Abstract B48: Minimally invasive classification of pediatric solid tumors using reduced representation bisulfite sequencing of cell-free DNA

AbstractIn the clinical management of pediatric solid tumors, histological examination of tumor tissue obtained by a biopsy remains the gold standard to establish a conclusive pathological diagnosis. The DNA methylation pattern of a tumor is known to correlate with the histopathological diagnosis across cancer types and is showing promise in the diagnostic workup of tumor samples. This methylation pattern can be detected in the cell-free DNA. Here, we provide proof-of-concept of histopathologic classification of pediatric tumors using cell-free reduced representation bisulfite sequencing (cf-RRBS) from retrospectively collected plasma and cerebrospinal fluid samples. We determined the correct tumor type in 49 out of 60 (81.6%) samples starting from minute amounts (less than 10 ng) of cell-free DNA. We demonstrate that the majority of misclassifications were associated with sample quality and not with the extent of disease. Our approach has the potential to help tackle some of the remaining diagnostic challenges in pediatric oncology in a cost-effective and minimally invasive manner.Translational relevanceObtaining a correct diagnosis in pediatric oncology can be challenging in some tumor types, especially in renal tumors or central nervous system tumors. Furthermore, the diagnostic odyssey can result in anxiety and discomfort for these children. By applying a novel technique, reduced representation bisulfite sequencing on cell-free DNA (cf-RRBS), we show the feasibility of obtaining the histopathological diagnosis with a minimally invasive test on either plasma or cerebrospinal fluid. Furthermore, we were able to derive the copy number profile or tumor subtype from the same assay. Given that primary tumor material might be difficult to obtain, in particular in critically ill children or depending on the tumor location, and might be limited in terms of quantity or quality, our assay could become complementary to the classical tissue biopsy in difficult cases.

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Minimally invasive classification of paediatric solid tumours using reduced representation bisulphite sequencing of cell-free DNA: a proof-of-principle study

Epigenetics ◽

10.1080/15592294.2020.1790950 ◽

2020 ◽

pp. 1-13

Author(s):

Ruben Van Paemel ◽

Andries De Koker ◽

Charlotte Vandeputte ◽

Lieke van Zogchel ◽

Tim Lammens ◽

...

Keyword(s):

Minimally Invasive ◽

Solid Tumours ◽

Cell Free Dna ◽

Reduced Representation ◽

Free Dna ◽

Bisulphite Sequencing ◽

Proof Of Principle

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A versatile method for circulating cell-free DNA methylome profiling by reduced representation bisulfite sequencing

10.1101/663195 ◽

2019 ◽

Cited By ~ 4

Author(s):

A. De Koker ◽

R. Van Paemel ◽

B. De Wilde ◽

K. De Preter ◽

N. Callewaert

Keyword(s):

Bisulfite Sequencing ◽

Preparation Method ◽

Cost Effective ◽

Cell Free Dna ◽

Reduced Representation ◽

Free Dna ◽

Genome Wide ◽

Reduced Representation Bisulfite Sequencing ◽

Methylation Profiling ◽

Circulating Cell Free Dna

AbstractThe methylation profile of circulating cell-free DNA (cfDNA) in blood can be exploited to detect and diagnose cancer and other tissue pathologies and is therefore of great diagnostic interest. There is an urgent need for a cost-effective genome-wide methylation profiling method that is simple, robust and automatable and that works on highly fragmented cfDNA. We report on a novel sample preparation method for reduced representation bisulfite sequencing (RRBS), rigorously designed and customized for minute amounts of highly fragmented DNA. Our method works in particular on cfDNA from blood plasma. It is a performant and cost-effective methodology (termed cf-RRBS) which enables clinical cfDNA epigenomics studies.

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The pitfalls and promise of liquid biopsies for diagnosing and treating solid tumors in children: a review

European Journal of Pediatrics ◽

10.1007/s00431-019-03545-y ◽

2020 ◽

Vol 179 (2) ◽

pp. 191-202 ◽

Cited By ~ 7

Author(s):

Ruben Van Paemel ◽

Roos Vlug ◽

Katleen De Preter ◽

Nadine Van Roy ◽

Frank Speleman ◽

...

Keyword(s):

Solid Tumors ◽

Liquid Biopsy ◽

Residual Disease ◽

Therapy Response ◽

Response Monitoring ◽

Liquid Biopsies ◽

Cell Free Dna ◽

Pediatric Solid Tumors ◽

Free Dna ◽

Tumor Types

AbstractCell-free DNA profiling using patient blood is emerging as a non-invasive complementary technique for cancer genomic characterization. Since these liquid biopsies will soon be integrated into clinical trial protocols for pediatric cancer treatment, clinicians should be informed about potential applications and advantages but also weaknesses and potential pitfalls. Small retrospective studies comparing genetic alterations detected in liquid biopsies with tumor biopsies for pediatric solid tumor types are encouraging. Molecular detection of tumor markers in cell-free DNA could be used for earlier therapy response monitoring and residual disease detection as well as enabling detection of pathognomonic and therapeutically relevant genomic alterations.Conclusion: Existing analyses of liquid biopsies from children with solid tumors increasingly suggest a potential relevance for molecular diagnostics, prognostic assessment, and therapeutic decision-making. Gaps remain in the types of tumors studied and value of detection methods applied. Here we review the current stand of liquid biopsy studies for pediatric solid tumors with a dedicated focus on cell-free DNA analysis. There is legitimate hope that integrating fully validated liquid biopsy–based innovations into the standard of care will advance patient monitoring and personalized treatment of children battling solid cancers. What is Known:• Liquid biopsies are finding their way into routine oncological screening, diagnosis, and disease monitoring in adult cancer types fast.• The most widely adopted source for liquid biopsies is blood although other easily accessible body fluids, such as saliva, pleural effusions, urine, or cerebrospinal fluid (CSF) can also serve as sources for liquid biopsies What is New:• Retrospective proof-of-concept studies in small cohorts illustrate that liquid biopsies in pediatric solid tumors yield tremendous potential to be used in diagnostics, for therapy response monitoring and in residual disease detection.• Liquid biopsy diagnostics could tackle some long-standing issues in the pediatric oncology field; they can enable accurate genetic diagnostics in previously unbiopsied tumor types like renal tumors or brain stem tumors leading to better treatment strategies

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ASO Author Reflections: Impact of Liquid Biopsy Using Plasma Cell-Free DNA in Solid Tumors in Japan

Annals of Surgical Oncology ◽

10.1245/s10434-021-09858-3 ◽

2021 ◽

Author(s):

Shotaro Matsudera ◽

Yoshihito Kano ◽

Sadakatsu Ikeda

Keyword(s):

Solid Tumors ◽

Plasma Cell ◽

Liquid Biopsy ◽

Cell Free Dna ◽

Free Dna

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Flow Cytometry Immunophenotyping for Diagnostic Orientation and Classification of Pediatric Cancer Based on the EuroFlow Solid Tumor Orientation Tube (STOT)

Cancers ◽

10.3390/cancers13194945 ◽

2021 ◽

Vol 13 (19) ◽

pp. 4945

Author(s):

Cristiane de Sá de Sá Ferreira-Facio ◽

Vitor Botafogo ◽

Patrícia Mello Ferrão ◽

Maria Clara Canellas ◽

Cristiane B. Milito ◽

...

Keyword(s):

Flow Cytometry ◽

Solid Tumors ◽

Solid Tumor ◽

Pediatric Cancer ◽

Diagnostic Procedures ◽

Hematologic Malignancies ◽

Multiparameter Flow Cytometry ◽

Diagnostic Screening ◽

Pediatric Solid Tumors

Early diagnosis of pediatric cancer is key for adequate patient management and improved outcome. Although multiparameter flow cytometry (MFC) has proven of great utility in the diagnosis and classification of hematologic malignancies, its application to non-hematopoietic pediatric tumors remains limited. Here we designed and prospectively validated a new single eight-color antibody combination—solid tumor orientation tube, STOT—for diagnostic screening of pediatric cancer by MFC. A total of 476 samples (139 tumor mass, 138 bone marrow, 86 lymph node, 58 peripheral blood, and 55 other body fluid samples) from 296 patients with diagnostic suspicion of pediatric cancer were analyzed by MFC vs. conventional diagnostic procedures. STOT was designed after several design–test–evaluate–redesign cycles based on a large panel of monoclonal antibody combinations tested on 301 samples. In its final version, STOT consists of a single 8-color/12-marker antibody combination (CD99-CD8/numyogenin/CD4-EpCAM/CD56/GD2/smCD3-CD19/cyCD3-CD271/CD45). Prospective validation of STOT in 149 samples showed concordant results with the patient WHO/ICCC-3 diagnosis in 138/149 cases (92.6%). These included: 63/63 (100%) reactive/disease-free samples, 43/44 (98%) malignant and 4/4 (100%) benign non-hematopoietic tumors together with 28/38 (74%) leukemia/lymphoma cases; the only exception was Hodgkin lymphoma that required additional markers to be stained. In addition, STOT allowed accurate discrimination among the four most common subtypes of malignant CD45− CD56++ non-hematopoietic solid tumors: 13/13 (GD2++ numyogenin− CD271−/+ nuMyoD1− CD99− EpCAM−) neuroblastoma samples, 5/5 (GD2− numyogenin++ CD271++ nuMyoD1++ CD99−/+ EpCAM−) rhabdomyosarcomas, 2/2 (GD2−/+ numyogenin− CD271+ nuMyoD1− CD99+ EpCAM−) Ewing sarcoma family of tumors, and 7/7 (GD2− numyogenin− CD271+ nuMyoD1− CD99− EpCAM+) Wilms tumors. In summary, here we designed and validated a new standardized antibody combination and MFC assay for diagnostic screening of pediatric solid tumors that might contribute to fast and accurate diagnostic orientation and classification of pediatric cancer in routine clinical practice.

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Technical Considerations for Reduced Representation Bisulfite Sequencing with Multiplexed Libraries

Journal of Biomedicine and Biotechnology ◽

10.1155/2012/741542 ◽

2012 ◽

Vol 2012 ◽

pp. 1-8 ◽

Cited By ~ 38

Author(s):

Aniruddha Chatterjee ◽

Euan J. Rodger ◽

Peter A. Stockwell ◽

Robert J. Weeks ◽

Ian M. Morison

Keyword(s):

Bisulfite Sequencing ◽

Flow Cell ◽

Throughput Capacity ◽

Sequencing Data ◽

Bioinformatics Pipeline ◽

Illumina Hiseq ◽

Reduced Representation ◽

Reduced Representation Bisulfite Sequencing ◽

Unique Mapping ◽

Nucleotide Resolution

Reduced representation bisulfite sequencing (RRBS), which couples bisulfite conversion and next generation sequencing, is an innovative method that specifically enriches genomic regions with a high density of potential methylation sites and enables investigation of DNA methylation at single-nucleotide resolution. Recent advances in the Illumina DNA sample preparation protocol and sequencing technology have vastly improved sequencing throughput capacity. Although the new Illumina technology is now widely used, the unique challenges associated with multiplexed RRBS libraries on this platform have not been previously described. We have made modifications to the RRBS library preparation protocol to sequence multiplexed libraries on a single flow cell lane of the Illumina HiSeq 2000. Furthermore, our analysis incorporates a bioinformatics pipeline specifically designed to process bisulfite-converted sequencing reads and evaluate the output and quality of the sequencing data generated from the multiplexed libraries. We obtained an average of 42 million paired-end reads per sample for each flow-cell lane, with a high unique mapping efficiency to the reference human genome. Here we provide a roadmap of modifications, strategies, and trouble shooting approaches we implemented to optimize sequencing of multiplexed libraries on an a RRBS background.

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