circulating tumor cells for comprehensive and multiregional non … · 2020. 1. 28. · circulating...
TRANSCRIPT
Circulating Tumor Cells for Comprehensive and Multiregional Non-Invasive Genetic
Characterization of Multiple Myeloma
Juan-José Garcés1, Gabriel Bretones2, Leire Burgos1, Rafael Valdes-Mas2, Diego Alignani1, Idoia Rodriguez1, Alberto
Orfao3, Carlos Lopez-Otin3, Jesús San Miguel1, Bruno Paiva1
1. Clinica Universidad de Navarra, Centro de Investigacion Medica Aplicada (CIMA); 2. Departamento de Bioquímica y Biología Molecular, Instituto Universitario de Oncología del Principado de
Asturias (IUOPA), Universidad de Oviedo; 3. Hospital Universitario de Salamanca, Instituto de Investigacion Biomedica de Salamanca (IBSAL), Centro de Investigación del Cancer (IBMCC-USAL, CSIC)
What’s Multiple Myeloma (MM)?
o Clonal expansion of plasma cells (PCs) inside the bone marrow (BM)
o CRAB symptoms:
o Elevated Calcium
o Renal failure
o Anemia
o Bone lesions
o Medical gold-standard technique: BM aspirate
2
cfDNA for non-invasive genetic characterization High concordance with BM aspirates but restricted information
3
Approach Genes n, paired* Applicability Concordance Reference
Targeted
BRAF, DIS3, DTX1, EGR1, FAM46C, IRF4, NRAS, KRAS, PRDM1, PSMD1, TP53, TRAF3, TRIP12
9 Not reported - Lohr, Sci Trans Med 2016
Targeted KRAS, NRAS, BRAF, EGFR, PIK3CA
30 80% samples successful (>80 ng total cfDNA, eq. >0.05% of tumor fragment)
96% Kis, Nat Comm 2017
Targeted KRAS, NRAS, CTNNB1, EGFR, PIK3CA, TP53, FOXL2, GNAS, BRAF
48 Not reported 30% Mithraprabhu, Leukemia 2017
WES - 9 (BM-cfDNA) + 4 (BM-CTC-cfDNA)
35% samples successful (>=10% tumor fraction):
• 107 cfDNAs > 16,8% • 56 CTCs > 21,4%
• 83% cfDNA-BM • 84% cfDNA-CTC
Manier, Nat Comm 2018
WES - 10 64% samples successful (>=5% tumor fraction)
• 90,5% (CNVs) • 93% (mutations)
Guo, Leukemia 2018
*only paired samples (cfDNA-BM or CTC-BM) were considered
CTCs in monoclonal gammopathies: random egress from BM to PB or in response to niche occupancy?
4 Sanoja-Flores et al. Blood Cancer Journal. 8, 117 (2018)
Multiple Myeloma MGUS Solitary Plasmocytoma (SP) Macrofocal MM (MMM) Smoldering MM (SMM)
Tumor cell starts to circulate when the BM plasma cell compartment is fully occupied
CTC numbers increase with disease aggressiveness
5 Sanoja-Flores et al. Blood Cancer Journal. 8, 117 (2018)
High numbers of CTCs correlate with increased risk of disease progression
Clinical significance of CTCs in MM stages
CTCs as key drivers of MM progression?
6 Ghobrial I. Blood. 120, 20-30 (2012)
The continuous circulation of clonal PCs leads to micrometastatic MGUS followed by more disseminated disease
To compare the genetic landscape of CTCs vs matched BM clonal plasma cells and extramedullary (EM) plasmacytomas,
and
to validate standardized assays for CTCs’ detection, isolation and genetic characterization
Aim
7
Experimental design (n = 51)
n = 8 Whole-exome seq
amplification, by triplicate (Genomi Phi-29)
Mutation if 2/3 triplicates Eu
roFl
ow
NG
F
Cytoscan HD (Affymetrix)
Variant calling
CNAs + translocations
Copy-number alterations (CNA)
Euro
Flo
w N
GF
Whole-exome seq with molecular barcoding (10X Genomics, ultra-low
input)
BM clonal PCs (n = 51)
CTCs (n = 51) EM plasmacytomas (n = 8)
8
CTCs harbor most mutations present in both medullary and extramedullary disease
9
60 mutations present in CTCs while undetectable in BM or EM disease
CTCs vs BM clonal PCs
Almost all MM classical mutations found in BM were also detected in CTCs
(86%)
CTCs vs EM plasmacytoma
(87%)
Most mutational burden found in BM clonal PCs were present in CTCs
10
84% of mutations
70% of CNA (↑ 94% at chromosomal-arm level)
74% of translocations (because stochastic fragmentation?)
Almost 100% concordance between chromosomal and interstitial CNA in CTCs and BM clonal PCs
11
This assay was applicable in 22/35 (63%) patients, typically when ≥ 5,000 CTCs were sorted
o Using two different standardized methods, we showed in the largest series in which CTCs were genetically characterized, that these are a reliable surrogate of MM patients’ genetic landscape inside and outside the BM
o Because next-generation flow cytometry is broadly used, quantification, isolation and genetic characterization of CTCs may emerge as an optimal and standardized approach for non-invasive risk-stratification of MM patients
Conclusions
12
13
Leire Burgos Diego Alignani Maria-José Calasanz Sonia Garate Idoia Rodríguez Ibai Goichoechea Paula Rodríguez-Otero Xabier Agirre Felipe Prosper Jesus San Miguel Bruno Paiva
Halima El Omri
Gabriel Bretones Rafael Valdés-Mas Diana Álvarez-Puente Miguel García Álvarez Carlos Lopez-Otin
María-Victoria Mateos Noemí Puig Juan Flores-Montero Luzalba Sanoja-Flores Alberto Orfao
Rafael Rios Joaquin Martinez-Lopez Pamela Millacoy Luis Palomera Rafel del Orbe Albert Perez Montaña Laura Rosiñol Joan Bladé Juan-José Lahuerta
Thanks!