cancers Article Analysis of Intratumoral Heterogeneity in Myelodysplastic Syndromes with Isolated del(5q) Using a Single Cell Approach Pamela Acha 1,† , Laura Palomo 1,† , Francisco Fuster-Tormo 1 , Blanca Xicoy 2 , Mar Mallo 1,3 , Ana Manzanares 1 , Javier Grau 2 , Silvia Marcé 2 , Isabel Granada 2 , Marta Rodríguez-Luaces 4 , María Diez-Campelo 5 , Lurdes Zamora 2 and Francesc Solé 1, * 1 MDS Group, Institut de Recerca Contra la Leucèmia Josep Carreras, ICO-Hospital Germans Trias i Pujol, Universitat Autònoma de Barcelona, 08916 Badalona, Spain; pacha@carrerasresearch.org (P.A.); lpalomo@carrerasresearch.org (L.P.); ffuster@carrerasresearch.org (F.F.-T.); mmallo@carrerasresearch.org (M.M.); amanzanares@carrerasresearch.org (A.M.) 2 Hematology Service, ICO-Hospital Germans Trias i Pujol, Institut de Recerca Contra la Leucèmia Josep Carreras, Universitat Autònoma de Barcelona, 08916 Badalona, Spain; bxicoy@iconcologia.net (B.X.); jgrau@iconcologia.net (J.G.); smarce@iconcologia.net (S.M.); igranada@iconcologia.net (I.G.); lzamora@iconcologia.net (L.Z.) 3 Microarrays Unit, Institut de Recerca Contra la Leucèmia Josep Carreras, ICO-Hospital Germans Trias i Pujol, Universitat Autònoma de Barcelona, 08916 Badalona, Spain 4 Hematology Service, ICO-Hospital Verge de la Cinta de Tortosa, 43500 Tarragona, Spain; marta.rodriguez@iconcologia.net 5 Hematology Service, Hospital Universitario de Salamanca, 37007 Salamanca, Spain; mdiezcampelo@usal.es * Correspondence: fsole@carrerasresearch.org; Tel.: +34-93-557-2806  † These authors contributed equally.  Citation: Acha, P.; Palomo, L.; Simple Summary: Myelodysplastic syndromes (MDS) are a heterogeneous group of clonal hematopoi- Fuster-Tormo, F.; Xicoy, B.; Mallo, M.; etic stem cell malignancies characterized by ineffective differentiation of one or more bone marrow Manzanares, A.; Grau, J.; Marcé, S.; cell lineages. Only 50% of patients with de novo MDS will be found to have cytogenetic abnormalities, Granada, I.; Rodríguez-Luaces, M.; of which del(5q) is the most common. In 10% of MDS cases, del(5q) is found as a sole abnormality. In et al. Analysis of Intratumoral this work, a single cell approach was used to analyze intratumoral heterogeneity in four patients Heterogeneity in Myelodysplastic with MDS with isolated del(5q). We were able to observe that an ancestral event in one patient can Syndromes with Isolated del(5q) appear as a secondary hit in another one, thus reflecting the high intratumoral heterogeneity in MDS Using a Single Cell Approach. with isolated del(5q) and the importance of patient-specific molecular characterization. Cancers 2021, 13, 841. https:// doi.org/10.3390/cancers13040841 Abstract: Myelodysplastic syndromes (MDS) are a heterogeneous group of hematological diseases. Among them, the most well characterized subtype is MDS with isolated chromosome 5q deletion Academic Editor: Adriano Venditti Received: 21 January 2021 (MDS del(5q)), which is the only one defined by a cytogenetic abnormality that makes these patients Accepted: 14 February 2021 candidates to be treated with lenalidomide. During the last decade, single cell (SC) analysis has Published: 17 February 2021 emerged as a powerful tool to decipher clonal architecture and to further understand cancer and other diseases at higher resolution level compared to bulk sequencing techniques. In this study, a Publisher’s Note: MDPI stays neutral SC approach was used to analyze intratumoral heterogeneity in four patients with MDS del(5q). with regard to jurisdictional claims in Single CD34+CD117+CD45+CD19- bone marrow hematopoietic stem progenitor cells were isolated published maps and institutional affil- using the C1 system (Fluidigm) from diagnosis or before receiving any treatment and from available iations. follow-up samples. Selected somatic alterations were further analyzed in SC by high-throughput qPCR (Biomark HD, Fluidigm) using specific TaqMan assays. A median of 175 cells per sample were analyzed. Inferred clonal architectures were relatively simple and either linear or branching. Similar to previous studies based on bulk sequencing to infer clonal architecture, we were able to Copyright: © 2021 by the authors. observe that an ancestral event in one patient can appear as a secondary hit in another one, thus Licensee MDPI, Basel, Switzerland. reflecting the high intratumoral heterogeneity in MDS del(5q) and the importance of patient-specific This article is an open access article molecular characterization. distributed under the terms and conditions of the Creative Commons Keywords: myelodysplastic syndromes; single cell; intratumoral heterogeneity Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). Cancers 2021, 13, 841. https://doi.org/10.3390/cancers13040841 https://www.mdpi.com/journal/cancers Cancers 2021, 13, 841 2 of 17 1. Introduction Myelodysplastic syndromes (MDS) are a heterogeneous group of hematologic malig- nancies characterized by ineffective hematopoiesis which ultimately derives from periph- eral blood (PB) cytopenias and dysplasia. Mainly based on those characteristics, the World Health Organization (WHO) distinguishes six MDS adult subtypes that differ on their risk of progression to acute myeloid leukemia (AML) [1]. Among them, the most well charac- terized subtype is MDS with isolated chromosome 5q deletion (MDS del(5q)), which is the only one defined by a cytogenetic abnormality [2]. Lenalidomide is an immunomodulatory drug that preferentially affects del(5q) cells, leading to a complete cytogenetic remission and transfusion independence in 50% and 70% of MDS del(5q) patients, respectively [3,4]. MDS patients are genetically heterogeneous, with approximately 90% of cases harbor- ing recurrent somatic mutations affecting around 40 different genes [5–7]. Despite none of them being specific of the disease, associations between mutations and prognosis or response to treatments have been described, such as the good prognosis of SF3B1 mu- tations and the association of TP53 mutations with adverse outcomes and resistance to lenalidomide [8,9]. Moreover, several studies have documented the complexity of clonal evolution in MDS describing both linear and branched evolutionary patterns even in low risk MDS cases [10,11]. These studies also corroborate that therapy induces a selective pressure capable of reshaping mutational architecture [12–14]. Previous studies use conventional or bulk sequencing approaches, which average information regarding mutational pattern in a sample using DNA from admixed cell populations comprised by different tumor clones as well as normal cells. Thus, clonal architecture (CA) of the sample can be inferred using the variant allele frequency (VAF) of each detected mutation: through statistical methods and bioinformatic algorithms, mutations with similar VAF are clustered together. However, this approach provides low resolution when there is not enough difference between VAF values, as mutations cannot accurately be separated into new clones and tend to be placed together in existing ones. This can also lead to miss small and rare cell subpopulations, which are often implicated in disease progression and relapse. In the last decade, single cell (SC) analysis has emerged as a powerful tool that might overcome these difficulties, allowing a higher resolution level to study cancer and to further understand the disease [15,16]. Herein, we performed SC studies in four patients with MDS del(5q) at diagnosis or before receiving any treatment (DX/PRE) and, for three of them, during follow-up (FU). For this purpose, CD34+CD117+CD45+CD19- bone marrow (BM) hematopoietic stem progenitor cells (HSPC) were studied. A median of 175 cells per sample were analyzed and considered for CA inference. 2. Results 2.1. Baseline Characteristics and Genetic Analysis of MDS Patients Four female patients with MDS del(5q), diagnosed according to the WHO 2017 classi- fication criteria, were included in the study [1]. Median age at DX was 78 years old (range: 69–83). Table 1 shows the main clinical and hematological characteristics of each patient at each studied time point. Cancers 2021, 13, 841 3 of 17 Table 1. Main clinical and hematological data of the four patients included in the study. Hb WBC Platelets ANC BM Blasts UPN Age at DX and Sex Sampling Time Points Karyotype, SNP-A and FISH (ISCN) IPSS-R Treatment (g/dL) (×109 /L) (×109 /L) (×109 /L) (%) 46,XX,del(5)(q13q33)[15]/46,XX[2] 1 DX 10.8 3.20 221 1.10 1.0 arr[hg19] 5q14.3q34(89575437-163450743)x1 Very low nuc ish(D5S1518Ex2,D5S1976x2,EGR1x1,RPS14x1)[63/100] 79 SINTRA-REV P1 Female 1st FU (14 months after 46,XX,del(5)(q13q33)[17]/46,XX[3] 1 (Placebo) 10.2 3.20 252 1.00 0.0 diagnosis) nuc ish(D5S1518Ex2,D5S1976x2,EGR1x1,RPS14x1)[80/100] Very low 2nd FU (33 months 46,XX,del(5)(q13q33)[20] 3 9.1 3.60 374 1.10 3.0 after diagnosis) nuc ish(D5S1518Ex2,D5S1976x2,EGR1x1,RPS14x1)[88/100] Low 46,XX,del(5)(q22q33)[6]/46,XX[14] PRE: 4 years after arr[hg19] 5q21.2q34(102986652-162755919)x1, 2 diagnosis (never 8.8 4.10 233 2.10 0.5 Xp22.31(6449753-8135644)x1 Low received treatment) 69 FISH: NA SINTRA-REV P2 Female (Lenalidomide) 1st FU (14 months after 46,XX[20] 2 9.3 4.60 262 1.52 0.5 diagnosis) nuc ish(D5S1518E,D5S1976,EGR1,RPS14)x2[100] Low 2nd FU (22 months 46,XX[20] 2 8.4 5.20 353 1.80 1.0 after diagnosis) FISH: NA Low PRE: 2 years after 46,XX,t(1;1)(p35;p36),del(5)(q12q33)[20] 77 2 P3 diagnosis (previously: 10.5 6.40 166 3.90 1.5 arr[hg19] 5q14.3q34(86255729-166126310)x1 Lenalidomide Female Low support treatment) FISH: NA 46,XX,del(5)(q13q32)[9]/46,XX[11] 3 DX 9.3 3.95 262 2.44 3.0 arr[hg19] 5q21.3q34(107937392-165840296)x1 83 Low P4 nuc ish(D5S1518Ex2,D5S1976x2,EGR1x1,RPS14x1)[32/100] Lenalidomide Female 1st FU (20 months after 46,XX,del(5)(q13q32)[9]/46,XX[11] 2 11.7 3.66 106 1.47 4.0 diagnosis) nuc ish(D5S1518Ex2,D5S1976x2,EGR1x1,RPS14x1)[29/100] Low FISH: XL 5q31/5q33/5p15 locus-specific deletion probe was used to detect 5q deletions (D-5081-100-TC, MetaSystems Probes). SINTRA-REV: phase III multicenter, randomized, double blind and controlled with placebo clinical trial and with two arms designed to assess the efficiency and toxicity of the scheme Lenalidomide versus observation in a series of 60 patients with low risk myelodysplastic syndrome associated to 5q deletion with anemia (Hb ≤ 12 g/dL) but without the need of transfusion. Abbreviations: ANC: absolute neutrophil count; BM: bone marrow; DX: diagnosis; FISH: fluorescence in situ hybridization; FU: follow-up; Hb: hemoglobin; IPSS-R: Revised International Prognostic Scoring System; NA: not available; PRE: pre-treatment; SNP-A: single nucleotide polymorphism arrays; UPN: unique patient number; WBC: white blood cells. Cancers 2021,2021, Cancers 13, x 13, FOR841 PEER REVIEW 1 of 18 4 of 17 At DX/PRE, BM and PB samples all were patients presented obtained fromwith low hemoglobin all patients at DX/PRElevels, which and from reflects the available FU. anemia. Risk waswere BM samples assessedusedaccording to thebulk to isolate Revised International tumoral DNA Prognostic and to sort Scoring System (IPSS-R) [17], withHSPC CD34+CD117+CD45+CD19− low and for very low riskSC subsequent forstudies. all patients. Conventional Matched germlinecytogenetics control DNA(CC) wasand fluorescence obtained in situlymphocytes from T-CD3+ hybridization (FISH) from werePB DX/PRE performed samples. by standard A general routine over- analysis. Three patients presented del(5q) view of the sample processing is shown in Figure 1. as an isolated alteration while patient P3 also harbored a reciprocal translocation between both chromosomes Whole exome sequencing (WES) analysis and single nucleotide polymorphism ar- 1. BM and rays (SNP-A) were PBused samples weremutations to detect obtained from all patients (single nucleotideat DX/PRE variants and from (SNVs) available and small FU. BM samples were used to isolate bulk tumoral DNA and to sort CD34+CD117+CD45+CD19 insertion/deletions) and copy number alterations (CNA) in DX/PRE samples, respectively. − HSPC for In addition, subsequent targeted SC studies.(TDS) deep sequencing Matched germline control was performed in DNADNA wasfrom samples obtained BM atfrom DX/PRE and last available FU samples using a custom capture-based panel targeting 40 pro- T-CD3+ lymphocytes from DX/PRE PB samples. A general overview of the sample cessing is shown in Figure 1. myeloid-related genes (Table S1). Figure Figure 1. General 1. General overview overview of sample of sample processing. processing. Abbreviations: Abbreviations: DX/PRE: DX/PRE: diagnosis/pre-treatment; diagnosis/pre-treat- FU: follow-up. ment; FU: follow-up. WESWhole exome revealed sequencing 29 somatic (WES) analysis mutations and across the single four nucleotide patients polymorphism at DX/PRE arrays where non (SNP-A) were used to detect mutations (single nucleotide variants (SNVs) synonymous SNVs were the most common alterations (25/29 mutations) (Table 2; Tables and small insertion/deletions) and copy number alterations (CNA) in DX/PRE samples, respectively. S2 and S3). Each patient harbored a median of 7 somatic mutations and, as expected, sim- In addition, targeted deep sequencing (TDS) was performed in DNA samples from BM ilar VAF values were observed by WES and TDS in common analyzed genes. Analysis of at DX/PRE and last available FU samples using a custom capture-based panel targeting FU samples by TDS revealed the same somatic mutations from the DX/PRE, except for 40 myeloid-related genes (Table S1). patient P4, in which an additional TP53 mutation was detected only in the FU sample, WES revealed 29 somatic mutations across the four patients at DX/PRE where suggesting that it was acquired during treatment with lenalidomide (TDS was also per- non synonymous SNVs were the most common alterations (25/29 mutations) (Table 2; formed at DX/PRE, confirming the absence of this mutation at this time point). Addition- Tables S2 and S3). Each patient harbored a median of 7 somatic mutations and, as expected, ally, SNP-A allowed a more precise definition of the del(5q) breakpoints (Table S4) and similar VAF values were observed by WES and TDS in common analyzed genes. Analysis revealed the presence of a 1.67 Mb microdeletion in the Xp chromosome of patient P2. of FU samples by TDS revealed the same somatic mutations from the DX/PRE, except for Based on the results of genetic analyses (WES and SNP-A), candidate SNVs and patient P4, in which an additional TP53 mutation was detected only in the FU sample, sug- CNAs were selected for each patient for posterior targeted qPCR analysis at SC resolution gesting that it was acquired during treatment with lenalidomide (TDS was also performed in DX/PRE and available at DX/PRE, FU samples. confirming SNVs the absence of highlighted this mutation in at bold thisintime Tablepoint). 2 and CNA de- Additionally, tected by SNP-A were selected for subsequent SC studies. SNP-A allowed a more precise definition of the del(5q) breakpoints (Table S4) and revealed the presence of a 1.67 Mb microdeletion in the Xp chromosome of patient P2. Cancers 2021, 13, 841 5 of 17 Table 2. Single nucleotide variants (SNVs) detected by whole exome sequencing (WES) and targeted deep sequencing (TDS) in diagnosis/pre-treatment samples and in the last available FU sample. VAF (%) UPN Gene Chr Transcript Type of Alteration Variant Aminoacid Change WES DX/PRE TDS DX/PRE TDS FU CUX1 7 NM_001202543 Stopgain SNV c.3019C>T p.Arg1007 * 3 SETBP1 18 NM_015559 Non-synonymous SNV c.2612T>C p.Ile871Thr 15 14 17 MAP7D2 X NM_001168465 Non-synonymous SNV c.116A>G p.Asn39Ser 24 TENM1 X NM_014253 Non-synonymous SNV c.3857G>T p.Cys1286Phe 27 P1 LRTOMT 11 NM_001145310 Non-synonymous SNV c.671G>A p.Arg224His 34 CCDC168 13 NM_001146197 Non-synonymous SNV c.122A>C p.Gln41Pro 34 TP53 17 NM_001126115 Non-synonymous SNV c.448C>T p.Arg150Trp 39 38 42 NUP93 16 NM_001242795 Non-synonymous SNV c.473C>A p.Ala158Asp 40 UNC79 14 NM_020818 Non-synonymous SNV c.7055T>A p.Val2352Glu 44 LRRC45 17 NM_144999 Non-synonymous SNV c.1670A>G p.Glu557Gly 7 CRIPAK 4 NM_175918 Non-synonymous SNV c.25A>C p.Asn9His 7 CACHD1 1 NM_020925 Non-synonymous SNV c.1161G>T p.Arg387Ser 9 P2 IL21R 16 NM_181078 Non-synonymous SNV c.179A>G p.Asp60Gly 27 SF3B1 2 NM_012433 Non-synonymous SNV c.2098A>G p.Lys700Glu 40 39 43 YLPM1 14 NM_019589 Stopgain SNV c.4087C>T p.Arg1363 * 46 CGNL1 15 NM_001252335 Non-synonymous SNV c.3481C>A p.Arg1161Ser 7 IBSP 4 NM_004967 Non-synonymous SNV c.231G>T p.Glu77Asp 8 SLC22A12 11 NM_144585 Non-synonymous SNV c.232C>T p.Pro78Ser 14 TRIM24 7 NM_015905 Non-synonymous SNV c.263A>G p.Tyr88Cys 14 P3 SETD2 3 NM_014159 Non-synonymous SNV c.6197A>G p.Asp2066Gly 21 FAT1 4 NM_005245 Non-synonymous SNV c.1507G>A p.Ala503Thr 23 TCHH 1 NM_007113 Non-synonymous SNV c.3770A>G p.Gln1257Arg 28 PPM1D 17 NM_003620 Stopgain SNV c.1434C>A p.Cys478 * 29 SS18L1 20 XM_005260390 Non-synonymous SNV c.604A>G p.Ser202Gly 29 TP53 17 NM_001276761 Non-synonymous SNV c.283T>C p.Phe95Leu ND ND 7 SCUBE1 22 NM_173050 Non-synonymous SNV c.1700C>T p.Ala567Val 8 P4 BMP7 20 NM_001719 Non-synonymous SNV c.908G>A p.Arg303His 14 NUP85 17 NM_024844 Non-synonymous SNV c.877G>C p.Ala293Pro 17 DNMT3B 20 NM_006892 Splicing variant c.1906-1G>T NA 21 SNVs in bold were selected for single cell analysis. Abbreviations: Chr: chromosome; DX: diagnosis; FU: follow-up; ND: not detected; PRE: pre-treatment; SNV: single nucleotide variant; TDS: targeted deep sequencing; UPN: unique patient number; VAF: variant allele frequency; WES: whole exome sequencing. * is to indicate that a stop codon is introduced. Based on the results of genetic analyses (WES and SNP-A), candidate SNVs and CNAs were selected for each patient for posterior targeted qPCR analysis at SC resolution in DX/PRE and available FU samples. SNVs highlighted in bold in Table 2 and CNA detected by SNP-A were selected for subsequent SC studies. 2.2. Clonal Architecture in CD34+CD117+CD45+CD19- HSPC We analyzed a median of 175 cells per sample (range: 94–201) that were considered for CA inference after analyzing sequential samples from each patient. Proposed CAs for each patient are represented in Figures 2–5. The number of identi- fiable subclones varied from one to three. Inferred CA were relatively simple and either linear or branching. Cancers 2021, 13, 841 6 of 17 Cancers 2021, 13, x FOR PEER REVIEW 4 of 18 Figure 2. Clinical and molecular characteristics of patient P1. (A) Bar plot depicts the number of cells with a given genotype Figure 2. Clinical and molecular characteristics of patient P1. (A) Bar plot depicts the number of for each clone that was detected at the moment of diagnosis (DX), 1st follow-up (FU-1) and 2nd follow-up (FU-2). Heatmap cells with a given genotype for each clone that was detected at the moment of diagnosis (DX), 1st below the bars indicates the genotype for each clone: dark blue and light blue are used to indicate the mutated or wild- follow-up (FU-1) type state, respectively. (B) Hematological and 2nd follow-up characteristics such as (FU-2). Heatmap hemoglobin (Hb), below plateletthe bars(Plt), count indicates the genotype leukocyte count for each clone: dark blue and light blue are used to indicate the mutated or wild-type (Leu) and absolute neutrophil count (ANC) are indicated for each analyzed time point. (C) Proposed clonal architecture, state, respectively. (B) Hematological karyotype and number of analyzed characteristics cells for each time point issuch as hemoglobin indicated. Color red(Hb), platelet count distinguishes new(Plt), leukocyte acquired count (Leu) alterations in each clone. and absolute neutrophil count (ANC) are indicated for each analyzed time point. (C) Proposed clonal architecture, karyotype and number of analyzed cells for each time point is indicated. Color red 2.2.2. Patient new distinguishes P2 acquired alterations in each clone. Five alterations were included for SC studies at 3 time points: PRE, FU after 14 and 22 months, respectively (Tables 1 and 2). In addition to del(5q), del(Xp), detected by SNP- A, was also included in SC studies. Results at PRE reveal a clone harboring SF3B1, YLMP1 and del(Xp) (Figure 3). Fol- lowing a branched evolutionary process, this clone gave rise to two other ones: the most prevalent clone harboring del(5q) and a small subclone without del(5q) that, instead, pre- sented with CACHD1 mutation. In contrast to patient P1, in this case del(5q) is not present Cancers 2021, 13, x FOR PEER REVIEW 6 of 18 Cancers 2021, 13, 841 7 of 17 Figure Figure 3. Clinical 3. Clinical and molecular and molecular characteristics characteristics of patient of patient P2. (A) P2. Bar (A) plotBar plot the depicts depicts the of number number cells of cells with a given genotype for each clone that was detected at the moment with a given genotype for each clone that was detected at the moment of pre-treatment (PRE), 1st of pre-treatment (PRE), 1st follow-up follow-up (FU-1) (FU-1) and 2ndand 2nd follow-up follow-up (FU-2). Heatmap (FU-2). Heatmap below thebelow the bars indicates bars indicates the genotype the genotype for each for clone: dark each bluedark clone: and light bluelight blue and are used blue to areindicate used tothe mutated indicate the or wild-type mutated state, respectively. or wild-type (B) state, respectively. Hematological characteristics (B) Hematological such assuch characteristics hemoglobin (Hb), platelet as hemoglobin countcount (Hb), platelet (Plt), (Plt), leukocyte countcount leukocyte (Leu)(Leu) and and absolute neutrophil absolute count neutrophil count(ANC) (ANC)areareindicated indicatedfor for each analyzed time each analyzed timepoint. point.(C) (C)Proposed Proposed clonal clonal architecture, karyotype and number of analyzed cells for each time point is indicated. Color architecture, karyotype and number of analyzed cells for each time point is indicated. Color red red distinguishes new acquired alterations in each clone. * homozygous mutation. distinguishes new acquired alterations in each clone. * homozygous mutation. 2.2.3. Patient P3 A total of 7 alterations were selected for SC studies (Tables 1 and 2). Two clones, which differed only in TRIM24 mutation, were detected at the moment of PRE (Figure 4). Two months after this sample was obtained, patient progressed to myelofibrosis and no evaluable BM samples could be further obtained for SC studies due to BM fibrosis. Unfor- tunately, the patient died before another sample could be obtained. No complex intratumoral heterogeneity was observed in this patient. This is con- Cancers 2021, 13, 841 8 of 17 sistent with the fact that MDS del(5q) patients are commonly grouped as low and very low risk categories. Figure 4. Clinical and molecular characteristics of patient characteristics of patient P3. P3. (A) Bar plot depicts the number of cells with a given genotype for each for each clone clone that thatwas wasdetected detectedatatthe themoment momentofofpre-treatment pre-treatment (PRE). (PRE). Heatmap Heatmap below below thethe bars bars indicates indicates the the genotype genotype for for each each clone: clone: darkdark blue blue andand light light blue blue areare used used toto indicatethe indicate themutated mutatedororwild-type wild-typestate, state,respectively. respectively.(B) (B) Hematological Hematological characteristics such characteristics such as as hemoglobin hemoglobin (Hb), (Hb), platelet platelet count count (Plt), (Plt), leukocyte leukocyte count count (Leu) (Leu) and and absolute absolute neutrophil neutrophil count count (ANC) (ANC) are indicated for the analyzed time point. (C) Proposed clonal architecture, karyotype and number of analyzed cells for are indicated for the analyzed time point. (C) Proposed clonal architecture, karyotype and number of analyzed cells for the the analyzed time point are indicated. Color red distinguishes new acquired alterations in the secondary clone. analyzed time point are indicated. Color red distinguishes new acquired alterations in the secondary clone. 2.2.4. Patient P4 2.2.1. Patient P1 Four alterations were selected for SC studies (Tables 1 and 2) at 2 time points: DX and A total FU after of six alterations were included for SC studies at 3 time points: DX, FU after 14 20 months. and 33SC analysisofatDX, months DXrespectively revealed the(Tables 1 and presence of a2).unique Figureclone 2 shows the detected harboring, clones besides and del(5q), proposed CA for each moment. NUP85 and BMP7 mutations (Figure 5). Subsequently, a FU sample obtained 20 months At DX, a clone harboring TP53, LRTOMT, NUP93 and del(5q) followed a linear after DX while the patient was under lenalidomide treatment, was analyzed. Results re- evolutionary process and gave rise to another subclone that acquired SETBP1 mutation. vealed that the initial clone gave rise to another one that additionally carried TP53 muta- These two clones could be detected in both FU samples. Additionally, 14 months after DX, tion and that represented the dominant clone in the CD34+CD117+CD45+CD19- HSPC a subclone harboring CUX1 mutation was detected. This subclone could not be detected compartment. The presence of TP53 mutations in MDS del(5q) is associated with lower in the sample obtained 33 months after the DX; however, another clone harboring TP53, response rates to lenalidomide, which would be consistent with the fact that the patient LRTOMT, NUP93 but without del(5q) was detected. It was inferred that this clone might did not achieve cytogenetic response even when hemoglobin levels improved after treat- precede the other ones. ment. Considering that this patient was included in the placebo arm in the SINTRA-REV In addition, cells with no alterations were also detected at this moment. It is highly clinical trial, our results would be reflecting the natural course of the disease. No significant probable that those cells correspond to a non-tumoral population (healthy HSPC). How- changes were observed at molecular level, nor were additional chromosomal lesions ever, the existence of small CNA or other SNV cannot be fully discarded because SNP-A detected in the FU samples. According to the IPSS-R criteria, the patient was categorized as and WES a very were only low-risk performed case at in DX the moment samples. of DX. However, considering that the hemoglobin level decreases and blast percentage increases, the IPSS-R increases in two points, recategorizing the patient in the low-risk group. Additionally, a TP53 mutation was present in all detected clones. There is evidence from previous studies that this mutation occurs in an early stage of the disease in at least one fifth of low-risk MDS patients with del(5q) and that it is associated with a shorter median overall survival and with an increased risk of disease evolution [18,19]. Therefore, the presence of this mutation might be contributing to clinical changes that were previously described. Cancers 2021, 13, Cancers 2021, 13, x841 FOR PEER REVIEW 8 of 18 9 of 17 Figure 5. Clinical and molecular characteristics of patient P4. (A) Bar plot depicts the number of cells Figure 5. Clinical and molecular characteristics of patient P4. (A) Bar plot depicts the number of with a given genotype for each clone that was detected at the moment of diagnosis (DX), 1st follow- cells up with (FU-1) a given and genotype 2nd follow-up for each (FU-2). clone below Heatmap that wasthe detected at thethe bars indicates moment genotype offor diagnosis (DX), 1st each clone: follow-up (FU-1) and 2nd follow-up (FU-2). Heatmap below the bars indicates the dark blue and light blue are used to indicate the mutated or wild-type state, respectively. (B) He- genotype for each clone:characteristics matological dark blue and light such asblue are used(Hb), hemoglobin to indicate plateletthe mutated count or wild-type (Plt), leukocyte state, count respectively. (Leu) and absolute neutrophil count (B) Hematological (ANC) are characteristics indicated such for each (Hb), as hemoglobin analyzed timecount platelet point.(Plt), (C) Proposed leukocyteclonal count (Leu) architecture, and absolute karyotype and neutrophil number count of analyzed (ANC) cells for are indicated for each each analyzed time pointtime are point. indicated. Color red clonal (C) Proposed distinguishes new acquired alterations in each clone. architecture, karyotype and number of analyzed cells for each time point are indicated. Color red distinguishes new acquired alterations in each clone. Cancers 2021, 13, 841 10 of 17 2.2.2. Patient P2 Five alterations were included for SC studies at 3 time points: PRE, FU after 14 and 22 months, respectively (Tables 1 and 2). In addition to del(5q), del(Xp), detected by SNP-A, was also included in SC studies. Results at PRE reveal a clone harboring SF3B1, YLMP1 and del(Xp) (Figure 3). Fol- lowing a branched evolutionary process, this clone gave rise to two other ones: the most prevalent clone harboring del(5q) and a small subclone without del(5q) that, instead, pre- sented with CACHD1 mutation. In contrast to patient P1, in this case del(5q) is not present in the inferred ancestral clone at the moment of PRE. Nonetheless, the secondary clone harboring del(5q) constitutes the predominant clone in CD34+CD117+CD45+CD19- HSPC compartment at that time point. After 14 months of being enrolled in the SINTRA-REV clinical trial, in contrast to normal karyotype results, SC studies revealed that the clone harboring del(5q) was still present but in a significantly lower proportion (only 5% of analyzed cells). However, in the second FU sample (22 months after the initial sample), del(5q) was not detected anymore by either CC or SC analysis. This complete cytogenetic response is consistent with this patient being in lenalidomide arm in the SINTRA-REV clinical trial. Notably, this is the only case where a homozygous mutation was detected. SNV in YLPM1 gene was previously detected by WES at 46% VAF, but only a mutated probe signal was detected by SC analysis in tumoral cells. Considering the VAF detected by WES, the presence of a homozygous mutation was not initially expected. However, the fact that WES was performed in bulk BM DNA and SC studies were done in a HSPC subpopulation of the BM might provide an explanation for the low VAF value. 2.2.3. Patient P3 A total of 7 alterations were selected for SC studies (Tables 1 and 2). Two clones, which differed only in TRIM24 mutation, were detected at the moment of PRE (Figure 4). Two months after this sample was obtained, patient progressed to myelofibrosis and no evaluable BM samples could be further obtained for SC studies due to BM fibrosis. Unfortunately, the patient died before another sample could be obtained. No complex intratumoral heterogeneity was observed in this patient. This is consistent with the fact that MDS del(5q) patients are commonly grouped as low and very low risk categories. 2.2.4. Patient P4 Four alterations were selected for SC studies (Tables 1 and 2) at 2 time points: DX and FU after 20 months. SC analysis at DX revealed the presence of a unique clone harboring, besides del(5q), NUP85 and BMP7 mutations (Figure 5). Subsequently, a FU sample obtained 20 months after DX while the patient was under lenalidomide treatment, was analyzed. Results revealed that the initial clone gave rise to another one that additionally carried TP53 mutation and that represented the dominant clone in the CD34+CD117+CD45+CD19- HSPC compartment. The presence of TP53 mutations in MDS del(5q) is associated with lower response rates to lenalidomide, which would be consistent with the fact that the patient did not achieve cytogenetic response even when hemoglobin levels improved after treatment. In addition, cells with no alterations were also detected at this moment. It is highly probable that those cells correspond to a non-tumoral population (healthy HSPC). However, the existence of small CNA or other SNV cannot be fully discarded because SNP-A and WES were only performed in DX samples. 3. Discussion Most of our current knowledge about intratumoral heterogeneity in MDS derives from bulk sequencing studies. However, part of this heterogeneity is the result of the coexistence Cancers 2021, 13, 841 11 of 17 of nonmalignant cells and diverse subpopulations of tumoral cells, each one harboring their own genetic characteristics, which might remain masked when bulk sequencing approaches are used. VAF values might give an idea regarding the size of a clone carrying a specific mutation and this might help to establish the order of acquisition of each mutational event when sequential samples are analyzed. Nonetheless, when there is not enough difference between VAF values, this approach could lead to mistakes when trying to resolve co- occurrence patterns of gene mutations and it is unsuccessful in resolving zygosity states. Because of this, rare cancer cells might remain masked and intratumoral complexity could be underestimated using bulk sequencing strategies [15,20]. All these difficulties might be overcome with SC strategies. Except for two recent studies [21,22], and unlike other diseases such as AML or multi- ple myeloma [20,21,23–27], DNA SC strategies have not been applied in the MDS research field. In the present study, SC was applied to a selected group of MDS del(5q) patients. Previous studies performed by Nilsson et al. [28,29] supported the view that del(5q) is an early event in the pathobiology of MDS del(5q), which was verified by Woll et al. in 2014 [30]. In our study, in 3 out of 4 studied patients, we could verify the presence of del(5q) in all SCs, suggesting that this alteration is present in the most ancestral clones that were detected at the moment of DX/PRE. Patient P2, in which del(5q) was detected in a secondary clone, was the exception. It is noteworthy that the clone preceding the one harboring del(5q) in patient P2 harbored a mutation in the SF3B1 gene. According to the literature, this mutation often represents an ancestral event in MDS and it is detected in approximately 20% of MDS del(5q) [8]. This finding is in line with the results from Mossner et al., who, contrary to what was reported before, demonstrated that del(5q) constitutes a secondary event in 62% of MDS del(5q), according to their patient cohort [12]. Similarly, Woll et al. reported four cases where SF3B1 mutation preceded del(5q), but these constituted cases of either refractory anemia with ring sideroblasts or refractory cytopenia with multilineage dysplasia. Mian et al. studied the role of SF3B1 mutation in a cohort of patients with MDS with ring sideroblasts and concluded that such alteration might have an initial role in the pathogenesis of that MDS subtype [31]. Co-occurrence of SF3B1 mutation and del(5q) was a matter of debate in a recent publication of the International Working Group for the Prognosis of MDS [32], in which they provided evidence supporting the recognition of SF3B1-mutant MDS as a distinct entity. Regarding those cases with SF3B1 mutation and del(5q), they argued that, although genetic ontogeny of these myelodysplastic clones might inform the classification process and determine whether a case with concomitant del(5q) and SF3B1 mutation should be more appropriately classified as MDS del(5q) or MDS with mutated SF3B1, in many cases, clonal hierarchy cannot be easily solved in the clinical practice. At present, these cases should be classified according to current WHO criteria within the category of MDS del(5q) [1]. To date, lenalidomide has been approved in Europe for the treatment of low-risk transfusion-dependent patients with MDS del(5q), when other therapeutic options are insufficient or inadequate [33]. Even when 56% to 67% of patients achieve transfusion independence, after 2–3 years of treatment, clinical and cytogenetic relapse might appear in 50% of cases [3,33–35]. In our study, patients P2, P3 and P4 received lenalidomide, while patient P1 did not receive treatment (placebo arm in the SINTRA-REV clinical trial). Clinical and molecular data were consistent with patient P2 being in lenalidomide arm considering that, after 14 months of being in the clinical trial, complete cytogenetic response was achieved with a slight improvement in hemoglobin levels. Surprisingly, SC studies revealed that del(5q) was still present at that FU moment in 5% of analyzed cells, despite a normal karyotype was observed by CC. However, in the sample analyzed 22 months after the diagnosis, this clone was undetectable by both CC and our SC approach. This finding highlights the elevated sensibility of SC techniques, which also depends on the number of analyzed cells. Regarding patient P1, del(5q) was present in all studied samples, and hemoglobin levels were gradually decreasing as the IPSS-R score increased. As previously mentioned, Cancers 2021, 13, 841 12 of 17 this is consistent with the patient being in the placebo group. Interestingly, in the last FU sample, we detected a clone without del(5q) but with TP53 mutation suggesting that, in this patient, the mutation might precede the deletion. On the contrary, even when CC results suggest that the proportion of cells with such deletion was increasing, those results are not comparable to SC analysis because the cell population that was studied by each technique is not the same and also because real BM composition might be underrepresented by the sampling process. Even when it is reported that nearly 20% of patients with MDS del(5q) harbor TP53 mutations [18], Scharenberg et al. suggested that, in some cases, this mutation is acquired during the disease evolution, especially after lenalidomide treatment [36]. This seems to correlate with patient P4’s situation, in which TP53 mutation was only detected in the FU sample by TDS at 7% VAF, suggesting that it constituted a secondary subclonal event. This was verified by SC analysis, which revealed that the single clone that was detected at DX gave rise to another one that acquired TP53 mutation and that, in proportion, represents the biggest clone in the FU sample. Besides, the presence of this mutation has been previously associated with lower rates of cytogenetic response [9,36], but not with lower hematologic response rates [2,12,18]. This also correlates with the clinical features of patient P4. Even when CC reveals the same number of metaphases with del(5q) in both studied samples, an improvement in hemoglobin levels was noticed (that impacted in decreasing IPSS-R score). SC analysis also revealed a pool of cells without any of the studied alterations. It is highly probable that those cells belong to a non-tumoral population, normal HSPC that might be part the pool of cells contributing to the clinical improvement mentioned before. Overall, few changes were observed in the CA of the studied patients. This is reason- able, considering that the studied MDS subtype is generally associated to IPSS-R low risk categories, patients often present no other cytopenia besides anemia, BM blast percentage is normally <5% and high-risk cytogenetic alterations are usually not observed [1]. In line with the aforementioned, it is common to find a low number of genetic alterations in such low risk patients in contrast to other MDS subtypes generally associated with a higher risk, such as MDS with excess of blasts [7,9]. Moreover, considering that the median overall survival of MDS del(5q) patients is around 66–145 months [1], such results could be expected, taking into account that the largest FU included sample was obtained 33 months after DX. During the last few years, SC techniques have rapidly evolved and part of this advance is due to its combination with sequencing techniques [37]. Most of the new high-throughput SC instruments are based on microfluidic systems, where thousands of barcoded droplets carry each SC for subsequent library generation in a single tube [20,38]. Unlike these new instruments, capture in the C1 system is limited by the number of capture sites in the chip [26,37]. As an advantage of this system, and contrary to the newest ones, with the C1 system, it is possible to verify (by microscope visualization) and select for subsequent analysis only those capture sites containing one cell. Our approach allowed us to study the intratumoral heterogeneity of four patients with MDS del(5q). Even when it is a limited subset of cases, compared to previous reports where larger cohorts were studied [11–13,30,39], clonal composition and evolution patterns were figured out in those studies based on mutation VAF values in bulk tumoral samples. Despite such differences, studies performed by Woll et al., Mossner et al. and ours were able to observe that an ancestral event in one patient can appear as a secondary hit in another one [12,30]. 4. Materials and Methods 4.1. Patients Four patients with MDS del(5q), diagnosed according to the WHO 2017 classification criteria, were included in the study [1]. Patients were diagnosed in Hospital Institut Català d’Oncologia (ICO)–Germans Trias i Pujol (Badalona, Spain, n = 3) and in Hospital Verge de la Cinta (Tortosa, Spain, n = 1). PB and BM samples were collected after written informed Cancers 2021, 13, 841 13 of 17 consent in accordance with the Declaration of Helsinki and after the approval from Hospital Germans Trias i Pujol Ethics Committee (Reference number: PI-21-058). CC and FISH were performed by standard routine analysis, as previously described [40]. Patients P1 and P2 were enrolled in SINTRA-REV clinical trial (registered at clinicaltrials. gov (accessed on 21 January 2021) as NCT01243476), which is a phase III multicenter, ran- domized, double blind and controlled with placebo trial. SINTRA-REV was designed to assess the efficacy and toxicity of the scheme lenalidomide (5 mg qd for 28 days) versus observation in low risk MDS associated with del(5q) with anemia (Hemoglobin ≤ 12 g/dL) but without transfusion requirements. 4.2. Sample Processing BM and PB samples were obtained from all patients at DX/PRE and from available FU. BM samples obtained at DX/PRE and at available FU were used to isolate bulk tumoral DNA using the Maxwell 16 Blood DNA Purification Kit (Promega, Madison, WI, USA), and to sort CD34+CD117+CD45+CD19− HSPC with the FACSAria™ II cell sorter (BD Biosciences, San José, CA, USA) (Figure 1; Supplementary Methods). Matched germline control DNA was obtained from T-CD3+ lymphocytes from DX/PRE PB samples. 4.3. Mutation and Copy Number (CN) Analysis WES analysis and SNP-A were used to detect mutations (SNVs and small inser- tion/deletions) and CNA in DX/PRE samples, respectively. WES libraries were prepared from 1.5 µg of genomic DNA using the SureSelect Human Exome Kit 51 Mb V5 (Agilent Technologies, Santa Clara, CA, USA) and sequenced on a HiSeq2500 instrument (Illumina, San Diego, CA, USA) following a standard 2 × 100 bp paired-end reads protocol at a minimum mean coverage of 140x for tumoral samples and 60× for control germline sam- ples. WES data were analyzed using an in-house bioinformatics pipeline (Supplementary Methods). Genomic microarrays were performed with the CytoScan®HD (Thermo Fisher Scientific, Waltham, MA, USA), following the CytoScanTM User Guide (P/N 703038 Rev. 4), and analyzed with the Chromosome Analysis Suite version 3.0.0.42 (Thermo Fisher Scientific, Waltham, MA, USA) software (Supplementary Methods). In addition, TDS was performed in DNA samples from BM at DX/PRE and last available FU samples using a custom capture-based panel targeting 40 myeloid-related genes (Table S1). Libraries were performed with the SureSelectQXT Target Enrichment for Illumina Multiplexed Sequencing chemistry (Agilent Technologies, Santa Clara, CA, USA). TDS was performed on a MiSeq instrument (Illumina) following a paired-end 2 × 75 bp reads standard protocol, with a mean coverage of 1000×. Data were analyzed using a previously reported in-house bioinformatics pipeline [41]. Based on the results of genetic analyses, candidate mutations and CNAs were selected for each patient for posterior targeted qPCR analysis at SC resolution in DX/PRE and available FU samples. 4.4. SC Isolation SC isolation and processing were performed with the Fluidigm C1 platform (Fluidigm, San Francisco, CA, USA) using the C1 Single-Cell Open App IFC microfluidic chip for tumoral HSPC isolation (10–17 µm IFC) or control cells isolation (5–10 µm IFC; healthy donor T-CD3+ lymphocytes served as wild type and normal CN control). Cell lysis and specific targeted DNA pre-amplification are part of SC processing that take place in the C1. Designed TaqMan assays (Thermo Fisher Scientific) or custom designed LNA prime time assays (Integrated DNA Technologies, Coralville, IA, USA) were used to test selected alterations of each patient. Protocols were based on those described by the manufacturer (PN 100-6117, Fluidigm) (Supplementary Methods). Cancers 2021, 13, 841 14 of 17 4.5. SC Multiplex qPCR for Genotyping and CNA Analysis SC analysis was performed following a previously described approach for SC multi- plex qPCR analysis [23,42]. As mentioned before, individual mutation-specific genotyping assays were custom designed for mutation analysis and three different TaqMan CN assays covering each chromosomal region of interest were used for CNA analysis (Tables S5–S7). A predesigned genotyping assay (rs346172) for loci in heterozygosis was used as a reference, while B2M locus, located in a diploid region of the genome, was used as a control for CN analysis. Additionally, BM bulk tumoral DNA from each tested patient was used as a positive control and T-CD3+ lymphocytes DNA from a healthy donor was used as a negative control. Genotyping assays were tested in triplicates whereas CN assays were tested in qua- druplicates. The specific target amplification product (from C1 pre-amplification step) was diluted (1:5) and qPCR was performed using the 96 × 96 Gene Expression Dynamic Array and the BioMarkTM HD (Fluidigm). PCR cycling conditions are detailed in Supplementary Table S8. Fluidigm Real-Time PCR Analysis software v.4.3.1. was used for mutations and CN analyses and CopyCaller v2.1 (Applied Biosystems, Thermo Fisher Scientific) was used to estimate the calculated CN values. Cycle threshold (Ct) was individually established for each assay, then cells without control amplification and with Ct > 30 were discarded. A heterozygous mutation was considered to be present if the signals from the mutant and wild-type sequence probes (FAM and VIC respectively) were present in an SC. A homozygous mutation was considered to be present if wild-type sequence signal was absent. To determine the CN for selected locus, the ∆∆Ct method with modifications was used to determine the relative CN for each locus, as previously described [42]. Results from all interrogated mutations and CNAs per cell were transformed into binary data (1, presence of mutation/CNA; 0, wild type) and combined. The total number of analyzable tumoral SCs and relative percentage of each population were calculated. The threshold to define subclonal populations was established in at least 5% of the total of analyzable tumor cells. 5. Conclusions Despite the limited subset of cases, intratumoral heterogeneity of four patients with MDS del(5q) was studied using a SC approach. As far as we know, this is the first time that an SC study reflects and confirms high intratumoral heterogeneity in MDS del(5q), reinforcing the importance of patient-specific molecular characterization. Although MDS del(5q) patients do not present with a very high molecular complexity, their intratumoral heterogeneity could be more complex than conventional studies have previously shown. SC studies with larger patient cohorts and analyzing a higher number of cells are desirable in order to explore deeper the complexity of this and other hematological malignancies. Supplementary Materials: The following are available online at https://www.mdpi.com/2072-6 694/13/4/841/s1, Supplementary Methods, Table S1: Genes with covered exons in the custom panel, Table S2: Annotation of variants detected by WES and TDS in the studied samples. Table S3: Associated biological process and molecular function of the genes in which variants were detected using WES and TDS. Table S4: CNA detected in each patient using SNP-A analysis. Table S5: Specific primers and assays designed for selected SNV for each patient included in the study. Table S6: Pre-designed TaqMan CNA. Table S7: Reference genotyping assay, used in all tested samples. Table S8: PCR cycling conditions in the BioMark HD. Author Contributions: Conceptualization, L.P., M.M. and F.S.; methodology, L.P. and P.A.; software, F.F.-T.; validation, P.A., L.P. and L.Z.; formal analysis, P.A., L.P., F.F.-T., M.M., J.G., I.G., S.M. and L.Z.; investigation, P.A. and L.P.; resources, B.X., M.R.-L., M.D.-C., L.Z. and A.M.; data curation, P.A., M.M. and L.P.; writing—original draft preparation, P.A., L.P.; writing—review and editing, P.A., L.P., and F.S.; supervision, F.S. and L.P.; project administration, F.S.; funding acquisition, F.S. All authors have read and agreed to the published version of the manuscript. Cancers 2021, 13, 841 15 of 17 Funding: This research was funded by Instituto de Salud Carlos III, grant number PI/14/00013. 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