a403ea69feeeedd54f09261d2a141ae4 pmc_33671317.pdf 5fe0a1519a81286c09e66fb3a5916f04f5626f4c pmc_33671317.pdf cf260e4210abb859994736fd413c1c98f819bc13a9cdd01ae269d994f8808dbf pmc_33671317.pdf Title: Analysis of Intratumoral Heterogeneity in Myelodysplastic Syndromes with Isolated del(5q) Using a Single Cell Approach Subject: Myelodysplastic syndromes (MDS) are a heterogeneous group of hematological diseases. Among them, the most well characterized subtype is MDS with isolated chromosome 5q deletion (MDS del(5q)), which is the only one defined by a cytogenetic abnormality that makes these patients candidates to be treated with lenalidomide. During the last decade, single cell (SC) analysis has 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 SC approach was used to analyze intratumoral heterogeneity in four patients with MDS del(5q). Single CD34+CD117+CD45+CD19- bone marrow hematopoietic stem progenitor cells were isolated using the C1 system (Fluidigm) from diagnosis or before receiving any treatment and from available 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 observe that an ancestral event in one patient can appear as a secondary hit in another one, thus reflecting the high intratumoral heterogeneity in MDS del(5q) and the importance of patient-specific molecular characterization. Keywords: myelodysplastic syndromes; single cell; intratumoral heterogeneity Author: Pamela Acha, Laura Palomo, Francisco Fuster-Tormo, Blanca Xicoy, Mar Mallo, Ana Manzanares, Javier Grau, Silvia Marcé, Isabel Granada, Marta Rodríguez-Luaces, María Diez-Campelo, Lurdes Zamora and Francesc Solé Creator: LaTeX with hyperref Producer: pdfTeX-1.40.21 CreationDate: Thu Feb 18 09:33:49 2021 CET ModDate: Thu Feb 18 09:43:13 2021 CET Custom Metadata: no Metadata Stream: no Tagged: no UserProperties: no Suspects: no Form: none JavaScript: no Pages: 17 Encrypted: no Page size: 595.276 x 841.89 pts (A4) Page rot: 0 File size: 2389518 bytes Optimized: no PDF version: 1.7 name type encoding emb sub uni object ID ------------------------------------ ----------------- ---------------- --- --- --- --------- YSPQJI+VnURWPalladioL-Bold Type 1 Custom yes yes no 10 0 UYBGVU+VnURWPalladioL Type 1 Custom yes yes no 15 0 JZLVLD+URWPalladioL-Ital Type 1 Custom yes yes no 19 0 EJFLEC+URWPalladioL-Bold Type 1 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yes no 220 0 AMIJII+TimesNewRoman,Bold CID TrueType Identity-H yes yes yes 230 0 AMIKKL+TimesNewRoman,Italic TrueType WinAnsi yes yes no 236 0 AMIJJJ+TimesNewRoman CID TrueType Identity-H yes yes yes 239 0 AMIJJN+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 245 0 AMIJJP+PalatinoLinotype TrueType WinAnsi yes yes no 248 0 OVNEXW+EURM10 Type 1 Builtin yes yes no 265 0 MPUDFL+PazoMath Type 1 Builtin yes yes no 272 0 Jhove (Rel. 1.28.0, 2023-05-18) Date: 2024-10-02 03:28:34 CEST RepresentationInformation: pmc_33671317.pdf ReportingModule: PDF-hul, Rel. 1.12.4 (2023-03-16) LastModified: 2023-03-31 12:09:04 CEST Size: 2389518 Format: PDF Version: 1.7 Status: Well-Formed and valid SignatureMatches: PDF-hul MIMEtype: application/pdf PDFMetadata: Objects: 475 FreeObjects: 1 IncrementalUpdates: 0 DocumentCatalog: PageLayout: SinglePage PageMode: UseNone Outlines: Item: Title: Introduction Destination: section.1 Item: Title: Results Destination: section.2 Children: Item: Title: Baseline Characteristics and Genetic Analysis of MDS Patients Destination: subsection.2.1 Item: Title: Clonal Architecture in CD34+CD117+CD45+CD19- HSPC Destination: subsection.2.2 Children: Item: Title: Patient P1 Destination: subsubsection.2.2.1 Item: Title: Patient P2 Destination: subsubsection.2.2.2 Item: Title: Patient P3 Destination: subsubsection.2.2.3 Item: Title: Patient P4 Destination: subsubsection.2.2.4 Item: Title: Discussion Destination: section.3 Item: Title: Materials and Methods Destination: section.4 Children: Item: Title: Patients Destination: subsection.4.1 Item: Title: Sample Processing Destination: subsection.4.2 Item: Title: Mutation and Copy Number (CN) Analysis Destination: subsection.4.3 Item: Title: SC Isolation Destination: subsection.4.4 Item: Title: SC Multiplex qPCR for Genotyping and CNA Analysis Destination: subsection.4.5 Item: Title: Conclusions Destination: section.5 Item: Title: References Destination: section.5 Info: Title: Analysis of Intratumoral Heterogeneity in Myelodysplastic Syndromes with Isolated del(5q) Using a Single Cell Approach Author: Pamela Acha, Laura Palomo, Francisco Fuster-Tormo, Blanca Xicoy, Mar Mallo, Ana Manzanares, Javier Grau, Silvia Marcé, Isabel Granada, Marta Rodríguez-Luaces, María Diez-Campelo, Lurdes Zamora and Francesc Solé Subject: Myelodysplastic syndromes (MDS) are a heterogeneous group of hematological diseases. Among them, the most well characterized subtype is MDS with isolated chromosome 5q deletion (MDS del(5q)), which is the only one defined by a cytogenetic abnormality that makes these patients candidates to be treated with lenalidomide. During the last decade, single cell (SC) analysis has 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 SC approach was used to analyze intratumoral heterogeneity in four patients with MDS del(5q). Single CD34+CD117+CD45+CD19- bone marrow hematopoietic stem progenitor cells were isolated using the C1 system (Fluidigm) from diagnosis or before receiving any treatment and from available 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 observe that an ancestral event in one patient can appear as a secondary hit in another one, thus reflecting the high intratumoral heterogeneity in MDS del(5q) and the importance of patient-specific molecular characterization. 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