Extent and Clinical Implications of Subclonal Diversity in Paroxysmal Nocturnal Hemoglobinuria

Paroxysmal Nocturnal hemoglobinuria (PNH) has been traditionally considered a monogenic disease due to somatic mutations in PIGA gene. While selective immune pressure was implicated in PNH evolution via expansion of a PIGA mutant clone in a privileged environment, history of aplastic anemia (AA) is not always present in a manifest hemolytic PNH. Similarly, expansion of PNH clone after AA therapy does not seem to correlate with the strength of immunosuppression and the quality of response. Our initial studies (J.Clin.Invest.2014; 124 :4529-4538) indicated that additional mutational events may be present in PNH and act as intrinsic factors modifying clinical features and leading to a differential expansion. We hypothesize that subclonal hits may be present in some patients with PNH to explain their expansion dynamics and clinical features. To that end we have collected 319 patients with bone marrow failure of whom 202 had PNH clone including 70 hemolytic PNH cases. For a subgroup of these patients (n=197), deep multi-targeted NGS has been performed to identify mutations in 78 myeloid genes of which 38 genes were found to be mutated (≥1) in this PNH spectrum disease. 116 serial samples for 33 patients were also sequenced. As a control group, we have included AA stably negative for PNH clone (n=113). For analytic purposes, on clinical grounds we have sub grouped patients with PNH clone into AA/PNH (31%) defined as patients with presence of bone marrow failure but no clinical or laboratory evidence of hemolysis and WBC PNH clone <20%, and PNH group (69%). The latter (69%) consisted of patients with primary hemolytic PNH (pPNH; 60%) or secondary PNH (sPNH;40%). sPNH is defined as a group showing hemolysis with an antecedent history of AA and clone size >20%.

Our analysis focused on driver mutations. In cross-sectional analysis, in addition to PIGA we have identified somatic hits in 10/26 (38%) of AA/PNH pts. (On average 2.1 hits/per positive patient) compared to 27/58(47%) of PNH patients (on average 1.22 hits per positive patients. Subdividing PNH patients, 20/35(57%) had a mutation in primary PNH compared to 7/23(30%) in the sPNH group. In contrast, 42/113(37%) patients had a mutation in the control AA group with a (2.1 mutations per positive patient). The mean VAF for AA is 29% compared to 23% in AA/PNH vs. 34% in PNH vs. 31%in sPNH.

In all patients with PNH clone, 44% had a mutation vs. 37% in the AA group. AA/PNH subcohort contained hits in cohesins genes (RAD21-SMC3-STAG2 (8 %), ZRSR2 (8%), BCORs (4%) and TET2 (4%). PNH group had mutations in BCOR/BCORL1 (BCORs) (11%), cohesions, ZRSR2 and TET2 (6% each). In sPNH, a similar trend was observed with the most common hits in EZH2-SUZ12 (9%), ZRSR2 (9%), and BCORs, TET2, LUC7L2 (4% each). In contrast, AA without PNH, the 5 most frequently mutated genes were TET2, ASXL1, EZH2-SUZ12 AND BCORs (5% each) and RUNX 1 (4%). BCORs mutations were more frequent in PNH group compared to AA, (11% vs. 5%). In summary, BCOR/L and ZRSR genes showed more mutations in PNH clone cohort while AA cohort did not show any dominant hits.

To determine whether somatic hits observed cross-sectionally led to establishment of stable subclones or were merely transient, we performed a longitudinal analysis. In PNH, 3/4 clones were transient which included EZH2, PHF6 and STAG2. In AA/PNH patients, 9/14 mutational hits appeared in the course of their disease (TET2, CEBPA, CUX1, STAG2, BCOR, SMC3) but 10/14 faded away in subsequent course and thus only 4 were stable clones. In AA cohort, 32 additional mutational hits were noted in 77 serial samples and 34 mutations vanished in subsequent course.

In sum, our results show that in PNH, similar to AA, additional somatic hits are common and involve some of the commonly mutated myeloid genes seen in MDS. While likely, many of these subclonal events are transient, seemingly lacking the proper ancestral context, some of the hits resulted in significant expansions (sweeping mutations) and thus may modify the clinical course including speed of the PNH clone evolution.