Clinical significance of cytogenetic and molecular genetic abnormalities in 634 Chinese patients with myelodysplastic syndromes

Abstract Purpose To explore the relevance of cytogenetic or molecular genetic abnormalities to clinical variables, including clinical and laboratory characteristics and prognosis in Chinese patients with myelodysplastic syndromes (MDS). Methods A total of 634 consecutive patients diagnosed with MDS at The First Affiliated Hospital, Zhejiang University School of Medicine from June 2008 to May 2018 were retrospectively included in this study. All patients had evaluable cytogenetic analysis, and 425 patients had MDS‐related mutations sequencing. Results 38.6% of patients displayed abnormal karyotypes. The most common cytogenetic abnormality was +8 (31%). Sole +8 was related to female (p = 0.002), hemoglobin >10 g/dL (p = 0.03), and <60 years old (p = 0.046). TP53 mutations were associated with complex karyotype (CK) (p < 0.001). DNMT3A mutations correlated with ‐Y (p = 0.01) whereas NRAS mutations correlated with 20q‐ (p = 0.04). The overall survival (OS) was significantly inferior in patients with +8 compared with those with normal karyotype (NK) (p = 0.003). However, the OS of sole +8 and +8 with one additional karyotypic abnormality was not different from NK (p = 0.16), but +8 with two or more abnormalities had a significantly shorter OS than +8 and +8 with one additional karyotypic abnormality (p = 0.02). In multivariable analysis, ≥60 years old, marrow blasts ≥5% and TP53 mutations were independent predictors for poor OS (p < 0.05), whereas SF3B1 mutations indicated better prognosis. Male IDH1 and IDH2 mutations and marrow blasts ≥5% were independent risk factors for worse leukemia free survival (LFS) (p < 0.05). Conclusion In this population of Chinese patients, trisomy 8 is the most common karyotypic abnormality. Patients with +8 showed a poorer OS compared with patients with NK. Sole +8 and +8 with one additional karyotypic abnormality had similar OS with NK, whereas +8 with two or more abnormalities had a significantly shorter OS. DNMT3A mutations correlated with ‐Y and NRAS mutations correlated with 20q‐. TP53 mutations were associated with CK and had a poor OS. SF3B1 mutations indicated a favorable OS. IDH1 and IDH2 mutations independently indicated inferior LFS.

With the use of next generation sequencing (NGS) technologies, 70-90% of MDS patients were detected with one or more genetic mutations. [20][21][22][23][24] Mutations were found to be associated with clinical phenotypes and prognosis in MDS patients. 25 The correlation between aberrant karyotypes and genetic mutations has been described previously. Mutations in U2AF1, ASXL1, IDH, and ZRSF2 were reported to be clustered with +8, [26][27][28][29] whereas SRSF2, ASXL1, and U2AF1 mutations were associated with 20q-. 30 However, the results were from single-center studies. Therefore, the conclusions needed to be confirmed.
This study aimed to analyze the relationships between aberrant karyotypes and genetic mutations in a cohort of 634 native born Chinese MDS patients, and explore their associations with clinical features and prognosis in MDS patients.

| Patients and diagnostic criteria
Six hundred and thirty-four patients were selected from the institutional database of patients with primary MDS from June 2008 to May 2018. Study eligibility criteria included the availability of bone marrow (BM) smear, BM histology, and cytogenetic information at the time of diagnosis/new referral to the hospital. Even though the patients at first referral were diagnosed at other hospitals, they were re-examined and not received treatment until hospitalized in our center. Clinical and laboratory data were acquired at the time of diagnosis. The diagnoses of MDS were according to the 2016 WHO classification. 1 The current study was approved by the ethics committee of The First Affiliated Hospital, Zhejiang University School of Medicine.
with NK, whereas +8 with two or more abnormalities had a significantly shorter OS.
DNMT3A mutations correlated with -Y and NRAS mutations correlated with 20q-.

| Prognostic criteria, response, and follow up
Patients were assigned into prognostic risk groups according to the IPSS-R. 14 The options of treatments included supportive care, low-intensity treatment approach hypomethylation agents (HMA)±chemotherapy (HMA±chemo), or allogeneic hematopoietic stem cell transplantation (allo-HSCT) according to NCCN guideline. Response to treatment was defined per the 2006 revised international working group (IWG) response criteria. 31 OS was measured from the time of diagnosis to the time of death from any cause. LFS was calculated from the date of diagnosis to the date of leukemia transformation.

| Statistics
Statistical significance was analyzed using Student's ttest to compare differences of the continuous variables
We further divided 76 patients with +8 abnormality into three groups: sole +8 group (tri8, 42), +8 with one karyotypic abnormality (tri8 +1 , 11), and +8 with two or more abnormalities (tri8 +≥2 , 23). Patients in tri8 +1 group did not have abnormalities concerning chromosome 7. Among 42 patients with sole +8, 5 patients were ranked as lower risk (LR) and received supportive care. The other 37 patients, in the higher risk (HR) group received various treatments as follows: supportive treatment, 22 HMA±chemo, 14 and allo-HSCT. 1 Eleven patients with tri8 +1 were all in the HR group, of which eight patients received supportive care, two patients received HMA±chemo, and one received allo-HSCT. Twenty-three patients with tri8 +≥2 were all in the HR group. Among them, 17 received supportive treatment, 6 received HMA±chemo.
There was a significant difference in the three groups with respect to age (p = 0.005; Table 4). By pairwise comparison of the three groups, it was found that tri8 +1 was more common in young patients (< 60 years old) than those with tri8 (p = 0.005) and tri8 +≥2 (p = 0.003). There were also significant differences in neutrophil count among the three groups (p = 0.02). Compared with tri8, patients in tri8 +1 were associated with neutrophil ≥0.8 × 10 9 /L (p = 0.02).

| Prognostic relevance
The study revealed that the OS of patients with mutated TP53 or TET2 was significantly shorter in comparison with wild type patients (p = 0.001 and p = 0.02 with TP53 and TET2 respectively; Figure 4A and B). Moreover, patients with mutated SF3B1 had significantly improved OS compared with wild type patients (p = 0.04; Figure 4C). In addition, when censoring the patients who received treatment with HMA, chemotherapy, or allo-HSCT, genetic mutations in TP53, TET2, SF3B1, U2AF1, EZH2 were found to be markedly associated with OS (p < 0.05; Figure 4D-H).
Trisomy 8 was considered an intermediate risk factor. Conflicting data exist about the impact of trisomy 8 on OS of patients with MDS. Consistent with prior reports, 15,16,18,39 we found that patients with +8 had a markedly shorter OS in comparison with those who had NK (median survival 26.8 months vs. 47.5 months, p = 0.003). The analysis of Zoe et al. included 496 MDS patients with karyotypic abnormalities from the Victorian Cancer Registry and showed that +8 was identified in 93 (18.75%) patients and independently predicted shorter OS in a multivariate analysis (p = 0.024). 7 Haase et al. analyzed 2124 MDS patients at eight institutions from Australia and Germany and found that +8 correlated with worse OS only in the patients with CK, which is consistent with our results. Median OS of +8 was 22 months and 44 months as an isolated abnormality and together with other abnormalities excluding CK, respectively. 6 As heterogeneous prognosis exists in patients with +8, we categorized +8 abnormality into three groups (tri8, tri8 +1 , and tri8 +≥2 ). The OS of the tri8 +1 group was similar compared with that of tri8 (p = 0.84). The median OS of patients with tri8&tri8 +1 was 32.1 months, which is not significantly different from those with NK (p = 0.16). Whereas survival was inferior in patients with tri8 +≥2 vs tri8&tri8 +1 patients (p = 0.02). This finding was similar to the phenomenon observed in MDS with 5q-, which demonstrated that del(5q) with one additional abnormality except −7/del(7q) had the same biological characteristics as sole 5q-, but not as 5qwith two or more abnormalities. 1 Seventy to ninety percent of MDS patients displayed at least one genetic mutation surveyed according to the NGS. [20][21][22][23] In this study, 204 out of 425 patients (48%) had at least one mutated gene. Consistent with previous investigations, the study showed that mutated SF3B1 was an independent predictor for improved survival. TP53 mutations were associated with CK and poor prognosis. 28,40 Mutations in IDH1 and IDH2 were recognized as independent factors for leukemia transformation. We also found that DNMT3A mutations were more likely in patients with -Y, and NRAS mutations in 20q-. We identified 44.9% of MDS patients with +8 had at least one mutation, but no significant association was found between +8 and distinct genetic mutations in this cohort.
There were several limitations to this study. Firstly, in this single-center retrospective study, 427 (67.4%) patients had grown 20 metaphases under cytogenetic analysis, whereas the rest 207 (32.6%) had grown 3-19 metaphases, and FISH was undertaken as a compensatory method to identity cytogenetic abnormalities only in 74 patients. Secondly, due to technical limitation and historical background, even though 223 (52.5%) patients used NGS to detect mutations in 15 most common genes, there were also 202 (47.5%) patients who had Sanger's sequencing with a small panel of 6 genes.
Notwithstanding the limitations, our study analyzed 634 Chinese MDS patients and showed that trisomy 8 is the most common karyotypic abnormality among Chinese MDS patients. Patients with +8 showed a poor OS compared with those with NK. Sole +8 and +8 with one additional karyotypic abnormality had a similar OS with NK, whereas +8 with two or more abnormalities had a significantly shorter OS. DNMT3A mutations correlated with -Y and NRAS mutations correlated with 20q-. TP53 mutations were associated with CK and a poor OS, SF3B1 mutations were associated with a favorable OS. [41][42][43] IDH1 and IDH2 mutations independently indicated a shorter LFS. 44 This study showed that cytogenetic and molecular genetic abnormalities had a significant influence on the prognosis of MDS.

ETHICS APPROVAL
This article does not contain any studies with animals performed by any of the authors. All procedure performed in studies involving human participants were in accordance with the ethical standards of the institutional and national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. Exclusively 5q-in 5q-/-5, 20q-in 20q-/-20, and −7 in −7/7q-.