When does a PNH clone have clinical significance?

Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired blood disease caused by somatic mutations in the phosphatidylinositol glycan class A (PIGA) gene required to produce glycophosphatidyl inositol (GPI) anchors. Although PNH cells are readily identified by flow cytometry due to their deficiency of GPI-anchored proteins, the assessment of the clinical significance of a PNH clone is more nuanced. The interpretation of results requires an understanding of PNH pathogenesis and its relationship to immune-mediated bone marrow failure. Only about one-third of patients with PNH clones have classical PNH disease with overt hemolysis, its associated symptoms, and the highly prothrombotic state characteristic of PNH. Patients with classical PNH benefit the most from complement inhibitors. In contrast, two-thirds of PNH clones occur in patients whose clinical presentation is that of bone marrow failure with few, if any, PNH-related symptoms. The clinical presentations are closely associated with PNH clone size. Although exceptions occur, bone marrow failure patients usually have smaller, subclinical PNH clones. This review addresses the common scenarios that arise in evaluating the clinical significance of PNH clones and provides practical guidelines for approaching a patient with a positive PNH result.

Is Nature Truly Healing Itself? Spontaneous Remissions and Clonal Replacement in Paroxysmal Nocturnal Hemoglobinuria

Paroxysmal nocturnal hemoglobinuria (PNH) is considered to be curable only through the means of allogeneic HSCT. One of the many fascinating and scientifically instructive aspects of the pathogenesis of this disease is the rare possibility of its spontaneous remission with disappearance of PNH clone and abatement of clinical symptoms, which has always captivated the research community. Due to the orphan nature of the condition, no clinical predictors have been identified so far as harbingers of this phenomenon. In a classical scenario, exhaustion of PNH clone may be associated with reappearance of aplastic anemia (AA), in which PNH clone reflects a semi-maladaptive attempt of recovery. Consequently, one could stipulate that the retraction of PNH clone(s) would have to be associated with a compensatory re-expansion of normal hematopoiesis should normal counts be maintained. The recent insights into the AA/PNH pathobiology shed light onto molecular underpinnings of polyclonal vs oligoclonal hematopoiesis and their dynamics. Here, through application of NGS we attempted to better discern the mechanism of PNH spontaneous remission taking advantage of our internal cohort of PNH patients. Among 92 patients with a diagnosis of hemolytic PNH (M:F ratio 0.88, median age 38 years, range 9-84) 41% were primary PNH (pPNH) while 59% were secondary to AA (sPNH). Overall, patients were clinically followed-up for a median time of 68 months (2-339). Median granulocyte clone size was 73% (22-99) with the majority of cases being classified as having a type III dominant red blood cells (RBCs) clone (80%) while 20% type II. Within this cohort, a total of 3 patients underwent spontaneous remission. UPN1 was a 69-year-old male diagnosed with pPNH at the age of 46 after an episode of deep vein thrombosis. He had been managed with prednisone, transfusions and anti-coagulation because of recurrent thrombotic episodes. Once available, he was started on eculizumab and later continued on ravulizumab. His initial flow cytometry study revealed the presence of a type III RBCs clone of 40% and a granulocyte clone of 89%. After 9 years of anti-complement therapy, the patient's clone started a slow decrease and the most recent study revealed a granulocyte clone of 0.02%. Molecular analysis performed at the time of eculizumab start showed a co-dominant mutational configuration by variant allelic frequency (VAF) with PIGA deletion (p.94_95del; VAF 29%) and a BCOR nonsense (p.Y1446X; VAF 27%). No HLA class I/II mutations were found in two longitudinal samples collected 1 year before and after eculizumab start. However, at the last sequencing performed after the complete disappearance of the PNH clone, the patient developed ASXL1 (p.E635Rfs*, VAF 26%) and ZRSR2 (p.E120Gfs*, VAF 42%) mutations along with retraction of the previous PIGA lesion. No decrease in blood counts was noted. UPN2 was a 58-year-old male initially diagnosed with severe AA at the age of 48 and treated with ATG + CsA. At that time, he had a co-existing PNH granulocyte clone of 28%. After 1 year from IST his PNH clone dropped to 1% and since then has been consistently below 1%. Patient has never received anti-complement therapy. At the time of PNH clone retraction, no HLA class I/II or myeloid driver mutations were found and PIGA mutations were not detectable. However, longitudinal molecular studies performed after disappearance of PNH clone revealed the acquisition of ASXL1 p.Q512X mutation at an initial VAF of 23%, which doubled (45%) at last follow-up 5 years later while normal counts persisted. UPN3 was instead a 59-year-old lady diagnosed with pPNH at the age of 30. She had a granulocyte clone as high as 43% with a type II RBCs clone of 17% and a typical PIGA splice site c.981+1G>A mutation (VAF 15%). She was initially treated with transfusions and steroids and her course was complicated by a cerebral venous sinus thrombosis. Patient was eventually given eculizumab and her PNH clone started decreasing until it vanished (last 0.04%) after 8 years. Analysis of samples prior to and after PNH disappearance did not show any HLA class I/II nor myeloid driver gene mutations with absence of PIGA alterations at last sequencing. PNH spontaneous remissions are rare events. In addition to be replaced by polyclonal hematopoiesis, PIGA clones may be swept by CHIP lesions in myeloid genes (e.g. ASXL1) characterized by improved fitness advantage in a process of Darwinian selection. Figure 1 Figure 1. Disclosures Maciejewski: Regeneron: Consultancy; Novartis: Consultancy; Bristol Myers Squibb/Celgene: Consultancy; Alexion: Consultancy.

Multipotent Mesenchymal Stromal Cells from the Bone Marrow of Untreated Aplastic Anemia Patients Preserve Their Ability to Support Hematopoietic Precursors However Display the Pronounced Changes in Gene Expression

Aplastic anemia (AA) is believed to be an autoimmune disorder characterized by the pancytopenia due to the depletion of hematopoietic stem and progenitor cells in the bone marrow. There are three forms of AA depending on the severity of pancytopenia: moderate, or non-severe AA (NAA), severe AA (SAA), and very severe AA (VSAA). Clones of cells typical for paroxysmal nocturnal hemoglobinuria (PNH-clones) are frequently present in AA patients in various proportions. We aimed to study stromal microenvironment of untreated AA patients depending on the AA severity, presence or absence of PNH-clone, and on the response to the therapy after 3 and 6 months of treatment. We analyzed the bone marrow (BM) multipotent mesenchymal stromal cells (MMSCs) in their ability to maintain hematopoietic precursors and examined relative expression levels (REL) of selected genes. The study included 17 patients with NAA (53% females, 47% males, 33.8±2.2 years old), 12 patients with SAA (33% females, 67% males, 29.3±3.8 years old). Among NAA patients 7 had PNH-clone, and among SAA - 6 patients. Control group consisted of 19 donors (42% females, 58% males, 30.4±3.1 years old). The ability to support hematopoietic precursors by MMSCs from the BM of AA patients was measured by cobble stone area forming cells (CAFC) assay, where BM cells from one healthy donor were seeded on different MMSCs; REL of selected genes was analyzed with TaqMan RT-PCR. Only the genes with statistically significant differences are presented. The data are presented as mean ± standard error of measures, the differences were statistically significant when p<0.05 when Student's unpaired t-test or Mann-Whitney test was applied. MMSCs from AA patients preserve their ability to maintain hematopoietic precursors. CAFC 7 frequency reflects the number of late hematopoietic precursors. CAFC 7 frequency was slightly higher on MMSCs from NAA patients (9.92±2.73 per 10 6 healthy BM cells) then on MMSCs from healthy donors (5.56±1.14 per 10 6 healthy BM cells), although the difference was not statistically significant. MMSCs from SAA patients maintained CAFC 7 as well as donors' MMSCs (6.75±1.96 per 10 6 healthy BM cells). The frequency of CAFC 28, reflecting the number of early hematopoietic precursor, displayed similar but more pronounced dynamics. CAFC 28 frequency on NAA patients' MMSCs was significantly higher than on donors' ones (2.17±0.34 versus 1.11±0.31 per 10 6 healthy BM cells, p=0.03), while on SAA patients' MMSCs it was also high (1.92±0.57 per 10 6 healthy BM cells) but the difference was insignificant (Table 1). The presence of PNH-clone does not affect the ability of stromal cells to maintain hematopoiesis. MMSCs from the patients that had responded to the therapy in 90 or 180 days did not differ in their ability to maintain hematopoietic precursors from the MMSCs of treatment resistant the patients. Therefore, we can assume that physiological function of stromal microenvironment is not affected deeply in the debut of AA. Gene expression analysis revealed statistically significant upregulation of FGFR1, PDGFRA, VEGFA and downregulation of ANG1 (in MMSCs from both NAA and SAA patients), and upregulation of FGFR2 and CFH (only in NAA patients' MMSCs) (Table 2). In MMSCs of AA patients (both NAA and SAA) without PNH-clone the upregulation of CFH gene was detected (Table 3). CFH is one of the players in the complement system which is disrupted in PNH. This fact needs to be further scrutinized. In addition, IL1R, SDF1 and VEGFA were statistically significantly downregulated in MMSCs from AA patients with PNH-clone compared with MMSCs from patients without PNH-clone. It seems that the presence of PNH-clone corresponds with the changes in stromal microenvironment. Gene expression of analyzed genes was the same in MMSCs of the patients that had responded or not responded to the treatment in 90 or 180 days since the therapy begun. Thus, MMSCs from the BM of untreated AA patients preserve their ability to support hematopoietic precursors however display the pronounced changes in gene expression. The work is supported by the RFBR, project 19-015-00280. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Incidential Findings of Mutations in the PIGA Gene Are Highly Specific for the Presence of PNH Clones

Background: Paroxysmal nocturnal hemoglobinuria (PNH) is a hemolytic anemia associated with severe thrombophilia and characterized by complement-mediated lysis of erythrocytes lacking glycosylphosphatidylinositol (GPI)-anchored proteins. In the majority of cases, GPI deficiency is caused by somatic mutations in the PIGA gene. Presence of PNH clones is associated with acquired aplastic anemia (AA) and can be found in patients with myelodysplastic syndrome (MDS) or rarely other myeloid neoplasms (MN). Flow cytometric analysis for deficiency of GPI-anchored proteins on multiple cell lineages detects PNH clones, and PIGA mutational analysis is not mandatory to establish the diagnosis. In contrast, molecular genetic analysis of targeted gene panels is widely used in the diagnostic workup of MN. We hypothesized that the inclusion of PIGA into the myeloid gene panel could identify obscure cases with PNH clones irrespective of the initial clinical suspicion. Aim: To assess the significance of incidental findings of mutations in PIGA in the diagnostic workup of MN. Methods: 20,320 consecutive patients undergoing sequencing analysis for a confirmed or suspected MN were analyzed for the presence of mutations in the PIGA gene. Patients with confirmed PNH analyzed only for PIGA were not included, and cases with previously known PIGA mutations were excluded from further analysis. DNA was isolated from peripheral blood (PB) or bone marrow, and sequencing was performed on NovaSeq after Illumina DNA Prep for Enrichment library preparation (Illumina, San Diego, CA) and hybrid capture of a 41 gene panel including the complete coding sequence of PIGA (IDT Inc., Coralville, IA); data was analyzed with Pisces and Pindel (BaseSpace, Illumina). Flow cytometry was performed on granulocytes, monocytes, and erythrocytes in PB using antibodies against GPI-anchored proteins (CD14, CD24, CD55, and CD59), fluorescein-labeled proaerolysin (FLAER) staining, and Navios cytometers; analysis was done using Kaluza software (both Beckman Coulter, Miami, FL). Results: PIGA mutations were newly identified in 67 patients (0.3%) undergoing targeted sequencing within the diagnostic workup of MN. 30 patients were excluded from further analysis as the gene panel had been requested for a MN associated with previously diagnosed PNH. From the remaining patients, PB for flow cytometry analysis could be obtained from 20 patients. Flow cytometry confirmed the presence of a PNH clone in 17 (85%) of these patients (median clone size: 41% for granulocytes, 54.5% for monocytes, and 12% for erythrocytes). In 3 patients (15%) with unexpected PIGA mutations, flow cytometry detected no PNH clone. The type of PIGA mutations differed significantly in those cases: Patients in whom a PNH clone was confirmed, showed protein-truncating frame-shift (41%) or nonsense (6%) mutations, splice site mutations (18%), or multiple mutations (35%) including at least one protein-truncating mutation at a median variant allele frequency (VAF) of 15.4% (range 2.0% to 50.1%). In contrast, patients without a PNH clone showed only singular missense mutations of PIGA with a VAF of 3.6% to 5.3% (Figure 1). Final diagnoses in patients with confirmed clones were sole PNH (n=9), or PNH clone associated with MDS (n=4), AA (n=3), and AML (n=1), and additional mutations in other genes were observed in 9 cases. While the initial clinical presentation included the differential diagnosis of PNH in some of the patients, flow cytometry was requested as a direct result of the PIGA mutation in 4 cases with an accompanying MN and in 3 patients without - the later showing a median latency of 6.5 years from the initial clinical presentation to the diagnosis. Con clusions: The inclusion of PIGA into a standardized targeted sequencing panel for MN helps to identify patients with PNH clones irrespective of the initial clinical suspicion but is not sufficient to rule out PNH. Protein-truncating PIGA mutations are highly specific for PNH clones whereas singular missense mutations may not necessarily effect GPI biosynthesis. Our data indicate that the incidental finding of a PIGA mutation in sequencing analysis shall entail flow cytometry of GPI-anchored proteins in PB. The potential clinical sequelae and the availability of specific treatment options such as complement inhibitors warrant the thorough exclusion of PNH in the diagnostic workup of suspected MN. Figure 1 Figure 1. Disclosures Hoermann: Novartis: Honoraria. Haferlach: MLL Munich Leukemia Laboratory: Other: Part ownership. Haferlach: MLL Munich Leukemia Laboratory: Other: Part ownership. Kern: MLL Munich Leukemia Laboratory: Other: Part ownership.

Long Non-Coding RNA FAM157C Contributed to Clonal Proliferation in Paroxysmal Nocturnal Hemoglobinuria

Background: Paroxysmal nocturnal hemoglobinuria (PNH) is a rare clonal disease of hematopoietic stem cells. However, the mechanism of proliferative advantage of PNH clone is unclear. Long noncoding RNAs (LncRNAs) have a wide range of biological functions, including regulation of gene expression, cell differentiation, and proliferation, while its role in PNH remains unclear. Methods: In our study, CD59－and CD59+ granulocytes and monocytes from 5 PNH patients were sorted, and LncRNAs and mRNAs were detected by RNA sequencing. The proliferation-related NF-κB pathway was focused on. A total of 8 mRNAs and 5 LncRNAs were verified by qRT-PCR, and analyzed the correlation with clinical data. Meanwhile, the function of LncRNA was studied.Results: LncRNA FAM157C were verified to be upregulated in PNH clone cells, which were positively correlated with LDH level and CD59- granulated and monocytes cells ratio. After knockdown of FAM157C gene in PIGA-KO-THP-1 cell line, we found that the cells were blocked in G0/G1 phase and S phase, and the apoptosis rate increased, while the proliferation ability decreased. Conclusions: LncRNA FAM157C was proved to promote PNH clone proliferation, which is the first time to explore the role of LncRNAs in PNH.

Ultradeep Sequencing Analysis of Paroxysmal Nocturnal Hemoglobinuria Clones Detected by Flow Cytometry: PIG Mutation in Small PNH Clones

Objectives We aimed to determine whether small paroxysmal nocturnal hemoglobinuria (PNH) clones detected by flow cytometry (FCM) harbor PIG gene mutations with quantitative correlation. Methods We analyzed 89 specimens from 63 patients whose PNH clone size was ≥0.1% by FCM. We performed ultradeep sequencing for the PIGA, PIGM, PIGT, and PIGX genes in these specimens. Results A strong positive correlation between PNH clone size by FCM and variant allele frequency (VAF) of PIG gene mutation was identified (RBCs: r = 0.77, P < .001; granulocytes: r = 0.68, P < .001). Granulocyte clone size of 2.5% or greater and RBCs 0.4% or greater by FCM always harbored PIG gene mutations. Meanwhile, in patients with clone sizes of less than 2.5% in granulocytes or less than 0.4% in RBCs, PIG gene mutations were present in only 15.9% and 12.2% of cases, respectively. In addition, there was not a statistically significant positive correlation between FCM clone size and VAF or the presence or absence of a PIG mutation. Conclusions Our results showed that in small PNH clones PIG gene mutations were present in only a small portion without significant correlation to VAF or the presence or absence of a PIG mutation.

A case report: paroxysmal nocturnal hemoglobinuria and systemic lupus erythematosus association

Paroxysmal nocturnal hemoglobinuria (PNH) is defined by acquired intravascular hemolytic anemia, thrombosis and bone marrow failure with pancytopenia. Systemic lupus erythematosus (SLE) also appears as an autoimmune disease. The coexistence of both is rarely reported. Here we report the case of a 30-year-old female presenting with pancytopenia and diagnosed as SLE, who also had a PNH clone. Bone marrow biopsy did not support hypoplastic anemia. The patient was then followed up with the consideration of the existence of a PNH clone with SLE. She was treated by the rheumatology department and complete blood count improved under immunosuppressive treatment. The coexistence of CD59–CD55 deficiency with autoimmune diseases has been reported. It is an important example in terms of receiving clinical response with SLE-specific treatment.

Immunogenomics of Aplastic Anemia: The Role of HLA Somatic Mutations and the HLA Evolutionary Divergence

Downregulation of class I human leukocyte antigen (HLA)-restricted antigen presentation has been identified as mechanism of immune-escape in many malignant and non-malignant disorders. In idiopathic aplastic anemia (AA), evolution of immune-privileged paroxysmal nocturnal hemoglobinuria (PNH) clones has been attributed to immune escape due to deficiency of GPI-anchored protein in the context of T-cell mediated autoimmunity. However, other mechanisms of clonal selection may also operate with or independently of PNH. Our group first described the presence of both somatic uniparental disomy (UPD) and microdeletions of the HLA region leading to loss of heterozygozity (LOH) and/or haploinsuffciency.1 Later the proof-of-concept of somatic mutations in HLA class I was provided.2 Mechanistically, HLA LOH leads to loss of an allele involved in the presentation of immune-dominant peptides, while haploinsufficiency may decrease the presentation threshold. Moreover, the general level of individual structural diversity of HLA molecules may determine the ability to present diverse targets, eventually derived from auto-antigens, and functionally would operate in the opposite direction to HLA LOH. In this scenario, we hypothesize that defects in both class I and II HLA loci may constitute different patterns of immune escape, reducing respectively CD8+ and CD4+ related activation and thus contributing to rescue hematopoietic stem cells from the immune attack. Furthermore, our idea is that the immune-escape environment may be related to the grade of HLA evolutionary divergence (HED), a metric that, accounting for the degree of structural diversity within a particular locus, represents an indirect measure of the antigenic landscape that the hematopoietic target cell is able to present (see abstract #:142693). Using a deep targeted HLA NGS panel and a newly developed in-house bioinformatic pipeline (characterized by stringent criteria for alignment, preprocessing and variant calling in the HLA region, based on the IPD IMGT/HLA database, Fig.A), we studied a large cohort of patients with idiopathic bone marrow failures (AA n=75, AA/MDS=10). In addition, we determined the impact of inter-loci HED on the probability to acquire somatic hits in HLA genes. Overall, 29 somatic HLA mutations were found in 16 patients (18%) at a median VAF of 11% (range: 2-93%):12 in class I (41%) and 17 in class II (59%), with 5 patients carrying mutations in both classes (Fig.B, C, D). The majority of those events (N=21, 72%) occurred in subjects also harbouring a PNH clone of small size (12 out 16 patients, median PNH clone size 1% [range:1-46%]). Most mutated loci were A and C for class I and DQB1 for class II (Fig. C, D); 9 mutations were identified as missense, with disruptive changes, 7 were intronic indels while 13 hits were localized in 5' or 3' untranslated regions (UTRs) (Fig.E, F). Through a computational prediction of the HLA regulatory domains involved in the UTR aberrations, we identified domains essential for the binding of GATA-1, RXRbeta, SP-1 and NFKB. The impairment of those regions may affect the transcription of HLA complexes. AA HLA mutant cases had more frequently a severe disease at diagnosis (severe AA: 81% vs. 60%, respectively in HLA mutated vs non mutated cases) and were in most part responders to immunosuppressive therapy (complete/partial responses: 75% vs 50% in HLA mutated vs non mutated patients). Within the AA/MDS group instead HLA mutations were found in 4 out of 10 patients (40%), including of note three -7/del7q cases. Using Pierini and Lenz algorithm3 to determine inter-class HED, we found that HLA mutations tended to occur more often in patients with a high inter-class mean HED (94% vs 72% in non mutated group, p=.001, Fig. G), consistent with the idea that higher structural diversity of HLA molecules may induce more pervasive auto-immune responses, stronger immune pressure and ultimately the establishment of immune-escape mechanisms. In summary, our results indicate the importance of class-I and -II HLA loci somatic hits as markers of autoimmunity and thereby the severity of the immune selection pressure, configuring possibly alternative mechanisms of immune-escape, in addition to immune privileged PNH clones. This environment may ultimately facilitate leukemic clonal expansion in AA-MDS setting. Disclosures Patel: Alexion: Other: educational speaker. Peffault De Latour:Apellis: Membership on an entity's Board of Directors or advisory committees; Alexion Pharmaceuticals Inc.: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Pfizer: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Amgen: Research Funding. Maciejewski:Novartis, Roche: Consultancy, Honoraria; Alexion, BMS: Speakers Bureau.