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Soverini, S.1; Martelli, M.1; Bavaro, L.1; Papayannidis, C.1; Sica, S.2; Sorà, F.2; Albano, F.3; Galimberti, S.4; Barate', C.4; Rondoni, M.5; Abruzzese, E.6; Annunziata, M.7; Russo, S.8; Mannina, D.9; Stulle, M.10; Imovilli, A.11; Curti, A.1; Bonifacio, M.12; Ferrero, D.13; Basilico, C.14; Reddiconto, G.15; Mineo, G.16; Laginestra, M. A.17; Pileri, S. A.18; Mignone, F.19; Percesepe, A.20; Martinelli, G.21; Cavo, M.1

doi: 10.1097/01.HS9.0000558904.20982.46
Poster Session I: Acute lymphoblastic leukemia - Clinical

1Hematology “L.e A. Seràgnoli”, UNIVERSITY OF BOLOGNA, BOLOGNA

2Hematology Department, Fondazione Policlinico Universitario A. Gemelli IRCCS - Università Cattolica del Sacro Cuore, Rome

3Hematology, Dept. of Emergency and Organ Transplantation - University of Bari, Bari

4Hematology, Department of Clinical and Experimental Medicine, University of Pisa, Pisa

5Hematology, Santa Maria delle Croci Hospital, Ravenna

6Hematology, S. Eugenio Hospital, Rome

7Hematology, Cardarelli Hospital, Naples

8Internal Medicine, AOU Policlinico di Messina, Messina

9Hematology and BMT Unit, San Raffaele Scientific Institute, IRCCS, Milan

10Hematology Unit, Trieste

11Hematology, Arcispedale Santa Maria Nuova IRCCS, Reggio Emilia

12Hematology, University of Verona, Verona

13A.O.U. Città della Salute e della Scienza, University of Turin, Turin

14ASST dei Sette Laghi, Presidio di Varese Osp Circolo Fondazione Macchi, Varese

15Hematology Unit, Vito Fazzi Hospital, Lecce

16Hematology Unit, Taormina

17Unit of Anatomic Pathology, University of Bologna, Bologna

18Hematopathology Unit, European Institute of Oncology, Milan

19Department of Science and Innovation Technology (DISIT), University of Piemonte Orientale, Alessandria

20Department of Medicine and Surgery, University of Parma, Medical Genetics Unit, Parma

21Istituto Scientifico Romagnolo per lo Studio e la Cura dei Tumori (IRST) IRCCS, Meldola, Italy

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Mutations in the BCR-ABL1 kinase domain (KD) are frequently detected in Philadelphia-positive (Ph+) Acute Lymphoblastic Leukemia (ALL) patients (pts) who are refractory or resistant to tyrosine kinase inhibitor (TKI) therapy. Emergence of mutant clones as early as during induction therapy supports the hypothesis that, at least in some cases, mutations may already be present at diagnosis. Routinely, Sanger sequencing (SS) is used for mutation screening but Next Generation Sequencing (NGS) may have considerable advantages.

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We aimed to assess the feasibility and informativity of NGS as compared to SS for routine BCR-ABL1 KD mutation screening of a prospective series of de novo and TKI-resistant Ph+ ALL leukemia patients (MutationALL study).

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Between May 2015 and February 2018, we used NGS in parallel with SS to analyze a consecutive series of 160 Ph+ ALL pts who were either newly diagnosed (n = 44) or had relapsed/refractory disease on TKI therapy (n = 116). NGS of ≈400 bp amplicons generated by nested RT-PCR was performed on a Roche GS Junior (until April 2017) or on an Illumina MiSeq (from May 2017 on). Read alignment and variant calling was done using the AmpSuite software (SmartSeq srl), with a lower detection limit set to 3%. Cis or trans configuration of mutation pairs, indicating compound mutations (CMs) or polyclonality, respectively, was determined correcting for the likelihood of PCR recombination.

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De novo pts positive for mutations were 0/44 by SS and 3/44 (7%) by NGS. All the 3 pts received TKIs effective against the low level mutations they had and achieved remission. Relapsed/refractory pts positive for mutations were 71/117 (61%) by SS and 89/117 (76%) by NGS. NGS identified low level mutations in 18 pts who were negative for mutations by SS. All of them had minimal residual disease (MRD)-positivity to 1st- (n = 10) or 2nd-line (n = 2) therapy or after transplant (n = 6). Most importantly, NGS provided a more accurate picture of BCR-ABL1 mutations status in 37/71 (52%) pts who turned out to have one or more low burden mutations in addition to the dominant mutation(s) detectable by SS. Each low burden mutation detected by NGS could be recognized as poorly sensitive either to the TKI the pt was receiving at the time of testing, or to the previous TKI(s). Out of 37 pts, 28 had hematological relapse and 9 had MRD-positivity. Overall, patients with multiple mutations were 25/71 (35%; up to 4 mutations) by SS and 56/89 (63%; up to 13 mutations) by NGS. Mutation complexity correlated with the number of lines of therapy received. T315I was the most frequent mutation; it was detected in 35 (50%) of the 71 mutated pts by SS and in 24 additional pts by NGS (66% of mutated pts). NGS could resolve the clonal complexity of 5 pts who had 2 base substitutions in the same codon so that the actual amino-acid change(s) were impossible to infer, and of 44/51 who had multiple mutations at different codons. Among these 44 pts, 36 were found to carry one or more (up to 4) CMs. CMs included most frequently T315I or F317L.

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In TKI-resistant Ph+ ALL, the underlying mutation landscape may be much more complex than it appears when SS is used for screening. Of note, approximately half of the pts positive for mutations by SS are found to harbor additional TKI-resistant low level mutations. Moreover, NGS may help resolve the subclonal complexity of Ph+ ALL cells. At diagnosis, NGS may identify low level TKI-resistant mutations in some pts, but the 3% lower detection limit might be a limit. More sensitive strategies like digital PCR should be explored in this setting.

Copyright © 2019 The Authors. Published by Wolters Kluwer Health Inc., on behalf of the European Hematology Association.