179 research outputs found
Rational management approach to pure red cell aplasia
Pure red cell aplasia is an orphan disease, and as such lacks rationally established standard therapies. Most cases are idiopathic; a subset is antibody-mediated. There is overlap between idiopathic cases and those with T-cell large granular lymphocytic leukemia, hypogammaglobulinemia, and low-grade lymphomas. In each of the aforementioned, the pathogenetic mechanisms may involve autoreactive cytotoxic responses. We selected 62 uniformly diagnosed pure red cell aplasia patients and analyzed their pathophysiologic features and responsiveness to rationally applied first-line and salvage therapies in order to propose diagnostic and therapeutic algorithms that may be helpful in guiding the management of prospective patients, 52% of whom were idiopathic, while the others involved large granular lymphocytic leukemia, thymoma, and B-cell dyscrasia. T-cell-mediated responses ranged between a continuum from polyclonal to monoclonal (as seen in large granular lymphocytic leukemia). During a median observation period of 40 months, patients received a median of two different therapies to achieve remission. Frequently used therapy included calcineurin-inhibitors with a steroid taper yielding a first-line overall response rate of 76% (53/70). Oral cyclophosphamide showed activity, albeit lower than that produced by cyclosporine. Intravenous immunoglobulins were effective both in parvovirus patients and in hypogammaglobulinemia cases. In salvage settings, alemtuzumab is active, particularly in large granular lymphocytic leukemia-associated cases. Other potentially useful salvage options include rituximab, anti-thymocyte globulin and bortezomib. The workup of acquired pure red cell aplasia should include investigations of common pathological associations. Most effective therapies are directed against T-cell-mediated immunity, and therapeutic choices need to account for associated conditions that may help in choosing alternative salvage agents, such as intravenous immunoglobulin, alemtuzumab and bortezomib
SARS-CoV-2 susceptibility and COVID-19 disease severity are associated with genetic variants affecting gene expression in a variety of tissues
Variability in SARS-CoV-2 susceptibility and COVID-19 disease severity between individuals is partly due to
genetic factors. Here, we identify 4 genomic loci with suggestive associations for SARS-CoV-2 susceptibility
and 19 for COVID-19 disease severity. Four of these 23 loci likely have an ethnicity-specific component.
Genome-wide association study (GWAS) signals in 11 loci colocalize with expression quantitative trait loci
(eQTLs) associated with the expression of 20 genes in 62 tissues/cell types (range: 1:43 tissues/gene),
including lung, brain, heart, muscle, and skin as well as the digestive system and immune system. We perform
genetic fine mapping to compute 99% credible SNP sets, which identify 10 GWAS loci that have eight or fewer
SNPs in the credible set, including three loci with one single likely causal SNP. Our study suggests that the
diverse symptoms and disease severity of COVID-19 observed between individuals is associated with variants across the genome, affecting gene expression levels in a wide variety of tissue types
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MICA Polymorphism Identified by Whole Genome Array Constitutes a Disease Predisposition Factor in T-Cell Large Granular Lymphocyte Leukemia
Abstract Large granular lymphocyte leukemia (LGL) is a disease of semiautonomous proliferation of cytotoxic T-cells (CTL) often accompanied by immune cytopenias, particularly neutropenia. LGL related cytopenias have been attributed to LGL cellular cytotoxicity or proapoptotic cytokines rather than intrinsic properties of the neutrophils. The association of LGL with autoimmunity suggests that genetic predisposition may contribute to disease pathogenesis. We studied 69 patients with LGL leukemia using a case-control approach; control populations included ethnically matched healthy individuals (N=82) and disease controls of aplastic anemia (N=48) and kidney transplant recipients (N=48). Initially, we applied the Illumina 12K non-synonymous SNP array to a subcohort of 36 LGL patients and 54 healthy controls (training set). Results were subjected to independent hypothesis-generating biostatistical algorithms. First, Exemplar automated analysis determined disease prediction based on independent χ2 analysis for each SNP. As expected, no SNP in this underpowered study reached Bonferroni corrected statistical significance, but our analysis allowed for ranking based on p-value. Second, Random Forests, a nonparametric tree method was applied, whereby all SNP information was calculated multivariately to predict disease. In a non-Mendelian inherited disease, this method more closely reflects the biology of complex polygenic traits; remarkably, those SNP identified by Random Forest were among the highest ranking SNP by Exemplar. Our initial hypothesis-generating set identified 1 SNP in unknown gene C8orf31 and 4 SNP within the MHC class I related-chain A (MICA) gene. We focused on MICA, a non-peptide presenting, tightly regulated stress response HLA molecule that could play a role in pathogenesis of neutropenia in LGL. To further substantiate our finding, the initial training set results were subjected to technical validation; fidelity was rechecked by PCR genotyping with 93% concordance. Biological validation was determined by confirmation in an independent test set consisting of 33 LGL patients and additional 28 controls. As only limited numbers of SNP were tested, there was no need for α-error adjustment. MICA SNP rs1063635 was found to have the most predictive value in both the training set (PPV=56%, NPV=89%) and test set (PPV=64%, NPV=86%). Overall, the control frequency of this SNP in homozygous form was 12% vs 60% in LGL (p<.01, OR=9.1). MICA alleles have been implicated in autoimmune diseases and malignancies. Although this SNP may not define a particular MICA genotype, it is possible that it is in linkage disequilibrium with genotype-defining polymorphisms. To study the functional consequences of our findings, flow cytometric analysis using anti-MICA antibodies was performed, which identified higher expression of MICA in neutrophils from patients as compared to controls (p=.04). MICA overexpression decreased after immunosuppressive therapy (p<.01). While the mechanism of MICA induction is unknown, we stipulate that the presence of MICA alleles leads to a persistent stimulatory signal in LGL predisposing to clonal outgrowth. In sum, our findings suggest that MICA polymorphisms may represent a predisposition factor in LGL and/or LGL-associated neutropenia
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Comprehensive Identification Of Germline Alterations In Telomerase Complex Genes By Whole Exome Sequencing Of MDS and Related Myeloid Neoplasms
Abstract
In addition to classical familiar forms of bone marrow failure, some cases of aplastic anemia (AA) have been linked to inherited germ line polymorphism/mutations of telomerase machinery, leading to excessive telomere shortening. Germline telomere maintenance machinery mutations have been also been found in a proportion of acute myeloid leukemia (AML) and Myelodysplastic syndromes (MDS) patients (pts). However, the molecular pathogenesis of adult MDS and AML is complex and determination of genetic risk factors in addition to established familial and congenital syndromes has been difficult. To date targeted sequencing has been used for mutational screens with the inherent limitations of limited exome coverage, empiric bias and labor intensity. New generation (NGS) whole genome approaches prioritize somatic mutations as initial discovery targets, but the availability of sequenced cohorts allows also for detection of germline lesions both in a targeted and an unbiased fashion.
Using NGS we studied 136 pts (mean age, 68.8 years, range 41-85) with MDS and related myeloid neoplasms for the presence of non-synonymous polymorphisms (SNV), which could affect telomerase machinery. These genes included TERT, DKC1, SMG6, NOP10, POT1, WRAP53, NHP2, GAR1, TINF2. No somatic defects of the telomerase complex were detected. We focused on novel sequence alterations or those described in available databases with a population allelic frequency of less than 5%. We identified 45 non-synonymous germline sequence alterations in 39 cases (32%). Most frequent SNV were found in TERT (n=15), DKC1 (n=7), SMG6 (n=6), NOP10 (n=4), POT1 (n=4), WRAP53 (n=4), while observations of NHP2 (n=3), GAR1 (n=1), TINF2 (n=1) were less prevalent. These variants were distributed in an almost mutually exclusive manner. Out of 3 variants in TERT, p.H412Y (n=3) and p.A279T (n=9) were reported to be pathogenic in bone marrow failure syndromes. In addition, p.A999T found in 8 cases in our cohort could also be pathogenic since it is less frequent in healthy controls. Similarly, p.441_442del (n=1), located in the N-terminal region, is a completely novel germline variant not detected in 6500 samples publicly available in ESP6500. In the pAML cohort (TCGA; n=197), the observations of germline variants for these telomerase complex genes were SMG6 (n=21), POT1 (n=19), NHP2 (n=1), NOP10 (n=1) GAR1 (n=1).
Next, we analyzed clinical characteristics, including treatment responsiveness as assessed per modified 2006 IWG response criteria. The mean age of the 39 patients with germline telomerase machinery alterations was 67 years, 24% (9/39) were younger (age<60 years) compared to 12% (12/97) of wild type (WT; p=.12). Of note, 58% of these cases had a family history of solid tumors including breast, gastrointestinal and prostate and 8% (3/36) had a family history of myeloid malignancies. 41% (16/39) of the telomerase mutants had higher-risk MDS/sAML at presentation compared to 23% in WT cases (23/97; p=.19). A higher percentage of mutants also had complex cytogenetics compared to WT (35% vs. 13%; p=.01). Response rates to common therapies, including hypomethylating agents were similar, but we noted that none of the carrier cases (n=16) treated with lenalidomide showed therapeutic responses (0% vs. 37%). The mean overall survival of the carrier cases was lower compared to the WT (36 vs. 39 months, p=.10). When we studied cases with telomerase alterations for the presence of coinciding somatic mutations, using a targeted deep sequencing panel of the 100 most common mutations acquired in pts with germline telomerase complex alterations, we found most common the acquisition of DNMT3 (18% vs. 6%, p.10) and cohesin mutations (13% vs. 4%,p=.11).
In sum, unbiased NGS sequencing approaches in MDS and related myeloid neoplasms allowed for identification of genetic germline alterations in telomerase maintenance machinery at higher rates than previously detected using targeted screening approaches, suggesting that such genetic defects may more frequently than previously thought contribute to cryptic and likely complex genetic predisposition to these diseases.
Disclosures:
Makishima: AA & MDS international foundation: Research Funding; Scott Hamilton CARES grant: Research Funding
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Multiple Mechanisms Leading to ARID2 defects in Myeloid Neoplasms
Abstract
Next generation sequencing (NGS) and single nucleotide polymorphism arrays (SNP-A) contribute to more precise identification of the spectrum of somatic mutations and karyotypic abnormalities in myeloid neoplasms. The diversity of individual defects and their combinations corresponds to the tremendous clinical heterogeneity. Identification of key driver genes remains a fundamental component to understanding the immense data generated from this technology and the contributions to leukemogenesis.
In this project, we evaluated 1200 cases of MDS and AML. Somatic mutations of AT rich interactive domain 2 (ARID2) were found in myelodysplastic syndrome (MDS), myeloproliferative neoplasms (MPN), primary acute myeloid leukemia (pAML) or secondary AML (sAML). All ARID2 mutations occurred in either frameshift (at p.S1489 and p.T1645) or nonsense (at p.E65, p.S154 and p.Q1637) configurations, consistent with loss of function. We have identified a total of 5 mutant cases, all of somatic origin. Study of clonal architecture elucidated that ARID2 mutations were ancestral events in 50% of mutant cases as defined by variant allelic frequencies. By SNP-A, a commonly deleted region on chr.12q was defined by mapping minimally affected lesions in 9 patients with MDS, MPN, sAML or pAML. Haploinsufficient expression of ARID2 was confirmed by expression array analysis in the cases with del(12q), which is compatible to the frequent incidence of heterozygous ARID2 loss-of-function mutations. Del(12q) was more frequent in high-risk phenotype with higher blast counts. In addition, significantly lower expression of ARID2 was also observed in 22 out of 183 patients with diploid 12q. Interestingly, amplification of locus was not found in any of the cases studied by SNP-A. Altogether, such lesions of defective ARID2 were pathogenic in more than 10% of cases with myeloid neoplasms.
ARID2 is encoding one of the components of SWI/SNF complex and involved in chromatin remodeling. Therefore, we stipulate that other genes which function together with ARID2 might be affected with somatic mutations or deletions. Further analyses demonstrated the presence of other somatic mutations and deletions affecting SWI/SNF complex, including ACTL6B (N=53) and SMARCD3 (N=66). While SWI/SNF complex lesions were mutually exclusive, concomitant subclonal mutations in such affected cases were commonly observed in RAS pathway genes, including K/NRAS, NF1 and PTPN11. To the contrary, ARID1B, which negatively regulates chromatin remodeling, is predominantly activated in the cohort with similar phenotype. While germline mutations of multiple genes in SWI/SNF complex are reported to be associated with Coffin-Siris syndrome, no family or past history characteristic of this congenital disorder was observed in our cohort.
Further clues into the function of ARID2 in myeloid neoplasms came from studying splicing dysfunction in U2AF1 mutant cases. Deep RNA sequencing in the cases with U2AF1 mutations (p.S43F and p.Q157P), showed a consistent loss of ARID2 exon 8 (predominantly noted in sAML). Two types (whole and partial) of exon skipping led to frameshift in the transcript creating a stop codon. Targeted RT-PCR confirmed the transcriptome sequencing results in primary bone marrow samples of the cases with U2AF1 but not in the corresponding wild-type cases. These results are compatible with the genetic finding that spliceosomal mutations were not concomitantly observed in the cases with SWI/SNF complex defects, suggesting misspliced transcript with nonsense decay consequences is enough pathogenic to preclude additional spliceosomal mutations.
To validate functional consequences of ARID2 loss, knockdown experiment using ARID2-shRNA transduction in K562 and HL60 cell lines were performed. Knockdown of ARID2 generally demonstrated cell cycle arrest in G2 phase prior to entry into the S-phase, which was partly caused by decreased expression of CDKL3 and CCND3. Hb staining with Benzidine showed impairment of erythroid differentiation in ARID2 knockdown K562, which was confirmed by cytological examination.
In sum, multiple mechanisms of defective ARID2 including somatic mutations, haploinsufficiency and phenocopy due to spliceosomal mutations can be involved in ARID2-mediated leukemogenesis. Together with the other components, novel lesions of SWI/SNF complex constitute a group of tumor suppressor genes in myeloid neoplasms.
Disclosures
No relevant conflicts of interest to declare
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Molecular Predictors Of Response To Lenalidomide In Myeloid Malignancies
Abstract
Lenalidomide (LEN) is effective in alleviating anemia in patients with MDS. Patients with del5q show particularly good responsiveness to LEN, but this drug has a significant activity in MDS patients without del5q and those with MDS/MPN and MPN. Currently, apart from del5q no routine molecular biomarkers have been identified to better predict response to LEN. Here, based on ability to perform a broad, unbiased mutational screen using generation sequencing (NGS) we hypothesized that somatic mutational patterns may help identify patients with myeloid malignancies who would benefit from LEN.
To that end, we identified a cohort of 92 patients with myeloid malignancies who were treated with LEN. Response criteria were by the 2006 International Working Group and only those who received at least 3 months of LEN were selected for outcome analysis. Sequencing was performed using targeted NGS for the 60 most common mutated genes (frequency >2%; 87% CI) identified by whole exome sequencing cohort (see another abstract from our group). Our approach focused on previously described and/or confirmed mutations, acknowledging that there may be some false negatives and our spectrum may not be entirely complete. For most of the cases confirmatory targeted Sanger sequencing for the top 19 mutations has been performed, in addition to NGS. For some analyses we also divided mutations into functional gene families; e.g., DNMT family (DNMT1/3A/3B), PRC2 family (EZH2/EED/SUZ12/JARID2/RBBP4/PHF1), IDH family (IDH1/2), RAS family (NRAS/KRAS/HRAS/NF1/RIT1/PTPN11), cohesin, various spliceosomal gene families among others.
Median age was 68 years (range, 20-84); according to IPSS there were 66 were lower (Low and Int-1) and 24 higher risk (Int-2; RAEB1/2 and some sAML) patients. According to cytogenetic risk groups there were 60% low, 18% intermediate, 22% poor risk patients; 31% had del5q. Overall response rate was 61%; rate of CR (including marrow/cytogenetic CR) was 19.5%, PR 5%, any HI 37%, SD 4%; 34% of patient were refractory. The cohort was then dichotomized into “responders” and “refractory,” with responders classified as those achieving CR, PR or any HI for the purpose of this analysis. Response rate was higher among del5q compared to non del5q (69% vs. 57%). Within the whole cohort the most frequently mutated genes were SF3B1 (24%), ASXL1 (18%), TET2 (17%), DNMT3A (14%), RUNX1 (12%), and U2AF1 (10%). At least one of the spliceosomal gene mutations was found in 37 (40%) cases and these seemed to be mutually exclusive.
We compared the mutations frequencies in responders vs. non responders and conversely response within mutant or wild type (WT) cases and prioritized them according to p value for subsequent analyses. For instance, ASXL1 (29% vs. 13%; p= .052), U2AF1 (20% vs. 4%; p= .025), DNMT3A (22% vs. 10%; p= .1), LUC7L2 (12.5% vs. 0%; p= .02), SETBP1 (12% vs. 2%; p= .071) and RAS family (12% vs. 4%; p= .2) mutations seem overrepresented among refractory cases. Albeit rare, mutations in ASXL1, U2AF1, and LUC7L2 were always associated with refractoriness. Among responders, mutations in RUNX1 (17% vs. 6%; p= .2), EZH2 (7% vs. 3%; p= .6), TP53 (6% vs. 3%; p= 1) were more prevalent. SETBP1 mutant cases were also more likely to be refractory. Interestingly, all 3 DDX41 mutant cases were responders. The results were augmented when mutations were grouped according to pathways showing e.g., that mutations in ASXL1/ASXL2 and SRSF2/ZRSR2/LUC7L2 were significantly associated with refractoriness (32%; p= .025 and 26%; p= .009, respectively). We next adopted the algorithm when multiple genes were combined in “either-or” fashion. Such an analysis revealed that patients with mutations in either/or ASXL1/U2AF1 (39% vs. 14%) were significantly associated with refractoriness (p= .007; OR 1.4). Similarly, patients harboring either/or RUNX1/EZH2 (22% vs. 6%) were significantly associated with response (p= .041, OR 3.8). Multivariate analyses confirmed U2AF1 mutation to be associated with refractoriness to LEN (OR 6.64; CI: 1.18-37.34; p= .032). Of importance is that presence of TP53 (4/5 mutant cases were responders) and RAS gene family mutations (4/6 refractory cases), usually associated to refractoriness to conventional chemotherapy, did not preclude response to LEN.
Our results demonstrate that analysis of somatic mutational patterns may be helpful in predicting patients who most likely benefit or those who will be refractory to LEN therapy.
Disclosures:
Makishima: AA & MDS international foundation: Research Funding; Scott Hamilton CARES grant: Research Funding. Sekeres:Celgene: Membership on an entity’s Board of Directors or advisory committees; Amgen: Membership on an entity’s Board of Directors or advisory committees. Maciejewski:Celgene: Honoraria, Speakers Bureau
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