_e7_fwd20KMT2A_e9_fwd20AF4_AFF1_e5_rev20AF9_MLLT3_e6_rev20AF6_AFDN_e2_rev20ENL_MLLT1_e7_rev20ELL_ELL_e3_rev20KMT2A_e7_FAMprobe5AF4_AFF1_HEXprobe5AF9_MLLT3_HEXprobe5AF6_AFDN_HEXprobe5ENL_MLLT1_HEXprobe5KMT2A_e9_FAMprobe5ELL_e3_HEXprobe5DNase-/RNase-free water25The stock primer and probes were 100 μmol/L. Each primer was 1000 nmol/L and each probe was 250 nmol/L in the final 20-μL ddPCR reaction.fwd, forward; rev, reverse. Open table in a new tab The stock primer and probes were 100 μmol/L. Each primer was 1000 nmol/L and each probe was 250 nmol/L in the final 20-μL ddPCR reaction. fwd, forward; rev, reverse. Cell lines harboring KMT2A fusions were serially diluted into OCI-AML3 cells (KMT2A wild type). Cells were quantified by automated cell counter (Nexcelom Bioscience, Lawrence, MA). Beginning at a 50% mixture, 1,000,000 cells harboring a KMT2A fusion (eg, THP-1) were mixed with 1,000,000 OCI-AML3 cells. From this mixture, 200,000 cells were removed and added to 1,800,000 OCI-AML3 cells to create the 5% mixture. The serial dilution was repeated to 0.0005%, at which point it was estimated there would be <10 cells harboring a KMT2A fusion remaining in the mixture. RNA was extracted and converted into cDNA as described above. The cDNA was diluted to 40 ng/μL and stored at −20 °C. For the 50%, 5%, 0.5%, and 0.05% dilutions, 80 ng of cDNA was used in a single ddPCR reaction well for fusion detection. For the 0.005% dilution, 320 ng of cDNA was used per well for four ddPCR reaction wells (1280 ng total). For the 0.0005% dilution, 320 ng of cDNA was used per well for eight ddPCR reaction wells (2560 ng total). Increasing the amount of cDNA included in the reaction was necessary at the 0.005% and 0.0005% dilutions to ensure that enough cells were assayed to enable detection of the rare KMT2A fusions. These serial dilution experiments simulated detecting rare leukemia cells harboring KMT2A fusions in patients and established the limit of detection for the assay. Prior work demonstrated that KMT2A fusions can be generated using clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 gene editing.24Jeong J. Jager A. Domizi P. Pavel-Dinu M. Gojenola L. Iwasaki M. Wei M.C. Pan F. Zehnder J.L. Porteus M.H. Davis K.L. Cleary M.L. High-efficiency CRISPR induction of t(9;11) chromosomal translocations and acute leukemias in human blood stem cells.Blood Adv. 2019; 3: 2825-2835Crossref PubMed Scopus (23) Google Scholar This fusion break point occurs between exons 10 and 11 of KMT2A.24Jeong J. Jager A. Domizi P. Pavel-Dinu M. Gojenola L. Iwasaki M. Wei M.C. Pan F. Zehnder J.L. Porteus M.H. Davis K.L. Cleary M.L. High-efficiency CRISPR induction of t(9;11) chromosomal translocations and acute leukemias in human blood stem cells.Blood Adv. 2019; 3: 2825-2835Crossref PubMed Scopus (23) Google Scholar Guide RNA sequences targeting the intronic regions of KMT2A and MLLT3/AF9 (Table 3) were synthesized (Synthego Corp., Redwood City, CA). Ribonucleoprotein complexes were formed by incubating guide RNAs (120 pmol) with Cas9 (Integrated DNA Technologies) for 20 minutes at room temperature.25Gundry M.C. Brunetti L. Lin A. Mayle A.E. Kitano A. Wagner D. Hsu J.I. Hoegenauer K.A. Rooney C.M. Goodell M.A. Nakada D. Highly efficient genome editing of murine and human hematopoietic progenitor cells by CRISPR/Cas9.Cell Rep. 2016; 17: 1453-1461Abstract Full Text Full Text PDF PubMed Scopus (182) Google Scholar Nucleofection was performed using the Neon Transfection System (Thermo Fisher Scientific). The ribonucleoprotein complex was combined with 250,000 cells in 10 μL of buffer R. Cells were electroporated using the settings 1700 V, 20 ms, and 1 pulse. After nucleofection, cells were cultured in appropriate media. From these cells, RNA was extracted using the above protocol and converted into cDNA. This cDNA was assayed using the KMT2A exon 9 and AF9 primer-probe combination to detect oncogenic fusions.Table 3sgRNA Molecules Designed to Introduce KMT2A-AF9 Rearrangements Using CRISPR/Cas9LabelsgRNA sequenceKMT2A_sgRNA5′-UUAGAAAUGGAGGCUGGGCG-3′AF9_sgRNA5′-AUCUACUUUGCCUGCGCUAU-3′sgRNA, single guide RNA. Open table in a new tab sgRNA, single guide RNA. No sequencing data were generated from this study. All reagents and protocols are listed in the Materials and Methods. The standard dual color ddPCR assay for mutation detection uses FAM- and HEX-labeled probes overlapping a region of interest that differ by a single nucleotide or small indel. Competitive annealing of the probes at the locus provides the specificity to distinguish between wild-type and mutated DNA molecules. This standard method for variant detection is not compatible with fusion detection. To enable low-frequency fusion detection, a novel cDNA-based dual color ddPCR assay was developed in which each fusion was identified by PCR primers spanning the fusion and nested fluorescently labeled probes flanking the fusion (Figure 1 and Table 1). In general, a FAM-labeled probe was designed to anneal within KMT2A upstream of the fusion and HEX-labeled probes were designed to anneal within the fusion partner downstream of the fusion. These primer-probe pairs were initially benchmarked in a dilution series experiment using bulk qPCR. Excellent performance was observed for both KMT2A FAM probe and fusion partner HEX probes across the panel (Supplemental Figure S2). For each qPCR experiment, a wild-type primer-probe pair was also incorporated into a separate reaction to estimate the abundance of the KMT2A wild-type transcript for comparison and ensure adequate sample preparation and loading. Once optimized, the primer-probe reagents were benchmarked on the ddPCR platform in a dilution series experiment (Supplemental Figure S3, Supplemental Table S1, and Materials and Methods). Cell lines harboring known KMT2A fusions were serially diluted into OCI-AML3 cells (KMT2A wild type) and analyzed using ddPCR. The appropriate cell type–specific KMT2A fusion was detected over 5 to 6 logs of dynamic range in this experiment. The fractional abundance of KMT2A fusion transcripts was determined by calculating the concentration of KMT2A fusion transcripts and dividing by the total number of KMT2A (fusion and wild-type) transcripts detected, and the expected KMT2A mutant cell line abundance was matched over the entire dilution series (Figure 2A). Primer-probe pairs designed to target a specific KMT2A fusion exhibited no off-target activity when assaying cell lines with different KMT2A fusions not targeted by the specific primer-pair pair (Supplemental Figure S4). As proof of principle for KMT2A fusion detection in a setting that mimics patients receiving TOP2 inhibitor therapy, HEL cells with wild-type KMT2A were edited with CRISPR/Cas9 to introduce a KMT2A fusion. KMT2A fusions were detected 4 days after CRIPSR/Cas9 treatment and persisted at 14 and 21 days in culture. Interestingly, the fractional abundance of KMT2A-AF9 fusion transcripts (relative to total KMT2A transcript abundance) remained stable for the duration of the experiment (Figure 2, B and C). Using a single primer-probe pair for tracking leukemic clones is useful when the oncogenic KMT2A fusion is known. However, in discovery settings when the KMTA2 fusion is unknown, for example, when screening for KMT2A fusions in patients receiving TOP2 inhibitors, simultaneous testing for all fusions is necessary. Because each reaction is limited by availability of patient sample RNA, a pooled primer-probe strategy was designed to enable discovery for the five most common KMT2A fusions in the same reaction (Table
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