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Letters to the Editor
P=0.043) were significantly higher in ICUS patients with CNA/CNLOH, and LOY was more preponderant (29% vs. 8% of males; P=0.028) than in ICUS patients without CNA/CNLOH (Online Supplementary Table S4). Notably, there was no significant correlation between the pres- ence of CNA/CNLOH and the mutational profile.
Of utmost importance for the clinical relevance is whether the presence of chromosomal lesions translates to more precise prognostication in ICUS patients. Median follow-up time was 25 months (range, 2-114 months). No patients were lost to follow-up, the only censoring was administrative at the study cut-off date. Twenty patients had a follow-up BM investigation performed for suspected MDS. A total of 15 of 153 patients (10%) pro- gressed to myeloid malignancy (MDS, n=12; AML, n=2; chronic myelomonocytic leukemia, n=1), and 35 died (23%) of any cause. All patients but one who progressed carried somatic mutation(s).
Survival of ICUS patients with CNA/CNLOH was sig- nificantly shorter than of patients without CNA/CNLOH for overall survival (OS) (median, 67 vs. 104 months; P=0.039; hazard ratio [HR]=2.26; 95% Confidence Interval [CI]: 1.04-4.93), and progression-free survival (PFS) (median, 49 vs. 66 months; P=0.04; HR=2.07; 95%CI: 1.04-4.14) (Figure 1A and B; Online Supplementary Table S5). In multivariable analysis adjusting for age, sex, severe anemia, mutational status and smoking, CNA/CNLOH were not associated with adverse out- comes in ICUS patients (Figure 1C; Online Supplementary Figure S6; Online Supplementary Table S5).
The presence of somatic mutation(s) was significantly associated with inferior PFS (HR=2.40; 95%CI: 1.30-4.43; P=0.004), but not OS (HR=1.60; 95%CI: 0.82-3.16; P=0.2) in univariable analysis. Somatic mutation(s) was a borderline significant independent factor for inferior PFS (adjusted HR=2.03; 95%CI: 0.97-4.26; P=0.06) (Online Supplementary Figure S6).
When we analyzed CCUS patients (n=64) as a sub- group the adverse effect of CNA/CNLOH on OS was more pronounced than in the entire study population of ICUS non-clonal and CCUS patients. After a median fol- low-up of 24 months (range, 2-109 months), median OS was 67 months in the group of CCUS patients with CNA/CNLOH compared with 104 months in the group of CCUS patients without CNA/CNLOH (P=0.013) (Figure 2A). The corresponding HR was 3.22 (95%CI: 1.22-8.51) for all-cause mortality in CCUS patients with CNA/CNLOH. Notably, also in multivariable analysis the presence of CNA/CNLOH in CCUS patients conferred a more than three times higher hazard of death, which was borderline significant (adjusted HR=3.56; 95%CI: 0.97- 13.15; P=0.056) (Figure 2B). Interestingly, this increased mortality hazard was not driven by progression to overt myeloid malignancy as an association with PFS was less evident (Online Supplementary Figure S7).
On the other hand, in a separate analysis of the patients with non-clonal ICUS, the presence of CNA/CNLOH was not associated with shorter survival (Online Supplementary Figure S8).
CCUS patients with CNA/CNLOH had a significantly higher variant allele frequency of somatic mutations than CCUS patients without CNA/CNLOH (median 36% vs. 24%; P=0.039) and were more likely to have LOY (P=0.035) and macrocytosis (P=0.022) (Online Supplementary Table S6). There was no significant differ- ence between the two CCUS groups with respect to age, sex, adverse mutations, number of mutations, smoking or hematological parameters.
Multiple studies have demonstrated worse survival of
patients with myeloid malignancies harboring cryptic chromosomal lesions compared with patients without cryptic lesions.3,4 Akin to our findings, the impact of CNA/CNLOH was generally more pronounced on OS than PFS, and SNP-A improved prognostic stratification in primarily lower-risk MDS patients including patients with a normal karyotype.7,8
To our knowledge, only three smaller previous studies have reported on CNA/CNLOH in patients with ICUS/pre-MDS with frequencies at 15-32%.9–11 However, their study designs did not enable distinction between ICUS non-clonal and CCUS or correlation to clinical out- comes.
SNP-A in large patient cohorts from genome-wide association studies including healthy controls showed that the frequency of mosaic CNA/CNLOH in peripheral blood increases to approximately 2-3% for age >75 years.12 Even when only considering clonal mosaicism (Online Supplementary Table S3), the frequency of autoso- mal mosaic CNA/CNLOH was 2-3-fold higher in our study population. This suggests that the loss of chromo- somal integrity found by SNP-A in the ICUS patients was related to their disorder, rather than their advanced age.
Our study has certain limitations. Firstly, follow-up was relatively short given the cohort size and the life expectancy of ICUS/CCUS patients. This may have influ- enced the lack of statistical significance in multivariable analysis. Obviously, our findings need validation in an independent cohort.
Secondly, DNA from BM was not available for SNP-A in all patients, hence, granulocytes from peripheral blood were the source of DNA in these cases (Online Supplementary Methods). We considered this feasible as previous studies have shown a high concordance (95%) for SNP-A karyotype between peripheral blood and BM as also seen for somatic mutations.8,13
Thirdly, germline DNA was not available as matched
DNA reference to allow definitive distinction between
acquired and constitutional aberrations. Some aberra-
tions, especially small CNA, appeared to be fully clonal
(i.e., not mosaic) (Online Supplementary Table S3) and
therefore could be germline variants potentially predis-
posing to disease development. However, extensive
MDS-associated aberrations and LOY were also present
as they do not occur in non‐clonal control DNA. Finally, due to the scarcity of surplus sample material we were unable to proceed with sequencing of myeloid malignancy-associated genes that were not included in our 20-gene panel and were found to be affected by dele- tion or CNLOH in a subset of patients (Table 1B). Sequencing of these genes was compelling as regions of acquired CNLOH may pinpoint homozygous loss of tumor suppressor genes or oncogenes with homozygosi-
ty of mutations.3,15
Besides these limitations, our data document that addi-
tional structural aberrations detected by SNP-A may influence the variability in the clinical course among CCUS patients and distinguish patients with a markedly worse OS. By contrast, in the group of ICUS non-clonal patients, CNA/CNLOH had no impact on survival. Newer technologies such as whole-genome sequencing, capable of simultaneously detecting mutations and CNA, are increasingly being used in the diagnostic setting. We believe our study emphasizes the importance of the com- pound analysis of mutations and structural aberrations in CCUS patients.
in a fully clonal state, as observed previously.14
Furthermore, it has been demonstrated that large (≥25
Mb) and/or telomeric CNLOH do not require verification
7,15
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