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Supplementary AppendixThis appendix has been provided by the authors to give readers additional information about their work.Supplement to: Foà R, Bassan R, Vitale A, et al. Dasatinib–blinatumomab for Ph-positive acute lymphoblasticleukemia in adults. N Engl J Med 2020;383:1613-23. DOI: 10.1056/NEJMoa2016272

SUPPLEMENTARY APPENDIXPageList of participating centers and investigators2Supplementary Methods4Supplementary Results8Supplementary Figures9- Figure S1. Treatment scheme9- Figure S2. Heat map of additional copy number aberrations10- Figure S3. Molecular clearance among p190 and p210 and p190/p210 patients11- Figure S4. DFS according to molecular response (A) and fusion type (B)12- Figure S5. Tregs cell percentages from paired samples measured before and duringblinatumomab treatment13Supplementary Tables14- Table S1. Sequences of primers used for the amplification of ABL1 KD mutations14- Table S2. Adverse events documented in the trial15References161

List of participating centers and investigatorsCenter086 - Ospedale dell’Angelo and Ospedale SS Giovanni ePaolo, Venice014 - Division of Hematology, Cardarelli Hospital,Naples028 - Division of Hematology, Department ofTranslational and Precision Medicine, SapienzaUniversity of Rome005 - Azienda Socio-Sanitaria Territoriale PapaGiovanni XXIII, Bergamo115 - Division of Hematology, Department ofTranslational Medicine, University of Eastern Piedmont,Novara019 - AO Ospedali Riuniti Villa Sofia Cervello Palermo024 - Department of Hematology, Ospedale Civile,Pescara043 - Department of Medicine, Section of Hematology,University of Verona045 -UOC Oncoematologia, Fondazione IRCCS Ca’Granda Ospedale Maggiore Policlinico di Milano,Università degli Studi di Milano004 - AOU consorziale Policlinico - Bari - UOEmatologia con Trapianto022 - AO di Perugia - Ospedale S. Maria dellaMisericordia - Ematologia e Trapianto Midollo Osseo044 - AOU San Luigi Gonzaga - SCDU EmatologiaGenerale e Oncoematologia133 - ASL Lecce - Ospedale ‘V. Fazzi’ - UO Ematologia170 - Ospedale Mauriziano Umberto I - Torino - SCDUEmatologia008 - CTC U.O di Ematologia con Trapianto di MidolloOsseo – Catania010 - AOU Careggi - Firenze - SOD Ematologia026 - Grande Ospedale Metropolitano ‘BianchiMelacrino-Morelli’ - Reggio Calabria - UOC Ematologia027 - Fondazione Policlinico Universitario AgostinoGemelli IRCCS - Roma - Area ematologica038 - AOU di Parma - SC Ematologia e Centro TrapiantiMidollo Osseo051 - IRCCS Ospedale S. Raffaele - Milano - UOOncoematologia055 - ASST degli Spedali Civili di Brescia - UOEmatologia087 - AUSL della Romagna - Ospedale ‘Santa Mariadelle Croci’ - Ravenna - Ematologia090 - Istituto clinico Humanitas - Rozzano - UOOncologia Medica ed Ematologia2PIRenato BassanFelicetto FerraraRobin Foà/Sabina ChiarettiAlessandro RambaldiGianluca GaidanoFrancesco FabbianoNicola Di Bartolomeo/Massimiliano BonifacioNicola FracchiollaFrancesco AlbanoBrunangelo FaliniGiovanna Rege CambrinNicola Di RenzoAlessandro CignettiFrancesco Di RaimondoAlberto BosiBruno MartinoSimona SicaMonica CrugnolaFabio CiceriErika BorlenghiMichela RondoniMatteo Della Porta

101 - ASL Salerno - Presidio Ospedaliero Tortora PaganiCatello Califano– Ematologia105 - AO Complesso Ospedaliero San GiovanniLaura CudilloAddolorata - Roma - UOC Ematologia301 - Hematology, Department of Biomedicine andAdriano VendittiPrevention, University Tor Vergata, Rome3

SUPPLEMENTARY METHODSRNA isolation and cDNA synthesisRNA extraction was performed from centrally prepared aliquots of cell dilutions lysed and homogenizedin guanydium isothiocyanate. At diagnosis, total RNA was extracted by columns following the instructionof the RNeasy Mini Kit (Quiagen, Hilden, Germany). During minimal residual disease monitoring, totalRNA was extracted by the automated Maxwell Instrument using the Total RNA Purification Kit (PromegaCorporation, Madison, WI). cDNA synthesis was performed using the SSIV Vilo Mastermix W/ez DNAse(Invitrogen, Carlsbad, CA) at diagnosis and by the Superscript Vilo CDNA Synthesis Kit (Invitrogen) forMRD monitoring samples.Multiplex-PCR at diagnosisAt diagnosis, a multiplex RT-PCR with a nested approach designed to detect the most common ALL fusiongenes - TCF3-PBX1, ETV6-RUNX1, SIL-TAL, NUP98-RAP1GDS1, SET-NUP214, BCR-ABL1 p190 (e1a2)and p210 (e13a2, e14a2), KMT2A/AFF1 (alias MLL-AF4) and KMT2A/MLLT1 (alias MLL-ENL) - wascarried out.1Q-RT-PCR for minimal residual disease monitoringThe number of BCR-ABL1 copies and the result interpretation were determined according to the“Standardization and consensus guidelines for minimal residual disease assessment in Philadelphia-positiveacute lymphoblastic leukemia by real-time quantitative reverse transcriptase”, the method developed by theEuro-MRD Consortium.2,3 All Q-RT-PCR reactions were performed on a 7500 fast ABI platform (LifeTechnologies, Carlsbad, CA). The number of amplification cycles was set at 50. Moreover, a commonthreshold set at 0.1 was selected in order to remain within the exponential phase. In determining the integrityof RNA, the criteria for excluding RNA samples was established as an ABL1 copy number 10000 fornegative samples and 1000 for those positive and quantifiable. The number of BCR-ABL1 copies wasnormalized with respect to the number of ABL1 copies and expressed as the number of BCR-ABL1/ABL1copies x 102.The quantity range was defined by the lowest dilution of standards with a reproducible Delta-CT of allreplicates ( 1.5 CT), a specific amplification (determined by shape and multicomponent graph), a meanCT value within 2.6-4.0 CT for a one log difference and 0.5-1.5 CT for two fold dilutions.Minimal residual disease-positive samples outside the quantitative range were scored as positivenonquantifiable. The sensitivity of the assay was defined as the lowest dilution of standards positive with aspecific amplification curve.2,34

All experiments were carried out in triplicate, with appropriate negative controls and a control that includeda mixture of all reagents without RNA, to rule out the possibility of cDNA contamination of RNA samples.Mutation analysis by Sanger sequencingBCR-ABL1 kinase domain (KD) mutation analysis was performed by Sanger sequencing (SS) in patientswith a MRD increase or at hematologic relapse. For ABL1 KD amplification, a nested-PCR approach wasused.4 The first amplification step was performed using 3 µL of cDNA and the following primers (TableS1): fwd-BCR-p210 (located on BCR mRNA exons 12/13) or fwd-BCR-p190 (located on BCR mRNA exon1a) and a rev common-ABL1 (located on ABL1 mRNA exon 8). This procedure ensured that the wild-type,non-rearranged ABL1 transcript was not analyzed. An initial denaturation step of 10 min at 95 C wasfollowed by an amplification for 35 cycles (denaturation for 30 s at 94 C, annealing for 1 min at 60 C,extension for 1 min at 72 C) and final extension for 10 min at 72 C. In the second amplification step, 2 µLof the first PCR product was then performed with 2 internal primer pairs: fwd-B-ABL1/Rev-B-ABL1 andfwd-C-ABL1/Rev-C-ABL1. In this way, the ABL1 kinase domain was divided into two partially overlappingfragments: fragment B (393 bp, spanning codons 206-335) and fragment C (482 bp, spanning codons 262421). For the second amplification step, the following PCR conditions were used: initial denaturation of 10min at 95 C, amplification for 30 cycles (denaturation for 30 sec at 94 C, annealing for 1 min at 58 C,extension for 1 min at 72 C), and final extension for 10 min at 72 C.All PCR experiments were performed in a 25 µL final volume containing 0.5U of AmpliTaq Gold(Invitrogen), 10X PCR Gold buffer (Invitrogen), 2.5 mM MgCl2 solution (Invitrogen), 2.5 mM each dNTP(Invitrogen), 25 pmoles of each primer for the first amplification step and 50 pmoles of each primer for thesecond amplification step.Specificity and efficiency of the amplification reactions were checked by electrophoresis of a 10 µL aliquoton ethidium bromide-stained 1.5% agarose gel.Copy number aberration analysisThe copy number status of IKZF1, CDKN2A or CDKN2B, PAX5, ETV6, EBF1 and BTG1 was investigatedby multiplex ligation-dependent probe amplification (MLPA) using the Salsa MLPA P335 ALL IKZF1 kit(MRC-Holland, Amsterdam, The Netherlands) and following the manufacturer’s instructions. This kitcontains probes for selected B-cell development and differentiation genes, and permits to detectabnormalities with a sensitivity of 20-30%. The full list and location of the MLPA probes are available atthe MRC Holland website (http://www.mrc-holland.com). One hundred ng of gDNA extracted from BMsamples of the patients were used, and gDNA extracted from 10 healthy donors was used as wild-typecontrol, at least 3 per experiment. Amplicons were separated on an ABI-Prism 3130 sequencer and analyzed5

by GeneMapper 4.0 software (Applied Biosystem, Life Technologies, Foster City, CA). The detection ofabnormal copy number values was performed using the Coffalyser.Net software and the manufacturer’sinstructions (www.coffalyser.net).Immunologic compartment evaluationA total of 197 samples of fresh PB were evaluated in a longitudinal analysis on paired samples from 43patients. Forty-three samples were collected prior to blinatumomab treatment (pre-blina), 38 after the firstcycle (1st blina), 40 after the second cycle (2nd blina), 29 after the third cycle (3rd blina), 25 after the fourthcycle (4th blina) and 22 after the fifth cycle (5th blina). To follow intra-individual variations, the T and NKcell compartments were investigated in a complete longitudinal analysis of 17 patients evaluated prior toblinatumomab treatment and at all time points.PB mononuclear cells were obtained by centrifugation on Ficoll-Histopaque (Axis-Shield, Oslo, Norway);for surface marker analysis, cells were incubated at room temperature for 30 min with the followingmonoclonal antibodies: CD45RA, CD45R0, CD3, CD4, CD8, CD16, CD56, CD57, CD62L, CD69 (allfrom Becton Dickinson [BD] Biosciences, San Josè, CA).To assess the presence of regulatory T (Tregs) cells (defined by the combined expression of CD3, CD4,CD25 and FoxP3), cells were stained with the following monoclonal antibodies: CD25-FITC, CD4-PerCPand CD3-APC, and then fixed and permeabilized (FIX and PERM kit BD) and stained with anti-humanFOXP3-PE (BD). For each analysis, 100000 events were acquired, and analyzed using the FACSDivasoftware (BD). Data were checked for normality with the Kolmogorov-Smirnov test. Due to non-parametricdata distribution, comparisons of continuous parameter in different groups were evaluated by Wilcoxonsigned rank test for paired samples as indicated. Data are reported as median percentages.Statistical analysisThe study was designed to evaluate the activity of dasatinib plus 2 cycles of blinatumomab in obtaining amolecular response in adult Ph ALL, considering the null hypothesis - 40% of patients (based on data ofthe historical control group - the GIMEMA LAL1509 protocol5) and the alternative hypothesis, fixed at60%, with a 90% power and a 5% significance level using a single stage phase II design. If the total numberof patients achieving a molecular response was at least 29, the primary endpoint was considered reached.Categorical data are presented as frequencies and percentages. For continuous data, median and ranges arepresented. Overall survival was calculated from the date of treatment start for all patients while diseasefree survival and cumulative incidence of relapse were calculated from the end of induction (day 85) forpatients in complete hematologic remission. The cumulative incidence of relapse was calculated using thecumulative incidence method, with death in complete hematologic remission being a competing risk. The6