Digital Sorting of Pure Cell Populations Enables Unambiguous Genetic Analysis of Heterogeneous Formalin-Fixed Paraffin-Embedded Tumors by Next Generation Sequencing

Chiara Bolognesi, Claudio Forcato, Genny Buson, Francesca Fontana, Chiara Mangano, Anna Doffini, Valeria Sero, Rossana Lanzellotto, Giulio Signorini, Alex Calanca, Maximilian Sergio, Rita Romano, Stefano Gianni, Gianni Medoro, Giuseppe Giorgini, Hans Morreau, Massimo Barberis, Willem E. Corver, Nicolò Manaresi

Research output: Contribution to journalArticle

17 Citations (Scopus)

Abstract

Precision medicine in oncology requires an accurate characterization of a tumor molecular profile for patient stratification. Though targeted deep sequencing is an effective tool to detect the presence of somatic sequence variants, a significant number of patient specimens do not meet the requirements needed for routine clinical application. Analysis is hindered by contamination of normal cells and inherent tumor heterogeneity, compounded with challenges of dealing with minute amounts of tissue and DNA damages common in formalin-fixed paraffin-embedded (FFPE) specimens. Here we present an innovative workflow using DEPArray™ system, a microchip-based digital sorter to achieve 100%-pure, homogenous subpopulations of cells from FFPE samples. Cells are distinguished by fluorescently labeled antibodies and DNA content. The ability to address tumor heterogeneity enables unambiguous determination of true-positive sequence variants, loss-of-heterozygosity as well as copy number variants. The proposed strategy overcomes the inherent trade-offs made between sensitivity and specificity in detecting genetic variants from a mixed population, thus rescuing for analysis even the smaller clinical samples with low tumor cellularity.

Original languageEnglish
Article number20944
JournalScientific Reports
Volume6
DOIs
Publication statusPublished - Feb 11 2016

Fingerprint

Paraffin
Formaldehyde
Population
Neoplasms
High-Throughput Nucleotide Sequencing
Precision Medicine
Workflow
Loss of Heterozygosity
DNA Damage
Sensitivity and Specificity
Antibodies
DNA

ASJC Scopus subject areas

  • General

Cite this

Digital Sorting of Pure Cell Populations Enables Unambiguous Genetic Analysis of Heterogeneous Formalin-Fixed Paraffin-Embedded Tumors by Next Generation Sequencing. / Bolognesi, Chiara; Forcato, Claudio; Buson, Genny; Fontana, Francesca; Mangano, Chiara; Doffini, Anna; Sero, Valeria; Lanzellotto, Rossana; Signorini, Giulio; Calanca, Alex; Sergio, Maximilian; Romano, Rita; Gianni, Stefano; Medoro, Gianni; Giorgini, Giuseppe; Morreau, Hans; Barberis, Massimo; Corver, Willem E.; Manaresi, Nicolò.

In: Scientific Reports, Vol. 6, 20944, 11.02.2016.

Research output: Contribution to journalArticle

Bolognesi, C, Forcato, C, Buson, G, Fontana, F, Mangano, C, Doffini, A, Sero, V, Lanzellotto, R, Signorini, G, Calanca, A, Sergio, M, Romano, R, Gianni, S, Medoro, G, Giorgini, G, Morreau, H, Barberis, M, Corver, WE & Manaresi, N 2016, 'Digital Sorting of Pure Cell Populations Enables Unambiguous Genetic Analysis of Heterogeneous Formalin-Fixed Paraffin-Embedded Tumors by Next Generation Sequencing', Scientific Reports, vol. 6, 20944. https://doi.org/10.1038/srep20944
Bolognesi, Chiara ; Forcato, Claudio ; Buson, Genny ; Fontana, Francesca ; Mangano, Chiara ; Doffini, Anna ; Sero, Valeria ; Lanzellotto, Rossana ; Signorini, Giulio ; Calanca, Alex ; Sergio, Maximilian ; Romano, Rita ; Gianni, Stefano ; Medoro, Gianni ; Giorgini, Giuseppe ; Morreau, Hans ; Barberis, Massimo ; Corver, Willem E. ; Manaresi, Nicolò. / Digital Sorting of Pure Cell Populations Enables Unambiguous Genetic Analysis of Heterogeneous Formalin-Fixed Paraffin-Embedded Tumors by Next Generation Sequencing. In: Scientific Reports. 2016 ; Vol. 6.
@article{69efdb25ae014325978ca668de271385,
title = "Digital Sorting of Pure Cell Populations Enables Unambiguous Genetic Analysis of Heterogeneous Formalin-Fixed Paraffin-Embedded Tumors by Next Generation Sequencing",
abstract = "Precision medicine in oncology requires an accurate characterization of a tumor molecular profile for patient stratification. Though targeted deep sequencing is an effective tool to detect the presence of somatic sequence variants, a significant number of patient specimens do not meet the requirements needed for routine clinical application. Analysis is hindered by contamination of normal cells and inherent tumor heterogeneity, compounded with challenges of dealing with minute amounts of tissue and DNA damages common in formalin-fixed paraffin-embedded (FFPE) specimens. Here we present an innovative workflow using DEPArray™ system, a microchip-based digital sorter to achieve 100{\%}-pure, homogenous subpopulations of cells from FFPE samples. Cells are distinguished by fluorescently labeled antibodies and DNA content. The ability to address tumor heterogeneity enables unambiguous determination of true-positive sequence variants, loss-of-heterozygosity as well as copy number variants. The proposed strategy overcomes the inherent trade-offs made between sensitivity and specificity in detecting genetic variants from a mixed population, thus rescuing for analysis even the smaller clinical samples with low tumor cellularity.",
author = "Chiara Bolognesi and Claudio Forcato and Genny Buson and Francesca Fontana and Chiara Mangano and Anna Doffini and Valeria Sero and Rossana Lanzellotto and Giulio Signorini and Alex Calanca and Maximilian Sergio and Rita Romano and Stefano Gianni and Gianni Medoro and Giuseppe Giorgini and Hans Morreau and Massimo Barberis and Corver, {Willem E.} and Nicol{\`o} Manaresi",
year = "2016",
month = "2",
day = "11",
doi = "10.1038/srep20944",
language = "English",
volume = "6",
journal = "Scientific Reports",
issn = "2045-2322",
publisher = "Nature Publishing Group",

}

TY - JOUR

T1 - Digital Sorting of Pure Cell Populations Enables Unambiguous Genetic Analysis of Heterogeneous Formalin-Fixed Paraffin-Embedded Tumors by Next Generation Sequencing

AU - Bolognesi, Chiara

AU - Forcato, Claudio

AU - Buson, Genny

AU - Fontana, Francesca

AU - Mangano, Chiara

AU - Doffini, Anna

AU - Sero, Valeria

AU - Lanzellotto, Rossana

AU - Signorini, Giulio

AU - Calanca, Alex

AU - Sergio, Maximilian

AU - Romano, Rita

AU - Gianni, Stefano

AU - Medoro, Gianni

AU - Giorgini, Giuseppe

AU - Morreau, Hans

AU - Barberis, Massimo

AU - Corver, Willem E.

AU - Manaresi, Nicolò

PY - 2016/2/11

Y1 - 2016/2/11

N2 - Precision medicine in oncology requires an accurate characterization of a tumor molecular profile for patient stratification. Though targeted deep sequencing is an effective tool to detect the presence of somatic sequence variants, a significant number of patient specimens do not meet the requirements needed for routine clinical application. Analysis is hindered by contamination of normal cells and inherent tumor heterogeneity, compounded with challenges of dealing with minute amounts of tissue and DNA damages common in formalin-fixed paraffin-embedded (FFPE) specimens. Here we present an innovative workflow using DEPArray™ system, a microchip-based digital sorter to achieve 100%-pure, homogenous subpopulations of cells from FFPE samples. Cells are distinguished by fluorescently labeled antibodies and DNA content. The ability to address tumor heterogeneity enables unambiguous determination of true-positive sequence variants, loss-of-heterozygosity as well as copy number variants. The proposed strategy overcomes the inherent trade-offs made between sensitivity and specificity in detecting genetic variants from a mixed population, thus rescuing for analysis even the smaller clinical samples with low tumor cellularity.

AB - Precision medicine in oncology requires an accurate characterization of a tumor molecular profile for patient stratification. Though targeted deep sequencing is an effective tool to detect the presence of somatic sequence variants, a significant number of patient specimens do not meet the requirements needed for routine clinical application. Analysis is hindered by contamination of normal cells and inherent tumor heterogeneity, compounded with challenges of dealing with minute amounts of tissue and DNA damages common in formalin-fixed paraffin-embedded (FFPE) specimens. Here we present an innovative workflow using DEPArray™ system, a microchip-based digital sorter to achieve 100%-pure, homogenous subpopulations of cells from FFPE samples. Cells are distinguished by fluorescently labeled antibodies and DNA content. The ability to address tumor heterogeneity enables unambiguous determination of true-positive sequence variants, loss-of-heterozygosity as well as copy number variants. The proposed strategy overcomes the inherent trade-offs made between sensitivity and specificity in detecting genetic variants from a mixed population, thus rescuing for analysis even the smaller clinical samples with low tumor cellularity.

UR - http://www.scopus.com/inward/record.url?scp=84958537604&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=84958537604&partnerID=8YFLogxK

U2 - 10.1038/srep20944

DO - 10.1038/srep20944

M3 - Article

VL - 6

JO - Scientific Reports

JF - Scientific Reports

SN - 2045-2322

M1 - 20944

ER -