Transcriptomic Profiling of Circulating Tumor

Cells (CTCs) in Multiple Myeloma (MM): A New

Model to Understand Disease Dissemination

Juan-Jose Garces,1 Michal Simicek,2 Marco Vicari,3 Lucie Brozova,4 Leire Burgos,1 Renata Bezdekova,5

Diego Alignani,1 Maria-Jose Calasanz,1 Katerina Growkova,2 Ludek Pour,5 Rafael Rios,6 Joaquin

Martinez-Lopez,7 Pamela Millacoy,8 Luis Palomera,9 Rafael Del Orbe,10 Sonia Garate,1 Laura Blanco,1

Patricia Maiso,1 Zuzana Chyra,2 Alexander Vdovin,2 Tomas Jelinek,2 Cirino Botta,11 Halima El Omri,12

Jonathan Keats,13 Xabier Agirre,1 Felipe Prosper,1 Roman Hajek,2 Jesus F. San Miguel,1 Bruno Paiva1

1University of Navarra, Pamplona, Spain; 2University Hospital of Ostrava, Ostrava, Czech Republic; 3Campus Bio-Medico University of Rome, Rome, Italy; 4Masaryk University, Brno, Czech Republic; 5University Hospital Brno, Brno, Czech Republic; 6Hospital Universitario Virgen de las Nieves, Granada, Spain; 7Hospital Universitario 12 de Octubre, Madrid, Spain; 8Complejo Hospitalario de

Navarra, Pamplona, Spain; 9Hospital Clínico Universitario Lozano Blesa, Zaragoza, Spain; 10Biocruces Health Research Institute, Barakaldo, Spain; 11Magna Graecia University, Catanzaro, Italy; 12National Center for Cancer Care and Research, Hamad Medical Corporation, Doha, Qatar; 13Translational Genomics Research Institute, Phoenix, AZ.

o Clonal expansion of plasmatic cells (PC)

o Clinical technique: bone marrow (BM)

aspirates.

What’s Multiple Myeloma?

Sanoja-Flores et al. Blood Cancer Journal. 8, 117 (2018)

CTCs in patients with monoclonal gammopathies: random egress from BM to PB or in response to niche occupancy?

Multiple Myeloma MGUS Solitary Plasmocytoma (SP) Macrofocal MM (MMM) Smoldering MM (SMM)

CTC numbers increase with disease aggressiveness

Tumor cell starts to circulate when the BM PC compartment is fully occupied

Clinical significance of CTCs in pre-malignant and

malignant monoclonal gammopathies

High numbers of CTCs correlate with increased risk of disease progression

Sanoja-Flores et al. Blood Cancer Journal. 8, 117 (2018)

Ghobrial I. Blood. 120, 20-30 (2012)

The continuous circulation of clonal PCs leads to micrometastatic MGUS followed by more disseminated disease

CTCs could be key drivers of myeloma progression

#1 #6

#9

#8

#12 #14

CTCs

BM PCs reference

Disease dissemination may depend on few tumor cells with unique features allowing them to egress from BM

Paiva B, et al. Blood. 122, 3591-8 (2013)

Myeloma dissemination is based in very low numbers of

circulating tumor cells

To identify gene regulatory networks related to MM

dissemination by comparing the transcriptional profile of

CTCs with patient-matched BM clonal PCs

Aim

*

matched BM and PB samples (FACS BM clonal PCs and CTCs)

Gene Expression Profiling (GeneChip Human Gene 2.0/1.0

ST array, Affymetrix)

enrichment analysis (GSEA software)

clinical validation (RNAseq from CoMMpass, IA11)

index single-cell RNA-seq (BD Genomics Precise WTA Single Cell)

anti-CD44 monoclonal antibody (CB17/Icr-PrKdcscid/Crl mice +

MM.1S inoculation)

shRNA knock-down (MM.1S & U266 cell lines)

n = 32

MMRF = Multiple Myeloma Research Foundation

*Modified from Papalexi & Satija, Nature Reviews Immunology 18, 35-45 (2018)

differential expression (limma package)

scRNA-seq analysis (Seurat package)

1 MGUS

38 newly-diagn. MM

12 relapse MM

Study design

Cytogenetics & Copy Number (CytoScan HD array, Affymetrix)

CTCs and BM clonal PCs shared almost all CNVs

BM PCs

CTCs

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22

#1

#2

#3

#4

#5

#6

#7

#8

#9

#10

#11

#12

#13

#14

#15

#16

BM PCs

CTCs

BM clonal PCs expression

CTC

s e

xpre

ssio

n

Transcriptional profiles of CTCs and BM clonal PCs are

highly overlapping at the single-cell level

BM clonal PCs expression

CTC

s e

xpre

ssio

n

infra-expressed (n = 7) over-expressed (n = 51)

log2FC

-lo

g2

(ad

j.P

.va

lue

) High correlation in gene expression and only 58 genes

emerged as significantly deregulated in CTCs

Gene expression bulk on BM clonal PCs and matched

CTCs

NES = Normalized Enrichment Score; DE = differentially expressed; # genes = number of genes

GSEA analysis uncovered distinct biological functions for

BM clonal PCs and CTCs

Inflammatory and interferon

response, EMT, angiogenesis or

hypoxia were more represented in

CTCs, whereas hallmarks related

with cell cycle were predominant

in BM clonal PC.

MM-recurrent genes

logFC

BMPCs

Cellcycle

Epithelial-mesenchymaltransition

Inflammation

CTCs

Pancancer genes

Filamin together with CD44, both overexpressed in CTCs, have an important role in the cytoskeleton dynamics

hsa05205, KEGG pathway

Validation of FLNA and CD44 as targets of cell

migration and invasion

Migration assay (transwell)

MM.1S U266

0.0

0.5

1.0

1.5

0.0

0.5

1.0

1.5

Adhesion assay (fibronectin plates) MM.1S

0.0

0.5

1.0

1.5

0.0

0.5

1.0

1.5

U266

Knock-down of CD44 and FLNA significantly reduce

migration and adhesion

day +10 day +19 day +26 day +33 0

2·107

1·107

Fuo

rese

cen

ce (

p/s

/cm

2/s

r)

day +10 day +19 day +26 day +33 0

1·104

5·103

1.5·104

2·104 6·105 8·105

Bio

lum

inis

cen

ce

(p/s

/cm

2/s

r)

Contr

ol

anti-

CD

44

anti-CD44

Control

MM.1S-tomato MM.1S-GFP (luc+)

(CB17/Icr-PrKdcscid/Crl)

control anti-CD44 control anti-CD44

Contr

ol

anti-

CD

44

anti-CD44

Control

Significant reduction in the plasmacytoma volume

and cell engraftment in mice treated with anti-CD44

log2FC

-lo

g2

(ad

j.P

.va

lue

)

Are overexpressed genes in CTCs related to MM

dissemination and worse outcome?

Hypothesis: Those myeloma patients that have high expression of genes overexpressed in CTCs have an enrichment in BM clonal PCs with a more similar CTC-behaviour and poor prognosis

Method: To analyze their expression in BM clonal PCs from patients enrolled in the CoMMpass study.

time (days)

In the multivariate cox (overexpressed genes & R-ISS) WEE1, LAMP3 and SAMD9 retained independent prognostic value

multivariate univariate

Expression levels of 23/51 over-expressed genes in

CTCs were associated with significantly different PFS

WEE1 + SAMD9 + LAMP3

o Gene expression of CTCs is almost identical to that of patient-matched BM

clonal PCs, except for a few genes that are involved in interferon and

inflammatory response, hypoxia, cell cycle and migration

o Some of these genes are related to more aggressive disease and modulating

their expression may impact migration and adhesion of clonal PCs

o Studying the transcriptome of CTCs may unveil novel prognostic markers

related to disease dissemination and therapeutic targets to overcome it

Conclusions

19

Leire Burgos Diego Alignani Maria-José Calasanz Sonia Garate Laura Blanco María Collantes Patricia Maiso Felipe Prosper Jesus San Miguel Bruno Paiva

Michal Simicek Lucie Brozova Renata Bezdekova Katerina Growkova Ludek Pour Zuzana Chyra Alexander Vdovin Tomas Jelinek Roman Hajek Rafael Rios

Joaquin Martinez-Lopez Pamela Millacoy Luis Palomera Rafel del Orbe

Marco Vicari

Cirino Botta

Halima El Omri

Acknowledgments