Precision diagnostics of Ewing's sarcoma by liquid biopsy: circulating EWS-FLI1 fusion transcripts

Matteo Allegretti, Beatrice Casini, Chiara Mandoj, Stefania Benini, Laurent Alberti, Mariangela Novello, Elisa Melucci, Laura Conti, Renato Covello, Edoardo Pescarmona, Giuseppe Maria Milano, Alessio Annovazzi, Vincenzo Anelli, Virginia Ferraresi, Roberto Biagini, Patrizio Giacomini

Research output: Contribution to journalArticle

Abstract

Background: Limited information is available on the applicative value of liquid biopsy (LB) in rare tumors, including Ewing's sarcoma (ES). The accepted precision diagnostics standards would greatly benefit from a non-invasive LB test monitoring pathognomonic gene rearrangements in the bloodstream.

Methods: Tissue and blood samples were collected from six and four ES patients, respectively. Plasma was cleared by two successive rounds of centrifugation and stored frozen until RNA extraction by the QIAmp CNA kit. RNA was retro-transcribed and subjected to real-time quantitative polymerase chain reaction (RT-qPCR) and digital polymerase chain reaction (dPCR). Reactions were set up using two custom primer sets identifying types 1 and 2 EWS-FLI1 fusion transcripts.

Results: The two prevalent types of EWS-FLI1 rearrangements could be identified using only two sets of polymerase chain reaction primers, regardless of patient-specific EWS-FLI1 DNA breakpoints. RT-qPCR and dPCR discriminated the two variants in five tumor tissue RNAs and in four circulating tumor RNAs (ctRNAs). Of note, EWS-FLI1 molecular diagnosis was possible using blood samples even when tumor tissue was not available. ctRNA levels correlated (p < 0.05) with volume-based positron emission tomography (PET) parameters (metabolic tumor volume and total lesion glycolysis), and allowed the fine tracking of disease course after surgery, during adjuvant as well as neoadjuvant chemotherapy, and at follow up in one patient.

Conclusions: To our knowledge, this is one of the few single-marker LB assays in solid tumors specifically designed to detect rearranged RNAs in blood, and the first study describing EWS circulating tumor RNAs in ES patients. Altogether, our results support the idea that LB may have a considerable impact on ES patient monitoring and management.

Original languageEnglish
Pages (from-to)1-9
Number of pages9
JournalTherapeutic Advances in Medical Oncology
Volume10
DOIs
Publication statusPublished - Jun 1 2018

Fingerprint

Ewing's Sarcoma
RNA
Biopsy
Neoplasms
Polymerase Chain Reaction
Real-Time Polymerase Chain Reaction
Gene Rearrangement
Physiologic Monitoring
Glycolysis
Tumor Burden
Centrifugation
Positron-Emission Tomography
EWS-FLI fusion protein
Drug Therapy
DNA

Keywords

  • EWS-FL1 gene rearrangements
  • Ewing’s sarcoma
  • PET imaging
  • circulating tumor RNA
  • liquid biopsy

Cite this

Precision diagnostics of Ewing's sarcoma by liquid biopsy: circulating EWS-FLI1 fusion transcripts. / Allegretti, Matteo; Casini, Beatrice; Mandoj, Chiara; Benini, Stefania; Alberti, Laurent; Novello, Mariangela; Melucci, Elisa; Conti, Laura; Covello, Renato; Pescarmona, Edoardo; Milano, Giuseppe Maria; Annovazzi, Alessio; Anelli, Vincenzo; Ferraresi, Virginia; Biagini, Roberto; Giacomini, Patrizio.

In: Therapeutic Advances in Medical Oncology, Vol. 10, 01.06.2018, p. 1-9.

Research output: Contribution to journalArticle

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T1 - Precision diagnostics of Ewing's sarcoma by liquid biopsy: circulating EWS-FLI1 fusion transcripts

AU - Allegretti, Matteo

AU - Casini, Beatrice

AU - Mandoj, Chiara

AU - Benini, Stefania

AU - Alberti, Laurent

AU - Novello, Mariangela

AU - Melucci, Elisa

AU - Conti, Laura

AU - Covello, Renato

AU - Pescarmona, Edoardo

AU - Milano, Giuseppe Maria

AU - Annovazzi, Alessio

AU - Anelli, Vincenzo

AU - Ferraresi, Virginia

AU - Biagini, Roberto

AU - Giacomini, Patrizio

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N2 - Background: Limited information is available on the applicative value of liquid biopsy (LB) in rare tumors, including Ewing's sarcoma (ES). The accepted precision diagnostics standards would greatly benefit from a non-invasive LB test monitoring pathognomonic gene rearrangements in the bloodstream.Methods: Tissue and blood samples were collected from six and four ES patients, respectively. Plasma was cleared by two successive rounds of centrifugation and stored frozen until RNA extraction by the QIAmp CNA kit. RNA was retro-transcribed and subjected to real-time quantitative polymerase chain reaction (RT-qPCR) and digital polymerase chain reaction (dPCR). Reactions were set up using two custom primer sets identifying types 1 and 2 EWS-FLI1 fusion transcripts.Results: The two prevalent types of EWS-FLI1 rearrangements could be identified using only two sets of polymerase chain reaction primers, regardless of patient-specific EWS-FLI1 DNA breakpoints. RT-qPCR and dPCR discriminated the two variants in five tumor tissue RNAs and in four circulating tumor RNAs (ctRNAs). Of note, EWS-FLI1 molecular diagnosis was possible using blood samples even when tumor tissue was not available. ctRNA levels correlated (p < 0.05) with volume-based positron emission tomography (PET) parameters (metabolic tumor volume and total lesion glycolysis), and allowed the fine tracking of disease course after surgery, during adjuvant as well as neoadjuvant chemotherapy, and at follow up in one patient.Conclusions: To our knowledge, this is one of the few single-marker LB assays in solid tumors specifically designed to detect rearranged RNAs in blood, and the first study describing EWS circulating tumor RNAs in ES patients. Altogether, our results support the idea that LB may have a considerable impact on ES patient monitoring and management.

AB - Background: Limited information is available on the applicative value of liquid biopsy (LB) in rare tumors, including Ewing's sarcoma (ES). The accepted precision diagnostics standards would greatly benefit from a non-invasive LB test monitoring pathognomonic gene rearrangements in the bloodstream.Methods: Tissue and blood samples were collected from six and four ES patients, respectively. Plasma was cleared by two successive rounds of centrifugation and stored frozen until RNA extraction by the QIAmp CNA kit. RNA was retro-transcribed and subjected to real-time quantitative polymerase chain reaction (RT-qPCR) and digital polymerase chain reaction (dPCR). Reactions were set up using two custom primer sets identifying types 1 and 2 EWS-FLI1 fusion transcripts.Results: The two prevalent types of EWS-FLI1 rearrangements could be identified using only two sets of polymerase chain reaction primers, regardless of patient-specific EWS-FLI1 DNA breakpoints. RT-qPCR and dPCR discriminated the two variants in five tumor tissue RNAs and in four circulating tumor RNAs (ctRNAs). Of note, EWS-FLI1 molecular diagnosis was possible using blood samples even when tumor tissue was not available. ctRNA levels correlated (p < 0.05) with volume-based positron emission tomography (PET) parameters (metabolic tumor volume and total lesion glycolysis), and allowed the fine tracking of disease course after surgery, during adjuvant as well as neoadjuvant chemotherapy, and at follow up in one patient.Conclusions: To our knowledge, this is one of the few single-marker LB assays in solid tumors specifically designed to detect rearranged RNAs in blood, and the first study describing EWS circulating tumor RNAs in ES patients. Altogether, our results support the idea that LB may have a considerable impact on ES patient monitoring and management.

KW - EWS-FL1 gene rearrangements

KW - Ewing’s sarcoma

KW - PET imaging

KW - circulating tumor RNA

KW - liquid biopsy

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DO - 10.1177/1758835918774337

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JO - Therapeutic Advances in Medical Oncology

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SN - 1758-8340

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