Phenotypic Identification Of Subclones In Multiple Myeloma With Different Genomic Profile, Clonogenic Potential and Drug Sensitivity
Authors: Paíno Teresa , Paiva Bruno , Sayagués José M , Mota Ines , Carvalheiro Tiago , Corchete Luis A , Gutiérrez Norma C , Aires-Mejia Irene , Sanchez Maria Luz , Barcena Paloma , Ocio Enrique M. , Oriol Albert , Hernandez Miguel T , Echeveste Maria Asunción , Paiva Artur , Blade Joan , Orfao Alberto , Mateos Maria Victoria , San Miguel Jesus F 
Published: October 2019

Abstract
The identification of subclones in multiple myeloma (MM) could have clinical implications since the complete eradication of all clones is required to prolong patients’ survival. Hence, there is an unmet need to fully characterize such subclones.
In the present study, we started by investigating using multidimensional (23-color) flow cytometry (MFC) the presence of ≥2 distinct bone marrow PC subclones in 116 newly-diagnosed (MM) patients included in the Spanish GEM2010 trial: sequential VMP (9 cycles) followed by Rd (9 cycles) vs alternating VMP with Rd (18 cycles). By principal component analysis (PCA) using the Infinicyt software, the immunophenotypic expression profile (based on the simultaneous evaluation of 23 antigens in single-clonal-PCs) showed the presence of two or more subclones in 35/116 (30%) newly-diagnosed MM patients. The most powerful antigens for the identification of subclonal heterogeneity were CD56, CD27, CD81, CD20, CD33, β7, CD138, CD49e, CD54, HLADR, CD19, CD44 and CXCR4.
Then, we investigated the genetic profile of distinct phenotypic subclones (sorted by FACS with purity ≥97%) through high-density Cytoscan750K (n=5) arrays and FISH (n=8). In 2/5 (40%) patients, specific copy number abnormalities (CNA; only those with minimum of 25 consecutive imbalanced markers per segment and minimum 100 Kb length were considered) were present in one clone [del(13q), del(14q), del(20p), +(20q), +(21q), or del(22q)] but absent in the other. Similar results were observed by FISH while investigating those probes used as part of the clinical workout. Noteworthy, in three cases del(17p13) was detected in only one subclone, whereas del(13q14) was restricted to one subclone in two patients.
To assess the clonogenic potential of distinct phenotypic subclones detected in the whole malignant PC compartment, these were sorted by MFC and seeded in coculture with the human mesenchymal cell line hTERT (with Methocult® supplemented with lymphocyte conditioned medium (10%) plus 20 ng/ml IL-6 plus 20 ng/ml IGF-1). In 2 out of 7 patients (29%) we observed different clonogenic potential between two patient-specific subclones (patient 1, 208 vs 0 colonies; patient 2, 5 vs 1 colony). Finally, we aimed to investigate if distinct phenotypic subclones display different chemoresistance. To address this question, we compared the immunophenotypic expression profile of paired baseline vs MRD clonal PC in 9 patients enrolled in the Spanish GEM2010 trial in which ≥2 subclones were identified at diagnosis. Our results show a phenomenon of clonal restriction in 7/9 (78%) patients after chemotherapy, indicating that among the initial tumor bulk, only specific subclones are primary chemoresistant (i.e.: MRD) in patients otherwise achieving a serological response. As an example, in one patient showing at diagnosis four clonal PC subsets (CD33-/CD117-, CD33+/CD117-, CD33+/CD117+ and CD33+/CD117+), only one subclone (CD33-/CD117-) persisted after 9 cycles of alterning Rd-VMP.
In conclusion, our results show that distinct phenotypic subclones translate into a different cytogenetic profile and clonogenic potential. Since the balance between different subclones may change over the course of treatment, so may change the extent of specific cytogenetic abnormalities and the patient risk. Thus, careful monitoring of specific subclones may have implications towards an adapted therapy to overcome chemoresistant cells.
This work was supported by funding from the RTICC-Hematology Group (RD12/0036/0058), the Asociación Española Contra el Cáncer (AECC) (GCB120981SAN) and Multiple Myeloma Research Foundation (MMRF) 2012 Research Fellow Award (B Paiva)

Disclosures:
No relevant conflicts of interest to declare.