Smaller Clone Size Research Articles

Chromosome Banding and SNP-Array Analysis Are Complementary Methods to Characterize Karyotype Complexity in High-Risk Chronic Lymphocytic Leukemia

Introduction: Karyotype complexity is of prognostic significance in chronic lymphocytic leukemia (CLL). It can either be assessed by chromosome banding analysis (CBA) or by chromosome microarray analysis (CMA). How to integrate both methods into CLL diagnostic procedures is not defined. Material and Methods: To compare both methods in their ability to detect chromosomal aberrations, samples from 110 patients enrolled on the CLL2O trial (NCT01392079) were analyzed in parallel by Affymetrix ® 6.0 single nucleotide polymorphism arrays (SNP-arrays, CMA) and CBA (CpG oligonucleotide and IL-2 stimulation). The trial included genetically complex high-risk CLL cases defined by TP53 loss (treatment-naïve or relapsed) or refractoriness to purine-analogue based treatment. Intraindividual reference DNA for paired CMA was available for 79 samples. CMA-defined gains and losses outside known structural variations were counted as aberration irrespective of their size. Discontinuous gains and losses were counted as one aberration whenever segments had a similar log2-ratio. Results: CBA identified 561 chromosomal aberrations with a median of 4 aberrations per case (range: 0-21). With 653 DNA copy number alterations, CMA identified more aberrations partly afforded by a high number of unbalanced translocations (median: 5 aberrations per case, range: 0-19). With regards to karyotype complexity (KC), CBA and CMA allocated 67/110 cases (61%) to comparable risk groups as defined by Baliakas et al. (2019) and Leeksma et al. (2021). CMA assigned more cases to higher complexity risk groups than CBA, which was not observed when excluding gains and losses <5Mb not previously deemed to be significant. However, this approach did not improve the congruency in KC risk group stratification between both methods (see Table). Only 14/110 cases (13%) had a complete overlap in the chromosomes found to be affected by CBA and CMA. Overall, CBA discovered more additionally affected chromosomes than CMA (p=0.0104). Considering all localizable breakpoints of the CBA-defined structural chromosomal changes (N=922), 532 coincided with a cytoband or a cytoband directly adjacent to one harbouring CMA-defined copy number alterations (N=299 and N=233, respectively), whereas 128 CBA-defined breakpoints located to other cytobands on the same chromosome arm (N=85) or on the other arm (N=43). For 50 of these 128 breakpoints the distance between the CBA-identified cytoband and the CMA-defined breakpoint was <20 Mb. About one quarter of CBA-defined breakpoints (N=237, 23%) affected chromosomes not found to be altered in CMA. Potential explanations for non-detection by CMA were small clone sizes (78/237, 33%; considering breakpoints found in <25% of metaphases), association with an ultra-complex karyotype (140/237, 59%; referring to cases with ≥10 affected chromosomes and/or a composite karyotype), and no or poor coverage of cytobands by SNP-array probes (72/237, 30%; referring to cytobands with stretches >1Mb covered by ≤5 probes). Besides, the vast majority of the 237 breakpoints belonged to alterations that can in principle be balanced: 209 translocations, 8 insertions and 2 inversions. Only 18/237 breakpoints defined deletions, 15 of which met at least one of the criteria mentioned above as potential explanations for non-detection by CMA. Overall, 134 chromosomal alterations were detected by CMA alone. Four of these alterations were copy neutral losses of heterozygosity. Most of the 130 DNA copy number alterations were <20 Mb in size (N=104) and 22/130 alterations have previously been associated with a shorter overall survival (affecting 18/110 cases; 16%). Regarding loss of CDKN2A/ B and gain of MYC as genetic features associated with an increased risk for Richter transformation, CMA identified three cases each lacking abnormalities on the respective chromosome in CBA. CBA identified three cases with a potential MYC rearrangement and one case with a potential CDKN2A/ B loss despite having normal results for 9p21 in CMA. Conclusions: In this cohort of high-risk CLL cases, CBA and CMA were complementary methods for karyotype characterization. CBA was more sensitive in uncovering chromosome involvement than CMA, whereas CMA was superior in determining specific DNA copy number alterations previously associated with an inferior prognosis. As to what extend these findings apply to standard-risk CLL cases needs to be investigated.

Clonal hematopoiesis has prognostic value in dilated cardiomyopathy independent of age and clone size

Abstract Background Clonal hematopoiesis (CH) gives rise to mutated leukocyte clones, that may induce cardiovascular inflammation, and thereby impact the disease course in cardiovascular diseases such as atherosclerosis and ischemic heart failure. The term clonal hematopoiesis of indeterminate potential (CHIP) is used when the variant allele frequency (VAF; a marker for clone size) in blood is ≥2%. The impact of CH clones in non-ischemic dilated cardiomyopathy (DCM) remains largely undetermined. Moreover, the impact of small clone sizes (VAF <0.5%) remains unknown in cardiovascular diseases in general. Objectives To establish the prognostic impact of CH in DCM down to small CH clone sizes. Methods CH was determined using an ultrasensitive single-molecule Molecular Inversion Probe technique that allows detection of clones down to a VAF of 0.01%. Cardiac death and all-cause mortality were predicted using receiver operating characteristic curve-optimized VAF cut-off values. Results 520 DCM patients were included. 109 patients (21%) had CH driver mutations, of which 45 had a VAF of ≥2% and 31 had a VAF of <0.5%. The median follow-up duration was 6.49 years [interquartile range 4.69 - 9.71]. DCM patients with CH had a higher risk of cardiac death (HR 2.33 using a VAF cut-off of 0.36%, 95% confidence interval 1.24 - 4.40) and all-cause mortality(HR 1.72 using a VAF cut-off of 0.06%, 95% confidence interval 1.10 - 2.69), independent of age, sex, left ventricle ejection fraction and New York Heart Association classification. Conclusion CH predicts cardiac death and all-cause mortality in DCM patients with an optimal threshold for clone size of 0.36% and 0.06%, respectively. Therefore, CH may be prognostically relevant independent of clone size in patients with DCM.Clonal hematopoiesis characteristicsClonal hematopoiesis prognosis

Clonal Hematopoiesis Has Prognostic Value in Dilated Cardiomyopathy Independent of Age and Clone Size

BackgroundClonal hematopoiesis (CH) gives rise to mutated leukocyte clones that induce cardiovascular inflammation and thereby impact the disease course in atherosclerosis and ischemic heart failure. CH of indeterminate potential refers to a variant allele frequency (VAF) (a marker for clone size) in blood of ≥2%. The impact of CH clones—including small clone sizes (VAF <0.5%)—in nonischemic dilated cardiomyopathy (DCM) remains largely undetermined. ObjectivesThe authors sought to establish the prognostic impact of CH in DCM including small clones. MethodsCH is determined using an ultrasensitive single-molecule molecular inversion probe technique that allows detection of clones down to a VAF of 0.01%. Cardiac death and all-cause mortality were analyzed using receiver-operating characteristic curve–optimized VAF cutoff values. ResultsA total of 520 DCM patients have been included. A total of 109 patients (21%) had CH driver mutations, of which 45 had a VAF of ≥2% and 31 <0.5%. The median follow-up duration was 6.5 years (IQR: 4.7-9.7 years). DCM patients with CH have a higher risk of cardiac death (HR: 2.33 using a VAF cutoff of 0.36%, 95% CI: 1.24-4.40) and all-cause mortality (HR: 1.72 using a VAF cutoff of 0.06%, 95% CI: 1.10-2.69), independent of age, sex, left ventricular ejection fraction, and NYHA functional classification. ConclusionsCH predicts cardiac death and all-cause mortality in DCM patients with optimal thresholds for clone size of 0.36% and 0.06%, respectively. Therefore, CH is prognostically relevant, independent of clone size in patients with DCM.

Mutational Landscape, Clonal Evolution and Prognostic Effect of Non-Driver Mutations during Disease Progression of Primary Myelofibrosis

Introduction: Primary myelofibrosis(PMF), including pre-fibrotic myelofibrosis(Pre-PMF) and overt-fibrotic myelofibrosis(Overt-PMF), may finally transform to the terminal stage, which was defined as primary myelofibrosis-accelerate phase/blast phase(PMF-AP/BP).In the past decade, high-throughput next-generation sequencing(NGS) technologies have been applied to identify a large range of genetic mutations in PMF. However, little is known about the difference in mutational landscape and enrichment of non-driver mutations between Pre-PMF, Overt-PMF and PMF-AP/BP patients, as well as the clonal evolution during disease progression of PMF. Therefore, we aimed to clarify the mutational landscape between Pre-PMF, Overt-PMF and PMF-AP/BP patients as well as the clonal evolution and prognostic effect of non-driver mutations during disease progression. Methods: This retrospective cohort included 69 Pre-PMF patients,145 Overt-PMF patients and 45 PMF-AP/BP patients from the Institute of Hematology and Blood Diseases Hospital in China from 2015 to 2021. The targeted panel that covered all exons of 137 genes involved in myeloid malignancies or previously described in MPN was used in patients to detect mutations. X-tile software was used to define a cut-off size of variant allele fractions (VAF) of mutation. ASXL1 mutation VAF≤7.5% was defined as a smaller clone size, while VAF>7.5% was defined as a larger clone size. Results: A total of 259 patients with Pre-PMF, Overt-PMF and PMF-AP/BP were included in this analysis. Driver mutation distribution was JAK2V617F 55.6%, CALRExon9 24.7%, MPLW515 3.9%,triple-negative 15.8%. Most frequent non-driver mutations were ASXL1 28.6%, TET2 18.9%, SRSF2 11.2%, U2AF1 10.1% and SETBP1 7.7%(Figure 1A-C). First, we compared the proportion of non-driver mutations between Pre-PMF, Overt-PMF and PMF-AP/BP. Mutations in the ASXL1(30.3%VS10.1%, P=0.001)and U2AF1 (15.2%VS1.4%, P=0.002)genes were more frequent in Overt-PMF compared with Pre-PMF, whereas mutation in the ARID1A (9.1% VS 0.7%, P=0.005)gene was more frequent in Pre-PMF(Figure1D); as mutations in ASXL1(51.1%VS30.3%,P=0.011), SRSF2(26.7%VS9.7%,P=0.004), RUNX1(22.2%VS5.5%,P=0.001), SETBP1(17.8%VS6.2%,P=0.018), NOTCH2(10.8%VS2.2%,P=0.038), NRAS(15.6%VS3.4%,P=0.004)and EZH2(13.3% VS 4.8%,P=0.048) genes were significantly more frequent in PMF-AP/BP compared with Overt-PMF(Figure 1E). Then, we analyzed the enrichments of non-driver mutations in different subtypes of PMF. ASXL1(OR=4.05,95%CI 1.59～10.31; P=0.003) and U2AF1(OR=12.16,95%CI 1.59～92.22; P=0.002) mutations were enriched in Overt-PMF compared with Pre-PMF, RUNX1 (OR=6.56,95%CI 1.84～23.40; P=0.004)and ASXL1(OR=2.94,95%CI 1.27～6.84; P=0.012) mutations were enriched in PMF-AP/BP compared with Overt-PMF(Figure 1F). Next, we explored clonal evolution during disease progression in 17 paired sequential samples. Non-driver mutations, especially ASXL1(Figure 2A)and RUNX1(Figure 2B)mutations tended to increase their clone sizes and were more likely to be newly acquired than lost during the transformation from the Overt-PMF phase to accelerate/blast phase. ASXL1 mutation was regarded as an adverse prognostic factor in PMF patients according to previous study. However, in Overt-PMF(Figure 2C) and PMF-AP/BP(Figure 2D) patients, ASXL1 mutation had an insignificant effect on overall survival (OS), so we examined the effect of clone size of ASXL1 mutation on OS. Among Overt-PMF patients with ASXL1 mutation, those with smaller clone size had a significantly shorter OS than those with larger clone size(HR=3.94,95%CI:1.09∽14.25; p=0.024) and those without ASXL1 mutation(HR=4.04,95%CI:1.35∽12.11; p=0.007)(Fig 2E). similarly, Among PMF-AP/BP patients with ASXL1 mutation, smaller clone size was associated with significantly inferior OS (HR=4.05,95%CI: 0.89∽18.47;p=0.045) compared with larger clone size (Figure 2F). Conclusion: This study clarified the mutational landscape and enrichments of non-driver mutations between different subtypes of PMF patients. The clonal architecture changes and increment of newly acquired pathogenetic mutations were associated with the disease progression of PMF. Smaller clone size of ASXL1 mutation had an adverse prognostic impact in Overt-PMF and PMF-AP/BP patients. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Detecting the Unusual without Compromising Diagnostic Accuracy - a Prospective WGS/Wts Pilot Study in Acute Leukemias Provided Additional Information for Diagnosis, Prognosis and Treatment

Background: Following WHO and ELN guidelines, standard diagnostics and risk stratification for acute leukemias (AML and ALL) are based on genetic information gathered by a combination of cytogenetics and targeted molecular profiling. Today, comprehensive techniques, i.e WGS and WTS (whole genome and transcriptome sequencing) and the accompanying clinical bioinformatics workflows may even refine and individualize diagnostics. Aim: To evaluate the clinical utility and added value of WGTS in a real-world setting in comparison to standard of care genetic diagnostics. Patients and Methods: The study enrolled 113 patients, 97 with AML and 16 with ALL, diagnosed according to WHO criteria based on cytomorphology, immunophenotyping, cyto- and molecular genetics. WGS (100x) and WTS (50 Mio reads) were performed on NovaSeq instruments. For selected AML cases CD3+ MAC sorted cells were used as normal controls (30x). Variants were called with Strelka2, Manta and GATK using a tumor w/o normal pipeline, fusions with Arriba, STAR-Fusion and Manta. Results: All patients were analyzed for structural variants (SV), copy number variations (CNV) and copy-neutral loss of heterozygosity (cnLOH) based on WGS data and the presence of fusion transcripts and expression patterns based on WTS data. Samples without a normal control (n = 74) were analyzed for 121 genes recurrently mutated in myeloid/lymphoid neoplasms, whereas for 39 AML cases with CD3+ normal controls the whole exome was assessed. Overall, 308/333 (92%) gene mutations were consistently identified by targeted sequencing and WGS. Due to the higher read depth, 25/333 (8%) sub-clonal mutations were only detected by targeted sequencing, with 23/25 showing a variant allele fraction <10%. However, 40 gene mutations were identified exclusively by WGS due to the unrestricted analytical scope (e.g. PTPN11, DDX54). Every patient carried on average 3.3 gene mutations within the 121 genes of interest. The number increased to 63 variants (mean; range: 46-116) when analyzing the exome. 52/63 variants are most likely germline variants based on occurrence in CD3+ control sample. Hence, the CD3+ control samples reduced the number of somatic variants in the exome to 11 (mean; range: 1-23) mostly patient-specific mutations that can be explored for personalized treatment options. Addressing cytogenetics with relevance to risk stratification and assignment to AML/ALL genetic subgroups, WTS identified all entity defining fusions and WGS and chromosome banding analysis (CBA) showed a very high concordance. In 10 patients WGS missed subclonal aberrations due to small clone size (median clone size detected by IP-FISH analysis: 6%). However, WGS provided additional information in 40 patients, mostly SV, CNV and cnLOH of unknown significance, but also alterations of biological interest and prognostic and/or therapeutic importance: in one AML case WGTS revealed a KMT2A::MLLT10 rearrangement that was cytogenetically cryptic and not detectable by FISH (KMT2A break apart probe), assigning the patient to the adverse risk group. WGS also identified a cytogenetically cryptic SV leading to an ETV6::ABL1 rearrangement in an ALL patient with complex karyotype. Retrospective analysis showed that the aberration had already been present when the patient presented with an MPN and a normal karyotype (CBA) two years earlier. Hence, WGS led to the diagnosis of an ETV6::ABL1-positive neoplasm in blast phase (poor prognosis), which might be treated with tyrosine kinase inhibitors. Of note, in 3 AML patients a 7q deletion or complex SV leading to a 7q deletion resulted in a CDK6::NOM1 fusion transcript and high MNX1 expression. In addition, WGS revealed in one patient with ETP-ALL a cytogenetically cryptic translocation t(7;14)(q36;q32) affecting the IGH locus and the NOM1 gene, accompanied by high MNX1 expression. Elevated MNX1 expression as a result of t(7;12)(q36;p13) is a very rare event in pediatric AML, but our findings might suggest a more important role of MNX1 expression in adult acute leukemia than previously known. Conclusions: This prospective study in a real-world setting 1) demonstrated the diagnostic power of WGTS with an accuracy comparable to gold standard techniques, 2) added important prognostic information, 3) identified potential new therapeutic targets and 4) gave new insights into disease biology. WGTS significantly will improve and refine diagnostics in the future.

Validation of a single tube 3-colour immature red blood cell screening assay for the detection and enumeration of small, medium and large paroxysmal nocturnal haemoglobinuria clones by flow cytometry.

The reliable diagnosis of paroxysmal nocturnal haemoglobinuria (PNH) by flow cytometry is based on mandatory analysis of the erythroid, neutrophilic and monocytic lineages. In this study, we have evaluated the performance characteristics of a recently published immature red blood cell (iRBC) assay as a potential screening test for PNH by flow cytometry. Intra- and inter-assay imprecision were determined in five replicates of small, medium and large PNH iRBC clones. Analytical and functional sensitivity was assessed by performing spiking tests for five replicates. Thirty healthy donors and 441 PNH patients were tested for evaluation of clinical specificity, sensitivity, positive and negative predictive values. Coefficients of variation (CV) for intra-/inter-assay imprecision analyses were 1.31/1.50, 3.19/2.61 and 3.99/1.58 for the big, medium and small clone sizes, respectively. Absolute values (100%) were found for both clinical specificity and sensitivity as well as for both positive and negative predictive values. The CV from 5 replicate results for 10 clustered events was 15.7%. The coefficient of determination (r2 ), Pearson's correlation coefficient (r) and Bland-Altman mean bias were 0.9436/0.9234/1.7 for PNH iRBC compared to PNH neutrophils and 0.9553/0.9387/2.1 for PNH iRBCs compared to PNH monocytes. Our results confirm very good performance characteristics, high analytical and functional sensitivity, absolute clinical specificity and sensitivity as well as favourable correlation between PNH iRBCs and both PNH neutrophils and monocytes, suggesting that this cost-effective 3-colour iRBC assay can be used as a reliable screening test for evaluation of small, medium and large PNH clones by flow cytometry.

Benchmarking of Whole Genome Sequencing (WGS) and Whole Transcriptome Sequencing (WTS) As Diagnostic Tools for the Genetic Characterization of Acute Myeloid Leukemia (AML) and Acute Lymphoblastic Leukemia (ALL) in Adults

Background: In AML and ALL the application of WHO classification and ELN guidelines requires a combination of cytogenetics and targeted sequencing for specific mutations to determine the diagnostic and prognostic subgroup. WGS and WTS have emerged as comprehensive techniques that allow the simultaneous analysis and identification of all genetic alterations in a single approach with possible turnaround times of 1 week.Aim: Evaluate the accuracy of WGS and WTS in providing all relevant genetic information in a clinical setting.Patients and Methods: The cohort comprised 738 AML, 293 BCP-ALL and 124 T-ALL. The diagnosis was established following WHO guidelines. WGS (100x, 2x151bp) and WTS (50 Mio reads, 2x101bp) were performed on a NovaSeq instrument. Variants were called with Strelka2, Manta and GATK using a tumor w/o normal pipeline, fusions with Arriba, STAR-Fusion and Manta.Results: The combination of WGS and WTS detected all chromosomal and molecular abnormalities in the AML and ALL cohorts relevant for disease stratification and prognostication as identified by chromosome banding analysis (CBA) and targeted panel sequencing (TPS). A very high concordance between CBA and WGS was revealed for the detection of balanced structural variants (SV) with the added benefit of WGS to also detect cytogenetically cryptic rearrangements (i.e.: ETV6-MN1, NUP98-KDM5A), which all were confirmed either by FISH or RT-PCR. Fusion calling by WTS identified 96% of the WHO subtype defining rearrangements and detected 20 additional fusion transcripts relevant for disease stratification (e.g. EP300-ZNF384, TCF3-HLF) including 9 fusion transcripts that led to prognostic reassignment or could serve as a potential treatment target. Breakpoints of unbalanced SV can occur in repetitive sequences of the genome, hampering the detection by WGS. However, adding copy number alteration (CNA) calls to the analyses allows also reliable identification of unbalanced SV.WGS outperformed CBA in cases with insufficient in vitro proliferation due to suboptimal pre-analytics (i.e. longer transport time) and identified 36 chromosomal aberrations in 12 cases with CBA not evaluable. WGS's independence of in vitro cell proliferation was most impactful in ALL: 40 T-ALL cases showed a normal karyotype according to CBA. WGS detected SVs in 16 (40%) and CNAs in 20 (50%) of these cases, confirming the normal karyotype for only 9 samples. In the BCP-ALL cohort, CNV analysis identified 29 low hypodiploid and 16 high hyperdiploid karyotypes, 6 of which were missed by CBA. Due to the higher resolution and unrestricted, genome-wide assessment, WGS detected relevant gene deletions (RB1, ERG, PAX5, CDKN2A, IKZF1, ETV6, BTG1) in 59% of ALL cases, providing additional diagnostic and prognostic information. In the AML cohort CBA and WGS detected 795 CNA concordantly. In addition WGS called 54 CNA with size 1-5 MB (below the detection limit of CBA), i.e. 3 BCOR deletions in inv(3)(q21q26) cases and 67 CNA with size > 5 MB, which were missed by CBA. 35 CNA were missed by WGS due to small clone sizes (median 6% as determined by FISH). WGS detected copy neutral loss of heterozygosity (CN-LOH) in AML most frequently on 21q (n=17), 4q (n=15), 13q (n=15), 11q (n=13) and in T-ALL on 9p (n=19), mostly encompassing CDKN2A/B deletions. Expression profiling provided additional diagnostic information for 57 ALL cases (41 BCR-ABL1-like, 16 DUX4 rearranged) that can only insufficiently be obtained by WGS or CBA. WGS reliably detected all gene mutations with a VAF > 15% (n = 647) identified by TPS encompassing especially all mutations in genes relevant for WHO diagnosis and prognostication. 26/171 mutations with a VAF < 15% were missed by WGS. Evaluation of WGS data for 121 genes recurrently mutated in hematologic neoplasms revealed an additional 2 mutations per sample on average (range: 0-9) which might qualify as targets for therapy.Conclusions: WGS and WTS provide all necessary genetic information to accurately determine the diagnostic and prognostic subgroup according to WHO and ELN guidelines in AML and ALL. Compared to today's gold standards, these novel methods provide a comprehensive genome wide characterization with higher resolution that directly identifies genes of impact, offering the basis for targeted treatment selection and monitoring of residual disease. Both can be implemented with automated analysis pipelines, consequently reducing time and error rates. [Display omitted] DisclosuresHaferlach: MLL Munich Leukemia Laboratory: Other: Part ownership. Kern: MLL Munich Leukemia Laboratory: Other: Part ownership. Haferlach: MLL Munich Leukemia Laboratory: Other: Part ownership.

Outcomes of Immunosuppressive Therapy for Pediatric Aplastic Anemia: A North American Pediatric Aplastic Anemia Consortium (NAPAAC) Study

Immunosuppressive therapy (IST) is effective treatment for severe aplastic anemia (SAA); however data on the quality of that survival for pediatric patients are limited. To investigate the clinical features and outcomes of children with SAA treated with IST, NAPAAC conducted a retrospective study of patients aged 1 to 20 years (yrs) treated with IST at 25 member institutions between 1/1/2002 and 6/30/2014 with a minimum of 24 months (mos) of follow-up.A total of 314 patients were treated with IST for SAA defined as a hypocellular marrow with at least two cytopenias: 1) absolute neutrophil count <500/uL, 2) platelet count <20,000/uL, and/or 3) hemoglobin <8 g/dL. The median age at diagnosis was 9.7 yrs and 51.6% were male. Patients were racially diverse with 58.3% white (15.9% overall Latino ethnicity), 16.6% Black, 7.3% Asian, and 17.8% mixed/other. A history of hepatitis was reported in 43 (13.7%) patients. Mean red cell volume (MCV) was elevated ≥100 fL in 54 of 272 pts (19.9%), median 89.1fL. Absolute lymphocyte count was <1,000/uL in 110 of 260 pts (42.3%). Lymphocyte telomere length was <1st percentile for age in 6 of 92 pts (6.5%). A granulocyte PNH clone was detectable in 55 of 140 pts (39.3%), all with a small clone size (75th quartile 0.12%).IST regimens included: horse ATG and cyclosporine (hATG/CSA; n=263), horse ATG/ tacrolimus (n=15), rabbit ATG/CSA (n=14), rabbit ATG/tacrolimus (n=1), cyclophosphamide (n=19) and sequential horse/rabbit ATG (n=2). Median time from diagnosis to IST was 24 days, with 168 (54.2%) treated within 4 weeks (wks).Response to IST was defined as follows: •Complete Response (CR): Hgb ≥ 10 g/dl, ANC ≥1,000/uL, Plts ≥100,000/uL•Very Good Partial Response (VGPR): Hgb ≥ 8 g/dl, ANC ≥ 500/uL, Plts ≥ 50,000/uL•Partial Response (PR): Hgb ≥ 8 g/dl, ANC ≥ 500/uL, Plts ≥ 20,000/uL•No Response (NR): Hgb < 8 g/dl, ANC < 500/uL, Plts < 20K/uLPatients receiving PRBCs within 6 wks, platelets or G- or GM-CSF within 2 wks of a timed endpoint were deemed to have no response.At a median follow-up of 61.5 mos post IST, 30 of 314 pts (9.6%) had died. Best response at any time after IST of 301 evaluable patients was CR (192; 61.1%), VGPR (25; 8%), PR (10; 3.2%) and NR (74; 23.6%). The median time to best response was 6 mos (range 3-48 mos), and did not differ with time between diagnosis and IST. Of the 227 pts with a CR, VGPR or PR at any time, 47 (20.7%) ultimately died or required additional therapy (IST and/or HSCT). Overall survival was 98% at 6 mos, 95% (95% CI: 92-97) at 24 mos, and 93% (95% CI: 90-96) at 60 mos. Survival did not differ between patients treated <4 vs ≥4 wks from diagnosis (p=0.533) with 24 mos estimate of 96% vs 94%. Survival without a subsequent therapy was 84% (95% CI: 80-88) at 6 mos, 68% (95% CI: 63-73) at 24 mos, and 58% (95% CI: 52-64) at 60 mos. Of the 261 evaluable pts treated with hATG/CSA, 21 pts (8.1%) died, overall survival was 94% (95% CI: 91-97) and one-third required at least one additional therapy.Cytogenetics were available at diagnosis for 271 of 314 (86.3%) pts, and 8 (3%) had clonal abnormalities. Of 171 patients with cytogenetics after IST, 13 (7.6%) additional patients developed clonal abnormalities at a median time of 28.5 mos (range: 4.3-143 mos). Monosomy 7, del 7q, and del 13q were the most common abnormalities. Only 4 patients with clonal abnormalities developed MDS, with one each progressing to ALL and AML. The other two patients received a HSCT soon after the MDS diagnosis. There was no significant association between hepatitis, cytogenetic abnormalities, short telomeres, or PNH clone at diagnosis with response to IST.In conclusion, we report a large multicenter retrospective analysis of ethnically diverse North American IST treated pediatric SAA patients. Acquisition of clonal abnormalities and progression to MDS/leukemia was rare. Outcomes included 69.1% of pts with a CR or VGPR and overall survival of 93%. Among the 314 patients, event-free survival was 58% at 60 mos with 130 (41%) having died or required subsequent therapies. Unexpectedly, neither clonal abnormalities at diagnosis or time between diagnosis and IST affected response. Clinical and laboratory features associated with survival, quality, and durability of response to IST are currently being ascertained. These data support the need for new approaches to treatment of pediatric SAA. DisclosuresRogers:Terumo BCT: Consultancy; Pfizer: Research Funding. Rothman:Agios Pharmaceuticals: Honoraria; Pfizer: Consultancy. Thompson:Celgene: Consultancy, Research Funding; Baxalta: Research Funding; Novartis: Consultancy, Research Funding; bluebird bio: Consultancy, Research Funding. Glader:Agios Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees. Huang:Baxter: Research Funding; Biogen: Research Funding; Bio Products Laboratory: Other: Study investigator; Novartis: Other: support to attend meeting. Shimamura:TransCellular Therapeutics: Other: Spouse is majority shareholder .

First Interim Analysis of the French National Observatory of Paroxysmal Nocturnal Hemoglobinuria Clones

Introduction: Following the published International Guidelines (Borowitz, 2010), the French-Belgian biological workshop was initiated in 2010 by the French Paroxysmal Nocturnal Hemoglobinuria (PNH) group (named HPNAFC). This effort to harmonize the diagnostic practices has been pursued through the development of an inter-laboratory comparison program, involving more than 50 French-speaking centers since 2013 (Debliquis, 2015). Thus, the French National Observatory of PNH clones opened for 5 years on the 9th International Rare Disease Day (February 29th 2016), in order to collect all PNH clone cases diagnosed by French cytometrists.Methods: The primary objective of the French National Observatory of PNH clones is to monitor all PNH clones ≥0.01%, along with two secondary objectives according to the PNH clone size: the evolution of minor clones <1% and the proportion and significance of type II and type III cells for clones ≥1%. All patients with a PNH clone ≥0.01% can be included, irrespective of age, provided that the center has validated a PNH flow cytometry quality control. For each patient, the inclusion time point always corresponds with the initial diagnosis date of the PNH clone, even if it was made before the opening of the Observatory, allowing the collection of all cases in the active French PNH file. This Observatory is based on a principle of non-opposition; inclusions are made by the referent cytometrist of each participating center by filling in the e-CRF form available on the HPNAFC website.Results: As of June 30th 2017, 39 cytometry laboratories have registered across France. Of these, 24 centers have submitted 163 inclusion points with a PNH clone above 0.01%, each corresponding to a patient. The cases were reviewed by the 2 principal investigators, who carefully re-examined all of the flow cytometry data. This method enabled the validation and inclusion of 126 cases, with 37 ongoing ones due to missing clinical or biological data. The first extraction led to the preliminary analysis of these 126 cases and showed that patient median age at diagnosis was 45 years [11-87], with 6 pediatric cases, and a M:F sex ratio of 0.83. Diagnosis was made between 1988 and 2017 and clinical information at diagnosis was available for 92% of patients. More than half of them had aplastic anemia (58.7%). Other diagnosis included: hemolytic anemia (with or without aplastic anemia) (15.9%), unexplained cytopenia(s)(7.9%), myelodysplastic syndrome (6.3%), hemoglobinuria (4.8%) and 2 cases (1.6%) of recurrent thrombosis without hemolysis. Biological data showed a median hemoglobin value of 9.3 g/dL [4.7-16.5], 1.4 x109/L [0.1-11] neutrophils and 60 x109/L [2-356] platelets. The median clone size at diagnosis was 1.9% on granulocytes with a very wide range from 0.01% to 99.4%, most of them being less than 10% and mainly composed of type III cells (median type III clone size: 1.6%). A similar distribution of PNH clones was observed on monocytes, with a good correlation between clone sizes on both lineages. RBC analysis, which was performed for less than half of the patients showed a majority of minor clones and no correlation was observed between PNH clone size on granulocytes and RBC, due to an underestimation of the clone size on RBC. Regarding the clinical context, the median PNH clone size on granulocytes in case of aplastic anemia was 1%, yet some patients had very large clones, higher than 99%. Conversely, PNH clones from patients with hemolytic anemia were very large with a median size of 88%, but some patients had smaller clones of less than 20%. The 2 patients with thrombosis displayed a much smaller clone size, below 0.05%.Conclusion: More than one year after its initiation, 126 confirmed patients were enrolled in the French National Observatory of PNH clones. Median PNH clone size was 1.9% on granulocytes and more than half of these patients had aplastic anemia. A good correlation was seen between clone sizes on granulocytes and monocytes, but not on granulocytes and RBCs. This collaborative work has established a strong network of motivated cytometrists as highlighted by the number of registered centers and the increasing inclusions since its recent opening. A comprehensive follow-up of a foreseeable larger number of PNH clones (so far 92 out of the 163 already have at least 1 follow up point) will lead to a better understanding of the physiopathology of minor PNH clones. DisclosuresDrenou:Alexion: Consultancy, Honoraria. Pruvot Debliquis:Alexion: Consultancy, Honoraria. Letestu:Alexion: Consultancy, Honoraria. Chatelain:Alexion: Consultancy, Honoraria. Socié:Alexion Pharmaceuticals, Inc.: Consultancy. Peffault De Latour:Pfizer: Consultancy, Honoraria, Research Funding; Alexion Pharmaceuticals, Inc.: Consultancy, Honoraria, Research Funding; Novartis: Consultancy, Honoraria, Research Funding; Amgen: Research Funding. Wagner Ballon:Alexion: Consultancy, Honoraria.

T-cell clones of uncertain significance are highly prevalent and show close resemblance to T-cell large granular lymphocytic leukemia. Implications for laboratory diagnostics

Benign clonal T-cell expansions in reactive immune responses often complicate the laboratory diagnosis T-cell neoplasia. We recently introduced a novel flow cytometry assay to detect T-cell clones in blood and bone marrow, based on the identification of a monophasic T-cell receptor (TCR) β chain constant region-1 (TRBC1) expression pattern within a phenotypically distinct TCRαβ T-cell subset. In routine laboratory practice, T-cell clones of uncertain significance (T-CUS) were detected in 42 of 159 (26%) patients without T-cell malignancy, and in 3 of 24 (13%) healthy donors. Their phenotype (CD8+/CD4−: 78%, CD4−/CD8−: 12%, CD4+/CD8+: 9%, or CD4+/CD8−: 2%) closely resembled that of 26 cases of T-cell large granular lymphocytic leukemia (T-LGLL) studied similarly, except for a much smaller clone size (p < 0.0001), slightly brighter CD2 and CD7, and slightly dimmer CD3 expression (p < 0.05). T-CUS was not associated with age, gender, comorbidities, or peripheral blood counts. TCR-Vβ repertoire analysis confirmed the clonality of T-CUS, and identified additional clonotypic CD8-positive subsets when combined with TRBC1 analysis. We hereby report the phenotypic features and incidence of clonal T-cell subsets in patients with no demonstrable T-cell neoplasia, providing a framework for the differential interpretation of T-cell clones based on their size and phenotypic properties.

Abstract P456: The Association Between Clonal Hematopoiesis Of Indeterminate Potential And Inflammatory Biomarkers Among Chronic Kidney Disease Patients

Introduction: Clonal hematopoiesis of indeterminate potential (CHIP) is an age-related disorder associated with increased risk of atherosclerotic cardiovascular disease (ASCVD). Mechanistic studies of CHIP implicate inflammatory pathways underlying this relationship. Although inflammation is a well-established component of chronic kidney disease (CKD), linked to both CKD progression and ASCVD in CKD patients, CHIP has not been investigated in a CKD setting. Objective: To estimate the prevalence of CHIP in patients with CKD and to examine the cross-sectional associations between CHIP and biomarkers that were previously related to CKD progression and ASCVD among CKD patients. Methods: The current study was conducted among 598 Chronic Renal Insufficiency Cohort (CRIC) study participants with whole-exome sequencing data and baseline measures of fibrinogen, interleukin (IL)-6, serum albumin, and tumor necrosis factor-α. CHIP was detected from sequencing data using MuTect2. CHIP was defined as the presence of a somatic hematologic malignancy-associated mutation with a variant allele frequency (VAF) of at least 2%. CHIP was also categorized ordinally by clone size as no CHIP, small CHIP clone (VAF&lt;10%), and large CHIP clone (VAF ≥10%). Ordinal logistic regression was used to test associations of CHIP with each biomarker in age- and multivariable-adjusted models. Multivariable models adjusted for age, sex, ancestry, body mass index, estimated glomerular filtration rate, systolic blood pressure, low-density lipoprotein cholesterol, fasting plasma glucose, and blood pressure, lipid, and glucose lowering medications. Results: CHIP was detected in a total of 28 individuals from the WES study (4.7%). Among those 70 years of age and older, we detected a CHIP prevalence of nearly 16%. As expected, participants with CHIP were older than those without CHIP (mean age: 67 and 58 years, respectively; P&lt;0.0001). Other demographic and clinical variables were similar between groups . Age-adjusted models showed strong associations between CHIP and both fibrinogen and IL-6 with odds ratios of 2.49 (95% CI: 1.09, 5.71) and 2.45 (95% CI: 1.05, 5.72), respectively. After multivariable adjustment, only fibrinogen remained associated with CHIP with an odds ratio of 4.70 (95% CI: 1.69, 13.0). Consistent with these findings, there was a strong dose-dependent association between CHIP clone size and fibrinogen in multivariable adjusted analyses (P for linear trend=0.009). Compared to those without CHIP, odds ratios for higher fibrinogen tertile were 3.0 (95% CI: 0.84, 11.3) and 4.7 (95% CI: 1.29, 16.7) among those with small and large CHIP clone sizes, respectively. Conclusions: CHIP prevalence was higher among CKD patients in the current study compared to previous estimates from the general population. Furthermore, a strong, dose-dependent association between CHIP and fibrinogen was identified.

Paroxysmal Nocturnal Hemoglobinuria Clones Are Infrequent in Hepatitis-Associated Aplastic Anemia at the Time of Diagnosis

Aplastic anemia (AA) is an immune-mediated bone marrow failure, resulting in reduced number of hematopoietic stem and progenitor cells and pancytopenia. The presence of paroxysmal nocturnal hemoglobinuria (PNH) clone in AA usually suggests an immunopathogenesis in patients. However, when and how PNH clone emerge in AA is still unclear. Hepatitis associated aplastic anemia (HAAA) is a special variant of AA with a clear disease course and relatively explicit immune pathogenesis, thus serves as a good model to explore the emergence and expansion of PNH clone. To evaluate the frequency and clonal evolution of PNH clones in AA, we retrospectively analyzed the clinical data of 90 HAAA patients that were consecutively diagnosed between August 2006 and March 2018 in Blood Diseases Hospital, and we included 403 idiopathic AA (IAA) patients as control. PNH clones were detected in 8 HAAA patients (8.9%,8/90) at the time of diagnosis, compared to 18.1% (73/403) in IAA. Eight HAAA patients had PNH clone in granulocytes with a median clone size of 3.90% (1.09-12.33%), and 3 patients had PNH clone in erythrocytes (median 4.29%, range 2.99-10.8%). Only one HAAA patients (1/8, 12.5%) had a PNH clone larger than 10%, while 24 out of 73 IAA patients (32.9%) had larger PNH clones. Taken together, we observed a less frequent PNH clone with smaller clone size in HAAA patients, compared to that in IAAs. We next attempted to find out factors that associated with PNH clones. We first split patients with HAAA into two groups based on the length of disease history (≥3 mo and &lt; 3mo). There were more patients carried PNH clone in HAAA with longer history (21.4%, 3/14) than patients with shorter history (6.6%, 5/76), in line with higher incidence of PNH clone in IAA patients who had longer disease history. Then we compared the PNH clone incidence between HAAA patients with higher absolute neutrophil counts (ANC, ≥0.2*109/L) and lower ANC (&lt; 0.2*109/L). Interestingly, very few VSAA patients developed PNH clone (5%, 3/60), while 16.7% (5/30) of non-VSAA patients had PNH clone at diagnosis. We monitored the evolution of PNH clones after immunosuppressive therapy, and found increased incidence of PNH clone over time. The overall frequency of PNH clone in HAAA was 20.8% (15/72), which was comparable to that in IAA (27.8%, 112/403). Two thirds of those new PNH clones occurred in non-responders in HAAA. In conclusion, PNH clones are infrequent in HAAA compared to IAA at the time of diagnosis, but the overall frequency over time are comparable between the two groups of patients. In SAA/VSAA patients who are under the activated abnormal immunity, longer clinical course and relatively adequate residual hematopoietic cells serve as two important extrinsic factors for HSCs with PIGA-mutation to escape from immune attack and to expand. Disclosures No relevant conflicts of interest to declare.

PIG-a Mutations in a Brazilian Cohort of Patients with Paroxysmal Nocturnal Hemoglobinuria

Introduction: Paroxysmal nocturnal hemoglobinuria (PNH) is a disorder due to an acquired loss-of-function mutation in the phosphatidylinositol glycan class A (PIG-A) gene. A large spectrum of acquired PIG-A mutations has been described, like insertions or deletions involving a single base or several bases, and single base substitution that are the most common. Usually there are more than one PIGA mutations and one clone is predominant. The clinical manifestations of PNH are intrinsically related to clonal expansion of hematopoietic stem cell deficient in GPI-anchored proteins. Some hypothesis failed to explain alone this clonal expansion. Here we try to identify and to correlate PIG-A gene mutations with clinical manifestations in a series of patients with PNH. Methods: We analyzed 31 patients with classical PNH (n=23) or aplastic anemia and PNH clone (n=8). The sequencing of the PIG-A gene was performed using the Sanger technique. The electropherograms were aligned against the reference sequence of the PIG-A gene deposited in GenBank (Accession number NG_009786), and analyzed using the Geneious R10 software (Biomatters). After analysis, a search was performed in the Clinvar, dbSNP and HGMD databases to verify the pathogenicity of the mutations. For variants without description in the literature, a pathogenicity prediction analysis was performed using Mutation Taster, Polyphen 2 and Human Splicing Finder software. Results: We found 29 different variants of the PIG-A gene in 27 patients: 23 were new mutations, with no previous description in the literature, 3 were previously described mutations, and 3 were single nucleotide polymorphism (SNP). There was great variation in the type and location of somatic mutations. Mutations were predominantly small deletions and simple base changes; 42% of the mutations were described as frameshift mutations and 31% missense mutations. We did not find any specific correlation between the clinical characteristics of hemolytic PNH patients and their mutations, due to the wide variety of mutations. According the pathogenicity prediction programs, the majority (22 of 29) of the variants found were classified as probably pathogenic. Among the 23 patients with hemolytic PNH, 19 patients had at least one mutation classified as pathogenic. In patients with subclinical PNH, only SNPs were found. Fifteen patients with hemolytic PNH had more than one concomitant mutation, most of which were probably pathogenic mutations associated with a polymorphism. Conclusion: We described PIG-A mutations in a series of PNH patients in Brazil and observed no correlation exists between mutation types and clinical features in hemolytic patients. Among subclinical PNH patients, only SNPs were observed, probably because of small clone sizes. Disclosures No relevant conflicts of interest to declare.

Prognostic Value of Clone Size in Paroxysmal Nocturnal Hemoglobinuria (PNH) for Thrombotic Events in Untreated Patients in the International PNH Registry

Background/Objective: The association between paroxysmal nocturnal hemoglobinuria (PNH) granulocyte clone size at disease onset and outcomes in patients with PNH remains unclear. Most but not all reports examining the relationship have shown a positive correlation between clone size and thrombotic events [TEs] in patients with PNH, but without a clear temporal association showing the prognostic value of clone size on the risk of TEs. The ongoing International PNH Registry (NCT01374360) is the largest prospective, observational study of patients with PNH conducted to date. The objective of this analysis was to examine the relationship between PNH granulocyte clone size at disease onset and risk of thrombosis after disease onset while untreated with a complement inhibitor in patients enrolled in the Registry.Methods: The current analysis included patients enrolled in the Registry as of April 2018 who had known demographics, were untreated with complement inhibitor therapy at enrollment, and had ≥12 months of untreated follow-up after disease onset. Baseline was defined as disease onset (earliest of a reported PNH clone, date of PNH diagnosis, or reported PNH symptom), and patients were stratified into 5 cohorts based on clone size at baseline (using the earliest reported clone prior to enrollment): cohort 1, clone size: 0.01-1%; cohort 2, clone size: >1-5%; cohort 3, clone size: >5-10%; cohort 4, clone size: >10-50%; cohort 5, clone size: >50%. Event rates for TEs and all major adverse vascular events (MAVEs; including TEs) were calculated for the time period from baseline to last follow-up. Other outcomes of interest included LDH ratio (LDH/LDH upper limit of normal [ULN]), hemoglobin levels, platelet counts, absolute neutrophil counts, and absolute reticulocyte counts at last follow-up.Results: A total of 2489 patients were eligible for the analysis. The majority of patients in the overall study population were female (54.0% [1343/2489]) and white (79.2% [1967/2484]). Mean (standard deviation [SD]) age at PNH start ranged from 36.4 (16.53) in cohort 5 to 44.2 (20.70) in cohort 1. Median time from baseline to last follow-up was 3.7 years in cohort 1, 4.3 years in cohort 2, 4.7 years in cohort 3, 5.6 years in cohort 4, and 6.8 years in cohort 5. Results for the outcomes of interest are summarized in the Table. All cohorts showed a risk of MAVE and TE during follow-up. Although estimated rates of MAVE and TE were highest in the >50% clone size cohort, there was no difference in the rate of MAVE or TE during follow-up across the 4 cohorts with clone size <50% at disease onset. Mean LDH ratio (LDH/LDH ULN) at last follow-up showed a statistically significant difference by clone size at baseline among the cohorts, ranging from a mean (SD) of 1.1 (1.34) in the patients with clone size <1% and increasing to 5.1 (3.81) in the patients with clone size >50% (P<0.0001). No difference in hemoglobin level at last follow-up was observed in the smaller clone size cohorts, although mean hemoglobin level was lower in patients with clone size >50% (P<0.0001). Similar trends were seen in mean platelet and absolute neutrophil counts across the smaller clone size cohorts, while patients with clone size >50% showed higher values (P<0.0001). Mean absolute reticulocyte count at last follow-up was lowest in patients with clone size 0.01-1%, and were incrementally higher in each successive clone-size cohort (P<0.0001).Conclusions: In this study, all patients with a PNH clone at disease onset were at risk for TEs and other MAVEs. Patients in the highest baseline PNH clone size strata (clone size >50%) had an approximately 2-times higher risk of TEs than patients with smaller clone sizes; Patients with small clone sizes (0.01-1%) were older and showed a higher prevalence of BMD. There was no difference in the prognostic value of clone size at disease onset on the risk of TEs and other MAVEs in patients with small (0.01-1% and 1-5%) versus medium-sized (5-10% or 10-50%) clones. [Display omitted] DisclosuresPeffault De Latour:Alexion Pharmaceuticals, Inc.: Consultancy, Honoraria, Research Funding; Novartis: Consultancy, Honoraria, Research Funding; Pfizer Inc.: Consultancy, Honoraria, Research Funding; Amgen Inc.: Research Funding. Maciejewski:Alexion Pharmaceuticals, Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Alexion Pharmaceuticals, Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Apellis Pharmaceuticals: Consultancy; Apellis Pharmaceuticals: Consultancy; Ra Pharmaceuticals, Inc: Consultancy; Ra Pharmaceuticals, Inc: Consultancy. Kulasekararaj:Alexion Pharmaceuticals, Inc.: Consultancy, Honoraria, Other: Travel Support . Larratt:Alexion Pharmaceuticals, Inc.: Honoraria, Research Funding. Dingli:Millennium Takeda: Research Funding; Alexion Pharmaceuticals, Inc.: Other: Participates in the International PNH Registry (for Mayo Clinic, Rochester) for Alexion Pharmaceuticals, Inc.; Millennium Takeda: Research Funding; Alexion Pharmaceuticals, Inc.: Other: Participates in the International PNH Registry (for Mayo Clinic, Rochester) for Alexion Pharmaceuticals, Inc.. Wilson:Alexion Pharmaceuticals, Inc.: Employment, Equity Ownership. Gustovic:Alexion Pharma GmbH: Employment, Equity Ownership. Kulagin:Alexion Pharmaceuticals, Inc.: Consultancy, Honoraria.

Selective Pharmacologic Inhibition of Paroxysmal Nocturnal Hemoglobinuria Clones

Paroxysmal nocturnal hemoglobinuria (PNH) is usually associated with reduced bone marrow (BM) capacity caused by acquired idiopathic aplastic anemia (AA). PIGA mutations lead to a partial or total deficiency of glycosylphosphatidyl-inositol (GPI) anchor proteins (AP). AA is characteristically accompanied by the presence of often tiny GPI-AP deficient clones, which in a significant proportion of patients (10-15%), irrespective of the initial success of immunosuppressive therapy, will evolve to produce manifest hemolytic PNH. Indeed in our cohort of BM failure patients (n=319), 41% of AA patients had a PNH clone present (0.02-20% of granulocytes) (AA/PNH), 14% of patients had primary PNH (primary PNH), and 8% had a history of PNH post AA (secondary PNH).To date, drug development for PNH has focused on designing supportive therapies to prevent transfusions due to hemolysis or thrombotic complications. In addition to the current FDA approved C5 inhibitor eculizumab, new, more convenient and effective complement blockers are under development. Apart from hematopoietic stem cell (HSC) transplantation, no direct strategies targeting basic pathophysiologic mechanisms of PNH have been ventured to prevent evolution of PNH clones and cure the disease. In early AA/PNH syndrome, the PIGA mutant HSCs are rare and unlikely contribute to significant blood cell production. While in later stages of manifest hemolytic PNH, hematopoiesis relies most frequently on mutant HSCs and thus elimination of these cells would result in AA.We hypothesized that if a selective inhibitor of GPI-AP-deficient [GPI-AP (-)] cells can be developed, it could be used primarily in AA/PNH patients with a small clone size. The hope would be to prevent both later expansion of GPI-AP d(-) cells and development of manifest PNH.To discover compounds acting selectively against GPI-AP (-) cells, we subjected wild type (WT) and GPI-AP (-) cell lines (K562, TF-1) to a high-throughput screen using a platform of 3000 bio-active molecules to identify hits and chemical compounds capable of selectively eliminating GPI-AP (-) cells. Our robotic screen yielded several top hits including GR -89696 fumarate, D-cycloserine and CGS-15943. Dose-response experiments confirmed CGS-15943 as a candidate growth inhibitor of GPI-AP (-) cells. CGS-15943 is an adenosine receptor antagonist and non-phosphodiesterase inhibitor which has previously been shown to inhibit cancer cell growth via PI3K/Akt pathway.Low range dose CGS-15943 (1uM) induced cell growth inhibition in K562 and TF-1 GPI-AP (-) cells by 4.7 fold and 3.2 fold, respectively. No cell growth arrest was observed in K562 WT and TF-1 WT cells, as the percentage of alive cells was >95% upon drug treatment. Mixed competition assays were conducted in vitro using equal ratios of K562 and TF-1 WT and GPI-AP (-) cells exposed to CGS-15943 (1uM). Six days after culture, flow cytometric analysis of CD59 surface expression revealed that CGS-15943 allowed for preferential survival of WT cells (84.7 % K562, 96.3% TF-1) vs. GPI-AP (-) cells (15.3% K562, 3.7% TF-1). CGS-15943 induced an increase in the % of AnnexinV+/PI- and AnnexinV+/PI+ in TF-1 GPI-AP (-) cells (12.04% and 44.82, respectively). Similar results were obtained in K562 GPI-AP (-) cells (15.84% and 21.08%). Mononuclear cells of a PNH patient were stimulated with CD3/28 beads in presence of CGS-15943. Flow cytometric analysis indicates a dose dependent growth inhibition effect on GPI-AP (-) lymphocytes after 3 days of culture. Previous reported observations from our group identified that the survival differences between GPI-AP (-) and WT cells largely depend on active PI3K signaling pathway. Our pilot investigation of CGS-15943 - indicates that CGS-15943 induces an decrease in the protein expression of the PI3K isoform - p110γ - exclusively in GPI-AP (-) cells possibly suggesting that CGS-15943 inhibits the catalytic subunit of- p110γ.In sum, we describe that the small molecule compound CGS-15943 selectively eliminates GPI-AP (-) cells in vitro, in both cell lines and in primary PNH cells most likely interfering with the PI3K/AKT survival pathway. DisclosuresMaciejewski:Alexion Pharmaceuticals, Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Ra Pharmaceuticals, Inc: Consultancy; Apellis Pharmaceuticals: Consultancy; Apellis Pharmaceuticals: Consultancy; Alexion Pharmaceuticals, Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Ra Pharmaceuticals, Inc: Consultancy.

Clinicopathological profile of paroxysmal nocturnal haemoglobinuria clone‐positive aplastic anaemia paediatric patients—A single centre study from North India

There is a paucity of literature related to the prevalence of Paroxysmal Nocturnal haemoglobinuria (PNH) clones in paediatric aplastic anaemia (AA) patients. We performed a retrospective analysis over a period of 42months to study the prevalence of PNH clones in paediatric (age less than 18years) AA cases, using Fluorescein-labelled proaerolysin-based flow cytometric screening and analysed their clinico-pathological features. PNH clone was identified in 100 (33.2%) of the 301 patients screened. These were comprised of 51 cases of non-severe AA, 33 cases of severe AA and 16 cases of very severe AA. The median age was 13years with an M:F ratio of 2.5:1. The median clone size (taken as the proportion of PNH-positive neutrophils) was 2.15% (range: 0.05%-93.1%). Although a majority of patients (n=77) had a clone size of less than 10%, a significant proportion (n=23) did harbour a clone size of more than 10%. Evidence of haemolysis was observed in 3 patients, all of them having a clone size of more than 10%. Interestingly, 1 patient with dural sinus thrombosis harboured a clone size of 1.25% only. Chromosomal breakage analysis was performed in 61 patients, none of which was positive. Complete and partial response to immunosuppressive therapy was found in 55.1% patients (16/29). There is a high prevalence of PNH clones in paediatric AA patients, which in a majority of cases are of small clone sizes. The use of immunosuppressive therapy does not show a better outcome as compared to PNH-negative cases.

Value of CD16/CD66b/CD45 in comparison to CD55/CD59/CD45 in diagnosis of paroxysmal nocturnal haemoglobinuria: An Indian experience.

Background & objectives:Diagnosis of paroxysmal nocturnal haemoglobinuria (PNH), a rare haematopoietic stem cell disorder, is challenging in patients with bone marrow failure (BMF) syndrome like aplastic anaemia (AA). This study was conducted with the aim to test the efficacy of the newly recommended markers viz. anti-CD16 and CD66b antibody over the existing anti-CD55 and CD59 antibody for PNH diagnosis in India.Methods:This study was conducted on 193 suspected cases of PNH by flow cytometry using lyse wash technique to stain the granulocytes with CD16/CD66b and CD55/CD59.Results:Of the 193 suspected cases, 62 patients showed the presence of PNH clone. Forty six patients were detected by CD55/CD59/CD45, whereas 61 were detected by CD16/CD66b/CD45. CD16/CD66b detected 16 (25.8%) additional patients over CD55/CD59 (P<0.05) and was more sensitive in detecting the PNH clone with higher negative predictive value. Most of the patients (11/16) who were picked up by CD16/CD66b were of AA who had small clone sizes. Further, the PNH clones were more discreetly identified in CD16/CD66b plots than by CD55/CD59. Clone size assessed by CD16/CD66b which reflects the clinical severity of classical PNH (thrombosis/haemolysis), was more representative of the underlying clinical condition than CD55/59.Interpretation & conclusions:In our experience of 62 patients of PNH, CD16/CD66b proved to be more efficacious in detecting PNH. The new panel was especially useful in monitoring PNH associated with BMF which had small clone sizes.

Fluorescent Aerolysin (FLAER)-based paroxysmal nocturnal hemoglobinuria (PNH) screening: a single center experience from India.

Fluorescent aerolysin (FLAER) has been recommended as an important part of antibody panel used for flow cytometric detection of paroxysmal nocturnal hemoglobinuria (PNH) clone. This study was aimed to observe the frequency of PNH-positive clones and their sizes in patients screened for various indications. A retrospective analysis of 624 patients screened over a period of 30 months. Frequency and size of clone sizes noted, and laboratory parameters were compared among different groups of patient being screened. There were 445 adults and 179 pediatric patients. Indications for screening included AA (n = 433), myelodysplastic syndrome (MDS) (n = 34), hemolytic anemia (n = 84), and thrombophilia workup group (n = 63). PNH clones were found in 39.03%, 5.88%, 26.19%, and 1.59% cases, respectively. No significant difference among adult or pediatric population was noted. The bone marrow failure (BMF) group [AA and MDS] with PNH clone had a significantly lower clone size (Median- 2.7%) as compared to classic PNH group (Median-77.2%). Most of the classic PNH cases (78.26%) and a small proportion of AA (9.9%) showed a large clone size (>50%). In spite of having large clone size, there was a significant difference between the median LDH values of these two groups (2511.5 vs 593 U/L). FLAER-based screening detects the presence of PNH clone in a high proportion of AA patients and some MDS patients. These patients usually have a small clone size. Even if they have a large clone, it does not get translated into a high LDH or severe clinical symptoms.

Abnormalities Detected By Array CGH and Fluorescence in Situ Hybridization in AML with Normal Karyotype Lacking Mutations in NPM1, CEBPA, RUNX1 and MLL Partial Tandem Duplications Are Associated with Unfavorable Outcome

Background: AML is a group of genetically distinct entities which have been defined by the presence of certain recurrent, mutually exclusive genetic abnormalities such as AML-specific fusion genes (e.g. PML-RARA, RUNX1-RUNX1T1) or recurrent molecular mutations (NPM1 mutations and CEBPA double mutations (dm)). In addition partial tandem duplications within the MLL gene (MLL-PTD) and RUNX1 mutations seem to play an important role in certain AML subtypes. A subset of AML with normal karyotype lacking the above-mentioned mutations is still poorly characterized. Array CGH and fluorescence in situ hybridization are able to detect abnormalities which are undetectable by chromosome banding analysis either due to a higher resolution, ability to detect copy neutral loss of heterozygosity (CN-LOH) and independence of in vitro proliferation.Aims: 1. Search for submicroscopic copy number changes and cryptic rearrangements in AML with normal karyotype lacking NPM1 and RUNX1 mutations, CEBPA dm, and MLL-PTD. 2. Determine whether submicroscopic cytogenetic changes impact on survival.Patients and Methods: For 1473 AML cases with normal karyotype complete data on mutation status of NPM1, CEBPA, RUNX1 and MLL -PTD was available. Of these 303 cases (21%) did not carry one of these mutations. Out of these 159 cases with de novo AML (median age: 68 years (range: 19-93)) were selected on the basis of availability of material for array CGH (SurePrint G3 ISCA CGH+SNP Microarray, Agilent, Waldbronn, Germany) and FISH screening with probes for MLL, RUNX1, CBFB, NUP98, MECOM/EVI1, NPM1, ETV6 and DEK-NUP214 (MetaSystems, Altlussheim, Germany; ABBOTT, Wiesbaden Germany).Results: In total in 67 of 159 patients (42%) abnormalities were identified by FISH and/or array CGH. In detail, 12 balanced rearrangements were detected by FISH screening involving NUP98 (n=7, in 6 of these a NUP98-NSD1 rearrangement was identified by PCR), MLL (n=2) and MECOM, RUNX1 and CBFB (one each). In addition, 27 gains, 42 losses and 41 copy neutral losses of heterozygosity (CN-LOH) were observed in 58 (37%) patients. Recurrent gains affected regions 6q23.3q23.3 (135.325.751-135.607.060) (n=2) and 8q24.21q24.21 (130.517.732-130.808.381) (n=2) while recurrent losses were found for the regions 21q22.12q22.12 encompassing the RUNX1 gene (36.228.735-36.303.952) (n=5), 5q31.2q31.2 including i.a. EGR1 and CTNNA1 (137.617.569-138.993.959) (n=3), 2q34q34 including i.a. IKZF2 (213.371.237-214.560.488) (n=2), 7q22.1q22.1 encompassing i.a. CUX1 (100.485.221-101.916.623) (n=2), and Yq11.223q12 (24.980.949-28.804.541) (n=2). Recurrent CN-LOH were observed on chromosomes 11q (n=10), 2p (n=5), 4q (n=4), 21q (n=3), 1p (n=2), 17q (n=2) and 18q (n=2). 20/27 gains and 38/42 losses were < 10 megabases in sizes and thus below the resolution of chromosome banding analysis. Only in 7 (4%) patients abnormalities (n=11) were identified which in principle are detectable by chromosome banding analysis. These were missed by chromosome banding analysis due to insufficient in vitro proliferation of the aberrant clone, small clone size or poor chromosome morphology. Comparing age, white blood cell count and bone marrow blast counts revealed no differences between patients with or without abnormalities detected by FISH and/or array CGH. However, FLT3-ITD and WT1 mutations were more frequent in cases with abnormalities (25% vs 7%, p=0.001; 9% vs 1%, p=0.021). Survival analysis was performed for 90 intensively treated patients. Overall survival (OS), event-free survival (EFS) and OS with censoring at time of allogeneic SCT (OSctx) were significantly shorter in patients with abnormalities detected by FISH and/or array CGH compared to those without (median OS: 19 vs 49 months, p=0.027; Figure 1; median EFS: 9 vs 21 months, p=0.016; median OSctx: 13 vs 26 months, p=0.018).Conclusions: 1. AML with normal karyotype based on chromosome banding analysis lacking a disease defining molecular mutation harbor balanced rearrangements, copy number gains and losses as well as CN-LOH at a high frequency (42%). 2. The presence of these abnormalities has a negative impact on survival demonstrating that FISH and array CGH can add prognostic information in the diagnostic work-up of AML with normal karyotype lacking a disease defining mutation. 3. Further investigations of mutations in genes located in regions of recurrent CN-LOH is necessary. [Display omitted] DisclosuresHaferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Zieschang:MLL Munich Leukemia Laboratory: Employment. Schnittger:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Alpermann:MLL Munich Leukemia Laboratory: Employment. Zenger:MLL Munich Leukemia Laboratory: Employment. Perglerová:MLL2 s.r.o.: Employment. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

Observational Study Of PNH Clone Size By High Sensitivity Flow Cytometry In High-Risk Patients InA Large Russian Population

IntroductionParoxysmal nocturnal hemoglobinuria (PNH) is a rare and life-threatening hematopoietic stem cell disease caused by a partial or absolute deficiency of proteins linked to the cell surface membrane via a glycosylphosphatidyl-inositol anchor, which leads to complement-induced intravascular hemolysis mediated via the membrane attack complex. Multiparameter high-sensitivity flow cytometric measurement of PNH clones is the method of choice for the diagnosis of PNH, as recommended by the International Clinical Cytometry Society (ICCS). After publication of the ICCS guidelines, screening of patients considered at high risk of PNH was commenced in Russia. Data are presented on PNH clone size distribution across patients with relevant ICD-10 diagnostic codes (based on patients′ initial assumed diagnoses). MethodsPatients were tested for the presence and size of PNH clones using high-sensitivity flow cytometry across nine laboratories. PNH clone evaluations were performed as described in the ICCS guidelines: CD59/CD235a monoclonal antibodies for RBC; CD45/CD15/CD24/FLAER for granulocytes and; CD45/CD64/CD14/FLAER or CD45/CD33/CD14/FLAER for monocytes. The sensitivity for PNH clone detection was 0.01%. Changes in PNH clone size were evaluated among patients who had follow-up studies after initial measurements. Results1889 patients were assessed between October 2011 and June 2013 (Table 1). Suspected PNH and bone-marrow disorders (AA, MDS, cytopenia) were the most common reasons for PNH testing. The greatest proportions of patients with PNH clones were among those with of an initial assumed diagnosis of AA or PNH. Notably, around 40% of patients with an initial assumed diagnosis of PNH actually had no detectable PNH clones. Most patients with small clone sizes (< 1%) were in the AA, MDS and hemolytic anemia groups. Overall, mean clone sizes were slightly higher in monocytes (31.5%) than in granulocytes (30.1%) across the diagnostic categories. While there was generally a good correlation between clone size measurements in granulocytes and monocytes (linear regression r2 = 0.9851), 10% of PNH-positive patients had detectable clones only in one of these leucocyte populations (i.e. either in monocytes or in granulocytes, but not both). PNH clones in RBCs were generally lower than in granulocytes. Repeat clone size measurements were performed in 316 patients over a mean follow-up period of 7.8 months. In patients with initial clone sizes <50% the PNH clones tended to decrease over time, whereas in patients with initial clone sizes >50%, clones tended to increase. PNH clones were not changed at all in 98 patients at follow-up, among whom 48% were patients with AA. ConclusionThese screening data confirm the utility of high-sensitivity flow cytometry testing in high-risk patient groups to ensure early and accurate diagnosis and to aid in the effective clinical management of patients. Disclosures:Babenko:Alexion: Research Funding. Sipol:Alexion: Research Funding. Borisov:Alexion: Employment. Naumova:Alexion: Research Funding. Boyakova:Alexion: Research Funding. Glazanova:Alexion: Research Funding. Chubukina:Alexion: Research Funding. Pronkina:Alexion: Research Funding. Popov:Alexion: Research Funding. Mustafin:Alexion: Research Funding. Fidarova:Alexion: Honoraria. Lisukov:Alexion: Honoraria. Kulagin:Alexion: Honoraria.