Powered by EHA Article OPEN ACCESS Influence of Telomere Length on the Achievement of Deep Molecular Response With Imatinib in Downloaded from http://journals.lww.com/hemasphere by BhDMf5ePHKav1zEoum1tQfN4a+kJLhEZgbsIHo4XMi0hCy Chronic Myeloid Leukemia Patients wCX1AWnYQp/IlQrHD3i3D0OdRyi7TvSFl4Cf3VC1y0abggQZXdgGj2MwlZLeI= on 04/20/2023 Natalia Estrada1, Blanca Xicoy1,2, Fabian Beier3, Olga Garcia1,2, Cristian Morales4, Concepción Boqué5, Miguel Sagüés6, Mónica S. Ventura Ferreira3, Rolando Vallansot7, Sílvia Marcé1,2, Marta Cabezón1,2, Tim H. Brümmendorf3, Lurdes Zamora1,2 Correspondence: Blanca Xicoy (bxicoy@iconcologia.net). Abstract Tyrosine kinase inhibitors have dramatically changed the outcome of chronic myeloid leukemia (CML), and nowadays, one of the main treatment goals is the achievement of deep molecular responses (DMRs), which can eventually lead to therapy discontinuation approaches. Few biological factors at diagnosis have been associated with this level of response. Telomere length (TL) in peripheral blood cells of patients with CML has been related to disease stage, response to therapy and disease progression, but little is known about its role on DMR. In this study, we analyzed if age-adjusted TL (referred as “delta-TL”) at diagnosis of chronic phase (CP)-CML might correlate with the achievement of DMR under first-line imatinib treatment. TL from 96 CP-CML patients had been retrospectively analyzed at diagnosis by monochrome multiplex quantitative PCR. We observed that patients with longer age-adjusted telomeres at diagnosis had higher probabilities to achieve DMR with imatinib than those with shortened telomeres (P = 0.035 when delta-TL was studied as a continuous variable and P = 0.047 when categorized by the median). Moreover, patients carrying long telomeres also achieved major molecular response significantly earlier (P = 0.012). This study provides proof of concept that TL has a role in CML biology and when measured at diagnosis of CP-CML could help to identify patients likely to achieve DMR to first-line imatinib treatment. Introduction so-called Philadelphia chromosome. This translocation encodes for the constitutively active tyrosine kinase BCR-ABL1, which Chronic myeloid leukemia (CML) is a clonal hematopoi- promotes exacerbated myeloproliferation, resistance to apopto- etic stem cell disease caused by the acquisition of a reciprocal sis and survival advantage.1 translocation between chromosomes 9 and 22, producing the Imatinib, the first tyrosine kinase inhibitor (TKI) developed, is rather selective for the BCR-ABL1 oncoprotein and has dra- matically improved the outcome of CML patients. This drug 1 Myeloid Neoplasms Research Group, Josep Carreras Leukemia Research is capable of inducing complete cytogenetic response in the Institute, Institut Català d’Oncologia-Hospital Germans Trias i Pujol, Badalona, majority of chronic phase CML (CP-CML) patients, and almost Universitat Autònoma de Barcelona, Spain all these patients normally achieve a major molecular response 2 Hematology Department, Institut Català d’Oncologia (ICO)-Hospital Germans (MMR), defined as BCR-ABL1 ≤0.1% on the international scale Trias i Pujol, Institut de Recerca Contra la Leucèmia Josep Carreras, Universitat (IS). However, with imatinib, only 30%–40% of patients achieve Autònoma de Barcelona, Spain 3 Department of Hematology, Oncology, Hemostaseology and Stem Cell a deep molecular response (DMR) defined as either MR4.0, BCR- Transplantation, Medical Faculty, Uniklinik RWTH Aachen University, Germany ABL1IS ≤0.01% or MR4.5, BCR-ABL1IS ≤0.0032%),2 while with 4 Core-Hematology Department, Clinical Laboratory ICS-Metropolitan Nord, the introduction of second and third generation TKI at first line, Hospital Germans Trias i Pujol, Badalona, Spain such as nilotinib, dasatinib, and bosutinib, higher rates as well 5 Hematology Department, Institut Català d’Oncologia – Hospital Duran i Reynals, as earlier achievement of DMR have been observed.3–5 Recent Hospitalet de Llobregat, Spain clinical trial data have demonstrated that TKI therapy can be 6 Hematology Department, Institut Català d’Oncologia-Hospital Universitari Doctor safely discontinued in patients with sustained DMR (sDMR), Josep Trueta, Girona, Spain leading to a successful long-term treatment-free remission (TFR) 7 Hematology Department, Institut Català d’Oncologia-Hospital Universitari Joan XXIII, Tarragona, Spain in 40%–50% of patients.6,7 In clinical practice, the TFR rate is Data that support the findings of this study are available from the corresponding slightly higher (~65%), likely due to longer duration of both author upon request. TKI exposure and sDMR, compared to clinical trials.8,9 Thus, Supplemental digital content is available for this article. the achievement of DMR has recently become one of the most Copyright © 2021 the Author(s). Published by Wolters Kluwer Health, Inc. important treatment goals in CML. on behalf of the European Hematology Association. This is an open-access Several clinical scores, such as Sokal, European Treatment article distributed under the terms of the Creative Commons Attribution-Non and Outcomes Study (EUTOS), Hasford and EUTOS long-term Commercial-No Derivatives License 4.0 (CCBY-NC-ND), where it is permissible survival (ELTS),10–13 are used to predict the outcomes of patients to download and share the work provided it is properly cited. The work cannot be changed in any way or used commercially without permission from the journal. diagnosed with CP-CML, although there are no clear recom- HemaSphere (2021) 5:12(e657). mendations regarding the most suitable TKI, especially in low- http://dx.doi.org/10.1097/HS9.0000000000000657. risk disease.14 Currently, the best way to adjust TKI treatment is Received: 7 May 2021 / Accepted: 11 October 2021 by assessing response at different milestones. One of these is the 1 Estrada et al Telomeres in CML and Deep Molecular Response to Imatinib “early molecular response”; a BCR-ABL1/ABL1IS ratio of >10% Duran i Reynals (n = 38), and ICO-Hospital Josep Trueta at 3 months from TKI initiation has been related to a worse (n = 22), between 2005 and 2016, according to the 2008 World response later on and even worse overall survival.15 Another Health Organization (WHO) classification.31 Retrospective example is the “halving time,” which evaluates the kinetics of DNA samples from PB and bone marrow (BM) at diagnosis decline of BCR-ABL1 transcripts during the first 3 months of were collected from all cases. treatment.16 Moreover, Sasaki et al17 evaluated the best-fit aver- Study approval was obtained from Institut Català d’On- age and minimum acceptable BCR-ABL1 levels within 1 year cología-Hospital Germans Trias i Pujol Ethics Committee (from 3 to 12 mo) of treatment to predict the achievement of (Ref. PI-17-261), and informed consent was provided by all Downloaded from http://journals.lww.com/hemasphere by BhDMf5ePHKav1zEoum1tQfN4a+kJLhEZgbsIHo4XMi0hCy sDMR at any point. However, all this prognostic information is the patients. The study was undertaken in accordance with the obtained while the patient is already under treatment (ie, “semi- Declaration of Helsinki. prospectively”) and not at diagnosis, thus clinical decisions All patients were selected according to the following criteria: wCX1AWnYQp/IlQrHD3i3D0OdRyi7TvSFl4Cf3VC1y0abggQZXdgGj2MwlZLeI= on 04/20/2023 must be suddenly changed once the TKI has been chosen and (1) first-line treatment with imatinib, 400 mg orally once a day; milestones established are not met. Knowing the probabilities to (2) a minimum of 1 year of follow-up; (3) absence of toxicity or achieve DMR with imatinib at diagnosis using molecular mark- intolerance to imatinib that required a change of TKI treatment ers could improve patient care and help to choose the best TKI or a dose reduction; and (4) the absence of mutations in ABL1 (imatinib versus a more potent second or third generation TKI) gene or additional cytogenetic abnormalities. when TFR is the main treatment goal. In this study, TL of CML patients was adjusted for age using Several studies have defined molecular markers that can pre- PB samples from 107 healthy subjects. dict response to imatinib at diagnosis, such as OCT-1, ABCB1, and PTCH1 expression levels or polymorphic variants,18–20 as well as a model based on a multigene expression signature, DNA extraction which is able to identify patients who are at very high risk DNA from PB (whole blood) samples at CML diagnosis was of early molecular response failure.21 However, none of these used, because positive correlation between TL measured with markers has been correlated with DMR. So far, the only bio- this type of sample has been observed.32 DNA from PB samples marker that has been related to higher rates of DMR achieve- was not available in 29 patients and in those cases DNA from ment in some but not all studies is the e14a2 transcript type.22–24 BM samples was used, as at diagnosis no TL differences between Telomeres represent a promising predictive molecular marker these 2 different samples have been shown.33 in CML. Telomeres are repeat DNA sequences (TTAGGG) DNA was extracted automatically using QIAcube (Qiagen, located at the end of the chromosomes. In somatic cells, telo- Germany) with the QIAamp Blood Mini Kit (Qiagen). DNA meres shorten with each cell division, reflecting the replicative concentration and quality status were assessed with a NanoDrop history of a cell, and can eventually lead to genetic instability spectrometer. and cellular senescence. In CML, increased cellular turnover of Fresh PB samples from 107 age-matched healthy subjects (age BCR-ABL1-positive stem and progenitor cells leads to signifi- range 16–84 y) were used for TL age-adjustment, applying lin- cantly shortened telomeres in peripheral blood (PB) myeloid ear regression analysis, and DNA was extracted as described cells.25 Mechanistically, an inflammatory phenotype called above. “telomere-associated secretory phenotype” has been suggested We used a PB sample from a healthy donor to generate a to contribute, via secretion of chemokines and interleukins, to standard curve for the monochrome multiplex (MM)-qPCR BCR-ABL1-mediated growth and thus CML onset.26 method. DNA was extracted manually using the Gentra Puregen Accelerated telomere shortening in CML has previously been Blood Kit (Qiagen) as it allows higher DNA concentrations to correlated with disease stage, clinical risk scores at diagnosis, be obtained. cytogenetic remission status as well as progression to acceler- ated phase or blast crisis.27,28 Moreover, telomere length (TL) at diagnosis of CML has been associated with MMR at 12 and 18 Definition of molecular response months of first-line nilotinib treatment.29 Altogether, these data provide further evidence for the cru- qPCR was used for measuring BCR-ABL1 transcripts, as cial role of telomere biology in CML, especially in terms of described previously.34 ABL1 was used as the control gene, and response and disease progression. To our knowledge, no study the results were reported as %BCR-ABL1/ABL1IS. Molecular has explored the association between TL at diagnosis and the monitoring was performed at diagnosis (baseline) and every 3–6 achievement of DMR to first-line imatinib treatment. months of imatinib treatment thereafter. Therefore, in this study, we retrospectively analyzed mean The 2020 ELN recommendations30 were considered to assess age-adjusted TL (referred as delta-TL) in CML patients at diag- MR to first-line imatinib treatment, that is: BCR-ABL1/ABLIS nosis. We evaluated whether delta-TL would be useful to iden- ≤10% at 3 months, <1% at 6 months and ≤0.1% (MMR) at tify individuals likely to achieve sDMR with first-line imatinib 12 months and later. Moreover, the primary study end-point treatment. Additionally, we aimed to (1) evaluate the association included the achievement of sDMR, defined as MR4.0 and/or between delta-TL at diagnosis and risk scores as well as BCR- MR4.5, confirmed on 2 or more consecutive determinations ABL1-p210 transcript type; (2) analyze if delta-TL at diagnosis has an impact on the achievement of optimal MR according to European LeukemiaNet (ELN) recommendations30; and (3) Telomere length measurements by MM-qPCR determine if there are differences in terms of delta-TL at diag- TL was analyzed by MM-qPCR, as previously described by nosis between patients that do achieve MMR but may or not Cawthon,35 using a CFX384 Touch Real-Time PCR Detection achieve MR4.0. System (Bio-Rad, Spain), and followed previously used proto- cols.36,37 All samples, standards, and controls were run in trip- licate, and the median value was used for the analyses. TL was Material and methods calculated as a ratio between fluorescence detected from telomere Patients, samples, and study design repeat copy number (T) and a single copy reference sequence (S) from the human beta globin gene. Primer pairs used for telomere A total of 96 adult patients with CP-CML were enrolled (T) amplification were telg 5′-ACACTAAGGTTTGGGTTTG in this study. Patients were consecutively diagnosed in ICO- GGTTTGGGTTTGGGTTAGTGT-3′ and telc 5′-TGTTAGGTAT Hospital Germans Trias i Pujol (n = 36), ICO-Hospital CCCTATCCCTATCCCTATCCCTATCCCTAACA-3′, and 2 (2021) 5:12 www.hemaspherejournal.com signal acquisition was at 74°C. Human beta-globin was patients were of low risk, 24 (29%) were of intermediate risk, used as single copy reference gene (S) using the primers hbgu and 10 (12%) were of high risk. Thirty-one (39%) patients had 5′-CGGCGGCGGGCGGCGCGGGCTGGG CGGCTTCATC the BCR-ABL1-p210 e13a2 transcript type, whereas 49 (61%) CACGTTCACCTTG-3′ and hbgd 5′-GCCCGGCCCGCCGC had the e14a2 transcript. The median follow-up of alive patients GCCCGTCCCGCCGGAG GAGAAGTCTGCCGTT-3′, and was 7.3 years (range, 1.3, 14.6 y). The median length of front- signal acquisition was at 88°C. CFX Manager Software (Bio- line imatinib treatment was 3.4 years (range, 0.3, 14.4 y). In Rad) was used to analyze the raw data and generate 2 standard total, 10 patients (10.4%) were exitus (Table 1). curves (one for each signal acquisition temperature; for the telo- Regarding TL, healthy controls showed the expected decline Downloaded from http://journals.lww.com/hemasphere by BhDMf5ePHKav1zEoum1tQfN4a+kJLhEZgbsIHo4XMi0hCy mere signal and for the single copy gene signal). Samples values with increasing age (R2 = 0.1693), a characteristic that was not were normalized and the telomere/single copy gene ratio (T/S detectable in CML patients (R2 = 0.0046) (Figure 1). ratio) was calculated for each DNA sample. We also tested the correlation of both sample sources used wCX1AWnYQp/IlQrHD3i3D0OdRyi7TvSFl4Cf3VC1y0abggQZXdgGj2MwlZLeI= on 04/20/2023 The age-adjusted T/S ratio (or age-adjusted TL), hereafter in our CML cohort and found no differences in the T/S ratio referred to as delta-TL, was calculated using data from 107 between PB and BM (median T/S ratio [95% confidence interval healthy subjects. Delta-TL represents the difference between the (CI)]: 0.60 (0.59, 0.71) versus 0.55 (0.51, 0.66), respectively, CML patient’s T/S ratio, calculated by MM-qPCR, and the cor- P = 0.28, Supplemental Digital Figure S1; http://links.lww.com/ responding TL expected according to age (calculated also by HS/A207). MM-qPCR using data from healthy subjects), with higher del- ta-TL values meaning shorter telomeres. All statistical analyses were performed using the delta-TL value for each patient. Correlation between delta-TL at diagnosis and the In this study, the average intra-assay variability for all sam- achievement of deep molecular response to first- ples (n = 203, control and CML samples included) was 8%. line imatinib treatment To monitor plate-to-plate variation, 4 reference samples (2 with known long telomeres and 2 with known short telomeres) were We examined the correlation between achieving or not MR4.0 included in each run and the resulting average inter-assay vari- and MR4.5 and the delta-TL at diagnosis as a continuous variable ability was 14%. and observed that lower delta-TL at diagnosis was significantly associated with the achievement of stable MR4.0 and MR4.5 (haz- ard ratio [HR] [95% CI]: 0.3 (0.1, 0.9), P = 0.035 and HR [95% Statistical analysis CI]: 0.22 (0.1, 0.66), P = 0.007, respectively). Then, we categorized delta-TL into quartiles (Q1: 0.06 ± 0.15; The study group characteristics were described as frequency Q2: 0.34 ± 0.03; Q3: 0.48 ± 0.05; Q4: 0.67 ± 0.08) and stud- and percentage for categorical variables and median and range ied the cumulative incidence of achieving MR4.0 at any time; for quantitative variables. Comparisons of continuous variables patients carrying longer telomeres at diagnosis (Q1) presented between groups were made using the median test, while cate- the highest rate of MR4.0 achievement, followed by patients gorical variables were compared using the χ2 test or Fisher exact allocated in Q2. Due to similar cumulative incidence of MR4.0 test, if necessary. curves between Q1 and Q2, and also between Q3 and Q4, we Due to the retrospective nature of this study including real- considered median delta-TL value (ie, delta-TL = 0.4) as the cut- world data and in line with previous studies,38,39 competing off for this variable. Descriptively, we found a dose-dependent risk analysis was used for all the time-dependent variables. The correlation, meaning that the longer the telomeres at diagnosis, achievement of MMR and/or DMR to imatinib was consid- the higher the probabilities of achieving DMR (Supplemental ered as the main event and the associated time was considered Digital Figure S2; http://links.lww.com/HS/A208). from imatinib start to documented MR (MMR, MR4.0 or MR4.5, Therefore, when patients were stratified according to median depending on the analysis). Patients who switched TKI without delta-TL value, the cumulative incidence (95% CI) of MR4.0 at achieving documented MR or those who did not change TKI any time between the 2 groups was 63% (45%, 76%) for del- but died without MR response, were considered as competitive ta-TL <0.4 and 46% (31%, 60%) for delta-TL ≥0.4 (P = 0.047). events; time from imatinib start to TKI change or death was Median cumulative incidence of MR4.0 in delta-TL <0.4 group considered for these competitive events. Alive patients without was at 27 months from imatinib start, while this point was not TKI change and without documented MR were considered as reached in delta-TL ≥0.4 group, since cumulative incidence for censures, and the associated time in these cases was the time MR4.0 in this subpopulation was <50% (Figure 2A). from imatinib start to last follow-up. Cumulative incidence Descriptively, 29 (62%) of 47 patients in delta-TL <0.4 group curves of MR were plotted and a multivariate analysis for MR4.0 and 22 (45%) of 49 of delta-TL ≥0.4 group achieved MR4.0 with was performed by the Fine and Gray model. a median (range) time of 14 months (6.2, 71.4) and 17 months Two-sided P values <0.05 were considered statistically sig- (5.5, 100.7), respectively. nificant. The statistical packages SPSS version 24.0 (SPSS Inc., Regarding MR4.5, the cumulative incidence (95% CI) was Chicago, IL) and R v4.0.1 software were used for all the anal- 59% (36%, 76%) for the delta-TL <0.4 group and 37% (22%, yses and the creation of graphics. GraphPad Prism 5 software 52%) for the delta-TL ≥0.4 group (P = 0.045), and for patients was also used for the creation of graphics and figures. allocated in the delta-TL <0.4 group, median cumulative inci- dence of MR4.5 was 77 months from imatinib start, while it was Results not reached in the delta-TL ≥0.4 group (Figure 2B). Moreover, cumulative incidences (95% CI) of MR4.0 were Clinical and biological characteristics of the studied establishing a cutoff at 4 years, being 58% (44%, 70%) patients of the series for the delta-TL <0.4 group and 39% (26%, 52%) for delta-TL ≥0.4 (P = 0.049). Cumulative incidences of MR4.5 at 4 years The series included a total of 96 CP-CML patients: 56 (58%) were 45% (30%, 59%) for the delta-TL <0.4 group and 29% males and 40 (42%) females, with a median age at diagnosis (17%, 42%) for delta-TL ≥0.4 (P = 0.08) (Supplemental Digital of 49 years (range 24–80 y). According to the Sokal score, 46 Figure S3; http://links.lww.com/HS/A209). (50%) patients were of low risk, 30 (32%) were of interme- We also explored if there were differences between CML diate risk, and 17 (18%) were of high risk. According to the patients that did achieve MMR (n = 64) but did or did not achieve EUTOS score, 77 (88%) and 11 (12%) were of low and high MR4.0 at any time. Analyzing the incidence of MR4.0 in this sub- risk, respectively. Finally, according to the ELTS score, 50 (59%) population (only patients that achieved MMR), we observed an 3 Estrada et al Telomeres in CML and Deep Molecular Response to Imatinib Table 1. Patient Characteristics Total Delta-TL<0.4 Delta-TL≥0.4 Clinical Characteristics n = 96 n = 47 n = 49 P Median age at diagnosis, y (range) 49 (24–80) 59 (30–80) 43 (24–77) ** Sex ns Downloaded from http://journals.lww.com/hemasphere by BhDMf5ePHKav1zEoum1tQfN4a+kJLhEZgbsIHo4XMi0hCy Female, n (%) 40 (42) 20 (43) 29 (59) Male, n (%) 56 (58) 27 (57) 20 (41) Sokal risk score, n (%) ns Low 46 (50) 22 (50) 24 (49) wCX1AWnYQp/IlQrHD3i3D0OdRyi7TvSFl4Cf3VC1y0abggQZXdgGj2MwlZLeI= on 04/20/2023 Intermediate 30 (32) 16 (36) 14 (29) High 17 (18) 6 (14) 11 (22) EUTOS score, n (%) * Low 77 (88) 40 (95) 37 (80) High 11 (12) 2 (5) 9 (20) ELTS score, n (%) ns Low 50 (59) 26 (63) 24 (56) Intermediate 24 (29) 11 (27) 13 (30) High 10 (12) 4 (10) 6 (14) p210 isoform, n (%) ns e13a2 31 (39) 14 (36) 17 (42) e14a2 49 (61) 25 (64) 24 (58) Imatinib treatment duration, y (range) 3.4 (0.3–14.4) 3.2 (0.3–14.4) 3.8 (0.3–14) ns Median follow-up, y (range) 7.3 (1.3–14.6) 6.7 (1.5–14.4) 7.1 (1.3–14.6) ns Exitus, n (%) 10 (10.4) 4 (8.5) 6 (12.2) ns ELTS = EUTOS long-term survival; EUTOS = European treatment and outcomes study; ns = not significant. *P < 0.05; **P < 0.001. earlier achievement of MR4.0 in the delta-TL <0.4 group, but dif- with TL at diagnosis. We did not observe any association with ferences were not statistically significant (median in months [95% the prognostic risk scores (Sokal, EUTOS, and ELTS) nor with CI]: 16.3 (12.4, 33.4) in delta-TL <0.4 versus 31.8 (16.2, 100.7) in transcript type when delta-TL was studied as a continuous vari- delta-TL ≥0.4, P = 0.144). In this subgroup of patients, cumulative able. However, when delta-TL was categorized by the median, incidence (95% CI) of MR4.0 was 84% (65%, 94%) for delta-TL we found a statistically significant correlation with the EUTOS <0.4 and 75% (54%, 87%) for delta-TL ≥0.4 (Supplemental score (P = 0.036), given that only two patients out of 42 (5%) Digital Figure S4; http://links.lww.com/HS/A210). with delta-TL <0.4 were classified within the EUTOS high-risk group (Table 1). Correlation between delta-TL at diagnosis, risk scores, and BCR-ABL1-p210 transcript type Multivariate analysis for MR4.0 achievement Associations between delta-TL and prognostic variables of Moreover, we performed a multivariate analysis to study the clinical relevance (Sokal, Eutos, ELTS, and BCR-ABL1-p210 role of delta-TL as an independent predictor of MR4.0. Variables transcript type) were examined to find any possible correlation significantly associated with MR4.0 in univariate analysis were Figure 1. Telomere length of chronic myeloid leukemia patients and healthy controls in relation to age. Relative TL expressed as T/S ratio plotted against age; healthy donors (gray circles) show the expected decline in TL with increasing age (r2 = 0.1693), whereas CML patients (black squares) runs nearly horizontally (r2 = 0.0046). CML = chronic myeloid leukemia; T/S = telomere/single copy gene ratio; TL = telomere length. 4 (2021) 5:12 www.hemaspherejournal.com Downloaded from http://journals.lww.com/hemasphere by BhDMf5ePHKav1zEoum1tQfN4a+kJLhEZgbsIHo4XMi0hCy wCX1AWnYQp/IlQrHD3i3D0OdRyi7TvSFl4Cf3VC1y0abggQZXdgGj2MwlZLeI= on 04/20/2023 Figure 2. Cumulative incidence (95% CI) of MR4.0 and MR4.5 according to delta-TL categorized by the median. (A) Cumulative incidence of MR4.0. (B) Cumulative incidence of MR4.5. CI = confidence interval; TL = telomere length. selected; delta-TL and ELTS score showed statistical significant molecular markers that could prospectively identify patients differences in MR4.0 between their categories, reaching higher with a high likelihood of achieving a DMR with a particular TKI, cumulative incidence of MR4.0 in delta-TL <0.40 and low risk thus allowing them to follow a strategy aimed at the achieve- ELTS score (cumulative incidence of MR4.0 [95% CI]: 63% [45%, ment of a TFR right from treatment initiation. In this study, 76%] for delta-TL <0.4 versus 46% [31%, 60%] for delta-TL we retrospectively evaluate if TL assessment by MM-qPCR at ≥0.4, P = 0.047, and 58% [42%, 71%] for low risk ELTS versus diagnosis of 96 CP-CML patients could be useful to identify 38% [21%, 55%] for intermediate-high risk ELTS, P = 0.041). those patients. In line with previous models, our results now However, in the multivariate analysis, delta-TL lost its signif- show clinically that patients with shortened telomeres at diag- icance (HR [95% CI]: 1.7 (0.92, 3.22), P = 0.089), whereas nosis (ie, a high delta-TL value) are likely to fail in achieving ELTS score retained its significance as an independent predictor DMR (both MR4.0 and MR4.5) compared to those with longer of MR4.0 (HR [95% CI]: 1.9 (1.02, 3.73), P = 0.045) (Table 2). telomeres (Figure 2). To our knowledge, the only study that has related TL with molecular response to TKI showed that TL at diagnosis of CML Correlation between delta-TL at diagnosis and was associated with MMR achievement at 12 and 18 months molecular response according to the 2020 ELN under first-line nilotinib treatment.29 Even though it was not the recommendations Finally, we studied the relation between TL at diagnosis and optimal molecular response to treatment according to the 2020 Table 2. ELN recommendations.30 Univariate and Multivariate Analysis for MR4.0 When analyzing delta-TL as a continuous variable, we did Univariate Analysis not find any association between delta-TL and the cutoffs estab- lished by ELN for BCR-ABL1/ABL1(IS) ratio at 3, 6, nor 12 Cumulative Incidence months after initiation of imatinib treatment (Supplemental Variable MR4.0 (95% CI) P Digital Table S1; http://links.lww.com/HS/A206). However, we delta-TL < 0.40 (n = 47) 63% (45%, 76%) 0.047 observed a trend toward a correlation between delta-TL and the ≥ 0.40 (n = 49) 46% (31%, 60%) achievement of MMR at 18 months (P = 0.075), indicating that Sokala Low (n = 46) 50% (37%, 61%) 0.312 patients with longer telomeres at diagnosis could have a higher Int-High (n = 47) 26% (14%, 39%) probability of achieving MMR at 18 months with imatinib than EUTOS Low (n = 77) 56% (43%, 66%) 0.414 patients with shorter telomeres. This association was confirmed High (n = 11) 36% (8%, 66%) when delta-TL was categorized by the median, with 78% and ELTSa Low (n = 50) 58% (42%, 71%) 0.041 56% of patients with delta-TL <0.4 and delta-TL ≥0.4, respec- Int-High (n = 34) 38% (21%, 55%) tively, achieving MMR at 18 months (P = 0.049) (Supplemental Isoform e13a2 (n = 31) 42% (23%, 60%) 0.151 Digital Table S1; http://links.lww.com/HS/A206). Finally, cumu- e14a2 (n = 49) 55% (40%, 68%) lative incidence analysis (95% CI) of MMR at 24 months from Multivariate Analysis imatinib start also supported this finding, being 68% (55%, 78%) for the delta-TL <0.4 group and 43% (30%, 56%) for Variable Reference Category HR (95% CI) P delta-TL ≥0.4 (P = 0.012) (Figure 3). delta-TL ≥ 0.40 1.7 (0.92, 3.22) 0.089 ELTS Int-High 1.9 (1.02, 3.73) 0.045 Discussion a Sokal and ELTS scores have been regrouped into 2 categories (low versus intermediate-high) in order to balance the number of patients between categories. Treatment with TKIs has dramatically improved CML out- CI = confidence interval; ELTS = EUTOS long-term survival; EUTOS = European treatment and comes up to the point of introducing the concept of TFR in outcomes study; HR = hazard ratio. optimal responders. However, there is still a lack of predictive Bold values mean they are statistically significant (P < 0.05). 5 Estrada et al Telomeres in CML and Deep Molecular Response to Imatinib However, only ELTS retained significance in multivariate analy- sis, although delta-TL showed a trend toward higher incidence of MR4.0 in low delta-TL patients. We also evaluated the impact of delta-TL at diagnosis on achieving the different degrees of MR at 3, 6, and 12 months, according to the 2020 ELN recommendations, but no correla- tion was observed. Only at 18 months of imatinib treatment, patients with delta-TL <0.4 at diagnosis significantly achieved Downloaded from http://journals.lww.com/hemasphere by BhDMf5ePHKav1zEoum1tQfN4a+kJLhEZgbsIHo4XMi0hCy MMR compared to those with delta-TL ≥0.4. In line with this observation, Wenn et al29 described a correlation between longer TL at diagnosis of CML and achievement of MMR at 12 and wCX1AWnYQp/IlQrHD3i3D0OdRyi7TvSFl4Cf3VC1y0abggQZXdgGj2MwlZLeI= on 04/20/2023 18 months with first-line nilotinib treatment, but they could not confirm this association at 3 or 6 months. As far as we know, the only study that has reported the use of a biomarker to predict DMR to imatinib therapy is the one recently published by Park et al.42 In this study, they described a polymorphism in HMGCLL1 gene that predicts intrinsic sensitivity to imatinib therapy which may be used to identify those patients at risk of not achieving DMR. Moreover, they postulated that HMGCLL1 blockade could potentially sensitize Figure 3. Cumulative incidence (95% CI) of MMR at 24 months from leukemic stem cells to TKI therapy, although no preclinical data imatinib start. CI = confidence interval are available. Despite being a promising marker, it needs further validation. In contrast, telomere biology has been widely related to CML pathology and has already been validated as a useful main endpoint of our study, we also observed statistically sig- marker in several studies.25–29 nificant differences in cumulative incidence of MMR between Despite our good results, we are aware that quantitative fluo- delta-TL <0.4 and delta-TL ≥0.4 groups, which started to be rescence in situ and flow cytometry (flow-FISH) based method- evident after 6 months of imatinib onset (Figure 3). Moreover, in our study we showed that longer telomeres (delta-TL < 0.4) ologies have shown to be more accurate than MM-qPCR for TL were positively correlated with the primary study endpoint, that measurement in vivo,36,43 but flow-FISH requires fresh viable PB is, a superior molecular response defined as MR4.0 and MR4.5. or BM samples and this material was not available because of the In this case, the largest differences between the 2 categorical retrospective nature of our study. Validation of our results in large groups (delta-TL <0.4 versus delta-TL ≥0.4) in terms of DMR prospective studies using flow-FISH would be of special interest. were mostly observed after 2 years of treatment (Figure 2), in In summary, telomere length measured by MM-qPCR in line with previous clinical trial data, in which cumulative rates either PB or BM samples at diagnosis of CML identified a of MR4.0 with imatinib were reported to be around 60% after previously unrecognized patient subgroup likely to achieve 10-year follow-up.2–5 both MMR and DMR with front-line imatinib treatment. We Caocci et al40 found a significant correlation between short believe that telomere length measurement could complement age-adjusted TL and higher rates of TFR. However, in this study other clinical prognostic scores at diagnosis (such as ELTS), TL was measured using PB samples obtained during the discon- providing additional information about the probability of tinuation phase (off TKI), when residual disease is minimal and DMR achievement. This molecular marker may be useful to often undetectable. In this scenario, in which normal hemato- guide the choice of TKI at diagnosis when DMR achievement poiesis predominates, this correlation may not be explained by is the objective as a road to treatment discontinuation, espe- CML itself. cially in young patients. Unlike other milestones (such as early We also confirmed that TL is significantly shortened in CML molecular response or halving time), this marker could pre- PB and BM leukemic cells at diagnosis compared to healthy dict long-term outcomes (in terms of MR) before any TKI is donors, and it is no longer age-dependent, consistent with previ- started. ous literature.28 Bouillon et al41 recently showed that the degree of TL shortening measured in the CD34+CD38− leukemic stem cell compartment of CML can discriminate between early from Acknowledgments late CP-CML and it can be directly related to the size of the The authors thank Josep-Maria Ribera and Diana Dominguez for leukemic clone. In line with this study, we observed that, even their help. though TL in CML patients was generally shortened compared to age-matched healthy controls, there was an important vari- ability in TL between patients at diagnosis, possibly indicating Disclosures distinct CP-CML disease substages. Of clinical relevance, we believe that TL measured in PB or BM could be used not only as B.X. and L.Z. received research funding from Novartis, Celgene, and a biomarker for TKI molecular response but also to estimate the Incyte (not related to this study). All the other authors have no conflicts duration of CP-CML from its onset. of interest to disclose. In our study, we did not find any correlation between prog- nostic risk scores and delta-TL at diagnosis. Only when delta-TL Sources of funding was categorized by the median, we did find a significant asso- ciation with EUTOS score (P = 0.036), but this finding would This work was in part supported by a grant from Instituto de Salud need to be validated. Moreover, the BCR-ABL1-p210 transcript Carlos III through the project “PI16/01200” (Co-funded by European type did not show any association with TL, suggesting that the Regional Development Fund/European Social Fund “A way to make type of transcript may not have any impact on the leukemic cell Europe”/”Investing in your future”) as well as by the grant “Bolsa de division rate. ayuda económica para la innovación científica” and the grant “Becas Univariate analysis showed statistically significant cumu- de Investigación” both from Fundación Española de Hematología y lative incidence of MR4.0 both for delta-TL and ELTS score. Hemoterapia (FEHH). 6 (2021) 5:12 www.hemaspherejournal.com References 22. Breccia M, Molica M, Colafigli G, et al. Prognostic factors associ- ated with a stable MR4.5 achievement in chronic myeloid leukemia 1. Savona M, Talpaz M. Getting to the stem of chronic myeloid leukae- patients treated with imatinib. Oncotarget. 2018;9:7534–7540. mia. 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