Prognostic Value of Antigen Expression in Multiple Myeloma (MM): A Large GEM/Pethema Study Based in Four Consecutive Clinical Trials

            Multiparameter flow cytometry (MFC) is commonly used to monitor minimal residual disease (MRD) in MM due to its widespread availability, fast turnaround, and the amount of information obtained upon enumeration of different cell populations and their corresponding antigen expression levels. Thus, MFC could potentially be used not only to monitor MRD, but also to offer additional prognostic information based on MM plasma cell (PC) phenotypes. However, in MM there is lack of consensus about which markers are prognostically relevant because of technical variability between centers and the paucity of studies in large series of patients.

            Before investigating the prognostic value of those antigens evaluated in MRD studies, we first demonstrated their stability over time by comparing the phenotypic profile of MM-PCs from diagnosis to the MRD stage using principal component analysis (PCA). Accordingly, PCA of merged diagnostic and MRD profiles showed phenotypic overlap between both (MM-PC references in red and blue, respectively; Panel A), that was also demonstrable at the individual patient level [Panel B, in which diagnostic and MRD phenotypic profiles from individual patients (n=14) are represented with the same color]. After demonstrating antigen stability from diagnosis to the MRD stage, we then investigated their prognostic value in a large series of 1265 newly-diagnosed patients enrolled in four consecutive GEM/PETHEMA clinical trials (GEM2000 and GEM2005MENOS65 for transplant-eligible, GEM2005MAS65 and GEM2010 for elderly patients). As compared to cases with bright CD38 expression, patients with aberrantly low CD38 had inferior PFS (medians of 38 vs 30 months; P<.001) and OS (medians of 92 vs 55 months; P<.001). Similar results were observed while comparing the outcome of patients with bright vs. low CD138 expression (median PFS of 34 vs 29 months; P=.003 / median OS of 67 vs 55 months; P=.05). CD81-positive patients had inferior survival than CD81-negative cases (median PFS of 25 vs 42 months; P<.001 / median OS of 55 vs 92 months; P<.001). Interestingly, CD117-negativity conferred inferior PFS and OS in transplant-eligible patients treated without novel agents (ie. GEM2000) but not in the era of novel agents (ie. GEM2005MENOS65). On the other hand, CD45-positive vs. negative expression resulted in inferior PFS (medians of 23 vs 31 months; P=.03) and OS (medians of 43 vs 61 months; P=.03) only among elderly (ie. GEM2005MAS65 and GEM2010) but not in transplant-eligible patients. Importantly, the prognostic value of CD38, CD138 and CD81 continued to be noted in patients with persistent MRD; furthermore, in a multivariate analysis with other prognostic factors such as ISS, FISH cytogenetics, CR and MRD status after therapy, CD38 and CD81 retained independent prognostic value for PFS and OS.

Subsequently, we determined the extent of antigenic heterogeneity in MM by enumerating how many different phenotypic profiles could be observed for the combination of markers consensually used in MRD studies: CD38, CD138, CD19, CD27, CD45, CD56, CD81 and CD117. Detailed analysis of 222 newly-diagnosed patients enrolled in the GEM2010MAS65 study and for which all the above markers were simultaneously evaluated, revealed that a total of 94 different phenotypes were identified (Panel D); the most common antigenic profile was CD38^+dCD138⁺CD19^-CD27⁺CD45^-CD56⁺CD81^-CD117⁺ in 13 of the 222 patients (6%). Since aberrant protein expression could result from specific genetic abnormalities, we subsequently investigated for a relationship between both. Interestingly, we found a phenotypic profile determined by combined CD45^-CD56⁺CD117^- expression that was present in 39% of the patients and that in comparison against other profiles was particularly enriched in high-risk cytogenetic abnormalities such as t(4;14) (19% vs 8%; P<.001), and del(17p) (13% vs 7%, P=.01).

            In summary, we unravel the heterogeneous phenotypic landscape of MM and shed light in the relationship between patients' phenotype and genotype. In addition, CD81, CD38 and CD138 were prognostically relevant in one of the largest series of MM patients ever studied; because their expression is stable over time, these results indicate that MRD monitoring by MFC can offer additional prognostic information based on the phenotypic profile of the chemoresistant PC clone.