Absence of circulating tumor cells (CTCs) defines a subtype of multiple myeloma (MM) patients (Pts) with unique clinical and biological features

Abstract Background: Evidence suggests that CTCs are responsible for MM spreading and therefore prognostic. However, some pts unexpectedly have undetectable CTCs and we hypothesized that this feature defines a distinct MM subtype. Aim: Investigate the clinical and biological features of MM pts with undetectable CTCs. Methods: This study included 3,146 pts. Disease characteristics and clinical outcomes associated with undetectable CTCs defined by next-generation flow (NGF) with a limit of detection (LOD) of 2x10-6 were investigated in 1,093 transplant-eligible and ineligible newly diagnosed MM (NDMM) pts enrolled in the GEM2012MENOS65, CLARIDEX and GEM2017FIT clinical trials. Validation was performed in a European pooled-analysis of 1,601 NDMM pts having CTC assessments by flow cytometry with LOD of ≤1x10-5. We further investigated the prognostic value of undetectable CTCs after 2 cycles of induction in the GEM2017FIT trial (n=251), and prior salvage therapy in 242 relapsed MM (RRMM) pts enrolled in the GEM KyCyDex and SeliBorDara trials. Risk of transformation in the absence of CTCs was investigated in 1,487 MGUS and 324 smoldering MM (SMM) pts. Molecular alterations associated with CTC egress were evaluated by exome and RNA sequencing. Mechanistic validation of selected genes was performed in MM5080 cells derived from Trp53-BIcγ1 mice, a model that recapitulates tumor dissemination from early to late stages of MM progression. Results: Among NDMM pts, 10% showed undetectable CTCs before treatment. Compared to those with detectable CTCs, they displayed significantly less anemia and tumor burden, only one had R-ISS 3 and MGUS-like profiles were more frequent. In contrast, the incidence of plasmacytomas was higher (33% vs 15%, p<.001). Altogether, pts with undetectable CTCs showed clinical features linked to macrofocal disease. When compared to NDMM pts with detectable CTCs, those with undetectable CTCs showed significantly higher 5y rates of PFS (80% vs 50%) and OS (92% vs 72%). In multivariate analyses of PFS and OS including transplant-eligibility and the R-ISS, undetectable CTCs showed independent prognostic value for PFS (HR: 0.5; p=.005) and OS (HR: 0.4, p=.02). Pts with undetectable CTCs achieving MRD negative CR displayed unprecedented 5y rates of PFS and OS (92% and 98%). In the European pooled analysis, undetectable CTCs was confirmed as an independent prognostic factor of PFS (HR: 0.5; p<.001) and OS (HR: 0.4, p<.001). Of note, a 2x10-6 LOD was required to define this group of pts with unprecedented prognosis since detection of CTCs in between ≥0.0002% and <0.001% was associated with inferior survival in the Spanish and European cohorts. Absence of CTCs was also associated with longer PFS after cycle 2 of induction in transplant-ineligible NDMM (HR: 0.47, p=.003) and before a new line of therapy in RRMM (HR: 0.5, p=.01). Although the absence of CTCs was associated with lower risk of progression in MGUS (HR: 0.1, p<.001) and SMM (HR:0.4, p<.001), 1% of MGUS and 10% of SMM pts with sustained undetectable CTCs progressed to active MM. These results suggest the presence of a subgroup of pts with undetectable CTCs throughout the disease course, which displays less aggressive tumors and favorable clinical outcomes by the time they progress to active MM. We next investigated the biology of bone marrow tumor cells in this subgroup. While the median number of coding mutations was similar, copy number alterations were less frequent in pts with undetectable vs detectable CTCs (29 vs 61, p=.03). The former displayed lower frequency of del(17p) (5% vs 11%, p=.03) and +1q21 (23% vs 48%, p<.001). RNAseq uncovered 66 differentially expressed genes, 9 of which were located on 1q, in line with the genetic findings. To gain more insights into the functional role of candidate genes we selected LGALS1, which was among the genes showing the highest correlation between mRNA expression and CTC levels, for CRISPR/Cas9 knockout experiments in MM5080 cells inoculated in Trp53-BIcγ1 mice. Compared to Lgals1 wild-type, biallelic loss of Lgals1 resulted in longer OS (median not reached vs 46 days). Conclusions: NDMMpts with undetectable CTCs have less genetic alterations and lower expression of genes linked to CTC egress, which results in less aggressive and macrofocal disease. Thus, this study recognizes a new myeloma subtype that accounts for 10% of NDMM pts, who show unprecedented survival and can be defined by undetectable CTCs with NGF.