Determinants of Phenotypic Commitment and Clonal Progression-- Conclusions from the Study of Clonal Architecture in CMML

CMML is heterogeneous clinically (a varying degree of dysplastic or proliferative clinical features) and in terms of its molecular pathogenesis. Analysis of the spectrum of genomic lesions in CMML may  contribute to understanding of the pathogenesis and help identify certain mutations as diagnostic biomarkers.  Apart of the mere presence of a somatic lesion, phenotypic features may be shaped by initial hits. Conversely sub-clonal events may determine the phenotype or progression. Finally, initial hits may predetermine e.g., mutator phenotype, or differentiation block and therefore selecting for specific sub-clonal hits.

We have selected a large CMML cohort to establish generalizable pathogenetic and clinical associations to account for molecular heterogeneity and serial samples have to be analyzed to assess clonal dynamics and hierarchy.  The study group consisted of 242 patients, including 150 CMML cases (96 CMML-1, 27 CMML-2, 27 post CMML sAML) and JMML (N=92); 15 patients were studied serially. We also used comparison cohorts of M4/M5 AML (N=64) and advanced (N=231) and low risk MDS (N=199)serving as risk adjusted match for CMML subtypes.  The CMML entity was further sub-classified based on clinical parameters and pathomorphologic features, 57% dysplastic (MD-CMML) and 43% proliferative form (MP-CMML). Analysis was performed using WES (paired germ line/tumor samples) and multiamplicon NGS targeting top 60 most commonly affected genes. For clonal architecture analysis, cross-sectional variant allelic frequency (VAF) concept based analysis was performed including assessment of affected genes by ranking of the corresponding clonal burden rather than the absolute cellular frequency. The results of this analysis were confirmed in serial samples to identify expanding, declining and stable subclones. 

Comparison of mutational spectra between the disease entity show profound differences in morphologically similar entities as particularly evident in comparison of CMML to JMML (TET2, ASXL1) or to lesser degree low risk MDS and CMML1 while progression in advanced cases was often associated with similar spectrum of additional subclonal events. However, the differences were more striking when clonal hierarchy was assessed to identify dominant/codominant and subclonal mutational events. We found that top 4 dominant/codominant clonal events, included TET2 (56%), SRSF2 (42%), ASXL1 (46%), DNMT3A (45% of patients), while in MDS corresponding frequency of these dominant events was TET2 (15%) SRSF2 (8%), ASXL1 (11%) and DNMT3A (8%), with most common ancestral events ranked SF3B1, TET2, ASXL1 etc. The clinical importance of these dominant events in CMML is highlighted by their impact on survival in KM analysis (p=.018).

Our analysis also demonstrated that for certain founding events not pathognomonic for CMML either codominant or subsequent subclonal events determine the phenotypic features (1^st generation) or progression (2^nd generation).  For instance initial TET2 in CMML was followed often by SRSF2 or in conjunction, RAS pathway mutations while MDS was driven by TET2, SF3B1, TP53, and many other events.  Progression in our cohort was driven in both CMML and MDS by ASXL1, RUNX1, NPM1. When other common mutations were categorized by their role in individual patients 27% EZH2, 20% of CBL and 22% of SETBP1 were dominant.  Serial analysis further qualified the cross-sectional analysis and allowed for categorization of subclonal events. For instance, CMML-1 cases initially presented with dominant TET2 followed by subtype specific subclonal SRSF2 and progression event IDH2 progressed to sAML with new NPM1 acquisition and expansion of IDH2 clone. In our serial sample analysis we observed that increasing ASXL1 and RUNX1 clones correspond to clinical progression, ancestral events may remain stable (TET2, SRSF2, SETBP1), while non-permissive subclones can smolder or even decline.

In sum, deep NGS allows for identification of specific ancestral events, which may determine the subsequent secondary mutational events in CMML. Classification of CMML based on ancestral events and subclonal events rather than on the global mutational spectrum correlates with clinical features and prognosis and may contribute to further clinical resolution of CMML based on the presence of specific founder mutations ultimately help establish therapeutic interventions.