Polycomb Complex Group Gene Mutations and Their Prognostic Relevance In 5-Azacitidine Treated Myelodysplastic Syndrome Patients

Abstract 125

Polycomb gene complex mutations have been implicated in the pathogenesis of myeloid neoplasms. ASXL1, a epigenetic regulator of transcription by recruiting polycomb and trithorax complexes to the chromatin domain and EZH2 (histone methyltransferase) is the catalytic domain of polycomb repressive complex (PRC) 2, causing gene silencing by trimethylation of lysine 27 of histone 3(H3K27).

To identify the role of mutations in ASXL1, EZH2 and their interaction with other mutations (P53, ras, c-cbl, IDH 1/2, BRAF), we analysed 63 MDS patients treated with 5-Azacitidine (Aza) at our institution. Mutation analysis was done by deep (454 FLX) and Sanger sequencing. SNP-6 karyotyping was also performed to correlate with mutation status. In 43 patients, samples were also analysed at various time points post aza treatment.

The median age was 67 years (range 36–86 years), median number of courses 7(range 2–109), 90% (57/63) of patients belonged to high risk IPSS category. WHO category subtypes were; RA/RARS-1; RCMD-7; RAEB-37; s-AML (evolved from pre-existing MDS) -9, therapy related myeloid neoplasm (t-MDS/t-AML) -7 and CMML-2.IPSS cytogenetic subgroups were, good risk: 19, intermediate: 6, and poor risk: 38. Thirty six patients had chromosome 7 abnormalities either isolated (12/36) or with additional aberrations. The median time from diagnosis to treatment with aza was 7 months (range 1–42). The overall response rate to Aza was 48% (30/63) with a better survival in responders compared with non-responders (17.3 vs 10.2 months p<0.001).

ASXL1 exon 12 mutations were present in 22/63 patients (35%), good risk cytogenetics (8/19), intermediate risk (4/6), isolated monosomy 7 patients (7/12) but none in the complex cytogenetics subgroup (p<0.02). EZH2 mutations were present in 4 of 18 patients with normal cytogenetics and 4/12 patients with isolated chromosome 7 abnormality. Five patients had both EZH2 and ASXL1 mutations. PRC mutant cases (25/63) had a better overall survival (OS) and progression free survival (PFS) compared with wild type cases (38/63) (OS;20.8 vs 11.9, p<0.007 and PFS 17.7 vs 8.5, p<0.004). Seven of 8 patients with EZH2 mutations responded to Aza (p<0.01) with 3 patients achieving CCyR, 2 CR and 2 marrow CR. There was no correlation with response to treatment and presence of ASXL1 mutations. The c1934dupG p. G646WfsX12 the frequent mutation (7/23, 30%) was not found in constitutional DNA

P53 mutations were detected and quantified by high throughput sequencing in poor risk IPSS cytogenetic group (14/38) and were absent in patients with normal cytogenetics and isolated chromosome 7 abnormalities. EZH2 mutations were not detected in patients with P53 mutations and only 2/22 patients with ASXL1 had a concurrent P53 mutation, suggesting a possible mutual exclusivity of polycomb gene mutations and P53. All 14 patients with P53 mutation died (p<0.007) and 43% of patients with P53 mutations showed response to Aza. Sequential samples were available in 7 patients and changes in clone sizes were correlative of treatment response, with 3 patients achieving cytogenetic response with concurrent reduction in clone size. Two different mutations (double clones) of P53 was found in 6/14 mutant patients. The P53 mutation status correlated strongly with finding aberration of chromosome 17p by SNP array and positive P53 antibody staining on bone marrow trephine. The P53 mutant patients had a worse OS (8.9 vs 17.7 months, p<0.0001) and PFS (8.1 vs 12.7 months, p<0.006).

IDH1and IDH2, C-Cbl, NRAS mutations occurred in 6/63 (10%), 4/63 (6%), 6/63 (10%) of patients respectively and did not correlate with clinical outcome.

Age, IPSS cytogenetic risk group, WHO subtypes, overall response rate (ORR),progression to AML, P53 mutation status and presence of EZH2 and/or ASXLI mutation were used as co-variables in multivariate analysis.P53 mutation status (HR=5.3(2.3-11.8), p<0.0001) ORR (HR=0.27 (0.13-0.57), p<0.001) were established as independent prognostic variables influencing OS.

This study shows that polycomb complex gene mutations are frequent particularly in relatively good cytogenetic subgroups of MDS. The presence of these mutations correlates with a significantly better OS and PFS following Aza therapy. P53 mutations are uncommon in patients that harbour polycomb complex gene mutations and the presence of P53 mutations do not correlate with response rate, however they remain a strong indicator of OS and PFS.