cancers Review Myelodysplastic Syndromes with Isolated del(5q): Value of Molecular Alterations for Diagnostic and Prognostic Assessment Pamela Acha 1 , Mar Mallo 1,2 and Francesc Solé 1,2, * 1 MDS Group, Institut de Recerca Contra la Leucèmia Josep Carreras, ICO-Hospital Germans Trias i Pujol, Universitat Autònoma de Barcelona, 08916 Badalona, Spain 2 Microarrays Unit, Institut de Recerca Contra la Leucèmia Josep Carreras, ICO-Hospital Germans Trias i Pujol, Universitat Autònoma de Barcelona, 08916 Badalona, Spain * Correspondence: fsole@carrerasresearch.org; Tel.: +34-93-557-2806 Simple Summary: Myelodysplastic syndromes with isolated del(5q) constitute the only MDS subtype defined by a cytogenetic alteration. The results of several clinical studies and the advances in new technologies have provided a better understanding of the biological basis of this disease. Specific genetic alterations have been found to be associated with prognosis and response to treatments. This review intends to summarize the current knowledge of the molecular background of MDS with isolated del(5q), focusing on the clinical and prognostic relevance of cytogenetic alterations and somatic mutations. Abstract: Myelodysplastic syndromes (MDS) are a group of clonal hematological neoplasms charac- terized by ineffective hematopoiesis in one or more bone marrow cell lineages. Consequently, patients present with variable degrees of cytopenia and dysplasia. These characteristics constitute the basis for the World Health Organization (WHO) classification criteria of MDS, among other parameters, Citation: Acha, P.; Mallo, M.; Solé, F. for the current prognostic scoring system. Although nearly half of newly diagnosed patients present Myelodysplastic Syndromes with a cytogenetic alteration, and almost 90% of them harbor at least one somatic mutation, MDS with Isolated del(5q): Value of Molecular isolated del(5q) constitutes the only subtype clearly defined by a cytogenetic alteration. The results of Alterations for Diagnostic and several clinical studies and the advances of new technologies have allowed a better understanding of Prognostic Assessment. Cancers 2022, the biological basis of this disease. Therefore, since the first report of the “5q- syndrome” in 1974, 14, 5531. https://doi.org/10.3390/ changes and refinements have been made in the definition and the characteristics of the patients with cancers14225531 MDS and del(5q). Moreover, specific genetic alterations have been found to be associated with the Academic Editor: Jorge Cortes prognosis and response to treatments. The aim of this review is to summarize the current knowledge of the molecular background of MDS with isolated del(5q), focusing on the clinical and prognostic Received: 30 September 2022 relevance of cytogenetic alterations and somatic mutations. Accepted: 7 November 2022 Published: 10 November 2022 Keywords: myelodysplastic syndromes; chromosome 5q deletion; somatic mutations; cytogenetic Publisher’s Note: MDPI stays neutral alterations with regard to jurisdictional claims in published maps and institutional affil- iations. 1. Introduction Myelodysplastic syndromes (MDS) are a group of clonal hematological neoplasms characterized by ineffective hematopoiesis in one or more bone marrow (BM) cell lineages. Copyright: © 2022 by the authors. Consequently, patients present with variable degrees of cytopenia and dysplasia, which are Licensee MDPI, Basel, Switzerland. This article is an open access article essential features for establishing a diagnosis according to the World Health Organization distributed under the terms and (WHO) classification [1,2]. conditions of the Creative Commons Although nearly half of newly diagnosed patients present a cytogenetic alteration and Attribution (CC BY) license (https:// almost 90% of them harbor at least one somatic mutation, MDS with isolated chromosome creativecommons.org/licenses/by/ 5q deletion (MDS-5q) constitutes the only subtype clearly defined by a cytogenetic alter- 4.0/). ation [3–6]. The results of several clinical studies and advances in new technologies have Cancers 2022, 14, 5531. https://doi.org/10.3390/cancers14225531 https://www.mdpi.com/journal/cancers Cancers 2022, 14, 5531 2 of 13 allowed better characterization of this entity. As a consequence, since the first report of the 5q- syndrome, changes and refinements have been made in the definition and characteris- tics of the patients that pertain to this subtype [7]. Moreover, specific genetic alterations have been found to be associated with prognosis and response to treatments [1,2,8,9]. While preparing this review, the overview of the next WHO classification and the proposal of the International Consensus Classification (ICC) of myeloid neoplasms and acute leukemias were published [2,8]. Consequently, MDS-5q will be renamed and the inclusion criteria will be slightly modified, as will be explained in the subsequent section. In the present review, we aimed to summarize the current knowledge of the molecular background of MDS-5q, focusing on the clinical and prognostic relevance of cytogenetic alterations and somatic mutations. 2. From “5q- Syndrome” to MDS-5q In 1974, Van den Berghe et al. reported a group of three patients with refractory anemia and interstitial deletion of the long arm of chromosome 5. Such cases were later recognized as the “5q- syndrome”. Features of the syndrome included macrocytic anemia, low-normal leukocyte counts, and normal to elevated platelet counts. The BM showed erythroid hypoplasia, hypolobulated megakaryocytes, and a blast count <15% [7]. Accord- ing to the French–American–British (FAB) cooperative group classification criteria, most of the patients with these characteristics pertain to the group of patients with refractory anemia [10]. It was not until the 2001 edition of the WHO classification that the 5q- syndrome was recognized as a unique and well-defined MDS subtype [11]. In addition to previously de- scribed characteristics of this syndrome, in this classification, the blast count threshold was redefined to <5%, and the absence of Auer roads was considered to define 5q- syndrome patients. In 2008, the subtype “MDS with isolated del(5q)” was introduced and the term 5q- syndrome remained restricted to a subset of cases within this category that presented with macrocytic anemia, normal or elevated platelet count, BM erythroid hypoplasia, and a blast count <5% in BM and <1% in peripheral blood (PB) [12]. In the 2017 WHO classification, these cases remained within the MDS with isolated del(5q) subtype. Additionally, the diagnosis of this subtype can be established even if there is one additional cytogenetic ab- normality besides the del(5q), unless this abnormality is monosomy 7 or del(7q) [1,13]. This is based on data showing that there is no adverse effect of one chromosomal abnormality in addition to the del(5q) in such patients [14]. As mentioned previously, the overview of the next WHO classification has recently been published [2]. In this new proposal, MDS with isolated del(5q) has been renamed as “myelodysplastic neoplasm with low blast and isolated del(5q)” (abbreviated MDS- 5q). The diagnostic criteria have not changed, and it is stated that although an SF3B1 or TP53 mutation (not multi-hit) may potentially alter the biology and/or prognosis of the disease, the presence of such mutations does not per se override the diagnosis of this entity. Regarding the ICC proposal for the classification of MDS, MDS with isolated del(5q) has been retained with no changes from the revised fourth edition of the WHO classification, although the name has been simplified to “MDS with del(5q)”. Similarly to the new WHO proposal, the ICC also specifies that TP53 mutations are admitted in this MDS subtype unless a multi-hit state is detected [8]. It is important to remark that although del(5q) is the most frequent cytogenetic alter- ation in MDS and is present in roughly 20% of cytogenetic abnormal cases, only about 5% are classified in the MDS-5q category. Features such as higher blast count, alterations in chromosome 7, or a multi-hit TP53 state impact disease prognosis and would reclassify these remaining patients into other, more aggressive, disease subtypes [2,8,15]. The changes in terms and inclusion criteria over time are produced as a consequence of the advances in technology and discoveries, which directly impact our knowledge and the management of this disease (Figure 1). Cancers 2022, 14, x FOR PEER REVIEW 3 of 13 The changes in terms and inclusion criteria over time are produced as a consequence Cancers 2022, 14, 5531 3 of 13 of the advances in technology and discoveries, which directly impact our knowledge and the management of this disease (Figure 1). Figure1.1.Timeline Figure Timelineshowing showingthethemain maindiscoveries discoveriesinvolving involving MDS-5q MDS‐5q and the changes changes in in nomencla‐ nomenclature tureinclusion and and inclusion criteria. criteria. Abbreviations:CDR, Abbreviations: CDR,commonly commonly deleted deletedregion; region;chr, chromosome; chr, ICC:ICC: chromosome; International Consensus Classification; MN, myeloid neoplasms; NGS, next‐generation sequencing. International Consensus Classification; MN, myeloid neoplasms; NGS, next-generation sequencing. 3.3.Role RoleofofConventional Conventional Cytogenetics Cytogenetics in inMDS‐5q MDS-5q Conventional cytogenetics Conventional cytogenetics (CC)(CC)constitutes constitutes thethe gold standard gold for the standard forgenetic diagno‐ the genetic diag- sis and nosis andprognosis prognosisofofMDS. MDS.However, However, fluorescence fluorescence in in situ hybridization situ hybridization (FISH) (FISH)of 5q31 of 5q31 couldbebeuseful could usefulinincases caseswithout withoutevidence evidence of of del(5q) del(5q) byby CC. CC. When When the thepresence presenceofofMDS‐ MDS-5q 5q is suspected and/or if the cytogenetic study shows is suspected and/or if the cytogenetic study shows no metaphases or no metaphases or an an aberrant aberrantkary‐kary- otype with chromosome 5 is involved (no 5q deletion), it is recommended to perform FISH otype with chromosome 5 is involved (no 5q deletion), it is recommended to perform analysis [16,17]. Figure 2 shows the genetic studies available for the diagnosis and char‐ FISH analysis [16,17]. Figure 2 shows the genetic studies available for the diagnosis and acterization of MDS‐5q. During follow‐up, genetics studies will be adapted to each pa‐ characterization of MDS-5q. During follow-up, genetics studies will be adapted to each tient, considering their comorbidities. A new BM aspiration, and the corresponding ge‐ patient, considering their comorbidities. A new BM aspiration, and the corresponding netic study, will be performed on suspicion of disease evolution and no response to treat‐ genetic study, will be performed on suspicion of disease evolution and no response to ment. In the case of clonal evolution, the approach can be decided according to the general treatment. In the case of clonal evolution, the approach can be decided according to the patient status. general patient status. The karyotype is a prognostic variable included in the International Prognostic Scor‐ The karyotype ing System (IPSS) and is a prognostic the revisedvariable edition of included the IPSSin(IPSS‐R) the International Prognostic [18,19]. Del(5q) aloneScoring has System (IPSS) and the revised edition of the IPSS (IPSS-R) [18,19]. always been considered a good prognostic variable and, in the IPSS‐R, a concomitant Del(5q) alone has always cy‐ been considered togenetic a good alteration has prognostic variable been included. and, in was This change the IPSS-R, based on a concomitant a scoring system cytogenetic pro‐ alteration posed by has been Schanz et included. al. based onThis change wasdata an international based on a scoring collection of 2902system patientsproposed [15]. De‐ by Schanz letion 5q is a classical alteration detected in around 15–20% of MDS patients, withDeletion et al. based on an international data collection of 2902 patients [15]. half 5qbeing is a classical alteration isolated, around 17%detected having an inadditional around 15–20% of and alteration, MDS36% patients, with being part of half a com‐being isolated, around[3]. plex karyotype 17% Thehaving an additional prognostic impact of alteration, and 36% the accompanying being part of abnormalities in adel(5q) complex karyotype is difficult[3]. The prognostic to determine becauseimpact double of the accompanying abnormalities are highlyabnormalities variable. In 2011,in del(5q) Mallo is et al. published difficult an international to determine collaborative because double study including abnormalities are highlya large series ofIn variable. del(5q) 2011,pa‐ Mallo ettients to determine al. published an the prognostic impact international of adjunct collaborative prognostic study includingabnormalities. a large seriesThe ofmulti‐ del(5q) variate analysis patients showed to determine thethat karyotype impact prognostic complexity was one prognostic of adjunct of the main prognostic factorsThe abnormalities. together with multivariate plateletshowed analysis count and BM that blasts [14]. karyotype The good prognosis complexity was one ofofthe del(5q) mainwith one prognostic factors together with platelet count and BM blasts [14]. The good prognosis of del(5q) with one accompanying alteration was included in the MDS with the del(5q) category of the 2017 WHO classification, excluding cases harboring a chromosome 7 alteration [1]. Cancers 2022, 14, x FOR PEER REVIEW 4 of 13 Cancers 2022, 14, 5531 4 of 13 accompanying alteration was included in the MDS with the del(5q) category of the 2017 WHO classification, excluding cases harboring a chromosome 7 alteration [1]. Figure 2. Genetic studies of MDS‐5q according to the new diagnostic and prognostic guidelines: Figure 2. Genetic techniques studies available of MDS-5q for correct according diagnostic to the new and prognostic diagnostic assessment and prognostic of MDS‐5q according guidelines: to the techniques criteria ofavailable for correct the next World Healthdiagnostic and prognostic Organization assessment (WHO) classification, theofproposal MDS-5qofaccording to the the Interna‐ criteria tionalofConsensus the next World Health Organization Classification (ICC) and the(WHO) classification, Molecular the Prognosis International proposal of the International Scoring System (IPSS‐M).Classification Consensus Abbreviations:(ICC) CGH‐A,andcomparative genomic the Molecular hybridization; International CNA, Prognosis copy number Scoring System altera‐ (IPSS-M). tion; FISH, fluorescence Abbreviations: in situ hybridization; CGH-A, comparative genomicOGM, optical genome hybridization; CNA,mapping; ROH,alteration; copy number region of ho‐ FISH, mozygosity; SNP‐A, single nucleotide polymorphism array; TDS, targeted gene sequencing (assum‐ fluorescence in situ hybridization; OGM, optical genome mapping; ROH, region of homozygosity; ing the use of probes that allow the detection of small CNA and ROH. Otherwise, SNP‐A would be SNP-A, single nucleotide recommended polymorphism to assess CNA and ROH inarray; TP53 TDS, targeted for accurate gene sequencing diagnostic (assuming and prognostic the use of assessment). probes that allow the detection of small CNA and ROH. Otherwise, SNP-A would be recommended to 3.1. assess CNA andDeleted Commonly ROH in TP53 for Regions accurate diagnostic in Chromosome 5q and prognostic assessment). Two “commonly deleted regions” (CDR) were originally described by Boultwood 3.1. Commonly Deleted Regions in Chromosome 5q and colleagues: a 1.5 Mb deletion encompassing 5q32–5q33, which was originally associ‐ Two ated “commonly with deletedand the 5q‐ syndrome regions” better (CDR) wereand prognosis, originally describedCDR a more proximal by Boultwood at 5q31. Theand colleagues: a 1.5 Mb deletion encompassing 5q32–5q33, which latter was associated with other MDS subtypes and cases of acute myeloid leukemia was originally associated with the 5q- syndrome and better prognosis, and a more proximal (AML) cases, with complex karyotypes and a worse prognosis [20–23]. High‐resolution CDR at 5q31. The latter was associatedsuch techniques, withasother genomicMDS subtypes and microarrays andcases optical ofgenome acute myeloid mapping leukemia (OGM), (AML) can detectcases, with complex cryptic karyotypes alterations and a worse accompanying the prognosis del(5q) and [20–23]. can help High-resolution techniques, define the breakpoint. How‐ such as ever, genomic sincemicroarrays high‐densityand optical genomic genome mapping microarrays work with(OGM), can detect DNA probes, cryptic alterations this approach has be‐ accompanying come the mostthe del(5q) suitable and cantohelp technique obtaindefine thedel(5q) precise breakpoint. However, breakpoint genomic since high-density coordinates. genomic microarrays Most patients havework with large DNA probes, deletions this approach that encompass has become both CDRs. This was the most suitable corroborated by subsequent technique to obtainstudies combining precise del(5q) conventional breakpoint genomiccytogenetics and single nucleotide poly‐ coordinates. morphism arrays,have Most patients withlarge Mallodeletions et al., describing a wider CDR that encompass both that CDRs.extended This was from q22.3 to corroborated q31.3. This region encompasses 14.6 Mb, while the median size of by subsequent studies combining conventional cytogenetics and single nucleotide polymor- the total deletion de‐ tected in most cases is around 70 Mb [17]. phism arrays, with Mallo et al., describing a wider CDR that extended from q22.3 to q31.3. Several This region studies have14.6 encompasses focused on thethe Mb, while study of thesize median 5q CDR, of thebut indeletion total 2012, Jerez et al., in detected published an article emphasizing most cases is around 70 Mb [17]. the importance of the common retained region (CRR) [24]. Their work Several studiesreinforces the ideaon have focused that theinstudy the 5q‐ofsyndrome, the 5q CDR, the proximal but in 2012, and terminal Jerez et al., regions are always retained. Thus, two CRRs were described: published an article emphasizing the importance of the common retained region CRR1 for the proximal (CRR) re‐[24]. gion (spanning 81.7 Mb and ending at band 5q14.2) and CRR2 for the Their work reinforces the idea that in the 5q- syndrome, the proximal and terminal regions distal region (5q34), arewith both being associated with disease subtypes. No CRR could be identified in other always retained. Thus, two CRRs were described: CRR1 for the proximal region forms of MDS and AML with del(5q). As was previously described, patients with CRR (spanning 81.7 Mb and ending at band 5q14.2) and CRR2 for the distal region (5q34), with both being associated with disease subtypes. No CRR could be identified in other forms of MDS and AML with del(5q). As was previously described, patients with CRR had a lower number of genomic lesions and correlate with better prognosis. Additionally, this study supports the idea that genomic microarrays can add prognostic information to prognostic Cancers 2022, 14, x FOR PEER REVIEW 5 of 13 Cancers 2022, 14, 5531 5 of 13 had a lower number of genomic lesions and correlate with better prognosis. Additionally, this study supports the idea that genomic microarrays can add prognostic information to prognostic scoring scoring systems assystems as was reported was reported by Arenillas by Arenillas et al., inet2013 al., in 2013Figure [25]. [25]. Figure 3 shows 3 shows the CDR the CDR and CRR identified for chromosome and CRR identified for chromosome 5q. 5q. Figure 3. Figure 3. Commonly Commonly deleted deletedregions regions(CDR) and (CDR) commonly and retained commonly regions retained (CRR) regions in chromosome (CRR) 5q. 5q. in chromosome ItIt isiswidely widelyknown knownthat thatadditional additionaltechniques techniquescancanhelp helptotodescribe describethe thekaryotype. karyotype.Most Most of the of the complex complex cases cases carrying carrying an apparent an apparent monosomy monosomy 5 have 5 have shownshown that,that, following following studies studies with with additional additional techniques techniques such such as FISH as FISH or genomic or genomic arrays, arrays, thisapparent this apparentmonosomy mono‐ somy presents presents a partially a partially retained retained 5q.previously 5q. As As previously mentioned, mentioned, genomicmicroarrays genomic microarrayscancan help help to accurately define the breakpoints to accurately define the breakpoints [24,26]. [24,26]. Molecular studies Molecular studies revealed revealedthatthathaploinsufficiency haploinsufficiency of of several genes several (particularly genes (particularly RPS14, CSNK1A1, EGR1, miR‐145, and miR‐14a) located in 5q RPS14, CSNK1A1, EGR1, miR-145, and miR-14a) located in 5q CDR contribute CDR contribute to the path‐ to the ogenesis and and pathogenesis hematological phenotype hematological associated phenotype withwith associated MDS‐5q [27–29]. MDS-5q For example, [27–29]. For example, miR‐145 and miR‐14a are micro RNAs that have been found to be responsible for the neg‐ miR-145 and miR-14a are micro RNAs that have been found to be responsible for the nega- ative regulation of effector molecules that regulate megakaryocytic differentiation. Thus, tive regulation of effector molecules that regulate megakaryocytic differentiation. Thus, a deficiency of these micro RNAs is responsible for the thrombosis and megakaryocytic a deficiency of these micro RNAs is responsible for the thrombosis and megakaryocytic dysplasia (hypolobulated megakaryocytes) which characterize MDS‐5q [30,31]. dysplasia (hypolobulated megakaryocytes) which characterize MDS-5q [30,31]. 3.2. Karyotyping: Present and Future Directions 3.2. Karyotyping: Present and Future Directions Point mutations have been described as a frequent event in MDS patients. In 2011, Point mutations have been described as a frequent event in MDS patients. In 2011, Bejar et al. described the clinical effects of these mutations and stated that the prognosis Bejar et al. described the clinical effects of these mutations and stated that the prognosis of these patients may be driven by the association of prognostic variables. Specific genes of these patients may be driven by the association of prognostic variables. Specific genes were found to be associated with specific risk groups such as TET2 in cases with a normal were foundand karyotype to be associated TP53 in cases with with aspecific complex risk groups such karyotype [32]. In TET2the as 2022, in cases with IPSS‐M, a normal a prog‐ karyotype nostic scoring TP53 based andsystem in cases with a complex on molecular data waskaryotype published.[32]. In 2022, This scoring the IPSS-M, system takes a prognostic scoring system based on molecular data was published. This scoring into account the mutational status of 31 genes; however, it still retains the karyotype as a system takes into account prognostic the[9]. parameter mutational status of 31 genes; however, it still retains the karyotype as a prognostic parameter [9]. OGM has emerged as a promising non-sequencing-based technique for high resolution genome-wide structural variant profiling. It can simplify lab workflow by reducing multiple tests. Parallel studies with standard-of-care tests have been performed in hematological neoplasms and have shown high concordance [33]. A recent study published by Yang et al. showed that OGM results changed the comprehensive cytogenetic scoring system and the Cancers 2022, 14, 5531 6 of 13 IPSS-R risk groups in 21% and 17%, respectively, of their MDS patient’s cohort with an improved prediction of prognosis. Although more studies especially focused on MDS-5q are needed, the combination of OGM and next-generation sequencing (NGS) seems to be a promising approach for the evaluation of prognosis [34]. 4. Prognostic Impact of Somatic Mutations in MDS-5q It has been described that one-third of MDS-5q patients present with no somatic alterations, while nearly half of patients (43%) can present with an isolated mutation [35,36]. The pattern of recurrently mutated genes is similar to other MDS subtypes, except for TP53 mutations that were found to be enriched in this subtype of patients [35,37,38]. In the subsequent section, the genes most frequently mutated in MDS-5q are described and Table 1 summarizes their biological and clinical associations and main characteristics and frequencies. Table 1. Recurrently mutated genes in MDS-5q: clinical and biological correlations. Gene Pathway/Function Frequency Clinical and Biological Correlations • Associated with RS SF3B1 Splicing factor 19–20% • Controvert data regarding outcome of concomitant SF3B1 mutation and del(5q) DNMT3A DNA methylation 18% • Recurrent founder lesion (DTA mutations) • Aggressive disease course • Higher risk of transformation to AML TP53 Checkpoint/cell cycle 18% • Shorter OS • Resistance to lenalidomide treatment TET2 DNA methylation 12% • Recurrent founder lesion (DTA mutations) Proliferation, apoptosis, • Associated with older age CSNK1A1 7–10% DNA damage response ASXL1 Chromatin modification 6% • Recurrent founder lesion (DTA mutations) JAK2 Tyrosine kinase 6% • Associated with elevated platelet counts Abbreviations: AML, acute myeloid leukemia; DTA, DNMT3A, TET2, and ASXL1; OS, overall survival; RS, ring sideroblasts. Based on data from Meggendorfer et al. [35], Malcovati et al. [39], Heuser et al. [40], and Mossner et al. [41]. 4.1. SF3B1 Mutation The SF3B1 gene encodes subunit 1 of the splicing factor 3b protein complex, which is a core component of the RNA splicing machinery. Mutations in SF3B1 have been reported in around 20% of MDS-5q cases and have been associated with a variable proportion of ring sideroblasts [5,6,39,42]. Evidence provided by several reports suggests that, in some cases of MDS-5q, the SF3B1 mutation might precede the cytogenetic alteration [41,43–45]. Despite the order of acquisition of such genetic events, cases with concomitant SF3B1 and del(5q) would still be classified within the category of MDS-5q in the WHO classification system, as well as in the ICC system [2,8]. Controversial data have been published regarding the prognosis of SF3B1 mutations in MDS-5q patients. On one hand, a study published by Meggendorfer et al., demonstrated Cancers 2022, 14, 5531 7 of 13 a significantly shorter overall survival (OS) in patients harboring both alterations compared with MDS-5q patients without SF3B1 mutation [35]. On the other hand, no significant difference in OS was reported by Malcovati et al. when analyzing the same in their respective cohort [39]. 4.2. DNMT3A, TET2 and ASXL1 Mutations Mutations affecting the genes DNMT3A, TET2, and ASXL1 genes—commonly known as DTA mutations—are frequently found in clonal hematopoiesis of indeterminate potential (CHIP), which is a non-malignant condition associated with increased risk of progression to hematologic neoplasia compared with individuals without detectable mutations [46,47]. In line with this, in mutational hierarchy studies performed by Mossner et al., DTA mutations were found to be recurrent “founder” lesions in MDS patients, including the MDS-5q cases analyzed [36,41]. DNMT3A codifies for DNA methyltransferase 3 alpha, which is required for genome- wide de novo methylation and is essential for the establishment of DNA methylation pat- terns during development [48]. On the contrary, TET2 codifies for tet methylcytosine dioxy- genase 2, which catalyzes the conversion of the modified genomic base 5-methylcytosine (5mC) into 5-hydroxymethylcytosine (5hmC) and plays a key role in active DNA demethy- lation. As mentioned previously, both genes are recurrently mutated in MDS. Specifically, in MDS-5q, DNMT3A mutations were found in roughly 18% of cases while TET2 mutations were described in nearly 12% of patients [35]. Some studies have reported that in MDS patients, DNMT3A mutations were associ- ated with a higher risk of leukemia transformation and shorter OS, but no specific study describing either phenotypic or survival associations was exclusively performed in MDS-5q patients [49,50]. A report by Scharenberg et al. described that progression in patients with low- and intermediate-1-risk del(5q) MDS is predicted by mutations in a limited number of genes, among which TET2 is included. Specifically, 6/13 patients with evidence of disease pro- gression presented mutations in the TET2 gene [51]. In MDS patient cohorts including all disease subtypes, TET2 mutations were found to be associated with shorter OS af- ter hematopoietic stem cell transplantation and lower response rate to hypomethylating agents [9,52,53]. Located in chromosome 20q, additional sex combs 1 (ASXL1) codifies for a protein involved in transcriptional regulation. Mutations of mostly the frameshift type have been described in MDS patients in variable frequencies ranging from 14–24% in different cohorts [5,6,9,32,53,54]. Concretely in MDS-5q, they are less abundant and most studies describe frequencies of around 6% [35,53–55]. While it is a common event in early disease, Fernandez-Mercado et al. reported higher frequencies of this mutation of up to 25% among advanced cases of the disease, suggesting a role in disease progression in MDS-5q [36]. Similarly to DNMT3A and TET2, ASXL1 mutations were mostly studied among MDS patient cohorts, including all subtypes, finding an association with worse prognosis and a shorter OS, but no specific associations were mentioned between ASXL1 mutations and outcomes in MDS-5q cases have been described. 4.3. TP53 Mutations The tumor-suppressor p53 gene (TP53) is located in chromosome band 17p13 and is essential for genome integrity. TP53 encodes for the p53 protein, which is a transcription factor involved in essential cell functions, such as DNA repair, cell cycle control, apoptosis, aging, and stemness [56,57]. TP53 gene mutations are detected in approximately 18% of MDS-5q [58,59]. It is the only mutation that was found to be significantly enriched in this MDS subtype compared with the other subtypes (18% vs. 6%) [35]. Data regarding the time of acquisition of this mutation are controversial. While it seems that there was a group of patients in which the mutation is already present in the early phases of the disease, there was another in Cancers 2022, 14, 5531 8 of 13 which the TP53 mutation arises during disease evolution, especially after treatment with lenalidomide [43,51,59]. Mutations in the TP53 gene in MDS patients are associated with generally unfavorable outcomes, aggressive disease course, higher risk of transformation to AML, shorter overall survival (OS), and resistance to lenalidomide treatment [17,37,51,59]. Double or even triple hits in the TP53 gene locus were already reported in 2013 by Kulasekararaj et al. [37]. A more recent study published by Bernard et al., provides new insights regarding the importance of the TP53 allelic state (multi-hit). After studying 3324 patients, four main TP53 mutational profiles were identified: (1) monoallelic muta- tions; (2) multiple mutations; (3) mutation and concomitant deletion affecting 17p; and (4) mutation and concomitant loss of heterozygosity of the 17p region. They found that two-thirds of patients with a TP53 mutation present with multiple hits, while only one- third present with monoallelic mutations. Associations with high-risk presentation and poor outcomes were only specific to multi-hit patients, while surprisingly, monoallelic patients did not differ from TP53 wild-type patients in outcome and response to therapy. The authors described that the TP53 allelic state segregates patient outcomes across WHO subtypes, despite monoallelic TP53 being enriched by MDS-5q. Moreover, they found that patients with monoallelic TP53 mutations had longer survival compared with multi-hit patients [58]. In the 2017 edition, the WHO recommended assessing TP53 mutational status in MDS- 5q to identify high-risk cases [1]. However, the upcoming edition of the WHO classification takes into consideration new insights regarding the allelic state of this gene to redefine a specific subtype of MDS associated with the presence of multiple alterations affecting the TP53 locus (Figure 2). This subtype is called MDS with biallelic TP53 inactivation (MDS-biTP53). However, the presence of a single TP53 mutation (unless it is multi-hit) does not per se exclude the diagnosis of MDS-5q [2]. Similarly, the ICC proposal takes into account the TP53 allelic state to define a new disease category called “myeloid neoplasms with mutated TP53”. In the case of MDS-5q, only single-hit TP53 mutations are admitted, otherwise, the diagnosis would change to the newly mentioned category [8]. 4.4. CSNK1A1 Mutations Located in the CDR 5q32, CSNK1A1 encodes for casein kinase 1A1 (CK1 α), a ser- ine/threonine kinase that participates in many cellular processes, including growth and proliferation via the β catenin and Wnt signaling pathway, apoptosis, and response to DNA damage [60–62]. In 2015, a study by Kronke et al., identified CK1α as a lenalidomide target in myeloid cells and found that heterozygous deletion of CSNK1A1 in del(5q) MDS provides a therapeutic window for selective targeting of the malignant cells [63]. Missense mutations have been reported in exons 3 and 4 in 7–10% of MDS-5q pa- tients [35,40,44,64,65]. Detected variant allele frequency values range from 3–78% and mimic a homozygous mutation status, which is consistent with the location of the CSNK1A1 gene and the CDR [35,40]. CSNK1A1 mutations were found to be associated with older age and some reports show a trend towards decreased response to lenalidomide, but no independent prognostic impact on OS has been described to date [40,60]. In a study performed by Meggendorfer et al., CSNK1A1 mutations were found to co-occur with SF3B1 mutations in 42% of the cases [35]. 4.5. JAK2 Mutations Janus kinase 2 (JAK2) encodes a non-receptor tyrosine kinase that plays a central role in cytokine and growth factor signaling. Somatic mutations in JAK2 constitute a major diagnostic criterion for myeloproliferative neoplasms (MPN) and are found in approximately 95% of polycythemia vera cases and 50% of essential thrombocythemia and primary myelofibrosis [2,9,66,67]. Cancers 2022, 14, 5531 9 of 13 Mutations in this gene, specifically the V617F hotspot, were reported in approximately 6% of patients with MDS-5q and were found to correlate with higher platelet counts when compared with JAK2 wild-type patients [35,55,68]. Sangiorgio et al., performed a detailed microscopic analysis of BM aspirates of MDS-5q cases with concomitant JAK2 mutations and found greater reticulin fibrosis in mutated cases. Additionally, they found a combination of hypolobulated megakaryocytes (typically found in MDS-5q) and large forms with hyperlobulated nuclei, which are commonly seen in MPN [69]. Although the phenotypic characteristics have been described, no significant differences in OS or disease progression were found in such MDS-5q JAK2 mutated cases when compared with JAK2 wild-type cases [55,69]. 5. Clonal Evolution As mentioned previously, several authors have described the commonly mutated genes and concomitant copy number alterations in MDS-5q, but few studies have explored clonal evolution in this specific subtype of MDS. The first systematic study providing molecular monitoring of long-term serial follow- up samples in a significant cohort of patients was by Mossner et al. [41]. As in most of the subsequent publications, such clonal evolution studies are based on bulk sequencing (exome or gene panel), in which clonal composition and evolutionary patterns are recon- structed based on variant allele frequency values of the detected mutations. The authors described that MDS “founder” lesions recurrently affected genes involved in the regulation of DNA methylation (e.g., TET2, DNMT3A), chromatin remodeling (e.g., ASXL1), or RNA splicing (e.g., SF3B1), and that del(5q) was acquired as a secondary lesion or constituted a minor independent clone in 62% of patients classified as MDS-5q. This is in contrast to previous studies proposing del(5q) as the initiating lesion in such patients [44]. In line with this, single-cell studies performed by our group demonstrated that in some MDS-5q cases, del(5q) can appear as the initiating lesion, while it can appear as a secondary hit in other cases [43]. As expected, the emergence and disappearance of specific clones in the BM are fre- quently correlated with changes in the clinical features in PB, such as hemoglobin and platelet levels. Moreover, it has been described that, in almost all cases, treatment with lenalidomide induced an effective reduction of cells carrying del(5q), however, it did not induce complete molecular remission of all clones carrying typical MDS mutations [41]. Furthermore, loss of response to lenalidomide is correlated with the gradual growth of a non-related clonal population already detectable at low levels before treatment or the expansion of a descendent from the original clone of the diagnosis [70]. In another longitudinal study, Scharenberg et al. described that 37% of their MDS-5q cohort progressed to either higher-risk MDS or transformed into AML in a median of 85 months after diagnosis. Interestingly, they found that all the cases harbored recurrent mutations in TP53, TET2, or RUNX1 in addition to del(5q) [51]. Thus, several patients showed an increased allele burden and gains of new mutations during the course of the disease and treatment. Particularly, the acquisition of TP53 mutations was relatively common in the progression of patients treated with lenalidomide, with some of them exhibiting more than one TP53 mutation. In general, all the above-mentioned studies agreed that both linear and branched evolutionary patterns occur with and without disease-modifying treatments, and sub- clones that acquire additional mutations associated with treatment resistance or disease progression can be detected months before clinical changes become apparent [41,43,51,70]. 6. Conclusions Based on our understanding of MDS-5q, together with the changes in the inclusion criteria, the evaluation of prognosis evaluation and the management of the disease are clearly in line with the progress of molecular genetics, which at the same time are linked to the advances in technology and scientific discoveries. Cancers 2022, 14, 5531 10 of 13 With the arrival of the IPSS-M and the newly proposed classifications for MDS, NGS techniques are mandatory for correct disease classification and assessment of prognosis. However, the approaches to financing health care are extremely diverse and are country- specific, and therefore, there may still be situations in which NGS remains restricted to potentially guiding therapeutic decisions, such as treatment intensity or hematopoietic stem cell transplantation. Although many advances have been achieved, especially in the last decade, unan- swered questions remain. 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