© blood advances Disc w wala a Molecular and cytogenetic characterization of myelodysplastic syndromes in cell-free DNA Nieves Garcia-Gisber,"? Sara Garcia-Avia,"® Brayan Merchén,’ Marta Saido,"® Concepeién Fernéndex-Rodkiguea,'* Joan Giber, Lierni Fernandez-Ibarrondo,"® Laura Camacho," Marta Latuente,""* Raquel Longarén,"* Blanca Espinet,*® Patricia Vélez," Ramon M. Pujol Marcio Andrade-Campos,’ Leonor Arenillas,"® Antonio Salar," Xavier Calvo,*® Carles Besses,’ and Beatriz Bellosillo'?° "Group of Applied CincalResearch in Homology, Hospital del Mar Medial Research atte (MIM, Barclon, Spin *Porpou Fabra Unies, Sarcsona Spin *Departnent of Heratsogy Horst dl Mar, Baresiona. Spain Group of Tranlatonal Reeeach on Hemalelogcl Neoplasms, MIM, Barcelona, Span; and "Deparment of Pathology, ard “Deparment of Dermatology, Hoop dl Mar, Baresi, Spa * Cell-free DNA mirrors the molecular profile of bone marrow cells in MDS. + The analysis of ciDNA is a promising method to characterize and monitor molecular and cytogenetic abnormal- ties present in MDS. ‘Molecular and cytogenetic studies are essential for diagnosis and prognosis in patients with myelodysplastic syndromes (MDSs). Cell-free DNA (cfDNA) analysis has been reported to be a reliable noninvasive approach for detecting molecular abnormalities in MDS; however, there is limited information about cytogenetic alterations and monitoring in cMDNA. We assessed the molecular and cytogenetic profile of a cohort of 70 patients with MDS by next-generation sequencing (NGS) of cfDNA and compared the results to sequencing of paired bone marrow (BMD DNA. Sequencing of BM DNA and c{DNA showed a comparable mutational profile (92.1% concordance), and variant allele frequencies (VAFs) strongly correlated between both. ‘sample types. Of note, SF3B1 mutations were detected with significantly higher VAFs in cfDNA than in BM DNA. NGS and microarrays were highly concordant in detecting, chromosomal alterations although with lower sensitivity than karyotype and fluorescence in situ hybridization. Nevertheless, all cytogenetic aberrations detected by NGS in BM DNA were also detected in cfDNA. In addition, we monitored molecular and cytogenetic alterations and observed an excellent correlation between the VAFs of mutations in BM DNA and cfDNA across multiple matched time points. A decrease in the cfDNA VAFs was detected in patients responding to therapy, but not in nonresponding patients. Of note, cfDNA analysis also showed cytogenetic evolution in 2 nonresponsive cases. In summary, although further studies with larger cohorts are needed, our results support the analysis of cfDNA as a promising strategy for performing molecular characterization, detection of chromosomal aberrations and monitoring of patients with MDS. Introduction 220 Pawar uo nb an sosecizoeeesRranc3e8L Lan amnR\eeomroE MEARE AH ID HERwEED Myclodysplastic syndromes (MDSs) are hematopoietic stem cell cisorders characterized by dysplasia and ineffective hematopoiesis driven by somaticaly acquired genomic alterations. Molecular studies and conventional cytogenetics are essential in MDS for establishing a corect diagnosis and setting up ‘Submit 12 Noverbr 2021; sceptd 3 aru 2022; prepubshed ore on tod ‘Adranas Fes Edin 22 Fey 2022; fa ors pubehod ea 23 My 2022 1901 10.1182/lendadvances 2021008865, ‘Sequencing deta canbe found in the EGA epost stos/ega achive. og 0208 ‘spr number EGADOO001008507) lato doctor ings to tho careapnding author: belositoBpredsclama. ‘The uta yrsion of i rl contains a data support. {© 2022 by Tho Amorcan Society of Hamatology. Lconsod under Crate Commons Atibuton NorConmarcal NeDealves 40 hiemsional (CC BY-NG NO 40), permiting only noncommercial. nondevatve use wth atbuton Al otheraccurate risk stratification.’ Routinely, these analyses are performed in bone marrow (BM) samples (in particular, cytogenetic analyses), 2s itis dffcut to obtain metaphases from peripheral blood (PB) samples? In recent years, it has been demonstrated that molecular profiing ‘can be performed robustly using cellftee DNA (cfDNA) analysis in solid tumors and lymphomas. cfDNA molocules are short DNA trag- ments present in plasma samples that are mainly released by imma ture hematopoietic and bone marrow (BM) cells°* As MDSs are characterized by an excessive apoptosis in BM,”® so an increased ‘olease of c{DNA into plasma is expected in these patients. Indecd, several groups have reported that itis possible to identify the ‘genetic aterations in MDS by analyzing cfDNA°'? Howover, there is limited information regarding the detection of eyto- ‘genetic aterations in patients with MDS by efDNA analysis. To this fend, we have designed @ targeted gene pane! to detect in a single test both molecular and cytogenetic alterations by next-generation ‘sequencing (NGS) and have investigated its potential use with ofDNA in comparison with BM samples in a cohort of patients with MDS. Patients and methods Patients BM aspirates and PB samples were prospectively colected from 70 patients with newly diagnosed MDS or pationts who received only erythropoietin with the folowing dagacses: MOS with single lineage dyeplaia (SLD; n= 1), MDS with mutiineage dysplasia (MLD; n = 35), MDS wath ring sidoroblasts (RS)SLD (n= 5), MDS-RS-MLD (n = 17), MDS with isolated dea) (n = 2), MDS. with excess basts EB)-1 (n = 8), MDS-EB-2 (n = 2), and MOS- unclassifiable (MDSU; n= 2; Table 1). The revised Intemational Prognostic Scoring System (PSS-R) score was calculated for each patent"? We analyzed PB samples from an addtional 21 healthy Control subjects and 18 pationts with acute mycoid leukemia (AML; supplemental Table 1), The study was approved by the Parc de Salut Mar Cinical Research Ethics Committoe (CEim, 2016/6768, and was conducted according tothe biomedical research guidelines of the Declaration of Helin PB and BM processing and DNA isolation BM aspirates were collected, and BM DNA was extracted with MagAttract DNA Blood Mini M48 Kit (Qiagen, Hiden, German). PB samples were collected in K3EDTA tubes and processed in the first 4 hours to isolate plasma (supplemental Figure 1). {DNA was isolated automaticaly by using AlAsymphony SP (lAsymphony DSP Virus/Pathogen Kit; Qiagen) and quentfied with Qubit 3.0 (Thermo Fisher Scientfic, Eugene, OR). The purity of the cIDNA ‘was assessed by electrophoresis (4200 TapeStation system; Agi lent, Santa Clara, CA) to discount the presence of genomic DNA. Allthe cfDNA samples analyzed in this project were free of genomic DNA contamination. NGS ‘Genomic characterization was performed in paired samples of BM DNA and cfDNA by NGS in all patents. Libraries were prepared by using a custom panel that included 48 myoloidassociated genes (ASXL1, ATM, BCOR, BCORL1, CALR, CBL, CEBPA, CHEK2, CSFSR, CSNKTAI, CUXT, DDX41, DLEU?, DNMT3A, EGR?, ‘© blood advances 24 MAY 2022 - VOLUME 6, NUMBER 10 EIV6, EZH2, FLTS, GATA2, IDH1, IDH2, JAK2, KIT, KMT2A, KRAS, MPL, NFI, NPM, NRAS, PHF6, PPM1D, PRPFB, PIPN11, RAD21, RUNX?, SETBPI, SF3B1, SH2B3, SRSF2, ‘STAG2, TET2, TNFSFI1, TPS3, TPSSRK, TPSSTGS, U2AF1, WTt, and ZRSR2) and genomic regions localized at the most frequently altered chromosomes in MDS (lAseq Custom DNA Pancls; Qiagen). Genomic regions included in tho NGS panel aro included in supplemental Table 2. Unique molecular identifiers were incorporated before targeted amplification, to tag individual DNA molecules, Libraries were sequenced with a 3000% minimum read depth in MiSeq/NexSeq (lumina, San Diego, CA. The GeneGiobe Data Analysis Center (Qiagen) was used for FASTO trimming, agement, and variant caling (smCounter2).* Variants were annotated and classed by lumina VariantStudio 3.0 software and wore visualized with Integrative Genomics. Viewer (GV) v2.11 software. Only pathogenic and Fkely pathogenic var iants wit a variant allele frequency (VAF) >2% were considered. Copy number variant anahsis was performed by NGS to detect cytogenetic alterations in both cDNA and BM DNA. Samples from heathy individuals (n= 24) were included in all sequencing runs and used as eoverage controls. Gone coverage was compared with teach sample by GeneGlobe Data Analysis Center, o identity regions affected by copy number varants, where the nomalzed coverage is significantly ditrent from the conto. Chromosomal microarrays. Cytoscan 750K Cytogenetic Solution (Thomo Fisher Scientific) was used to obtain a genetic ain, loss, and regions of homozygos- ity profile according to the manufacturer's recommendations. This chip consists of more than 750.000 markers for copy number analy 8 with 650.000 unique nonpolymorphic probes and ~200000 ‘SNPs that fully genotype with greater than 99% accuracy. Chromo some Analysis Suite v4.1 (ChAS) software (Thermo Fisher Scien- tiie) and the hg88 genome version (NASB annotations) was used to analyze the results. Gains with a minimum of 26 altered markors in a 150:kb region, losses with at least 35 altered markers in a 75:kb region, ad regions with telomeric copy neutral loss of hetero zygosity (CNLLOH) greater than 10 Mb or affecting relevant gones have been collected. Fluorescence in situ hybridization analyses Fluorescence in stu hybridization (FISH) was performed according to the standard methods used in our laboratory? FISH was per formed on BM cols from cytogenetic citures using the following probes: Vysis CEPB, ysis EGFR FISH probe kit (Abbott Molocu- lar, Abbott Park, IL), and XL 20q12/20qter (Metasystems, Altus- sheim, Germany) Statistical analysis IBM SPSS software was usod for statistical analysis, For categorical data, comparisons. of proportions wore evaluated by x? test or Frshors exact test as appropriate, For continous variables, compat sons wore assessed by nonparametiie Mane- Whitney or Wilcoxon tost when appropriate. We assessed the Spearman's rank corel tion coefficient to evaluate the strength of association between 2 variables. P< 08 was considered statistical signtcant. Coverage metice were obtained from the DeCovA libra." Variant analysis was pertormed in R version 3.6.2 using the Mattools packago."* CELLFREE ONAIN MDS 9170 i 5 : & i : 3‘able 1. Clinical and biological features of study pationts, a cry) Mae) 31729) Fomal, (i) 19.270) Managobin median fangs. 1175 (78178) WC coun, mesan (nga, 108%, ‘sp (1441228), Nowopht coun, masta angel, 10% 241 (031-76) Pt count, mean ange, 107 Presence Pat (6) {DH mean ane! pon 11497) EM tants %, mein ngs) 2019) Se, macnn (01050) ‘Alored karyotype, m0) 20,288) Vey good eogenac PSS rok group 9) e068) cd erogenate PBS rsh 048 98) 7499) Inamedate cognac ISS. rok group) aon Poor open IPSS ek rue.) 229) ey poor esopntt SSR rsk pup) 108 1PS5-R sk group er ow (8) 25,40) own) 3443) ered) 740) Hn (i) 209) or Has 9 (8) 209) Mos subtype cwHO 2017) MDs sto. 9%) naa MDsIAD,n 38000 MOSRS-SLD, 90) san MDS dela» 209 MosEB3, 908) eee MosEB2,9(%) 209) MDs. »() 209) amber of patente wth muaton, (8) 8 (043) Matos par pater, median (208) 3(010) ‘ted genes par patos, median (ange) 208 CEONA cancertaton, median (age a4 EB pcs bt LDH acl ener NLD, rloage pn SLD. ore ‘en ppt Ueto, WO ae ond oe WHO, eth Ort Tho cade usod in R 3.6.2 to creat the figures is desplayod in supplo- ‘mental Methods, and the files necossary to gonorate the figures and {ull ist of variants identified are shown in supplemental Data 1 to 3. Results The amount of cell-free DNA in plasma Is higher in Patients with MDS than in controls. ‘A total of 70 plasma samples from pationts with MDS at diagnosis fF in the absence of any therapy were analyzed, The clinical and 3190 GARCIAGISBERT et st ‘900 250 200 150 100 ‘ADA pla 60 Control MoS aM Figute 1. cIDNA concentration ln healthy controls, MDS patents and AML patients. Loves ae shown (ng DNA plasa in pasa samples rom heathy controls and pation wih MDS or AML *P = 05; %P = 01 biological features of patients are shown in Table 1. The amount of total cIDNA obtained in patients with MDS (median, 58.4 ng/mL) was significantly higher than that obtained from healthy controls (modian, 32.4 ng/mL; P = 028, Mann-Whitney) (Figure 1). No sig- nificant differences were observed in efDNA concentration among Patients with MDS when comparing by disease subtype or by risk ‘category according to the IPSS-R. Nevertheless, even lower risk patients with MDS had a significantly higher cfDNA concentration than the healthy control group (P = .023). On the contrary, a signf- ‘cantly lower cDNA concentration was observed in the MDS group than that inthe cohort of patients with AML (P = .017) We analyzed the correlation of the concentration of c{DNA with cin- ical and biological characteristics, A postive corrolation was ‘observed between the amount of cIDNA and the serum lactate dohydrogenase levels (P = .027; r, = 0.275). No statistically Signt- ccant association was observed with hematological parameters (hemoglobin, leukocytes, monocytes, platelets, or blast percentage). cfDNA and BM DNA show an equivalent mutational profile Mutational profiing of BM DNA and cfDNA showed comparable results: 187 mutations were detected in 8M DNA and cfDNA, with 2 82.1% concordance (Figure 2). The most frequently mutated genes wore TET2 (45.7%), SFSB1 (37.1%), ASXLT (21.4%), DNMT8A (20.098), SRSF2 (15.796), ZRSR2 (11.4%), and UZAY (11.496). A strong correlation was observed between the VAFS of BM and cIDNA (7, = 0.797; P< .001, Spearman; Figure 3). There were 16 discordant mutations: 8 were detected only in c{DNA, and 8 were detected only in BM (Figure 4A). These discordant © blood advances ‘24 MAY 2022 - VOLUME 6, NUMBER 10 i z t : a i i i i a i 5 i i i : : 2 i roe amthdd hon Sue Mutation type Wi Nonsense Moser vet I spce ste ame niDet Wtrenclaion 8S Role onyam ony rDNA Figure 2. Distribution of mutation identified in BM DNA and cIONA in 70 patients with MDS, Resits of he sequencing re shown nthe plot, where each colar represents patent an each row epes ene. The rub of mutations ented per patois represented as columns above the top Yow. Ganes ae ‘rune by function anda in od om the mast to the est requ mated, Frequencies foreach gane ae delayed atthe ght a wl a8 the mutation ype (nonsense, missense, insortonldeltn, spice ste or vasation cat site). Oscordant mutans ae rpresote with 2 square, a chown thelogend: ed equree show ‘mutations cal iene in ONA and une squares chow mutations ely eatiied a BM DNA. Dal, dln; ne, insrion, SS, tat sta ‘mutations presented a lower VAF (median 5.60%; range, 2.5% to 25.53%) when compared with the VAF observed in the whole cohort (median 28.2796; range 0.74% to 98.28%; P < 001; Figure 48). These cases showed that the cortelation between BM DNA end ofDNA mutations may decrease when studying low: incidence subclones. ‘SF3B1 mutations present a higher VAF in cfDNA than in BM DNA. ‘We compared the VAF ofthe detected mutations in cDNA and BM DNA grouped by gone and observed that VAFSs of SF3B1 mutations ‘were sigeificantly higher in cfDNA than in BM DNA (P = .016, Wicoxon; Figure A). No significant diferences were observed in tho concentration of total {DNA betwoen the SF3B1- mutated ‘and the patient with the SF2B1 witype gene. Mutations in exon 16 of SF3B1 (K700E; NP_036565.2: p.(Lys700Giu) in al cases) presented a tendency toward a higher VAF c/DNA/BM ratio than mutations in other SF3B1 exons (median ratio, 1.82 vs 1.09; P = .08; Figure 58). In this contort, we assessed the representation ‘© blood advances 24 MAY 2022 - VOLUME 6, NUMBER 10 ‘of SF3B1 exons in cDNA, as it has been reported that nucleosome strbution affects DNA fragmentation, and as a consequence, ‘some genomic regions are overrepresented in ciDNA® We com: pared the dopth of coverage obtained for the exons, and we ‘observed a higher representation of SF387 exon 15 in cfDNA braves than in BM libraries (Figure 5C). This finding was not observed in other SF3B1 exons, which suggests that exon 15 is better represented in ciDNA from patients with MDS, thus producing a higher VAF in cfDNA of the SF3B1 K7OOE mutation, In line with previous studios, we observed that the percentage of RSs in BM correlated with the VAF of SF381 mutations, in both BM DNA and ciDNA (7, = 0.684, P< .001 in BM DNA and 1 = 0.602, P = .002 in cfDNA; supplemental Figure 2). Moreover, we identified an SFSB1 K700E mutation detectable only in CIDNA in 1 pationt In this case, the quantification of RSs was rot assessable because of the lack of collularty in the BM aspirato, boing the analysis of cIDNA a useful noninvasive alternative to iden tiy the presence of this pathological clone. This SF3B7 mutation \was later confirmed by NGS in a subsequent PB sample. CCELLFREE ONAIN MDS 2181 = i a i : i : a 5 i i s : : q i zene pawnscAOHA AF (4) ° rr a a eT) a VAF Figure 3. Comelation ofthe VAF In cfONA and BM DNA, cate pt of the 187 vacant detected in ONA and BM DNA showing the corti betwen the ‘arate frequency (WAR), = 0.797, P=.00%, Spearman ‘Chromosomal microarray and NGS were highly concordant for detect cytogenetic aberrations In addition to gene mutations, we assessed the detection of cytoge- nec alterafions by NGS. Cytagenetic/FISH alterations were detected at the time of ciagnosis in 20 of 70 (28.6%) patients with MDS (Figure 6A). Of those, 2 of 20 were infrequent alterations in MDS and were not covered by the design of the NGS panel and in 6 of 20, chromosome Y loss was the only alleration detected that ‘was also not covered by the NGS panel. So, the cohort included cytogenetic alterations potentially detectable by our gone panel in 12 of 70 patients. NGS analysis detected abnormalities in 10 and 70 patients with MDS, in both BM DNA and cfDNA. Interestingly, in pationt without ‘analyzable metaphases in BM karyotype, del(20q) was found by NGS and further confirmed by chromosomal microarray (CMA). Overall CMA and NGS were highly concordant to detect chromo: ‘somal aberrations although they did not reach the sensitivity achioved by conventional cytogenetic analysis (karyotypo/FISH; Figure 68; supplemental Figure 9). Nevertheless, as previously Stated, all cytogenetic aberrations detected by NGS in BM DNA ‘wore also detected in c/DNA. cfDNA is useful to predict transformation and monitor response to treatment Molecular and cytogenetic alterations were moritored in sequential ‘samples from 7 cases (median folow up, 13 months; range, 10-30). We observed an excellent correlation betwoon the VAFs of mutar tions in BM and cfDNA across mutiple matched time points. Both sample types showed similar clonal dynamics irespective of the treatment and allowed for the monitoring of both mutations and ‘chromosomal aberrations (Figure 7). In those cases treated with hypomethylating agents (i, azacitcine), 1 VAF decrease was detected in patients responding to therapy, but not in nontesponding patients. Of note, cIDNA analysis also 3182 GARCIAGISBERT ets showed cytogenetic evolution in 2 paints who did not respond to azaciténe (del(12p) and +21) and who had to stop treatment because of lack of response, In the patient treated with FLAG-DA followed by hematopoietic cell transplantation, the 5 mutations identified at diagnosis wore undetectable in cDNA in a sample col lected 7 months after the HCT. One patient treated with hypoxia: inducible factor inhibitor showed a VAF decrease in DNMT3A and ‘SF3B1 mutations and a concomitant increase in the RUNX1 and 'SETBP1 VAFs during the follow-up and later transformed to chronic: myelomonocytic leukemia. In addition, the emergence of a mutation in ASXL1, undetectable at diagnosis, was identfied in the latest ‘sample avallablo of both ofDNA and BM DNA. Two patients who were not receiving treatment were also moni- tored. One patient, who progressed to AML, showed a clonal ‘expansion of the NF1 mutant clone at the time of AML transforma- tion. The second patient acquired a subclonal del(7q) not detected by NGS and observed only by karyotype in 2 of 20 metaphases, ‘Although our cohort of patients with AML (16 of 18 wore de novo ‘AML presented a higher c{DNA concentration than of MDS at diag- ros, we did not observe an increase in the concentration of efDNA in the 2 patients with MDS who progressed to AML. Discussion Inthe present study, we assessed the genomic charactoization of MDS by targeted NGS of plasma cfDNA compared with BM DNA This is to the best of our knowledge, the largest series of cIDNA analyses in patients with MDS. Of note, all samples were taken at siagnosis or betore treatment, thus excluding any potentially modi. ing effect on the results. We designed an NGS gene panel to detect both molecular and cytogenetic alterations with a single test and investigated its potential use in cfDNA, which would be particu- ley useful in several cases, such as nonfit or fragile elderly pationts, Patients with fibrotic or hypocelular BM, and patients with contrain- cation of difficul-to-access BM. Our data demonstrate that the analysis of cDNA roprosents a novel strategy that would be usoful ‘or routine testing, as cfDNA is obtained fast and easly from blood plasma, when compared with BM aspirates or purified CD94~ cols.” In pationts with solid tumors, cfONA has been incorporated as a noninvasive strategy to assess molecular alterations in routine clic cal practice. it has been reported that most of the cfDNA is released by hematopoietic cells in health and disease.*® However, Patients with MDS showed a signficanty higher amount of {DNA than healthy controls, indicating a higher rolease of cIDNA into PB plasma from MDS clonal cell OF note, even calls in lower risk MDS contained a higher quantity of cfDNA than the control cells, The inefctive hematopoiesis in the stem cell niche and the increasod apoptosis of BM cols in MDS®? i in ine with this Fighor shedding of {DNA into PB. A significant correlation was observed botween the ciDNA concentration and lactate dehydrogenase val ues, in accordance with previous studies"? However, contrary to previous findings," wo did nt find a higher concentration of DNA in IPSS higher risk groups than in lower risk groups. This discor dant observation could be explained by the lower risk IPSSR scores of most patients with MDS included inthe study In our study, we observed a similar mutational profile in cfDNA and BM DNA (93% concordance) and the VAFS of the mutations © blood advances 24 MAY 2022 - VOLUME 6, NUMBER 10 i 5 : & i : 3Cee ary erry ‘cfDNA only 100 E 0 i: CANA BMDNA SKA BM DNA ony oni Figure 4, Discordant mutations in BM DNA and eIDNA. (A) Discordant muons ented in M DNA ond DNA. #Patlon presented 2 muttons detected ony ‘DNA: "12 paints showing 2 mutations detected on in BM. Tho 10 remaining discordant alxatons were identi 10 diferent pains. (8) VAS identiied a concordent (be) and discordant (nh) mutations in BM ONA and IDNA “P = .05; "P= 001 identiod in both sample types correlated highly. However, some iscordant mutations were also identified in a small proportion of patients, in some cases mutations that may have prognostic rele- vance, such as SF3B1 mutations or mutations in damage DNA repair genes. Cases with mutations detected in cfDNA and not in BM DNA were all those in which plasma and BM samples had boon collected at different time points. In adltion, some of those discordant cases showed low VAFs that could reflect small clones emerging or slowly expanding or de novo-acquired mutations. Overal, cfDNA and BM DNA showod a high concordance, though they may have had a worse correlation in subclonal atera- tions, as has been reported for cDNA in AML" Studies of MDS ‘comparing the reliability of cfDNA and total PB collar DNA analy- sis for detecting molecular abnormaltios by NGS”? showed that ‘IDNA analysis was a better option, as additional mutations were dotected in cfONA and the VAFS in cfONA were significantly higher than those in PE DNA? Interestingly, the VAFs of SFSB1 mutations were significantly higher in cDNA than in BM DNA, especialy for exon 15 SF3E1 mutations (io, K700E}. This obsoration is clinically rolovant, as the analysis of ‘CADNA could be the best altemative for detecting these mutations, ‘when low-quality BM aspirates aro obtained, of for detecting smal ‘mutant clones. We identified a SFSB1 mutation in cfDNA and not in BM DNA in a patient in whom the presence of RSs was not assessable because of lack of cellularity in the BM aspirate. We hypothesizo that the postion of the nucleosomes in exon 15 of ‘SF3B1 could facitate the detection of the SFSB7 K700E mutation, ‘as wo observed that exon 15 of SF8B1 was more represented in ‘ADNA than in BM DNA. In view of our resuits, the sensitive detection of genomic altrations in cIDNA observed in myeloid malignancies suggests that this non invasive tool could provide useful information in the diagnostic ‘workup of cases with unclear eytopenias, given thal the absence of ‘genetic and cytogenetic alterations has demonstrated a high nega- tive predictive value in these cases.*° However, the sole pres- ‘ence of genetic alterations should not be interpreted as unequivocal ‘® blood advances 24 MAY 2022 - VOLUME 6, NUMBER 10 rot sigan, evidence of MDS diagnosis; these alterations could represent clonal hhematopoiosis of indaterminate potential that reinforces the need to integrate the molecular information with the patients’ morphological studies and clinical context.2°?” Regarding the detection of cytogenetic alterations in {DNA of patents wih MDS, our rosuls confim that gains or losses of gonotc matoral are reflected in tho IDNA, and thus we can idontity most of them by NGS, including a d!(20q) in a patent without ana Iyaablo metaphases at diagnosis, which was confimed by CMA. This isthe frst study to assess the detection of oytogenati altrar tions in cIDNA by NGS in a cohort of patients with MDS. Other studios have used NGS to detect these chromosomal alteration in PB or BM samples.” and only 1 study used cIDNA to identi the loss of chromosome & in efDNA in patient with MDS." However, it should be noted thatthe design of the NGS panel covered only a selected part of the genome, so those altora- tions occurring in uncovered regions wil net be detected. Th chromosome Y deletion was not included in the NGS panel design, because it has boon associated with normal aging” and isnot a defining alteration for MDS." As NGS comprohen- sive molecular profiing with broad NGS+targeted panels or {even exome of whole-genome analyses are implemented in clin ical practice, these limitations wil be overcome. We also observed that subclonal cytogenetic alterations could be dotected only in pationts by karyotype or FISH, because of tho limitations of sensitivity of NGS or CMA. We must recall that, at present, cytogenetic techniques are stil, besides morphology, the backbone elements of MDS diagnosis, Further validation of these results including higher risk cytogenetic subgroups would support the value of cIDNA analysis as a useful tool to be implemented in routine ctinical practice that could improve the identification of alterations required for accurate risk classification ‘One of the plausible applications of cfDNA is disease monitoring To evaluate this approach, we used NGS to analyze sequential sam- ples from 7 pationts with MDS, Previous studies had shown that CCELLFREE ONAN MDS 189 220 Pawar uo nb an sosecizoeeesRranc3e8L Lan amnR\eeomroE MEARE AH ID HERwEEDAF ofNA/VAF BU Pa aches i VAF IAAF BI SFB) exon 14 ‘$F281 exon 15, ‘SF2a1 exon 16 c ‘SFS8) exon 14 ‘9F81 exon 15 ‘SF981 exon 16 oo 05 10 15 20 tit ol panel coeape Figure 5. Comparison between VAF in cDNA and BM DNA for each gent characterization of SF3B1 mutations (A) Ratio ofthe dct ves ofthe ‘wos equenty mutated gene nou cor. The red Snes inde the median VAF ai foreach ge, Vasants stud ith la above the ine hada higher VAF Press NA) et + CUT p ArgtseTer (DNA) ° 5 OUKTpAgtSeTer BM “= GURY pAla720TINSTer18 (DNA) “CUNY pAla7aOTnTer18 (2M) “+ SRSF2pPrea8Aeg (fDNA) “© SRSF2pPreo5A9 (BM) + ASML pGYBAeTipsTert2 (DNA) ASXL1 pGyeaeTpistert2 (2M) + TPS Arg248tIp (CDNA) © TPeopArgaastip GM) 1% 28 Monts Azactiaine clnealospons) a am 6 ar a) 20 += sTAG2pSers20ValsTora BM TAG. p S020 asx? (cDNA) © ASAL I pGiyS4sTpisTer12 BM) “= ASIL p.Giy646T pier? (rDNA) 1 ZRSROpSer8a7 high 48aup BM) = RRO Senta ig 4p (AONA) “© U2AF pSacPhe (EM |= U2AFpSer4Phe (@ONA) © ASML pGysssTipsTer!2 EM) 1+ ASAL I p.lyS46Tiptcort2 (ONAL +> SF901 pyesseThe BM) ~ SF281 pLys666Th (IDNA) © ASAL piySesTipisTr2 (3M) “+ ASML pGy64sTipcTer2 (DNA) “© NF pag 276Te (OM) + NEY pArgh276Ter CDNA) WP orursAp ayer (A0NAI 1 DNMUTSAp Gye42Ter EM) “= F981 p.Aen82Asp (IDNA) 2 SFB pAan628Ae9 (BM) + ASILT pSee10280yssTer2 (DNA) + ASKL pSer1028yTor12 (BN “+ RUDI Tip 106L2u (CONA) “> RUN pptOeLeu (EM) + SETEPI pAeps8Asn IDNA) ‘+ SETBPY p AspeEBAsn (EM) Azactiging (be Simeaeesponse) “¢ KRAS pi 2kep (BM “+ KRAS PGI 2Aep (DNA) © SRSFZpPr0EAG EM) “+ SRSFZp.ProBSAeg CDNA) = RUN p1y287T “= RUNS pT} 287Tr (DNA) 08 6 8 2 Figure 7. Monitoring of molecular and cytogenetic alterations in 7 pationts with MDS. Five patn's ecsiingvestmont (9 azacidn, 1 FLAGIDAHCT, and 1 HF typoninindvible tater nhs) and 2 untested cave were nuded. 8M VAF dari ee ehown wih doe nes and DONA dynamics ae shown with od nes ‘CL, crore myelomonocytic leukama: CNA, copy runber eaten; HCT,hanatapoit cel vansplanttion; ND ot detected 3196 GARCIAGISBERT ets! © blood advances ‘24 MAY 2022 - VOLUME 6, NUMBER 10 vn srnersomperocain sua nL ape, i 4 : : q t 2 i iAcknowledgments ‘This work was supported by Institut de Salud Carlos IhFederacién Espariola de Enfermedades Raras (SCIKFEDER) grants F116/0163, 19/0005, 2017SGR206, and PT20/00023, and Xara do Bane cde Tumors de Catalunya, Authorship Contribution: N.G-G. performed the research and the statistical analysis, analyzed and interpreted the results, and wrote the man- uscript; C.B, and BB. designed the research study, analyzed ‘and intorproted the results, and wrote the manuscript; SGA, BM, MS, CF-R, and 1G. performed the research, collected the data and interpreted the results; LF, LC, ML, RL, BE, PN. RMP,, and MA-C. collected and analyzed the data; LA, AS, and X.C. collected the data and analyzed and interpreted tho results; and all authors reviewed and approved the final ver~ sion of the manuscript Confictofinterest disclosure: AS. has received research funding ‘rom and serves on the speakers bureau of Roche; is a consultant to References and serves on the speakers bureau of Janssen Pharmaceuticals and Gilead; and has been a consultant to Celgene. CB. has received research funding fom Gilead. B.B. has served on the speakers bureau, received resaarch funding from, and served ae a consultant to Thormo Fisher Scientific; has been a consultant to and served on the spoakers bureau of Qiagen, and has received research funding ‘rom and served on the speakers bureau of Roche. The remaining authors declare no competing financial interests. ORCID profiles: N.G-G., 0000-0002:8185-9786; BM, 0000- (0002-6733-6008; MS, 0000-0001-9988-6077; CFR, 0000- (0002-5859-0843; 1G, 0000-0002-0749-0759; LF-1, 0000-0003 2612-0484; LC., 0000-0002-7981-8486; RL, 0000-0002-0515- 9278; BE, 0000-0002.4204.8145; RMP, 0000-0002-5622- 6055; MAC, 0000-0003-1637-7112; LA, 0000-0002.8176- 7179; AS., 0000-0002:4652-4825; XC, 0000-0001-7934-9130; CB, 0000-0003-1806:2440; BB, 0000-0002-5335-2726, Corespondence: Beatie Bellosillo, Hospital del Mar, Passelg Maritim 25-28, Barcelona 08003, Spain; e-mail: bbellosilo@ parcdesalutmar.cat. 1. Arber DA, Oras A, HasseranR, at al. The 2016 revision tothe Werld Heath Organization classification of myeloid neoplasms and acute leukemia Blood. 2016;127(20):2801-2408, 2 Choryy AM, Siovak ML, Campbell Lota. Wil a periphoral blood (PS) sample yield the same dagnostic and prognostic cytogenetic data asthe ‘concomitant Bone marow (BM) in myelodysplasia? Leuk Res. 2012;38(7)'832-840, 8. LUiYYN, Chik K-W, Chiv RWK, Ho C-¥, Lam CWK, Lo YMD. Predominant hematopoietic origin of cole DNA in plasma and serum after ‘sexmismatched bone marrow transplantation. Clin Chem, 2002;48(3):421-427, 4. ryder MW, Kicher M, Hil Al, Daza RM, Shondure J. Col-ree DNA comprises ann vivo nucleosome footprint that informs its tssves-oF- ogi, Cel 2016;164(1-2)57-68, 5. UlzP,Thalingor GG, Auor M, et al. Inering exprossod gones by whole-genome eoquancing of plasma DNA. Nat Genet 2016;48(10):1273-1278. 8. Mess J, Magenheim J, Neiman 0: etal Comprehensive human cal-type methylation atlas reveals origins of circulating cellree DNA in health and sisoase. Nat Commun, 2018;(1):5068, 7. Clark DM, Lampert IA. Apoptosis i @ commen histopathological nding in myelodysplasa: the correla Lymphoma, 1980;2(6:415-418. 8 Shetty V, Hussaini S, Ali S, eta. Excessive apoptosis, increased phagocytosis, nuclear inclusion bodies and cyindrcal confronting cistemae in bone mantow biopsies of myelodysplastic syndrome pations. BJ Haematol 2002;116(4):817-825. 9. yam ©, Tomita A, Hoshino He al. Using peripheral blood circulating DNAs to detect CpG global methylation status and genetic mutations in patonts with myelodysplastic syndrome, Biochem Biophys Res Commun, 2012;819(8):662-669, 10. Nakamura 8, Yokoyama K, Shimizu E, et al. Prognostic impact of circulating tumor DNA status postallogeneic hematopoietic stem cell ‘ransplantation in AML and MDS. Blood. 2018;133)25):2682-7605, 11. Yeh P, Dickinson M, FouniS, et a Molecular disease monitoring using circulating tumor DNA in myelodysplastic syndromes. Blood, 2017; 129(12):1885-1690. 12, Sumi, Tomta A, Nakamura F, etal Peripheral blood cal-ree DNA isan altematve tumor DNA source reflecting disease status in myelodysplastic syrdomes. Cancer Sei 2016;107(0)"1329-1337 13. Groonberg PL. Tuochior H, Schar2 J, ot al. Revised international prognostic scoring system for myolodysplasic sckomes. Blood. 2012:120(12): 2458-2465, of inet heomatopsiesis. Leuk 14 XuC, Gu X, Padmanabhan Rota. snCounter?: an accurate low-frequency variant cllor fr targeted sequencing data with unique molecular identiers. Bioinformatics. 2019:95(8)'1299-1308, 15, Reinecke F, Satya RV, DiCarlo J. Quanttative analysis of efrences in copy numbers using read depth obtained from PCReentched contol. BMC Bioinfermaves. 2015;:18(1)17. 16. Solé F, Sado M, Espinet 8, ota. Splonic marginal zone B-cell ymphomas: two cytogenetic subtypes, one with gain of Sq and the othor with loss (01 74, Haematologica. 2001;88(1):71-77. ples and ©@ blood advances 24 may 2022 - voLUME 6, NUMBER 10 CELL-FREE DNAIN MOS 3107 i z t : a i i i i a i 5 i i i : : 2 i roe amtvv. 18, 19, 20, 21 22 23, 24, 25, 26, 27 29, 29, 20. Dimaasi S, Smmonet T, Labalme A, etal. Comparison of two next ganeration sequencing lit for diagnosis of epileptic disorders with a user tendly too or displaying gore coverage, DeCovA. App Transl Genomics. 2015;7:19-28. Mayakonda A Lin D.C, Assonov ¥, Plass C, Koetlr HP. Mattoo: Ros. 2018;26(11):1747-1766, Marin R, Acha P, Ganster C, eta. Targeted deep sequencing of CD34" cells trom peripheral blood can reproduce bone marrow molecular protle in myelodysplastic eyndromes. Am J Homatol. 2016:98(6)£152-£164. Garcia-Gisbert N, Feméndez brrondo L, Femandez-Rocriguer C, tal. Circulating call4ree DNA improves the molecular characterisation of Phenegatve myeloprolferave neoplasms. Br J Haematel 2021;102(2):300-308. ‘Short NJ, Patel KP, Abitar M, etal. Targeted next-generation sequencing of circulating cellree DNA vs bone marrow in pationts with acute myeloid leukemia, Blood Adv. 2020;4(8):1670-1677. ‘AbtarF, Ma W, Diop K, eal. Deop sequencing of collfoe peripheral blood DNA as arolable method for confirming the diagnosis of myelodysplastic syndrome Genet Test fo! Biomarkers, 2016;20(7):241-245, Zhao P, Gin J, Liu W, etal: Using circulating tumor DNA to monitor myelodysplastic syndromes status. Hematol Oncol. 2018;38531-533, Maloovati L, Gali A, Travagine E, etal. Circa significance of somatic mutation in unexplained blood eytopenia, Blood. 2017;120(25):2971-9378, ‘Steonsma DP. How | use molecular gonetic tests to evaluate pationts who have or may have myclodyeplasic syndromes [published conection appears in Blood, 2018;132(22):2419). Blood, 2018;132(16):1657-1663. ‘Steensma DP. Cinical consequences of clonal hematopoiesis of indeterminate potential. Blood Ady. 2018;2(22):3404-9610, ‘Gutirrez-Rediques F, Borman |, Groarko EM, et al: Utity of plasma calfroa DNA for de nova datection and quantification of clonal hemstopoie- ‘si [published onine ahead of print 80 September 2021], Haematologica 2021, doi 10.8824/naomatol 2021.278230 Liguori A, Lesend | Palomo L a al. singlorun next ganoration sequencing (NGS) assay forthe simultaneous detection of bath gene mutations ‘and large chromosomal abnormalities in ationts with myolodyeplastic syndromes (MDS) and related myolid neoplasms. Cancers (Basel). 2021 1948):1847, ‘Stone JF, Sandberg AA. Sex chromosome ancupleidy and aging. Mutat Res, 1996;388(1-6):107-113, Jabbour E, Takahashi K, Wang X, etal. Acquistion of cytegenetic abnormalities in pationte with IPSS defined lowerak myelodyeplaatc eyndrome is ascociafed with poor prognosis and transformation to acute myelogenous leukemia. Am J Hematol 2013'88(10)-831-837. ficient and comprehensive analysis of somatic variants in cancer. Genome 3108 GARCIA-GISBERT et si ‘24 MAY 2022 - voLUME 6, nuMBeR 10 ® blood advances