Immunogenetic, Molecular and Clinical Determinants of Clonal Evolution in Aplastic Anemia and Paroxysmal Nocturnal Hemoglobinuria

Despite therapeutic successes, AA patients (pts) exhibit a much higher risk of leukemic evolution than the general population. Secondary myeloid neoplasia (sMN) remains the most serious AA complication with major therapeutic and prognostic implications. Historically, multiple theories have been proposed as to the origin of leukemic evolution. For instance, sMN may be the consequence of a relentless autoimmune attack producing a maladaptive response to immunosuppression (IST). Alternatively, occurrence of leukemogenic drivers may be an event setting in motion initially successful and overshooting tumor surveillance reactions. Finally, sMN pathogenesis may be related to either CHIP evolution or acceleration of its progression. Each of these theories is supported by clinical-molecular features (e.g. HLA mutations, secondary PNH evolution, somatic mutations at presentation etc.). Here, we took advantage of a multicentric cohort of pts with AA (n=1010; to our knowledge the largest yet explored) and primary MN (n=3599) to define immunogenetic, iatrogenic and molecular determinants of MN progression.

Among AA pts (M:F ratio 0.98; median age 34 years, IQR 20-54, median follow-up 89 months), the 5 and 10 years cumulative incidences of sMN were 6% and 11% respectively, with a median time to progression of 56 months (IQR 23-96). Analysis of available data showed that younger pts (<40 years, HR=0.27 [95%CI 0.1-0.5] p<.001), IST responders (HR=0.36 [95%CI 0.1-0.8] p=.01) and moderate AA (HR=0.33 [95%CI 0.1-0.8] p=.02) had lower risk of malignant evolution. MDS was the most frequent diagnosis at progression (80%), followed by AML (17%, of which 71% MRC subtype) and MPN (3%). Whereas only 27% of MDS pts were classified as EB-1/2, high-risk R-IPSS scores (>3.5) were observed in 59% (vs 43% in pMDS, p=.02) due to the enrichment in poor/very poor cytogenetic risk groups. In particular, chr.7 abnormalities were the most frequent (54%, of which 88% were del7). By comparison, del7/7q was present in 8% of pMN cases (p<.001). Among treated pts, chemotherapy was administered to 37%, HMA to 63% and, overall 36% received HSCT. Disease progression was the main cause of death (42%). When compared to pMN, sMN had poorer survival outcomes (p=.01) especially among del7/7q carriers (p=.004).

At the time of AA onset only 18% of pts harbored somatic myeloid mutations with their presence/absence not influencing evolution, whereas mutations were found in 81% of sMN (1.7 mutations/patient, n=86/101). No difference in mutational burden was observed according to presence/absence of del7/7q, which constituted the founder lesion in 60% of cases, when the analysis was possible. ASXL1 (24% vs 14%, p=.01), RUNX1 (21% vs 11%, p=.008), SETBP1 (14% vs 3%, p<.001) and U2AF1 (13% vs 6%, p=.01) mutations were more frequent in sMN, while TET2 (8% vs 26%, p<.001) and SF3B1 (1% vs 12%, p<.001) were less common as compared to pMN. Del7/7q pts were enriched in SETBP1 (22% vs 4% in pMN, p<.001), ASXL1 (29% vs 12%, p=.007) and RUNX1 (29% vs 12%, p=.003) lesions while TP53 mutations were by far less common (5% vs 31%, p<.001).Remarkably, CUX1 hits at AA onset heralded malignant progression (p<.001) and longitudinal analysis showed their loss in patients who eventually acquired del7/7q. In aggregate, the low CUX1 expression in 70% of primary del7/7q MDS and its function in DNA repair, may argue for a role of CUX1/chr.7 during AA to MN progression. ¹ When we studied immune-selected somatic events, PIGA mutations were the most frequent lesions at AA onset (33%). However, only 5% of cases at MN evolution (p<.001) had PNH clones, consistent with a reciprocal expansion of PNH clones/evolution to secondary PNH in non-progressors, and clonal sweeping in sMN. HLA class I/II mutations or loss were instead identified at a similar rate in AA and sMN (~27%) pts. No HLA alleles were identified as harbingers of malignant evolution, which instead associated with a lower HLA class II evolutionary divergence (HR=2 [95%CI 1-4] p=.03) possibly hampering efficacious surveillance responses. ²

AA malignant evolution is characterized by an orchestra of molecular events with an invariant genomic signature (e.g. CUX1, SETBP1, ASXL1). Immunogenetic and immune escape mechanisms may also play a role in shaping the fate of individual patients' trajectories towards PNH vs sMN progression, which may be considered a maladaptive escape event resulting from a bottlenecked hematopoiesis.