Journal of Clinical Medicine Article Impact of BCR-ABL1 Transcript Type on Response, Treatment-Free Remission Rate and Survival in Chronic Myeloid Leukemia Patients Treated with Imatinib Sílvia Marcé 1, *,† , Blanca Xicoy 1,† , Olga García 1,† , Marta Cabezón 1,† , Natalia Estrada 1,† , Patricia Vélez 2,† , Concepción Boqué 2,† , Miguel Sagüés 3,† , Anna Angona 3,† , Raúl Teruel-Montoya 4,† , Francisca Ferrer-Marín 4,† , Paula Amat 5,† , Juan Carlos Hernández-Boluda 5,† , Mariana M. Ibarra 6,† , Eduardo Anguita 6,† , Montserrat Cortés 7,† , Andrés Fernández-Ruiz 2,† , Sandra Fontanals 8,† , Lurdes Zamora 1,† and on behalf of the Grupo Español de Leucemia Mieloide Crónica (GELMC) † 1 Myeloid Neoplasms Group, Hematology Department, ICO Badalona-Hospital Germans Trias i Pujol, Josep Carreras Leukaemia Research Institute (IJC), 08916 Badalona, Spain; bxicoy@iconcologia.net (B.X.); olga.garcia.calduch@fundacionpethema.es (O.G.); mcabezon@iconcologia.net (M.C.); nestrada@carrerasresearch.org (N.E.); lzamora@iconcologia.net (L.Z.) 2 Hematology Department, ICO Hospitalet-Hospital Duran y Reynals, 08908 l’Hospitalet de Llobregat, Spain; pvelez@iconcologia.net (P.V.); c.boque@iconcologia.net (C.B.); frizandres@gmail.com (A.F.-R.) 3 Hematology Department, ICO Girona-Hospital Josep Trueta, 17007 Girona, Spain; msagues@iconcologia.net (M.S.); angona.figueras@iconcologia.net (A.A.) 4 Hematology-Oncology Department, Hospital General Universitario Morales Meseguer-CIBERER, UCAM,  30008 Murcia, Spain; raulteruelmontoya@hotmail.com (R.T.-M.); fferrermarin@gmail.com (F.F.-M.)  5 Hematology Department, Hospital Clínico Universitario-INCLIVA de Valencia, 46010 Valencia, Spain; Citation: Marcé, S.; Xicoy, B.; García, paulaamat@yahoo.es (P.A.); hernandez_jca@gva.es (J.C.H.-B.) 6 Hematology Department, Hospital Clínico San Carlos (HCSC), Instituto de Medicina de Laboratorio (IML), O.; Cabezón, M.; Estrada, N.; Vélez, P.; Boqué, C.; Sagüés, M.; Angona, A.; Instituto de Investigación Sanitaria San Carlos (IdISSC), Department of Medicine, Universidad Complutense de Madrid (UCM), 28040 Madrid, Spain; marianamim@gmail.com (M.M.I.); Teruel-Montoya, R.; et al. Impact of eduardo.anguita@salud.madrid.org (E.A.) BCR-ABL1 Transcript Type on 7 Hematology Department, Hospital General de Granollers, 08402 Granollers, Spain; mcortes@fphag.org Response, Treatment-Free Remission 8 Pharmaceutics Department, ICO Hospitalet-Hospital Duran y Reynals, 08908 l’Hospitalet de Llobregat, Spain; Rate and Survival in Chronic Myeloid sfontanals@iconcologia.net Leukemia Patients Treated with * Correspondence: smarce@iconcologia.net; Tel.: +34-934-978-868 (ext. 3697) Imatinib. J. Clin. Med. 2021, 10, 3146. † Membership of the Grupo Español de Leucemia Mieloide Crónica (GELMC) is provided in the https://doi.org/10.3390/jcm10143146 Acknowledgments. Academic Editor: Domenico Russo Abstract: The most frequent BCR-ABL1-p210 transcripts in chronic myeloid leukemia (CML) are e14a2 and e13a2. Imatinib (IM) is the most common first-line tyrosine–kinase inhibitor (TKI) used to Received: 15 June 2021 treat CML. Some studies suggest that BCR-ABL1 transcript types confer different responses to IM. Accepted: 13 July 2021 The objective of this study was to correlate the expression of e14a2 or e13a2 to clinical characteristics, Published: 16 July 2021 cumulative cytogenetic and molecular responses to IM, acquisition of deep molecular response (DMR) and its duration (sDMR), progression rate (CIP), overall survival (OS), and treatment-free Publisher’s Note: MDPI stays neutral remission (TFR) rate. We studied 202 CML patients, 76 expressing the e13a2 and 126 the e14a2, and with regard to jurisdictional claims in correlated the differential transcript expression with the above-mentioned parameters. There were published maps and institutional affil- no differences in the cumulative incidence of cytogenetic responses nor in the acquisition of DMR iations. and sDMR between the two groups, but the e14a2 transcript had a positive impact on molecular response during the first 6 months, whereas the e13a2 was associated with improved long-term OS. No correlation was observed between the transcript type and TFR rate. Copyright: © 2021 by the authors. Keywords: chronic myeloid leukemia; BCR-ABL1 transcripts; response to imatinib; survival; discon- Licensee MDPI, Basel, Switzerland. This article is an open access article tinuation; relapse-free survival distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). J. Clin. Med. 2021, 10, 3146. https://doi.org/10.3390/jcm10143146 https://www.mdpi.com/journal/jcm J. Clin. Med. 2021, 10, 3146 2 of 12 1. Introduction Chronic myeloid leukemia (CML) is characterized by a reciprocal translocation be- tween chromosomes 9 and 22, known as the Philadelphia chromosome (Ph), containing the BCR-ABL1 fusion gene. The most frequent breakpoint of chromosome 22, known as the major breakpoint cluster region (M-bcr), comprises exon 13 or exon 14, generating the e13a2 (b2a2) or the e14a2 (b3a2) transcripts. Both are translated into a constitutively active protein of 210 kDa that differs in 25 amino acids more from the e14a2 transcript [1–3]. Imatinib (IM) is a tyrosine–kinase inhibitor (TKI) that inhibits the BCR-ABL1 protein in Ph-positive CML [4]. IM is the most widely used TKI for first-line therapy in CML patients, with most patients achieving optimal hematologic, cytogenetic, and major (MR3.0 ) and deep molecular responses (MR4.0 or better) [5]. However, IM fails in some patients and very few of them progress to acute leukemia. The causes of resistance to IM are heterogeneous and have been investigated in depth. On the other hand, in some non- resistant patients, minimal residual disease persists during TKI therapy. One hypothesis is that the difference in M-bcr breakpoint may be a cause of this persistence [3]. Some studies carried out prior to the IM era did not find any influence of BCR-ABL1 transcript type on the clinical outcome [6–8]. However, in the IM era, a Brazilian study, including a small number of patients at different phases of the disease, suggested that patients with the e13a2 transcript were more sensitive to IM in terms of molecular response [9], whereas other studies with larger number of patients demonstrated a better molecular response to IM in patients with the e14a2 transcript [3,10–12]. The achievement of deep molecular response (DMR) and its duration (sDMR) for at least 2 years are the current goal for discontinuing TKI treatment in CML. Approximately 50% of patients with sDMR can remain relapse-free after discontinuing TKI treatment in the so called treatment-free remission (TFR) phase, defined as the time from TKI discon- tinuation to the date of restarting therapy or the date of last control if treatment is not restarted. Therefore, there is a need to identify factors at diagnosis that will be associated with the acquisition of DMR, sDMR, and TFR. In this respect, a negative effect of the e13a2 transcript on the probability of achievement of TFR has been reported [13–15]. The aim of the present study was to evaluate the influence of the e13a2 and e14a2 BCR- ABL1 transcripts on outcomes of IM treatment in a multicenter series of 202 CML patients. 2. Material and Methods 2.1. Patient Samples We selected patients from databases from seven Spanish centers diagnosed of chronic phase CML from 1999 to 2016 and treated with IM at first-line according to the local standard of care and with a minimum follow-up of 18 months in the majority of cases. Samples were collected after obtaining informed consent in accordance with approved Institutional Research Boards protocols and the Declaration of Helsinki (Ref. CEI: PI-15-007, Code: ICO-ITK-2015-01 (ISOF-p210-LMC)). Peripheral blood (PB) and bone marrow (BM) (when applicable) were obtained at diagnosis and after 3, 6, and 12 months of IM treatment and during the follow-up. The main demographic and clinical characteristics were collected from the records of all patients. Cytogenetic and molecular responses were analyzed according to the 2013 European Leukemia Net (ELN) guidelines [16]. 2.2. Cytogenetic Studies Conventional chromosome G-banding (CG-banding) was performed at diagnosis and at 3, 6, and 12 months. The analyses at 6 and 12 months were performed in patients who did not achieve optimal cytogenetic response at 3 and 6 months, respectively. Fluorescent “in situ” hybridization (FISH) at diagnosis was performed in cases where CG-banding was not possible, according to the manufacturer’s instructions using LSI BCR/ABL dual-color, dual-fusion (Vysis-Abbott Molecular). At least 200 interphase nuclei were analyzed from each case. J. Clin. Med. 2021, 10, 3146 3 of 12 2.3. Determination of BCR-ABL1 Transcript Type Whole PB or BM samples were collected in 10 mL or 3 mL EDTA tubes, respectively. RNA was isolated from bone marrow or peripheral blood total leukocytes using TRIzol® reagent (Invitrogen; Carslbad, CA, USA) according to the manufacturer’s protocol. RNA concentration was quantified, and 1 µg of total RNA was reverse transcribed to cDNA using SuperScript IV or MMLV Reverse Transcriptase with random hexamers/primers according to the manufacturer’s protocol (Invitrogen; Thermo Fisher Scientific, Inc, Waltham, MA, USA). BCR-ABL1 was amplified using PCR primers as previously described [17] using 3 µL of cDNA. PCR products were transferred to a QIAxcel (QIAGEN Inc, Hilden, Germany) to identify the type of transcript expressed depending on its size. 2.4. Real-Time Quantitative PCR Real-time quantitative PCR was carried out in an ABI7900 PCR thermal cycler (Applied Biosystems, Foster City, CA, USA). Messenger RNA (mRNA) expression of BCR-ABL1 was calculated as a percentage relative to ABL1 expression and adjusted to the international scale (IS). The ratio was calculated at diagnosis and at 3, 6, and 12 months and at different points during follow-up of IM treatment to verify the acquisition of molecular response and deep molecular response. 2.5. Statistical Analysis Baseline characteristics were described as frequency and percentage for categorical variables and median and range for quantitative variables. Comparisons of categorical variables between groups were calculated using the Chi-square or Fisher exact test, when necessary, while the median test was used to compare continuous variables. Cumulative incidence of cytogenetic and molecular response was analyzed taking into account competing risks. Achieving a response to IM during the first 12 months after IM onset was considered, as main event and time was defined as months from IM onset to response. Patients who died or changed TKI during the first 12 months without achieving response were considered as competing events [4], and time was defined as months from IM onset to date of death or date of IM stop. Patients alive or dead after 12 months without response were considered as censures, and time was defined as months from IM onset to date of last follow-up. The DMR was defined as the time from diagnosis until the time of achievement of MR4.0 or MR4.5 and was analyzed by competing risks. OS was defined as time from diagnosis to the last follow-up or death from any cause. Survival probabilities were calculated using the Kaplan–Meier method, and the log-rank test was used for comparisons between groups. The CIP to acute leukemia was defined as the time from diagnosis to progression or the last follow-up and was analyzed by competing risks, considering patients alive without progression as censures, patients who progressed as events, and patients who died without progression as competing events. Both cumulative incidence curves for response and CIP were plotted, and comparison analyses between groups were performed by Gray test. The relationship between TKI discontinuation and isoform type was studied by logistic regression. Treatment-free remission (TFR) was defined as time from IM discontinuation to the occurrence of loss of MMR, restart of TKI treatment, progression, or death from any cause, the earliest of these events. TFR probabilities were calculated using the Kaplan–Meier method, and a log-rank test was used for comparisons between groups. Two-sided p values < 0.05 were considered as statistically significant. The statistical package SPSS, version 24.0 (SPSS Inc., Chicago, IL, USA), and R software (version 3.3.2) were used for all analyses. J. Clin. Med. 2021, 10, 3146 4 of 12 3. Results 3.1. Patients We studied 202 CML patients from seven Spanish centers diagnosed from 1999 to 2016, most of them with a minimum follow-up of 18 months on IM treatment (median 6 years; 0.3–11.6). The molecular analysis at diagnosis showed that 76 (37.6%) patients expressed the e13a2 BCR-ABL1-p210 transcript, while 126 (62.4%) expressed the e14a2 transcript. All patients were treated with IM at standard doses as first-line TKI therapy. The demographic and clinical characteristics were not significantly differing between the two groups. Patient distribution according to Sokal [18], EUTOS [19], and ELTS [20] scores was also comparable (Table 1). Table 1. Demographic and baseline clinical characteristics of the study series. e13a2 e14a2 Total p Value (n = 76) (n = 126) (n = 202) Sex (male/total) (%) 37/76 (49) 67/127 (53) 104/202 (52) 0.536 Age, median (min, max) 54 (15, 83) 57 (12, 84) 56 (12, 84) 0.828 Splenomegaly, n (%) 23/75 (31) 39/120 (33) 62/195 (32) 0.789 Median cm (min, max) 4 (2, 20) 5 (1, 30) 5 (1, 30) 0.542 Platelets (x10e9/L), median (min, max) 314 (37, 2236) 386 (21, 1872) 364 (21, 2236) 0.241 Blasts PB (%), median (min, max) 0 (0, 11) 0 (0, 11) 0 (0, 11) 0.918 t(9;22) 61/68 (90) 99/116 (85) 160/184 (87) Cytogenetics, n (%) 0.397 t(9;22) + others 7/68 (10) 17/116 (15) 24/184 (13) EUTOS, median (min, max) 28 (0, 143) 28 (0, 148) 28 (0, 148) 0.358 EUTOS > 87, n (%) 3/75 (4) 13/120 (11) 16/195 (8) 0.091 Low risk 39/75 (52) 46/120 (38) 85/195 (44) Sokal, n (%) Int risk 26/75 (35) 54/120 (45) 80/195 (41) 0.173 High risk 10/75 (13) 20/120 (17) 30/195 (15) Low risk 43/75 (57) 61/120 (51) 104/195 (53) ELTS, n (%) 0.376 Int/High risk 32/75 (43) 59/120 (49) 91/195 (47) PB: peripheral blood; ELTS: EUTOS long-term survival score; Int: intermediate. Some patients showed suboptimal response or were intolerant to IM (Table 2). Nine- teen patients required an increase in IM dose (five with e13a2 and 14 with e14a2), while a change to a second-line treatment was needed in 96 patients (37 with e13a2 and 59 with e14a2). Most of these patients switched beyond 12 months of treatment, and patients that did so earlier were intolerant. The reason for switching to a second-generation TKI was not influenced by the transcript types (Table 2). Table 2. Intolerance, suboptimal response, and failure to imatinib treatment according to the tran- script type. e13a2 e14a2 Total p Value (n = 76) (n = 126) (n = 202) Intolerance, n (%) 12/76 (16) 22/126 (18) 34/202 (17) 0.758 Suboptimal 13/76 (17) 19/126 (15) 32/202 (16) 0.702 Response, n (%) Failure, n (%) 19/76 (25) 28/126 (22) 47/202 (23) 0.651 J.J.Clin. Clin.Med. 2021,10, Med.2021, 10,3146 x FOR PEER REVIEW 55of of12 12 3.2. Cytogenetics 3.2. Cytogenetics According According toto the the Transcript TranscriptType Type Cytogenetic studies Cytogenetic studies were were available available in in 182 182 patients patients at at diagnosis. diagnosis. In In 154 154 out out of of 182 182 (84.6%) patients, the (84.6%) the t(9;22) t(9;22) was wasthetheonly onlyalteration alterationfound. found.Additional chromosomal Additional chromosomal ab- normalities did abnormalities didnot notdiffer between differ between the two the twogroups groups (Table (Table1).1). Weanalyzed We analyzedthethe cumulative cumulative incidence incidence of cytogenetic of cytogenetic response response duringduring the firstthe first 12 12 months months of of treatment treatment and differences and differences in the two ingroups the tworegarding groups regarding major and major and complete complete cy- cytogenetic togenetic(MCyR response responseand(MCyR CCyR)andwereCCyR) were comparable comparable (Figure 1A).(Figure 1A). Figure1.1. Cumulative Figure Cumulative incidence incidence of of cytogenetic cytogeneticresponse responseatat12 12months months(A) (A)and andmolecular molecularresponse MR response 3.0 at 12 months (B) MR3.0 at 12 months and at 6 months (C) according to the transcript type in our series of patients. (B) and at 6 months (C) according to the transcript type in our series of patients. 3.3. Molecular 3.3. Molecular Response Response by by Transcript TranscriptType Type Thenumber The numberofofpatients patients with with molecular molecular response response during during follow-up follow-up was similar was similar in in both groups (Figure 1B), but a trend of better cumulative incidence of MR 3.0 both groups (Figure 1B), but a trend of better cumulative incidence of MR was observed was 3.0 observed at 6atmonths in patients 6 months expressing in patients e14a2 expressing (Figure e14a2 1C).1C). (Figure 3.4. Effect of Transcript Type on Deep Molecular Response (DMR) One hundred and four out of 202 patients (51.4%) who started IM as first-line obtained a DMR within two years. No statistically significant differences were observed in the cumulative incidence of DMR 4.0 and 4.5, respectively, at 2 years between the e13a2 and J. Clin. Med. 2021, 10, x FOR PEER REVIEW 6 of 12 3.4. Effect of Transcript Type on Deep Molecular Response (DMR) J. Clin. Med. 2021, 10, 3146 6 of 12 One hundred and four out of 202 patients (51.4%) who started IM as first-line ob- tained a DMR within two years. No statistically significant differences were observed in the cumulative incidence of DMR 4.0 and 4.5, respectively, at 2 years between the e13a2 andgroups e14a2 e14a2 groups (Figure(Figure 2A,B). On2A,B). the On thehand, other otherahand, betteraDMR better(4.0 DMRand(4.0 4.5)and 4.5) in patients in patients with withand a low a low and intermediate intermediate Sokalasscore Sokal score as compared compared to patients to patients with Sokal with a high a highscore Sokalwas score was observed observed (33%43%] (33% [23%, [23%,vs.43%] 22%vs. 22%30%], [15%, [15%,p 30%], p = and = 0.0169 0.0169 27% and 27%37%] [17%, [17%,vs.37%] 14%vs. 14% [8%, [8%,p 21%], 21%], p =respectively) = 0.018, 0.018, respectively) (Figure (Figure 2C,D). 2C,D). Figure Figure 2. Cumulative 2. Cumulative incidence incidence of deep of deep molecular molecular response response 4.04.5 4.0 and and 4.5 according according to the to the transcript transcript type ((A) type ((A,B)) and and (B)) and according according to Sokal score ((C) and (D)) at to Sokal score ((C,D)) at 2 years, respectively.2 years, respectively. 3.5.3.5. Prognostic Prognostic Impact Impact of Transcript of Transcript Type Type on Overall on Overall Survival Survival andand CIPCIP TheThe 8-year 8-year OS OS probability probability (95%(95% CI) CI) of theofwhole the whole cohortcohort was 87%was(82–92%) 87% (82–92%) (Figure(Figure 3A), being 3A),significantly better in better being significantly the e13a2 group in the e13a2(Figure group 3B). At the3B). (Figure standard At theIM dose, 25% standard IMand dose, 22% of and 25% patients 22% in of the e13a2inand patients thee14a2 e13a2groups, and e14a2 respectively, did not achieved groups, respectively, did notthe optimalthe achieved response optimaltoresponse treatment to during treatmentfollow-up. Eight of these during follow-up. Eightpatients of these(two in the patients e13a2 (two group in the e13a2 and six inand group the e14a2 six in group) progressed the e14a2 group) to accelerated progressed tophase without accelerated significant phase without differences significant in differences the 8-year CIPin thebetween 8-year CIPthe between two groups (Figure the two 3C),(Figure groups and with3C),one and out withofonetwo out(50%) of two and four and (50%) out of sixout four (67%), respectively, of six dying afterdying (67%), respectively, progression to blast phase after progression due phase to blast to CML due causes (twocauses to CML patients(twodied due to died patients cerebral duehemorrhage, two due to infection, to cerebral hemorrhage, two dueand one due to to infection, and multi-organ failure). one due to multi-organ failure). 3.6. Analysis of TKI Discontinuation and Treatment-Free Remission (TFR) According to the Transcript Type Data of TKI discontinuation and TFR were available in 104 patients that acquired DMR. Only 66 of these patients achieved sDMR (25 with the e13a2 and 41 with the e14a2 transcript) during at least two years, and 36 of them discontinued IM treatment after a J. Clin. Med. 2021, 10, 3146 7 of 12 minimum of five years of treatment (Supplementary Figure S1). Within this group, 16 (44%) had the e13a2 transcript and 20 (56%) the e14a2. There was no correlation between J. Clin. Med. 2021, 10, x FOR PEER REVIEW the 7 of 12 transcript type and the fulfillment of discontinuation criteria (16/24 e13a2 (67%) and 20/36 e14a2 (56%), p = 0.389). Figure 3. Figure 3. Overall Overallsurvival survivalininthe thewhole wholeseries (A) series and (A) according and accordingto the transcript to the typetype transcript (B). (B). Cumulative incidence Cumulative of pro- incidence of gression (CIP) according to the transcript type in our series of patients (C). progression (CIP) according to the transcript type in our series of patients (C). 3.6. Analysis TFR (95% of TKI Discontinuation CI) of the whole seriesandofTreatment-Free discontinued Remission patients (n(TFR) = 44,According to the and 36 with sDMR Transcript eight TypesDMR due to toxicity or patient’s decision) was 63% (48%, 78%) (Figure 4A). without Fifteen Data ofofTKI out 44 cases relapsed, being discontinuation andthe TFRmedian follow-upinafter were available 104discontinuation of non- patients that acquired relapsed patients of 28 months (range 0, 65), but all of them regained molecular DMR. Only 66 of these patients achieved sDMR (25 with the e13a2 and 41 with the e14a2 response when reintroducing transcript) during atthe TKI. least twoDifferences years, andin36the of TFR thembetween e14a2 and discontinued e13a2 transcript IM treatment after a were not statistically significant (55% [32%, 78%] vs. 69% [49%, 89%], minimum of five years of treatment (Supplementary Figure S1). Within this group, respectively, p = 0.265) 16 (Figure (44%) had4B).the e13a2 transcript and 20 (56%) the e14a2. There was no correlation between Duration type the transcript of IMand treatment and DMR the fulfillment of duration before discontinuation discontinuation did not criteria (16/24 e13a2 signifi- (67%) and cantly correlate with TFR 20/36 e14a2 (56%), p = 0.389). in our cohort (HR [95% CI]: 0.99 [0.98, 1.01], p = 0.237 and 0.99 [0.98,TFR 1.01], p = CI) (95% 0.372, respectively). of the whole series of discontinued patients (n = 44, 36 with sDMR and eight without sDMR due to toxicity or patient’s decision) was 63% (48%, 78%) (Figure 4A). Fifteen out of 44 cases relapsed, being the median follow-up after discontinuation of non- relapsed patients of 28 months (range 0, 65), but all of them regained molecular response when reintroducing the TKI. Differences in the TFR between e14a2 and e13a2 transcript were not statistically significant (55% [32%, 78%] vs. 69% [49%, 89%], respectively, p = 0.265) (Figure 4B). J. Clin. Med. J. Clin. Med. 2021, 2021, 10,10, x FOR PEER REVIEW 3146 8 of8 12 of 12 Figure 4. Analysis of treatment-free remission (TFR) in the global series (A) and in the two groups according to the tran- Figure 4. Analysis of treatment-free remission (TFR) in the global series (A) and in the two groups according to the transcript script type (B). type (B). Duration 4. Discussion andof Conclusions IM treatment and DMR duration before discontinuation did not signifi- cantly correlate with TFR in This multicenter study our cohort performed in(HR 202 [95% CI]:with patients 0.99 chronic [0.98, 1.01], phasep =CML 0.237treated and 0.99 [0.98, with IM1.01], p = 0.372, as first-line respectively). therapy showed a higher percentage of major molecular response at 6 months in the e14a2 group that also presented a descriptively tendency to better TFR 4. Discussion rates, even though and noConclusions differences in the cumulative incidence of cytogenetic and molecular Thisnor responses multicenter study performed in the acquisition of DMR and in 202 sDMR patients with between thechronic phasewere two groups CMLfound. treated with IM transcript Although as first-linetypetherapy had no showed impact a higher on CIP,percentage patients with of major molecular the e13a2 response transcript had at 6 months better OS. in the e14a2 group that also presented a descriptively tendency to better TFR rates, Theeven though majority of no CMLdifferences patientsinhavethe cumulative a BCR-ABL1-p210 incidence withof cytogenetic an e13a2 orand an molecu- e14a2 lar responses transcript [1,21].nor in the studies Previous acquisition haveof DMRthat shown andthe sDMR e13a2between theistwo transcript more groups were frequent infound. youngAlthough men andtranscript is associatedtype with had no impact a blast on CIP, crisis patientsphenotype of myeloid with the e13a2 transcript [8,12,22,23]. The hade14a2 bettertranscript OS. has been associated with higher platelet count [12,24], while its relationship with lower The majority of CMLwhite cell counts patients have is controversial [11,12,25]. a BCR-ABL1-p210 with an In ouror e13a2 series, we did an e14a2 tran- not observe script anyPrevious [1,21]. of these correlations, studies havenot even that shown withthesex,e13a2 age, splenomegaly, transcript is more or platelet and in frequent blast youngcount. men Patients and is with CML were associated with stratified a blast crisisin low, intermediate, of myeloid or high-risk phenotype groups [8,12,22,23]. The according to EUTOS e14a2 transcript hasorbeen Sokal scores [18,19]. associated The recently with higher plateletdeveloped ELTS count [12,24], scoreitsdivides while relation- the patients ship in three with lower different white risk groups cell counts according[11,12,25]. is controversial to the probability of dying In our series, we did of CML not ob- improving serve anyprognostication compared of these correlations, not to EUTOS even with andsex,Sokal scores [20]. Nevertheless, age, splenomegaly, or platelet andpatient blast distribution according to Sokal, EUTOS, and ELTS scores between count. Patients with CML were stratified in low, intermediate, or high-risk groups accord- the two groups was comparable ing to EUTOS in ourorstudy Sokalso that transcript scores [18,19]. The type did notdeveloped recently affect the patients’ ELTS scorerisk divides stratification. the pa- We did not find a correlation between the presences of additional tients in three different risk groups according to the probability of dying of CML improv- chromosomal abnormalities at diagnosis ing prognostication in the to compared two groups. EUTOS andIntolerance, Sokal scoressuboptimal response, patient [20]. Nevertheless, or failure dis- totribution achieve optimal response to IM were also similar in both according to Sokal, EUTOS, and ELTS scores between the two groups was groups. Our results arecom- in accordance parable inwith previous our study studies so that that did transcript notdid type findnotany correlation affect between the patients’ riskthe transcript stratification. type and clinical characteristics and prognostic scores [26–28]. We did not find a correlation between the presences of additional chromosomal ab- In the pre-IM normalities era, noindifferences at diagnosis the two groups.in outcomes Intolerance,weresuboptimal found between response,patients with to or failure CP-CML expressing the e13a2 or e14a2 transcripts [6–8,24,29]. In achieve optimal response to IM were also similar in both groups. Our results are in ac-the IM era, some studies have addressed cordance withthe influence previous of the studies BCR-ABL1-p210 that did not find any transcript type between correlation with response to IM the transcript treatment and CML outcome with controversial type and clinical characteristics and prognostic scores [26–28]. results [9–12]. Lucas et al. [2] found that the one-year CCyR rate in 78 patients treated with IM 400 mg daily was higher in e14a2 In the pre-IM era, no differences in outcomes were found between patients with CP- patients than in those with e13a2. In a larger study, Hanfstein et al. [25] demonstrated CML expressing the e13a2 or e14a2 transcripts [6–8,24,29]. In the IM era, some studies that the probability of achieving MR3.0 or MR4.0 was significantly higher in e14a2 than in have addressed the influence of the BCR-ABL1-p210 transcript type with response to IM e13a2 patients. Baccarani et al. [3] described no statistically significant differences in CCyR treatment and CML outcome with controversial results [9–12]. Lucas et al. [2] found that achievement, while the major molecular response rate was lower in e13a2 patients in seven the one-year CCyR rate in 78 patients treated with IM 400 mg daily was higher in e14a2 of the eight studies analyzed. In our analysis, the cumulative incidence of cytogenetic and patients than in those with e13a2. In a larger study, Hanfstein et al. [25] demonstrated that J. Clin. Med. 2021, 10, 3146 9 of 12 molecular responses during the first 12 months of IM treatment was similar in both groups of patients, with optimal responses at 3 and 6 months being also similar. Nevertheless, we observed a better cumulative incidence of major molecular responses at 6 months in the e14a2 group, comparable to previously reported data, and supporting the concept that this transcript type may be more sensitive to IM [3,11,12,30,31]. Correlation between BCR-ABL1 transcript type and the probability of achieving DMR and its duration has less frequently been investigated. However, some studies demon- strated a significantly lower rate of DMR in the e13a2 subgroup [3,14,23], while few studies showed that patients with the e14a2 transcript have prolonged sDMR [14,15]. In our study, we did not observe differences in the number of patients that acquired DMR between the two groups nor in the time of the achievement such DMR and the sDMR, maybe because some patients changed to a second TKI and were censored for the analysis at that time. On the contrary, we found a tendency and a significant correlation between ELTS and Sokal scores, respectively, and the acquisition of DMR according to what was published by Breccia et al. [15] who found an association between a stable DMR and Sokal risk in univariate and multivariate analysis. It has also been suggested that the transcript type not only affected response outcomes, but also long-term OS and CIP to acute leukemia [11]. Additionally, the e14a2 transcript has been correlated with longer OS, and on the contrary, patients with e13a2 may have an aggressive course. Although previous studies did not show any difference in event-free survival (EFS) or transformation-free survival (TFS) according to transcript type among patients treated with IM [22,32], Jain et al. [11] analyzed the impact of transcript types in 481 patients with CML treated with different TKI regimens and suggested that the e13a2 transcript may negatively impact EFS and TFS, but these results were not statistically significant. Castagnetti et al. [4] demonstrated a better 7-year OS in patients with the e14a2 transcript. In contrast, Baccarani et al. [3] did not observe differences in the impact of transcript type in PFS and OS, and Pagnano et al. [32] observed a superior 10-year OS in patients with e13a2 transcript, although they could not find differences at 5-year OS. In our study, we found significant differences in OS between the two groups, with the e13a2 transcript having a better OS than the e14a2 transcript despite a tendency to better molecular response at 6 months in the e14a2 group. One plausible explanation of this discrepancy might be that due to the efficacy of TKI treatment most patients die in the long term from causes other than CML due to older age, toxicities of TKIs, or co-morbidities especially under treatment with second- or third-generation TKI. Other factors such as the resistance to more than one TKI might also play a role in the long-term OS. All these data suggest several discrepancies between studies about OS and transcript type relationship that should be investigated in larger trials [12]. Patients with e14a2 transcript predicted for longer transformation-free survival and lower progression to acute leukemia [11]. We did not observe differences in CIP between the two groups with only eight patients who progressed to accelerated phase and five out of eight who died due to CML. Although these patients who died were young patients with high-risk Sokal and ELTS scores, and nowadays, they would be treated with second-generation TKI, they were treated with imatinib because no other options as first line were possible at the moment of their diagnosis. Some limitations of our study are the limited number of cases available for analyzing the endpoints of our study, its retrospective and multicenter nature, and the different treatment approaches that have emerged throughout the years since IM became available. There may be also technical limitations, because the same PCR assay is used to amplify both transcripts variants, and some studies have suggested that e14a2 amplification may be technically limited during RT-qPCR quantification [33,34]. Moreover, a polymorphism in exon 13 of BCR that reduced the efficiency of the primers used to amplify e13a2 has been described [3]. There are few studies focused on the effect of transcript type and the maintenance of a long-term TFR after TKI discontinuation. D’Adda et al. [14] reported the negative effect J. Clin. Med. 2021, 10, 3146 10 of 12 of e13a2 transcript on a durable TFR in a larger series of 173 patients and suggested that differences in immunogenic ability between the two transcript types may be associated with the risk of relapse after TKI discontinuation [14], and other studies postulated that IM treatment and DMR duration may modulate the immunogenic system [35,36]. Our results, with a lower number of cases, are in line with D’Adda et al.’s study suggesting a negative effect of e13a2 transcript on TFR. In summary, in this CP-CML series of patients treated with IM in first line, the e14a2 transcript type may have a positive impact on cumulative incidence of major molecular response at 6 months, whereas the e13a2 transcript type might be associated with improved long-term OS. The influence of transcript type on outcomes in CML remains controversial and needs further investigations with a larger series to consider transcript type as a prognostic factor associated with TFR. For the time being, we think that the transcript type (e13a2 vs. e14a2) should not guide treatment decision making in CML. Supplementary Materials: The following are available online at https://www.mdpi.com/article/10 .3390/jcm10143146/s1, Figure S1: Diagram of CML cases in each analysis performed. Author Contributions: S.M., B.X., M.C. (Marta Cabezón), N.E., P.V., C.B., M.S., A.A., R.T.-M., F.F.-M., P.A., J.C.H.-B., M.M.I., E.A., M.C. (Montserrat Cortés), A.F.-R., S.F., L.Z. made substantial contri- butions to the conception of the work and acquisition of data. Interpretation, formal analysis and investigation of data, S.M. and O.G.; writing—original draft preparation, S.M.; writing review, B.X., J.C.H.-B., and L.Z.; software, O.G. All authors have read and agreed to the published version of the manuscript. Funding: This work was supported in part by a grant from Instituto de Salud Carlos III through the project “PI16/01200” as well as 2017 SGR288 (GRC) Generalitat de Catalunya. Institutional Review Board Statement: The study was conducted according to the guidelines of the Declaration of Helsinki, and approved by the Institutional Research Boards protocols and the Ethics Committee of Hospital Germans Trias i Pujol (Ref. CEI: PI-15-007, Code: ICO-ITK-2015-01 (ISOF-p210-LMC), 24 April 2015) Informed Consent Statement: Information was collected after obtaining informed consent from all patients. Data Availability Statement: The data present in the study can be partialy available upon the request to corresponding author, under national regulations for data sharing. Acknowledgments: The authors would like to thank Diana Domínguez for her technical support, the Grupo Español de Leucemia Mieloide Crónica (GELMC) for its project support and the CERCA Programme/Generalitat de Catalunya, Josep Carreras International Foundation and “La Caixa” Foundation for institutional support. Conflicts of Interest: The authors declare no conflict of interest. References 1. Deininger, M.W.; Goldman, J.M.; Melo, J.V. The molecular biology of chronic myeloid leukemia. Blood 2000, 96, 3343–3356. [CrossRef] 2. Lucas, C.M.; Harris, R.J.; Giannoudis, A.; Davies, A.; Knight, K.; Watmough, S.J.; Wang, L.; Clark, R.E. Chronic myeloid leukemia patients with the e13a2 BCR-ABL fusion transcript have inferior responses to imatinib compared to patients with the e14a2 transcript. 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