cancers Article Real-World Data on Chronic Myelomonocytic Leukemia: Clinical and Molecular Characteristics, Treatment, Emerging Drugs, and Patient Outcomes Sandra Castaño-Díez 1,2,3 , Mónica López-Guerra 1,3,4 , Cristina Bosch-Castañeda 2 , Alex Bataller 1,2,3,5 , Paola Charry 1 , Daniel Esteban 1 , Francesca Guijarro 1,2,3 , Carlos Jiménez-Vicente 1 , Carlos Castillo-Girón 1 , Albert Cortes 1,6 , Alexandra Martínez-Roca 1,3 , Ana Triguero 1 , José Ramón Álamo 1 , Silvia Beà 1,3,4 , Dolors Costa 1,3,4 , Dolors Colomer 1,3,4 , María Rozman 1,3 , Jordi Esteve 1,2,3,5 and Marina Díaz-Beyá 1,3,5, * 1 Hematology and Hematopathology Departments, Hospital Clínic Barcelona, 08036 Barcelona, Spain 2 Medical School, University of Barcelona, 08036 Barcelona, Spain 3 August Pi i Sunyer Biomedical Research Institute (IDIBAPS), 08036 Barcelona, Spain 4 Centro de Investigación Biomédica en Red de Cáncer (CIBERONC), 28029 Madrid, Spain 5 Josep Carreras Leukemia Research Institute, 08916 Badalona, Spain 6 Hematology Department, Hospital de la Santa Creu i Sant Pau, 08025 Barcelona, Spain * Correspondence: diazbeya@clinic.cat; Tel.: +34-9-227-54-28 Simple Summary: Chronic myelomonocytic leukemia (CMML) is an infrequent disease with poor prognosis and risk of progression into acute myeloid leukemia (AML). Stem cell transplantation (alloSCT) is the only potentially curative option. New targeted drugs (NTDs) directed at specific gene Citation: Castaño-Díez, S.; mutations are useful in AML, but little is known about how CMML progresses to AML and if these López-Guerra, M.; Bosch-Castañeda, drugs are effective in CMML. In our study, 38% of patients received hypomethylating agents but less C.; Bataller, A.; Charry, P.; Esteban, D.; than half of them responded. Six patients received NTDs and responded well. AlloSCT was possible Guijarro, F.; Jiménez-Vicente, C.; in only 10% of patients. Progression to AML occurred in 25% of patients, and there were changes in Castillo-Girón, C.; Cortes, A.; et al. their gene mutations between the time of diagnosis and the time of progression. Although prognosis Real-World Data on Chronic is poor in CMML, analyzing gene mutations can help to better stratify the risk of each patient and to Myelomonocytic Leukemia: Clinical identify potentially effective NTDs for each patient. and Molecular Characteristics, Treatment, Emerging Drugs, and Abstract: Despite emerging molecular information on chronic myelomonocytic leukemia (CMML), Patient Outcomes. Cancers 2022, 14, 4107. https://doi.org/10.3390/ patient outcome remains unsatisfactory and little is known about the transformation to acute myeloid cancers14174107 leukemia (AML). In a single-center cohort of 219 CMML patients, we explored the potential correlation between clinical features, gene mutations, and treatment regimens with overall survival (OS) and Academic Editor: David Wong clonal evolution into AML. The most commonly detected mutations were TET2, SRSF2, ASXL1, and Received: 25 July 2022 RUNX1. Median OS was 34 months and varied according to age, cytogenetic risk, FAB, CPSS and Accepted: 22 August 2022 CPSS-Mol categories, and number of gene mutations. Hypomethylating agents were administered to Published: 25 August 2022 37 patients, 18 of whom responded. Allogeneic stem cell transplantation (alloSCT) was performed in Publisher’s Note: MDPI stays neutral 22 patients. Two-year OS after alloSCT was 60.6%. Six patients received targeted therapy with IDH or with regard to jurisdictional claims in FLT3 inhibitors, three of whom attained a long-lasting response. AML transformation occurred in published maps and institutional affil- 53 patients and the analysis of paired samples showed changes in gene mutation status. Our real- iations. world data emphasize that the outcome of CMML patients is still unsatisfactory and alloSCT remains the only potentially curative treatment. However, targeted therapies show promise in patients with specific gene mutations. Complete molecular characterization can help to improve risk stratification, understand transformation, and personalize therapy. Copyright: © 2022 by the authors. Licensee MDPI, Basel, Switzerland. Keywords: chronic myelomonocytic leukemia; CMML; AML transformation; clonal evolution; This article is an open access article prognosis; treatment; hypomethylating agents; targeted therapy; gene mutations; survival distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). Cancers 2022, 14, 4107. https://doi.org/10.3390/cancers14174107 https://www.mdpi.com/journal/cancers Cancers 2022, 14, 4107 2 of 21 1. Introduction Chronic myelomonocytic leukemia (CMML) is a clonal hematopoietic stem cell disor- der consisting of persistent monocytosis in peripheral blood (≥1 × 109 /L monocytes over 3 months), ≥10% of monocytes in the white blood cell count (WBC), dysplasia involving one or more myeloid lineages, and an acquired clonal cytogenetic or molecular abnormality in hematopoietic cells [1]. CMML is classified as a distinct entity in the myelodysplas- tic/myeloproliferative (MDS/MPN) category [1] and subdivided according to leukocyte cell count by the French-American-British (FAB) classification [2] and by blast and promono- cyte percentage in blood and bone marrow by the World Health Organization (WHO) [1]. CMML is a rare disease in general but with an incidence of 2.5 cases per 100,000 individuals > 70 years [3]. Median age at CMML diagnosis is 72 years and almost 90% of cases occur in patients > 60 years [4,5]. Around 20–30% of patients with CMML have cytogenetic abnormalities at diagnosis [6] and almost 90% harbor at least one somatic mutation, most frequently of TET2, SRSF2, and ASXL1 [7]. Median overall survival (OS) of CMML patients is 29 months [8,9], and about 25% of patients progress to acute myeloid leukemia (AML). The most frequently used risk stratification scales for CMML are the CMML Prognostic Scoring System (CPSS) and the CPSS-Molecular (CPSS-Mol). The CPSS includes the FAB classification, the WHO category, transfusion dependence, and cytogenetic risk, while the CPSS-Mol adds the negative prognostic value of ASXL1, NRAS, SETBP1, and/or RUNX1 mutations. In approximately half of CMML patients, the CPSS-Mol calculates a higher estimate of individual risk than the CPSS [10]. Both scales recognize four risk groups (low, intermediate-1, intermediate-2, and high), each with a different OS and AML transformation rate [10,11]. In fact, the pathophysiology of AML transformation in CMML patients remains a challenge [12,13]. Sequential studies with paired samples of CMML at diagnosis and at the time of AML transformation [14–16] have suggested a key role for the RAS pathway, but there are still blind spots to be elucidated, and there is a crucial need for a better understanding of the molecular mechanisms involved in AML transformation in order to design effective targeted treatments. In real-world clinical practice, patients are generally classified into two risk groups: low (low or intermediate-1 risk in CPSS or CPSS-Mol) or high (intermediate-2 or high CPSS or CPSS-Mol). Low-risk patients are generally treated with non-disease-modifying treatments, such as erythropoietin-stimulating agents for anemia and hydroxyurea for leukocytosis in patients with the MPN phenotype, while high-risk patients receive disease-modifying treatments, including hypomethylating agents (HMAs), chemotherapy, and allogeneic hematopoietic stem cell transplantation (alloSCT) [4]. AlloSCT is the only treatment option with proven curative capacity, although 5-year OS after alloSCT is still only 20–50% [17–21]. Moreover, in fact, due mainly to the older age and comorbidities of most CMML patients, alloSCT is only feasible in around 10% of patients [22,23]. In recent years, several targeted therapies have been used and approved for other myeloid neoplasms, including AML. For example, patients with FLT3 mutations can be treated with FLT3 inhibitors (midostaurin, gilteritinib, sorafenib), and those with IDH1/2 mutations can receive IDH1/2 inhibitors (ivosidenib, enasidenib). However, there is still very limited clearly defined data on the efficacy of these treatments in CMML [24–27] since CMML patients are commonly not included in clinical trials of MDS/AML, due both to the low incidence of CMML and the older age of the patients [24,28]. Given this scarcity of clinical trials in CMML, there is increasing interest in the use of real-world data to guide and improve disease management and to refine risk stratification. Here we report our retrospective study of clinical features, gene mutations, treatments (including novel targeted therapies), AML transformation, and patient outcome in a large cohort of CMML patients with long follow-up at a single center. Cancers 2022, 14, 4107 3 of 21 2. Materials and Methods 2.1. Patients We included a consecutive series of 219 patients diagnosed with CMML at Hospital Clínic of Barcelona, Spain from 1997 to 2021. CMML was morphologically defined accord- ing to the 2017 FAB and WHO criteria [1,2]. Clinical and molecular data were collected from patient records. Cytogenetic and mutational analyses were performed during patient treatment and follow-up, and results were examined retrospectively for the present study. 2.2. Cytogenetics Metaphase cytogenetic profiling was carried out on samples from bone marrow as- pirates. Chromosomal preparation was performed on G-banded metaphase cells using standard techniques. Karyotypes were reported according to the International System for Human Cytogenetic Nomenclature [29], and cytogenetic risk was calculated according to the Spanish cytogenetic risk-stratification system: low risk (normal karyotype or loss of chromosome Y as a single abnormality); intermediate risk (all chromosomal abnormali- ties except those included in the low- and high-risk categories); and high risk (trisomy 8, chromosome 7 abnormalities or complex karyotype [≥3 chromosomal abnormalities]) [9]. 2.3. Gene Mutations Bone marrow samples were collected at the time of CMML diagnosis and, when possible, at the time of AML transformation. Targeted next generation sequencing (NGS) was carried out on genomic DNA using Ion AmpliseqTM AML Research Panel (n = 17) or OncomineTM Myeloid Research Assay panel (ThermoFisher Scientific, Waltham, MA, USA) (n = 78) (Table S1). Sequencing data were analyzed using the Ion Reporter software (ThermoFisher Scientific). The minimum VAF for calling single nucleotide variants was 1%. Synonymous, intronic, and polymorphic variants were ruled out. Selected variants were reviewed in COSMIC (Catalog of Somatic Mutations in Cancer), ClinVar, and Var- some databases to finally select the clinically relevant variants. Molecular screening of FLT3-ITD, FLT3-TKD, and NPM1 mutations was performed by conventional PCR-based techniques and the FLT3-ITD allelic ratio was estimated using PCR DNA fragment analysis, as previously described [30,31]. 2.4. Treatment and Response Patient prognosis was estimated with CPSS and CPPS-Mol [10,11] and treatment was based on patient risk group. Treatment response was determined according to the criteria of Savona et al. [32]: no response, complete response, hematological response, partial response, and clinical benefit. Transfusion dependency was defined as needing at least one red blood cell transfusion every 8 weeks over a period of 4 months [11,33]. 2.5. Statistical Analyses Categorical variables were summarized as numbers and percentages and continuous variables as median (range). Comparisons between groups were done with the Chi-square test, Fisher’s exact test, Student’s t-test, or Mann–Whitney U test, as appropriate. OS was defined as the time from CMML diagnosis to death from any cause or last follow- up. OS after alloSCT was defined as the time from alloSCT to death from any cause or last follow-up. OS was calculated with the Kaplan–Meier method and compared with the log-rank test. Median follow-up was calculated with the Kaplan–Meier estimate of potential follow-up [34]. Time to AML transformation was defined as the time from CMML diagnosis to AML transformation. The cumulative incidence of AML transformation was estimated with a competing risk approach, considering death from any cause without AML transformation as a competing event, and the effect of quantitative covariates was estimated with the Fine-Gray regression model [35]. Non-relapse mortality was defined as the probability of dying after alloSCT without previous occurrence of a relapse, which is a competing event [36]. Patients with no events were censored at the time of last follow-up. Cancers 2022, 14, 4107 4 of 21 All analyses were performed using SPSS version 25 (SPSS Inc., Chicago, IL, USA) and R version 4.2.0. (R Core Team, Vienna, Austria) Two-sided p-values < 0.05 were considered statistically significant. 3. Results 3.1. Patient Characteristics Baseline patient characteristics are shown in Table 1. Of the 219 patients included in the study, 144 (65.8%) were men and median age at diagnosis was 74.1 years (range, 28–99). Fifty-three patients (24.3%) were transfusion-dependent at diagnosis. Table 1. Clinical characteristics of CMML patients. Characteristics at Diagnosis Patients (n = 219) Age, years—median (range) 74.1 (28–99) Men—n (%) 144 (65.8) WBC count—median (range) 9.47 (3–119) × 109 /L Neutrophil count—median (range) 4.7 (0.49–57.12) × 109 /L Monocyte count—median (range) 1.91 (1–33.72) × 109 /L % monocytes—median (range) 20 (10–60) Platelets—median (range) 126 (7–1037) × 109 /L Hemoglobin—median (range) 11.6 (4.9–16.1) g/dl Blast percentage 1 —median (range) 3% (0–19) Transfusion dependence—n (%) 53 (24.3) Presentation—therapy related n (%) 14 (7.3) Cytogenetic risk—n (%) n = 156 low 110 (70.6) intermediate 23 (14.7) high 23 (14.7) Cytogenetic profiling—n (%) n = 156 normal 106 (67.9) abnormal 50 (32.1) FAB classification—n (%) n = 218 myelodysplastic 148 (67.9) myeloproliferative 70 (32.1) WHO classification—n (%) n = 213 CMML-0 151 (70.9) CMML-1 35 (16.4) CMML-2 27 (12.7) CPSS classification—n (%) n = 153 low 57 (37.5) intermediate-1 44 (28.8) intermediate-2 43 (28.1) high 9 (5.9) CPSS-Mol classification—n (%) n = 60 low 17 (28.3) intermediate-1 12 (20) intermediate-2 17 (28.3) high 14 (23.3) Patients receiving disease-modifying n = 50 treatment—n (%) 2 Only HMA 34 (68) Only chemotherapy 13 (26) Both HMA and chemotherapy 3 (6) Patients receiving alloSCT—n (%) 22 (10) Patients progressing to AML—n (%) 53 (24.2) 1 Bone marrow blasts. 2 The first patient was treated with HMA in 2011. From then on, 96 patients were diagnosed with CMML, 37 (38.54%) of whom received HMA. Of the three patients who received both treatments, two received first HMA and then chemotherapy, and the other received first chemotherapy and then HMA. Cancers 2022, 14, 4107 5 of 21 Patients were classified according to cytogenetics, FAB classification [2], WHO cate- gories [1], CPSS [11], and CPSS-Mol [10] when these data were available. Of the 156 patients (71.2%) with informative karyotype results, 106 (67.9%) had a normal karyotype. One- hundred-and-ten patients (70.6%) were classified as cytogenetic low-risk, 23 (14.7%) as intermediate-risk, and 23 (14.7%) as high-risk [9]. Cytogenetic abnormalities are shown in Table S2. FAB classification identified 148 patients (67.9%) as MDS subtype and 70 (32.1%) as MPN subtype. According to the WHO categories, 151 patients (70.9%) were CMML-0, 35 (16.4%) were CMML-1, and 27 (12.7%) were CMML-2. CPSS classified 57 patients (37.5%) as low, 44 (28.8%) as intermediate-1, 43 (28.1%) as intermediate-2, and nine (5.9%) as high. CPSS-Mol classified 17 (28.3%) as low, 12 (20%) as intermediate-1, 17 (28.3%) as intermediate-2, and 14 (23.3%) as high (Table 1). 3.2. Gene Mutations NGS was performed in 72 patients at diagnosis and in 23 patients at the time of AML transformation. Fourteen patients had available paired NGS data at diagnosis and at AML transformation. Of the 72 patients with NGS data at diagnosis, 69 (95.8%) had at least one mutation, with a median of three mutations (range, 0–7) per patient. Three patients (4.2%) had no mutations; 13 (18.1%) had one; 18 (25%) had two; 14 (19.4%) had three; and 24 (33.3%) had four or more mutations. (Table 2). The most commonly detected mutation was in TET2 (59.7%), followed by SRSF2 (30.2%), ASXL1 (22.2%), RUNX1 (20.8%), CBL (19.4%), ZRSR2 (15.9%), EZH2 (15.9%), and SETBP1 (14.3%). Other genes were mutated in <10% of patients (Table 2 and Figure 1). The TET2 mutation was associated with older age (≥70 years, p < 0.001), while KRAS and SETBP1 mutations were associated with younger age (<70 years; p = 0.04 and p = 0.026, respectively). TET2 and ZRSR2 mutations were associated with the MDS phenotype (p < 0.001 and p = 0.01, respectively) and ASXL1 and SETBP1 with the MPN phenotype (p = 0.019 and p = 0.001, respectively). Table 2. Gene mutations in CMML patients. At CMML Diagnosis At AML Transformation Gene Mutations Patients (n = 72) n (n/N, %) 1 Patients (n = 23) n (n/N, %) 1 Number of mutated genes per patient 0 3 (3/72, 4.2) 1 (1/23, 4.3) 1 13 (13/72, 18.1) 1 (1/23, 4.3) 2 18 (18/72, 25) 3 (3/23, 13) 3 14 (14/72, 19.4) 3 (3/23, 13) ≥4 24 (24/72, 33.3) 15 (15/23, 65.1) Most frequently mutated genes TET2 43 (43/72, 59.7) 8 (8/23, 34.8) SRSF2 19 (19/63, 30.2) 10 (10/21, 47.6) ASXL1 16 (16/72, 22.2) 7 (7/23, 30.4) RUNX1 15 (15/72, 20.8) 5 (5/23, 21.7) CBL 14 (14/72, 19.4) 3 (3/23, 13) ZRSR2 10 (10/63, 15.9) 2 (2/21, 9.5) EZH2 10 (10/63, 15.9) 1 (1/21, 4.8) SETBP1 9 (9/63, 14.3) 4 (4/21, 19) IDH2 2 (2/72, 2.8) 6 (6/23, 26.1) NRAS 7 (7/72, 9.7) 5 (5/23, 21.7) DNMT3A 7 (7/72, 9.7) 4 (4/23, 17.4) U2AF1 3 (3/63, 4.8) 3 (3/21, 14.3) SF3B1 2 (2/63, 3.2) 3 (3/21, 14.3) JAK2 6 (6/72, 8.3) 3 (3/23, 13) FLT3 3 (3/72, 4.2) 3 (3/23, 13) Cancers 2022, 14, 4107 6 of 21 Table 2. Cont. At CMML Diagnosis At AML Transformation Gene Mutations Patients (n = 72) n (n/N, %) 1 Patients (n = 23) n (n/N, %) 1 PHF6 5 (5/63, 7.9) 2 (2/21, 9.5) KRAS 4 (4/72, 5.6) 2 (2/23, 8.7) Genes in signaling pathways NRAS 7 (7/72, 9.7) 5 (5/23, 21.7) KRAS 4 (4/72, 5.6) 2 (2/23, 8.7) FLT3 3 (3/72, 4.2) 3 (3/23, 13) CSF3R 0 (0/63, 0) 1 (1/21, 4.8) JAK2 6 (6/72, 8.3) 3 (3/23, 13) CBL 14 (14/72, 19.4) 3 (3/23, 13) PTPN11 2 (2/71, 2.8) 1 (1/23, 4.3) Epigenetic regulators TET2 43 (43/72, 59.7) 8 (8/23, 34.8) IDH2 2 (2/72, 2.8) 6 (6/23, 26.1) DNMT3A 7 (7/72, 9.7) 4 (4/23, 17.4) ASXL1 16 (16/72, 22.2) 7 (7/23, 30.4) EZH2 10 (10/63, 15.9) 1 (1/21, 4.8) IDH1 1 (1/72, 1.4) 1 (1/23, 4.3) Transcription factors RUNX1 15 (15/72, 20.8) 5 (5/23, 21.7) SETBP1 9 (9/63, 14.3) 4 (4/21, 19) CEBPA 3 (3/72, 4.2) 0 (0/23, 0) Spliceosome complex SRSF2 19 (19/63, 30.2) 10 (10/21, 47.6) SF3B1 2 (2/63, 3.2) 3 (3/21, 14.3) ZRSR2 10 (10/63, 15.9) 2 (2/21, 9.5) U2AF1 3 (3/63, 4.8) 3 (3/21, 14.3) DNA damage response genes TP53 1 (1/72, 1.4) 1 (1/23, 4.3) PHF6 5 (5/63, 7.9) 2 (2/21, 9.5) Others Cancers 2022, 14, x FOR PEER REVIEW 7 of 22 NPM1 4 (4/72, 5.6) 2 (2/23, 8.7) 1 n, number of patients with the mutation; N, number of patients with informative NGS results. Genemutations Figure1.1.Gene Figure mutationsdetected detectedatatCMML CMMLdiagnosis. diagnosis. 3.3. Overall Survival With a median follow-up of 10.2 years (IQR, 5.3–12.3), median OS for the entire co- hort was 34 months (95% CI, 28–40) (Figure 2), while it was 28.9 months for patients ≥70 Cancers 2022, 14, 4107 7 of 21 Figure 1. Gene mutations detected at CMML diagnosis. 3.3. Overall 3.3. Overall Survival Survival Withaamedian With medianfollow-up follow-upof of 10.2 10.2 years years (IQR, (IQR, 5.3–12.3), 5.3–12.3), median median OS for OSthe forentire the entire cohortco- was 34 months (95% CI, 28–40) (Figure 2), while it was 28.9 months for patients ≥70 years≥70 hort was 34 months (95% CI, 28–40) (Figure 2), while it was 28.9 months for patients years at at diagnosis diagnosis and 51 and 51 months months for patients for patients < 70(pyears < 70 years (p =(Table = 0.002) 0.002)3).(Table 3). Figure 2. Overall Figure 2. Overallsurvival survivalofofthe theentire entirecohort cohortofof CMML patients CMML (n (n patients = 219). = 219). Table 3. Overall survival according to age, risk categories, and gene mutations. Table 3. Overall survival according to age, risk categories, and gene mutations. Factor Factor No. Patients OS (Months)OS (Months) No. Patients 95% CI 95% CIp p Age Age 0.002 0.002 ≥70 ≥70years years 142 142 28.9 28.9 21.0–37.3 21.0–37.3 <70years <70 years 77 77 51.0 37.9–64.1 51.0 37.9–64.1 Cytogenetic Cytogeneticrisk risk 0.001 0.001 Low 110 51.0 40.7–61.4 Low 110 51.0 40.7–61.4 Intermediate 22 30.4 23.6–37.2 High 24 19.4 9.1–29.8 FAB 0.04 MDS 148 40.3 32.3–49.3 MPN 70 21.3 14.7–27.8 WHO 0.227 CMML-0 151 37.7 29.4–45.9 CMML-1 35 25.0 19.1–30.9 CMML-2 27 18.3 11.3–25.4 CPSS 0.01 Low 57 57.2 35.4–78.8 Intermediate-1 44 34.8 10.3–59.3 Intermediate-2 43 19.4 13.8–25.2 High 9 13.7 5.6–21.8 Dichotomized CPSS 0.01 Low 101 51.6 39.9–63.2 High 52 19.1 13.3–24.9 Cancers 2022, 14, 4107 8 of 21 Table 3. Cont. Factor No. Patients OS (Months) 95% CI p CPSS-Mol 0.09 Low 17 85.1 37.6–132.7 Intermediate-1 12 64.3 20.3–108.3 Intermediate-2 17 46.8 12.8–80.8 High 14 19.1 6.7–31.5 Dichotomized CPSS-Mol 0.03 Low 29 67.3 49.6–85.1 High 31 28.7 17.2–40.0 No. gene mutations 0.006 <2 mutations 16 85.2 51.3–119.0 ≥2 mutations 56 25.0 17.6–32.5 RUNX1 mutation 0.02 Detected 15 16.6 10.0–23.0 Not detected 57 51.1 21.5–80.8 Mutations in 0.001 transcription factors ≥1 mutation detected 25 21.3 12.0–30.4 No mutation detected 41 64.3 31.8–96.9 ASXL1/EZH2 co-mutation 0.001 Detected 3 2.5 0.3–4.7 Not detected 68 35.2 10.3–60.2 OS was 51 months for patients with low cytogenetic risk, 30.4 months for those with intermediate risk, and 19.4 months for those with high risk (p < 0.001). OS was 40.3 months for patients with the FAB MDS phenotype and 21.3 months for those with the MPN phenotype (p = 0.04). OS was 37.7 months for the WHO CMML-0 category, 25 months for the CMML-1 category, and 18.3 months for the CMML-2 category (p = 0.227) (Table 3). OS was 57.2 months for CPSS-defined low-risk patients, 34.8 months for intermediate-1 patients, 19.4 months for intermediate-2 patients, and 13.7 months for high-risk patients (p < 0.01) (Table 3 and Figure 3A). According to the dichotomized CPSS categories used in clinical practice (low and intermediate-1 vs. intermediate-2 and high), patients in the low-risk group had an OS of 51.6 months compared to 19.1 months for those in the high-risk group (p < 0.01) (Table 3 and Figure 3B). OS was also different according to the CPSS-Mol-defined risk groups but these differences did not reach statistical significance: 85.1 months for low-risk patients; 64.3 months for intermediate-1 patients; 46.8 months for intermediate-2 patients; and 19.1 months for high-risk patients (p = 0.09) (Table 3 and Figure 3C). According to the dichotomized CPSS-Mol categories, OS was 67.3 months for the low-risk group and 28.7 months for the high-risk group (p = 0.03) (Table 3 and Figure 3D). Nearly half of our patients had a higher individual risk according to CPSS-Mol than according to CPSS. In fact, 25% of patients increased their risk from CPSS low to CPSS-Mol high (Figure S1). OS was 85.2 months for the 16 patients with fewer than two mutations and 25 months for the 56 with two or more mutations (p = 0.006) (Table 3 and Figure S2). The RUNX1 mutation at CMML diagnosis was associated with shorter OS: 51.1 months for patients with wild-type RUNX1 vs. 16.6 months for those with the RUNX1 mutation (p = 0.02) (Table 3). Patients harboring at least one mutation in transcription factors (RUNX1, GATA2, SETBP1, CEBPA) had an OS of 21.3 months, compared to 64.3 months for those without any of these mutations (p = 0.001) (Table 3 and Figure S3). The three patients with the ASXL1/EZH2 co-mutation had an OS of 2.5 months vs. 35.2 months for those without the co-mutation (p = 0.001) (Table 3). Cancers 2022, 14, x FOR PEER REVIEW 9 of 22 Cancers 2022, 14, 4107 according to CPSS. In fact, 25% of patients increased their risk from CPSS low to CPSS- 9 of 21 Mol high (Figure S1). (A) (B) (C) (D) Figure 3. Overall survival according to (A) CPSS risk score; (B) CPSS risk score dichotomized into Figure 3. Overall survival according to (A) CPSS risk score; (B) CPSS risk score dichotomized into low risk (low and intermediate-1) and high risk (intermediate-2 and high); (C) CPSS-Mol risk score; low risk (low and intermediate-1) and high risk (intermediate-2 and high); (C) CPSS-Mol risk score; and (D) CPSS-Mol dichotomized into low risk (low and intermediate-1) and high risk (intermediate- and (D) 2 and CPSS-Mol dichotomized into low risk (low and intermediate-1) and high risk (intermediate-2 high). and high). OS was 85.2 3.4. Response months for the 16 patients with fewer than two mutations and 25 months to Treatment for the 56 with two or more mutations (p = 0.006) (Table 3 and Figure S2). The RUNX1 Fifty patients (22.8%) received disease-modifying treatment: 34 with only HMA, 13 mutation at CMML diagnosis was associated with shorter OS: 51.1 months for patients with only chemotherapy, and 3 with both treatments (Table 1). The median time from with wild-type RUNX1 vs. 16.6 months for those with the RUNX1 mutation (p = 0.02) diagnosis to initiating disease-modifying treatment was 6.8 months (range 0.1–111.7). (Table 3). Patients harboring at least one mutation in transcription factors (RUNX1, HMA was first implemented in our center in 2011, after which CMML was diagnosed in 96 patients, 37 (38.5%) of whom received HMA and 8 (8.3%) of whom received chemother- apy. Data on response to HMA were available for 36 patients: 10 patients (27.8%) attained a complete response; 6 (16.7%) a partial response; 2 (5.6%) a hematologic response; 1 (2.8%) Cancers 2022, 14, 4107 10 of 21 had clinical benefit; and 17 (47.2%) did not respond. Data on response to chemotherapy was available for 15 patients: eight (53.3%) achieved a complete response; two (13.3%) achieved a partial response; and five (33.3%) did not respond. The ASXL1 mutation was associated with a lower response rate to chemotherapy: 0% in the two patients with ASXL1 mutations vs 100% complete response in the five without ASXL1 mutations (p = 0.05). OS for patients receiving HMA calculated from the start of treatment was 23.8 months (95% CI, 13–34). OS for all 96 patients treated from 2011 on was 35.8 months (95% CI, 22–49), compared to 33 months (95% CI, 24–43) for the 123 patients treated before 2011 (p = 0.63). 3.5. AlloSCT Twenty-two patients (10%) received alloSCT, six of whom had progressed to AML at the time of alloSCT. Prior to alloSCT, 9 of these 22 patients (40.9%) had received chemother- apy (3 with chemotherapy plus midostaurin due to FLT3 mutations), 11 (50%) had received HMA, and 2 (9.1%) had not received any prior treatment. Three patients had received both HMA and chemotherapy prior to alloSCT. Two did not respond to HMA and then received chemotherapy, while the third did not respond to chemotherapy and then received HMA plus venetoclax. Both 2- and 4-year OS after alloSCT were 60.6% (95% CI, 36–78) (Figure 4A). Non-relapse mortality at both 2 and 4 years after alloSCT was 13.6% (95% CI, 3.2–31.4). Ten patients relapsed after alloSCT, with a 2- and 4-year cumulative incidence of relapse after alloSCT of 37.6 % (95% CI, 17–58) and 43.7% (95% CI, 21–64), respectively (Figure 4B). At relapse, 7 of the 10 patients received salvage treatment: 1, donor lymphocyte Cancers 2022, 14, x FOR PEER REVIEW 11 of 22 infusion; 1, enasidenib; 3, sorafenib; 1, HMA plus venetoclax; and 1, chemotherapy. OS calculated from the time of relapse was 15 months (95% CI, 0.03–32). (A) (B) Figure Figure 4. 4. (A) (A) Overall Overall survival survival and and (B) (B) cumulative cumulative incidence incidence of of relapse after alloSCT. relapse after alloSCT. 3.6. Targeted Therapies A total total of ofsix sixpatients patientswere treated were with treated targeted with therapies. targeted FourFour therapies. patients who relapsed patients who re- after alloSCT lapsed were salvaged after alloSCT with novel were salvaged targeted with novel therapies: one with one targeted therapies: enasidenib and three with enasidenib withthree and sorafenib. Three of these with sorafenib. Three patients attainedattained of these patients a prolonged complete a prolonged remission, complete with a remission, median with duration a median of response duration of 11 months of response (95% CI,(95% of 11 months 8–44).CI, An8–44). additional patient received An additional patient sorafenibsorafenib received post-alloSCT as a preventive post-alloSCT measure measure as a preventive and a sixthandpatient a sixthreceived sorafenibso- patient received at AML transformation. rafenib at AML transformation. A 61-year-old A 61-year-old manman with with CMML-2 CMML-2 and and an IDH2 mutation an IDH2 mutation relapsed relapsed 4.5 4.5 months months after after alloSCT. He was treated with enasidenib, attained a complete response, and alloSCT. He was treated with enasidenib, attained a complete response, and is still in re- is still in remissionafter mission aftermore more than than 3.5 3.5 years. years. The Theother otherfive patients five received patients FLT3 received inhibitors FLT3 (mi- inhibitors dostaurin or sorafenib). (midostaurin A 55-year-old or sorafenib). woman A 55-year-old with CMML-2 woman and FLT3-TKD with CMML-2 was treated and FLT3-TKD was treated with chemotherapy plus midostaurin followed by alloSCT. She relapsed 2.5 months after alloSCT, immunosuppressive therapy was reduced, and she started treat- ment with sorafenib. She achieved a complete remission for a year while continuing with sorafenib but died due to non-relapse mortality while still in complete remission. A 56- Cancers 2022, 14, 4107 11 of 21 Cancers 2022, 14, x FOR PEER REVIEW 12 of 23 with chemotherapy plus midostaurin followed by alloSCT. She relapsed 2.5 months after al- loSCT, immunosuppressive therapy was reduced, and she started treatment with sorafenib. She achieved a complete remission for a year while continuing with sorafenib but died due 3.7.toAML non-relapse mortality while still in complete remission. A 56-year-old man with Transformation CMML-2 and TET2, NPM1,(24.2%) Fifty-three patients and FLT3-ITD (ratioto0.17) progressed AML,mutations receivedtime with a median chemotherapy to transfor- plus midostaurin followed by alloSCT. He relapsed 3 months after alloSCT, mation of 16.6 months (95% CI, 0.7–124.9). The 2-year and 4-year cumulative immunosup- incidence of pressive therapy was reduced, AML transformation and (95% were 19% he achieved a complete CI, 13.9–24.7%) remission and with CI, 23.3% (95% negative minimalre- 17.7–29.4%), residual disease (MRD). spectively (Figure 5A). After 6 months, MRD reappeared, he started sorafenib, and he continues to be in complete remission with decreasing MRD kinetics. A 69-year-old man with AML transformation after CMML, an IDH2 mutation, and FLT3-ITD (ratio 0.3) was treated with chemotherapy plus midostaurin followed by alloSCT. Nine months after al- loSCT, he relapsed and received salvage treatment: azacytidine plus sorafenib (no response) and decitabine plus venetoclax (no response). He is currently included in a clinical trial of an IDH2 inhibitor. A 67-year-old man with AML transformation after CMML-2 and FLT3-ITD received chemotherapy plus midostaurin followed by alloSCT. He then received maintenance sorafenib for 2 years and is still in complete remission 3 years after alloSCT. Finally, a 69-year-old man with AML transformation after CMML-2, FLT3-ITD, and an IDH2 mutation, who was not eligible for alloSCT, received three different treatments: aza- cytidine (achieving a partial response), sorafenib plus azacytidine (achieving sustained complete remission for 7 months), and gilteritinib (achieving sustained complete remission for 1 year). However, he then relapsed and died. 3.7. AML Transformation Fifty-three patients (24.2%) progressed to AML, with a median time to transformation of 16.6 months (95% CI, 0.7–124.9). The 2-year and 4-year cumulative incidence of AML transformation were 19% (95% CI, 13.9–24.7%) and 23.3% (95% CI, 17.7–29.4%), respectively (Figure 5A). When the CPSS score was dichotomized into low and high risk, the 2-year cumulative incidence of AML transformation was 9.6% for low-risk patients vs 39.2% for high-risk patients (p < 0.001) (Figure 5B). When the CPSS-Mol score was dichotomized into low and high risk, the 2-year cumulative incidence of AML transformation was 0% for low-risk patients and 25.9% for high-risk patients (p < 0.001) (Figure 5C). (A) Figure 5. Cont. Cancers2022, Cancers 14,x4107 2022,14, FOR PEER REVIEW 1312ofof23 21 (B) (C) Figure Figure5.5. Cumulative Cumulative incidence incidence of transformation to of transformation to AML AML(A) (A)for forthe theentire entirecohort cohort(n(n = 219), = 219), (B)(B) ac- according to CPSS prognostic score, and (C) according to CPSS-Mol prognostic score. cording to CPSS prognostic score, and (C) according to CPSS-Mol prognostic score. When At thethetimeCPSS of AMLscoretransformation, was dichotomized the into mostlow and highdetected commonly risk, themutation 2-year cumula- was in tive incidence SRSF2 (47.6%),offollowed AML transformation by TET2 (34.8%), wasASXL1 9.6% for low-risk (30.4%), IDH2patients vs 39.2% (26.1%), RUNX1 for(21.7%), high- risk NRASpatients (p < SETBP1 (21.7%), 0.001) (Figure (19%),5B). When the DNMT3A CPSS-Mol (17.4%), U2AF1 score was dichotomized (14.3%), SF3B1 (14.3%), intoJAK2 low and high (13%), CBLrisk, the 2-year (13%), cumulative FLT3 (13%), ZRSR2incidence (9.5%), PHF6of AML(9.5%),transformation and KRAS (8.7%). was 0% for genes Other low- risk werepatients mutated and at 25.9% lower for high-risk(Table frequencies patients 2). (p < 0.001) (Figure 5C). At the time of AML transformation, The two patients harboring an IDH2 mutation the most commonly detected mutation at CMML diagnosis was in both progressed SRSF2 to AML (47.6%), followed bythis and maintained TET2 (34.8%),but mutation ASXL1with(30.4%), IDH2 (26.1%), an increased VAF at the RUNX1 time (21.7%), of AML NRAS (21.7%), SETBP1 transformation. The two (19%), DNMT3A patients with SF3B1(17.4%), U2AF1at(14.3%), mutations diagnosisSF3B1also(14.3%), progressed JAK2 to (13%), AML. CBL Of the(13%), FLT3 (13%), four patients ZRSR2NPM1 harboring (9.5%), PHF6 (9.5%), mutations and KRAS at diagnosis, two(8.7%). Othertogenes progressed AML were mutated and two were at loweras treated frequencies AML from(Table 2). the beginning. The two patients Fourteen patientsharboring had paired anbone IDH2marrow mutation at CMML samples diagnosis at CMML both progressed diagnosis and at pro- gression to AML and to AML (n = 13) this maintained or tomutation myelofibrosis (n = 1) but with an(Figures increased 6 and VAF 7).atAmong theofpatients the time AML who progressedThe transformation. to AML, 10 hadwith two patients not previously SF3B1 mutationsreceived disease-modifying at diagnosis treatment also progressed to and they AML. maintained Of the four patientsmostharboring of the same NPM1mutations mutations withatan increasedtwo diagnosis, VAF. Six of these progressed to 10 patients AML and two had alsotreated were acquired new mutations as AML at the time of AML transformation: NRAS from the beginning. 2), FLT3-ITD (n = Fourteen patients hadFLT3-TKD (n = 2), paired bone 1), PTPN11 (n =marrow (n = 1), samples RUNX1diagnosis at CMML (n = 2), ASXL1 and at(npro- = 1), and BCOR (n = 1). gression to AML (n = 13) or to myelofibrosis (n = 1) (Figures 6 and 7). Among the patients Three patients who progressed had received to AML, 10 had not disease-modifying previously received treatment prior to AML disease-modifying transforma- treatment and tion.maintained they One patientmost maintained the SETBP1 of the same mutations ASXL1 and with an mutations increased detected VAF. Sixatofdiagnosis these 10 pa-and tients hadRUNX1, acquired CBL, and also acquired newNF1 mutations. mutations at theThe second time of AML patient harbored BCOR transformation: NRAS and(nSF3B1 = 2), mutations(nat=diagnosis FLT3-ITD 2), FLT3-TKD and acquired an NRAS (n = 1), PTPN11 (n mutation = 1), RUNX1 at the(n time = 2), of AML(n ASXL1 transforma- = 1), and tion. The BCOR (n = 1).third patient maintained the ASXL1, SETBP1, and SRSF2 mutations detected at diagnosis, but with an increased VAF, and also acquired RB1, Three patients had received disease-modifying treatment prior to AML transfor-ETV6, and STAG2 mutations. The patient mation. Onewho progressed patient maintained to myelofibrosis the SETBP1 harbored and ASXL1 SRSF2, TET2, detected mutations and MPLat mutations diagnosisat CMML diagnosis and acquired a JAK2 mutation at the time and acquired RUNX1, CBL, and NF1 mutations. The second patient harbored BCOR andof myelofibrosis transformation. SF3B1 mutations at diagnosis and acquired an NRAS mutation at the time of AML trans- formation. The third patient maintained the ASXL1, SETBP1, and SRSF2 mutations de- tected at diagnosis, but with an increased VAF, and also acquired RB1, ETV6, and STAG2 mutations. The patient who progressed to myelofibrosis harbored SRSF2, TET2, and MPL and acquired RUNX1, CBL, and NF1 mutations. The second patient harbored BCOR and SF3B1 mutations at diagnosis and acquired an NRAS mutation at the time of AML trans- formation. The third patient maintained the ASXL1, SETBP1, and SRSF2 mutations de- tected at diagnosis, but with an increased VAF, and also acquired RB1, ETV6, and STAG2 mutations. The patient who progressed to myelofibrosis harbored SRSF2, TET2, and MPL Cancers 2022, 14, 4107 13 of 21 mutations at CMML diagnosis and acquired a JAK2 mutation at the time of myelofibrosis transformation. Figure6.6. Mutational Figure landscape in Mutational landscape in14 14CMML CMMLpatients patients who who transformed transformed to AML to AML (n = (n 13)=or13) or to to myelo- myelofibrosis fibrosis (n = (n1).=Each 1). Each rowrow corresponds corresponds to one to one gene. gene. Each Each blue blue square square correspondstotoone corresponds onepatient patientat atCMML CMMLdiagnosis, diagnosis, andand each square corresponds each orange square corresponds totoone onepatient patientatattransformation. transformation.White White squares indicate unanalyzed genes. Gray squares indicate unmutated genes. squares indicate unanalyzed genes. Gray squares indicate unmutated genes. Red squares indicate Red squares indicate thatthe that theoriginal originalmutation mutationwas wasretained retainedatat transformation.Light transformation. Lightgreen greensquares squaresindicate indicatemutations mutations acquired at transformation. Yellow squares indicate mutations not retained at transformation. Dark acquired at transformation. Yellow squares indicate mutations not retained at transformation. Dark green squares indicate a mutation present at transformation but not analyzed at diagnosis. Ten pa- green squares indicate a mutation present at transformation but not analyzed at diagnosis. Ten tients (1, 2, 4, 5, 6, 7, 8, 10, 12, and 13) had not previously received disease-modifying treatment and patients (1, 2, 4, 5, 6, they maintained 7, 8,of10, most the12,same and mutations 13) had notwithpreviously received an increased disease-modifying VAF. Patients 4, 6, 7, 8,treatment 10, and 12 and they maintained most of the same mutations with an increased VAF. Patients 4, 6, 7, 8, 10, and 12 had acquired new mutations at the time of AML transformation. Three patients (3, 9, and 11) had received disease-modifying treatment prior to AML transformation. Cancers 2022, 14, x FOR PEER REVIEW 1 Cancers 2022, 14, 4107 14 of 21 had acquired new mutations at the time of AML transformation. Three patients (3, 9, and 1 received disease-modifying treatment prior to AML transformation. Figure 7. Cont. Cancers 2022, 14, 4107 Cancers 2022, 14, x FOR PEER REVIEW 15 of 21 15 Figure 7. Variant allele7.frequency Figure (VAF) Variant allele plots of mutated frequency (VAF) plotsgenes in 14 CMML of mutated genespatients who trans- in 14 CMML patients who formed to AML formed (n = 13) to or AML (n = 13) or (n to myelofibrosis to=myelofibrosis (n = 1). Disease-modifying 1). Disease-modifying treatments treatments are shown. are shown. Symbols bols are used are used to differentiate to differentiate variants of mutationsvariants of mutations (*, ˆ, ´ and ¨). (*, ^, ´ and ¨). 4. Discussion 4. Discussion CMML is a rare disease CMML is awith rarehighly diseaseheterogeneous morphologicalmorphological with highly heterogeneous features, clinicalfeatures, cl manifestations, cytogenetic abnormalities, molecular alterations, and manifestations, cytogenetic abnormalities, molecular alterations,treatment responses. and treatmen Advances in molecular techniquesin sponses. Advances have made it techniques molecular possible to have improve theitrisk made stratification possible to improve the of CMML and to better understand stratification of CMML and its pathogenesis by taking to better understand into account by its pathogenesis nottaking only into ac cytogenetic abnormalities but also molecular alterations. We retrospectively reviewed not only cytogenetic abnormalities but also molecular alterations. We retrospective the real-world data on 219 CMML patients who were diagnosed and treated at our viewed the real-world data on 219 CMML patients who were diagnosed and treat institution. Our results confirm the poorOur our institution. prognosis results of the disease, confirm point the poor out theofcrucial prognosis importance the disease, point out the c of alloSCT in improving OS, clarify details of clonal evolution to AML, and importance of alloSCT in improving OS, clarify details of clonal evolutionhighlight the to AML feasibility of using new targeted therapies in these patients. highlight the feasibility of using new targeted therapies in these patients. Cancers 2022, 14, 4107 16 of 21 Many of our findings are along the lines of previous reports, including patient charac- teristics [8,10,11,22,32,37–40] and median OS (34 months) [8,9,40]. OS was longer in younger patients (p = 0.002), those with low cytogenetic risk (p < 0.001), and those with the FAB MDS phenotype (p = 0.04), as previously reported [8,40]. Also in line with other studies [41], differences in OS according to the current WHO classification (CMML-0/CMML-1/2) were not significant. The mutational landscape of our patients was also similar to that of previous studies [7,15,42,43]. For example, the vast majority of our patients harbored at least one somatic mutation. While the most frequently mutated gene in previous studies was TET2, followed by SRSF2, ASXL1, and the RAS pathway genes (NRAS, KRAS, and CBL) [15,42], in our study, the most frequently mutated genes were TET2, SRSF2, ASXL1, RUNX1, and CBL. In line with a previous study [15], we identified a higher incidence of TET2 mutations in patients ≥70 years, suggesting that older age is related to a higher frequency of clonal hematopoiesis [44]. In contrast, KRAS and SETBP1 mutations were more frequent in younger patients. Although several studies have explored the clinical impact of mutations in CMML, the issue remains unresolved. In the present study, patients with fewer than two gene mutations at CMML diagnosis had longer survival (p = 0.006), which is in line with some previous reports [10,15]. The negative prognostic impact of ASXL1, NRAS, SETBP1, and/or RUNX1 mutations has been incorporated in the CPSS-Mol prognostic score [7,42,45], and we found that half of our patients were classified as higher risk by CPSS-Mol than by CPSS. In fact, CPSS-Mol may be a superior method for calculating risk and deciding on treatment, as patients classified as low-risk by CPSS may well benefit from the treatment that would be indicated by CPSS-Mol. In line with previous reports, the EZH2/ASXL1 co- mutation [22,46,47], present in three of our patients, and RUNX1 mutations [10], detected in 20.8% of our patients, were also associated with shorter OS (p = 0.001 and p = 0.02, respectively). Interestingly, patients harboring a mutation in at least one of the transcription factors (RUNX1, GATA2, SETBP1, and CEBPA) had shorter OS (p = 0.001); to the best of our knowledge, this phenomenon has not been explored in previous studies. The MDS phenotype of CMML has been associated with mutations of TET2, U2AF1, and SF3B1 [48], and the MPN phenotype with mutations that activate RAS family members, ASXL1, JAK2, SETBP1, SRSF2, and EZH2 [10,22,46]. In our patients, TET2 and ZRSR2 mutations were associated with the MDS phenotype and ASXL1 and SETBP1 with the MPN phenotype. The percentage of patients who progressed to AML in our study was also along the lines of previous reports [11,22,47,49]. The risk of AML transformation depends on the transformative aggressiveness of CMML and on the competing risk of dying before trans- formation, usually from comorbidities or consequences of cytopenia [37]. Findings in our cohort confirm the capacity of dichotomized CPPS and CPSS-Mol scores to discriminate be- tween patients with low and high risk of AML transformation [10,11]. The most frequently mutated genes at the time of AML transformation in our cohort were SRSF2, TET2, ASXL1, IDH2, RUNX1, NRAS, SETBP1, and DNMT3A. Comprehensive information about clonal evolution from CMML to AML is still lack- ing. A few sequential studies comparing paired patient samples at the time of CMML diagnosis and at AML transformation have postulated a key role for the RAS pathway and for mutations in EZH2, IDH1/2, NPM1, and FLT3-ITD [14,50]. Patel et al. [16] suggested that TET2, ASXL1, and SRSF2 were ancestral mutations present in early stages of CMML and that KRAS, NRAS, RUNX1, U2AF1, and CBL mutations were secondary events leading to AML transformation. In our study, we observed newly acquired mutations at AML transformation, including in genes involved in signaling pathways (NRAS, FLT3, JAK2, PTPN11, and CBL), as well as in RUNX1 and ASXL1. While we cannot draw definitive conclusions from our findings in only 14 patients, we can speculate that, in line with the suggestion by Patel et al. [16], there is a connection between the acquisition of new muta- tions and AML transformation. If this hypothesis is borne out in further studies, we can Cancers 2022, 14, 4107 17 of 21 recommend serial analyses during follow-up of CMML patients to predict progression to AML and adapt treatment accordingly. Importantly, FLT3 mutations, which are druggable, appeared at AML transformation in two patients. Interestingly, the two patients in our cohort who had an IDH2 mutation at diagnosis and two of the four patients with mutated NPM1 at diagnosis progressed to AML, while the other two patients with NPM1 mutations received chemotherapy followed by alloSCT despite having <20% blasts, following previous recommendations [51] that prognosis is improved with more intense therapy. Only a quarter of our patients diagnosed from 2011 that received disease-modifying treatment (HMAs or chemotherapy) and less than 50% of these achieved any type of re- sponse. These findings are in line with other studies. A large retrospective non-randomized study indicated that HMAs conferred a survival advantage over hydroxyurea in high-risk, but not low-risk, CMML patients [38]. In contrast, the DACOTA trial (EudraCT 2014- 000200-10) [52] failed to demonstrate a benefit for the HMA decitabine over hydroxyurea in proliferative CMML. HMAs in combination with venetoclax have improved outcomes in AML patients who are ineligible for intense chemotherapy [53]. However, their role in CMML and AML transformation remains unclear, though they have been proposed as a feasible and effective bridge to alloSCT [25,26]. In fact, no available drug has been able to improve the natural course of CMML and alloSCT remains the only potentially curative therapy. In our cohort, only 22 patients (10%) underwent alloSCT, which is similar to previous reports [18,54], mainly because the majority of CMML patients are elderly and not eligible for alloSCT. However, the outcomes of our patients after alloSCT were slightly better than those of other studies [55,56]. Our patients achieved a 4-year OS of 61%, and many of our patients were long survivors despite a cumulative incidence of relapse at four years of 43.7%. This may be due, at least in part, to the fact that three patients who relapsed after alloSCT and were salvaged with new targeted therapies attained a prolonged complete remission, with a median duration of response of 11 months. In the last few years, several targeted therapies have been approved for treatment of other myeloid diseases, such as AML, but their efficacy in CMML is largely unknown [25–28]. In fact, six of our patients harbored FLT3 or IDH mutations, which are druggable, and after treatment with FLT3 and IDH inhibitors, some attained a long-lasting response. These findings indicate that novel targeted therapies are feasible in CMML and suggest that new drug combinations could induce quicker and deeper responses and thus improve alloSCT outcomes [26,55]. In this line, there are several targeted drugs on an early phase of development (preclinical models or clinical trials) being tested in myeloid pathology that could be of utility on CMML in a foreseeable future. Some examples includes the orally bioavailable modulator of the SF3b complex H3B-8800 for patients with splicing mutations as SRSF2 [57–60]; MEK kinase inhibitors, for patients harboring a RAS Pathway-Activating Mutation [61–63]; or cotreatment with Omacetaxine (an inhibitors of protein translation) and a BCL2 inhibitor for targeting RUNX1 mutations (preclinical model study) [64]. Further, they highlight the importance of exhaustive molecular characterization at diagnosis and at progression. NGS is an extremely useful tool that makes it feasible to perform this molecular characterization. It can provide valuable information on gene alterations that can help determine the diagnosis and prognosis of CMML patients, improve patient risk stratification, use available treatments more rationally, and identify potential therapeutic targets. The present study has several limitations, including its retrospective nature and the limited number of patients in whom NGS was performed. However, since CMML patients are generally not included in clinical trials because of the low frequency and heterogeneity of the disease, at present we must rely on real-world data for information on prognosis, molecular characterization, and treatment response. However, the DACOTA trial [52] proved that randomized clinical trials are feasible in CMML, and clinical trials in CMML are now ongoing. Enrollment in CMML-specific clinical trials or in exploratory trials of specific tumor biomarkers should be encouraged as the best way to clarify the Cancers 2022, 14, 4107 18 of 21 molecular mechanisms behind CMML and to explore the efficacy of new drugs in this disease [22,24,47,65,66]. Nevertheless, until such time where we have reliable data from clinical trials, studies such as ours, based on real-world data, can provide valuable insight into the CMML mutational landscape and treatment outcomes. 5. Conclusions Our comprehensive clinical and molecular characterization of a large cohort of CMML patients shows that despite advances in risk stratification and the increasing use of HMAs, the prognosis of CMML continues to be poor. AlloSCT is the only potentially curative treatment, but only a small proportion of CMML patients are eligible candidates, mainly due to older age and comorbidities. There is clearly an unmet need for improved treatment for these patients. Complete molecular characterization can help us to understand the pathophysiology of CMML, to improve risk stratification, and to better understand trans- formation to AML. The incorporation of novel therapies targeting specific gene alterations could contribute to a more precise treatment, with a higher response rate, lower toxicity, and greater survival benefit. Since CMML-specific clinical trials are difficult to perform due to the low frequency and the heterogeneity of the disease and the age of the patients, more studies of real-world data similar to the present one can provide information on prognosis in CMML. Supplementary Materials: The following supporting information can be downloaded at: https:// www.mdpi.com/article/10.3390/cancers14174107/s1, Table S1: NGS panels used for gene mutation analysis; Table S2: Cytogenetic abnormalities; Figure S1: Alluvial plot of the patients included in the study according to CPSS and CPSS-Mol scores; Figure S2: Overall survival according to the number of mutations detected at CMML diagnosis; Figure S3: Overall survival according to the mutational status of transcription factors. Author Contributions: Conceptualization, M.D.-B.; methodology, S.C.-D., J.E. and M.D.-B.; vali- dation, S.C.-D., J.E., and M.D.-B.; formal analysis, S.C.-D., M.L.-G., C.B.-C., F.G., J.R.Á., S.B., D.C. (Dolors Costa), D.C. (Dolors Colomer), M.R., J.E. and M.D.-B.; investigation, S.C.-D., J.E., and M.D.-B.; resources, S.C.-D., M.L.-G., C.B.-C., F.G., J.R.Á., S.B., D.C. (Dolors Costa), D.C. (Dolors Colomer), M.R., J.E. and M.D.-B.; data curation, S.C.-D., C.B.-C., A.B., P.C., D.E., C.J.-V., C.C.-G., A.C., A.M.-R., A.T. and M.D.-B.; writing—original draft preparation, S.C.-D., J.E., and M.D.-B.; writing—review and editing, S.C.-D., M.L.-G., C.B.-C., A.B., P.C., D.E., F.G., C.J.-V., C.C.-G., A.C., A.M.-R., A.T., J.R.Á., S.B., D.C. (Dolors Costa), D.C. (Dolors Colomer), M.R., J.E. and M.D.-B.; visualization, S.C.-D., J.E. and M.D.-B.; supervision J.E. and M.D.-B.; project administration, J.E. and M.D.-B.; funding acquisition, J.E. and M.D.-B. All authors have read and agreed to the published version of the manuscript. Funding: This research was supported by grants from resident award Contractes Clínic de Recerca Emili Letang-Josep Font 2021, granted by Hospital Clínic de Barcelona; and it was funded by Instituto de Salud Carlos III (ISCIII) through the project “FIS PI19/01476” and co-funded by the European Union. Institutional Review Board Statement: The study was conducted in accordance with the Declaration of Helsinki and approved by the Institutional Review Board (or Ethics Committee) of Hospital Clínic Barcelona (2021-1230; on 20 July 2022). Informed Consent Statement: Written informed consent was obtained from all subjects involved in the study. Data Availability Statement: Due to privacy and ethical concerns, the data that support the findings of this study are available on request from the corresponding author. Conflicts of Interest: A.M.-R.: consultant or advisory role (Bristol Myers Squibb, Abbvie, Kite Gilead), travel grants (Kite Gilead, Roche, Takeda, Janssen, Abbvie), speaker (Abbvie, Gilead); J.E.: consultancy-honoraria (Abbvie, Novartis, Astellas, Jazz Pharmaceuticals, BMS-Celgene, Pfizer, Daichii-Sankyo), research grants (Novartis, Jazz Pharmaceuticals); M.D.-B.: consultant or advisory role, travel grants or speaker (Bristol Myers Squibb, Abbvie, Astellas, JazzPharma, Takeda, Novartis). The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results. Cancers 2022, 14, 4107 19 of 21 References 1. Swerdlow, S.H.; Campo, E.; Harris, N.L.; Jaffe, E.S.; Pileri, S.A.; Stein, H.; Thiele, J.; Arber, D.A.; Hasserjian, R.P.; Le Beau, M.M. 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