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!