Clonal Heterogeneity and Evolutionary Phylogeny of Critical Cytogenetic Aberrations in Multiple Myeloma

Single-cell analysis is of significant importance in delineate the exact phylogeny of subclonal population and in discovering subtle diversification. So far studies of intratumor heterogeneity and clonal evolution in multiple myeloma (MM) were largely focused at the bulk tumor population level. Here, we performed quantitative multi-gene fluorescence in situ hybridization (QM-FISH) in 129 longitudinal samples of 57 MM patients. All the patients had newly-diagnosed and relapsed paired samples. An expanded cohort of 188 MM patients underwent conventional FISH (cFISH) to validate the cytogenetic evolution in bulk tumor level. 43 of 57 patients (75.4%) harbored three or four cytogenetic clones at diagnosis. We delineated the phylogeny of subclonal tumor population and derived the evolutionary architecture in each patient.13q deletion and the first 1q gain tended to be earlier cytogenetic alternation, whereas 16q and 17p deletion were acquired later. Patients with clonal stabilization had a significantly improved OS than those with other evolutionary patterns (median OS, 71.2 vs. 39.7 vs. 35.2 vs. 25.5 months, for stable, differential, branching and linear patterns, respectively, p=0.001). Besides, a high degree of consistency and complementarity across QM-FISH and cFISH was observed in evaluation of cytogenetic evolution pattern in MM. In total, at least two time-point cytogenetic evaluations by cFISH were underwent in 188 MM patients. The proportion of patients with high-risk cytogenetic features was 33% at diagnosis and 49% at relapse. The prognostic value of the presence of high-risk aberrations at diagnosis were attenuated over time (HR=1.79, p=0.002 for survival from diagnosis; HR=1.55, p=0.026 for survival from relapse, ). Survival from relapse were greater influenced by the presence of high-risk aberrations at relapse (HR=2.07, figure 5E) rather than present at diagnosis (HR=1.55). The present study investigated the prognostic value of evolution in copy number or clone size of 1q21 gain/amplification during follow-up. The incidence of patients carrying at least three copies of 1q21 was higher after relapse than at diagnosis (69% vs. 55%, p=0.004).Patients were categorized as six groups according to the change patterns in copy number and clone size of 1q21 gain between the two time-point samplings. Patients without 1q21 gain/amplification at both time points (group B) and patients who had obvious decrease in clone size or loss of 1q21 gain at relapse (group A) experienced similar superior outcome (Failure free survival after relapse (2 nd FFS), 18.1 vs. 27.8 months, p=0.469), whereas patients carrying 1q21 gain/amplification at both time points with or without increase (group C,D) in clone size relatively worse survival (2 nd FFS 12.4 and 10.5 months, respectively, p<0.05 compare to group A and B). The remaining patients who had an increase in copy number of 1q21 and those who developed de novo 1q21 gain at relapse were observed poorest outcome (group E and F,2 nd FFS 6.7 and 8.9 months). The interval time between two time-point samplings were similar among groups, whereas the different evolution pattern of 1q21 gain could clearly stratify both overall survival and post-relapse survival (p<0.001). This study shows that QM-FISH is a valuable tool to elucidate the clonal architecture at single cell level. Clonal evolution pattern is of prognostic significance, highlighting the need for repeated cytogenetic evaluation in relapsed MM. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Clonal Phylogeny and Evolution of Critical Cytogenetic Aberrations in Multiple Myeloma at Single Cell Level

Introductions Although intratumor heterogeneity and clonal evolution have been inferred in multiple myeloma (MM), this was largely focused at the bulk tumor population level. Single-cell analysis is of significant importance in delineating the exact phylogeny of subclonal population and in discovering subtle diversification. Here, we identified the clonal architecture of different time points using multi-gene fluorescence in situ hybridization (mFISH) at single cell level, and explored the prognostic values of different clonal evolution patterns in MM. Methods We performed mFISH in 129 longitudinal samples of 57 MM patients. All the patients had newly-diagnosed and relapsed paired samples, and 12 patients had cytogenetic evaluation for more than two time points. An expanded cohort of 188 MM patients underwent conventional FISH (cFISH) to validate the cytogenetic evolution in bulk tumor level. Results 43 of 57 patients (75.4%) harbored three or four cytogenetic clones at diagnosis. We delineated the phylogeny of subclonal tumor population in each patient and established robust trends for the timing of temporal acquisition in the whole cohort using the pairwise precedence. 13q deletion and the first 1q gain tended to be earlier cytogenetic alternation, whereas 16q and 17p deletion were acquired later. The sequence of 13q deletion and 1q21 gain occurrence was identified in 23 patients by the single-cell analysis. 1q21 gain and 13q deletion each occurred first in 12 and 11 patients respectively. Strikingly, patients in whom 13q deletion was acquired first showed a significantly worse survival than 1q21 gain-first patients (median OS 32.9 vs. 71.2 months, p=0.010). We inferred the most likely ancestral relationships between subclones and derived the evolutionary architecture in each patient. Four distinct evolutionary patterns were identified (Figure 1). 18 of 57 (31.6%) patients showed clonal stabilization. These patients were characterized by no novel subclones emerging and no existed subclones disappearing at relapse. Differential evolution was observed in 12 patients, where clonal dynamics resulted from a change in predominant clone from presentation to relapse. The major clone at diagnosis disappeared or decreased to a minor clone while a subclone showed growth advantage and turned to be a major clone at relapse. We found evidence of branching evolution in 9 patients. Here, one or more clones harboring novel cytogenetic abnormalities emerged between the early and late time points, whereas some disappeared. The remainder of patients demonstrated a linear evolution pattern (18/57, 31.6%). The predominant clones acquired one or more novel cytogenetic abnormalities at the later time point. Patients with clonal stabilization had a significantly improved OS than those with other evolutionary patterns (median OS, 71.2 vs. 39.7 vs. 35.2 vs. 25.5 months, for stable, differential, branching and linear patterns, respectively, p=0.001). However, there is no difference in sampling interval among four evolutionary patterns (p=0.131). Therefore, the survival differences were mostly attributable to a significantly shorter failure free survival from relapse (p<0.001). In order to evaluate the accuracy of abnormalities detection by mFISH, we performed cFISH in these 57 MM patients. Cell fractions of cytogenetic abnormalities detected by mFISH were significantly correlated with that detected by cFISH (p<0.001). Besides, a high degree of consistency and complementarity across cFISH and mFISH was observed in evaluation of cytogenetic evolution pattern in MM. Then we expanded our cohort to 188 patients to further discuss the prognostic value of cytogenetic evolution. Survival from relapse were greater influenced by the presence of high-risk aberrations at relapse (HR=2.07) rather than present at diagnosis (HR=1.55). There was no difference in OS for patients who had primary high-risk aberrations at diagnosis compared with those who developed high-risk aberrations after relapse (p=0.800). Conclusions These findings suggest that mFISH is a valuable tool for the analysis of clonal phylogeny and evolution pattern of critical cytogenetic aberrations. Patients may benefit from the repeated cytogenetic evaluation, especially for the risk stratification of survival after relapse. Personalized treatment strategy is required for MM patients based on their clonal evolution patterns. Figure 1 Disclosures No relevant conflicts of interest to declare.

3D Telomere Structure Analysis to DetectGenomic Instability and Cytogenetic Evolutionin Myelodysplastic Syndromes

The disease course of myelodysplastic syndromes (MDS) features chromosome instability and clonal evolution, leading to the sequential acquisition of novel cytogenetic aberrations and the accumulation of these abnormalities in the bone marrow. Although clonal cytogenetic abnormalities can be detected by conventional cytogenetics in 50% of patients with MDS, such distinguishing patterns are lacking in the other 50%. Despite the increase in the prognostic value of some biomarkers, none of them is specific and able to discriminate between stable and unstable patients that subsequently progress to acute myeloid leukemia. This pilot study aimed to investigate the potential use of the 3D telomere profiling to detect genomic instability in MDS patients with or without clonal cytogenetic evolution. The comparison between different time points in patients with cytogenetic changes showed that in the CD34+ MDS cells, there was a significant decrease in the total number of telomeric signals, the average intensity of signals and the total intensity of telomeres. By contrast, the number of aggregates increased during cytogenetic evolution (p < 0.001). This pattern was observed only for MDS patients with cytogenetic evolution but was absent in patients without cytogenetic changes. In conclusion, we demonstrated that the 3D nuclear telomere organization was significantly altered during the MDS disease course, and may have contributed to cytogenetic clonal evolution.

Fulminant blast crisis with de novo 11q23 rearrangement in a Philadelphia-positive CML patient undergoing treatment with dasatinib

Background: Progression of chronic myeloid leukemia (CML) is frequently accompanied by cytogenetic evolution, with an extra copy of the Philadelphia chromosome, trisomy 8 and 19, and isochromosome (17p) commonly detected. Translocations involving 11q23 chromosomal region have been rarely reported in CML. The few reported patients with blast crisis (BC) of CML carrying an 11q rearrangement have insufficient responses to tyrosine kinase inhibitors (TKIs) and possess a poor prognosis. Case report: We report the case of a 30-year-old man with CML who had a fulminant myeloid BC 4 months after initiation of first-line therapy with the TKI dasatinib, despite showing an optimal response at the 3-month timepoint. Despite cytoreductive therapy with hydroxyurea and 3rd-generation TKI ponatinib, the patient died within 10 days after the diagnosis of BC. Cytogenetic analyses revealed additional genetic aberrations including trisomy 8 and t(9;11)(p21;q23) involving the mixed lineage leukemia ( MLL) gene. Conclusion: The presence of 11q23 rearrangements in the relapse clone in BC of CML most likely accounts for the adverse clinical outcome. Thus, in the case of rapid and unexpected BC, the presence of 11q rearrangements should be tested together with other additional chromosomal alterations, and immediate addition of chemotherapy to the TKIs should be evaluated.