Telomere length and clonal chromosomal alterations in peripheral blood of patients with severe aplastic anaemia.

Severe aplastic anaemia (SAA) is a rare and life-threatening bone marrow failure disorder. We used data from the transplant outcomes in aplastic anaemia study to characterize mosaic chromosomal alterations (mCAs) in the peripheral blood of 738 patients with acquired SAA and evaluate their associations with telomere length (TL) and survival post-haematopoietic cell transplant (HCT). The median age at HCT was 20.4 years (range = 0.2-77.4). Patients with SAA had shorter TL than expected for their age (median TL percentile for age: 35.7th; range <1-99.99). mCAs were detected in 211 patients (28.6%), with chr6p copy-neutral loss of heterozygosity (6p-CNLOH) in 15.9% and chr7 loss in 3.0% of the patients; chrX loss was detected in 4.1% of female patients. Negative correlations between mCA cell fraction and measured TL (r = -0.14, p = 0.0002), and possibly genetically predicted TL (r = -0.07, p = 0.06) were noted. The post-HCT 3-year survival probability was low in patients with chr7 loss (39% vs. 72% in patients with chr6-CNLOH, 60% in patients with other mCAs and 70% in patients with no mCAs; p-log rank = 0.001). In multivariable analysis, short TL (p = 0.01), but not chr7 loss (p = 0.29), was associated with worse post-HCT survival. TL may guide clinical decisions in patients with SAA.

PLCß4 driven by cadmium-exposure during gestation and lactation contributes to cognitive deficits by suppressing PIP2/PLCγ1/CREB/BDNF signaling pathway in male offspring.

The fetus and infants are particularly vulnerable to Cadmium (Cd) due to the immaturity of the blood-brain barrier. In utero and early life exposure to Cd is associated with cognitive deficits. Although such exposure has attracted widespread attention, its gender-specificity remains controversial, and there are no reports disclosing the underlying mechanism of gender­specific neurotoxicity. We extensively evaluated the learning and cognitive functions and synaptic plasticity of male and female rats exposed to maternal Cd. Maternal Cd exposure induced learning and memory deficits in male offspring rats, but not in female offspring rats. PLCß4 was identified as a critical protein, which might be related to the gender­specific cognitive deficits in male rats. The up-regulated PLCß4 competed with PLCγ1 to bind to PIP2, which counteracted the hydrolysis of PIP2 by PLCγ1. The decreased activation of PLCγ1 inhibited the phosphorylation of CREB to reduce BDNF transcription, which consequently resulted in the damage of hippocampal neurons and cognitive deficiency. Moreover, the low level of BDNF promoted AEP activation to induce Aß deposition in the hippocampus. These findings highlight that PLCß4 might be a potential target for the therapy of learning and cognitive deficits caused by Cd exposure in early life.

Hemophagocytic Lymphohistiocytosis Gene Variants in Severe Aplastic Anemia and Their Impact on Hematopoietic Cell Transplantation Outcomes.

Germline genetic testing for patients with severe aplastic anemia (SAA) is recommended to guide treatment, including the use of immunosuppressive therapy and/or adjustment of hematopoietic cell transplantation (HCT) modalities. Hemophagocytic lymphohistiocytosis (HLH) is a life-threatening hyperinflammatory condition often associated with cytopenias with autosomal recessive (AR) or X-linked recessive (XLR) inheritance. HLH is part of the SAA differential diagnosis, and genetic testing may identify variants in HLH genes in patients with SAA. The impact of pathogenic/likely pathogenic (P/LP) variants in HLH genes on HCT outcomes in SAA is unclear. In this study, we aimed to determine the frequency of HLH gene variants in a large cohort of patients with acquired SAA and to evaluate their association(s) with HCT outcomes. The Transplant Outcomes in Aplastic Anemia project, a collaboration between the National Cancer Institute and the Center for International Blood and Marrow Transplant Research, collected genomic and clinical data from 824 patients who underwent HCT for SAA between 1989 and 2015. We excluded 140 patients with inherited bone marrow failure syndromes and used exome sequencing data from the remaining 684 patients with acquired SAA to identify P/LP variants in 14 HLH-associated genes (11 AR, 3 XLR) curated using American College of Medical Genetics and Genomics/Association of Molecular Pathology (ACMG/AMP) criteria. Deleterious variants of uncertain significance (del-VUS) were defined as those not meeting the ACMG/AMP P/LP criteria but with damaging predictions in ≥3 of 5 meta-predictors (BayesDel, REVEL, CADD, MetaSVM, and/or EIGEN). The Kaplan-Meier estimator was used to calculate the probability of overall survival (OS) after HCT, and the cumulative incidence calculator was used for other HCT outcomes, accounting for relevant competing risks. There were 46 HLH variants in 49 of the 684 patients (7.2%). Seventeen variants in 19 patients (2.8%) were P/LP; 8 of these were loss-of-function variants. Among the 19 patients with P/LP HLH variants, 16 (84%) had monoallelic variants in genes with AR inheritance, and 3 had variants in XLR genes. PRF1 was the most frequently affected gene (in 8 of the 19 patients). We found no statistically significant differences in transplantation-related factors between patients with and those without P/LP HLH variants. The 5-year survival probability was 89% (95% confidence interval [CI], 72% to 99%) in patients with P/LP HLH variants and 70% (95% CI, 53% to 85%) in those with del-VUS HLH variants, compared to 66% (95% CI, 62% to 70%) in those without variants (P = .16, log-rank test). The median time to neutrophil engraftment was 16 days for patients with P/LP HLH variants and 18 days in those with del-VUS HLH variants or without variants combined (P = .01, Gray's test). No statistically significant associations between P/LP HLH variants and the risk of acute or chronic graft-versus-host disease were noted. In this large cohort of patients with acquired SAA, we found that 2.8% of patients harbored a P/LP variant in an HLH gene. No negative effects of HLH gene variants on post-HCT survival were noted. The small number of patients with P/LP HLH variants limits the study's ability to provide conclusive evidence; nonetheless, our data suggest that there is no need for special transplantation considerations for patients with SAA carrying P/LP variants.

JAK2 V617F mutation and associated chromosomal alterations in primary and secondary myelofibrosis and post-HCT outcomes.

JAK2 V617F is the most common driver mutation in primary or secondary myelofibrosis for which allogeneic hematopoietic cell transplantation (HCT) is the only curative treatment. Knowledge of the prognostic utility of JAK2 alterations in the HCT setting is limited. We identified all patients with MF who received HCT between 2000 and 2016 and had a pre-HCT blood sample (N = 973) available at the Center of International Blood and Marrow Transplant Research biorepository. PacBio sequencing and single nucleotide polymorphism-array genotyping were used to identify JAK2V617F mutation and associated mosaic chromosomal alterations (mCAs), respectively. Cox proportional hazard models were used for HCT outcome analyses. Genomic testing was complete for 924 patients with MF (634 primary MF [PMF], 135 postpolycythemia vera [PPV-MF], and 155 postessential thrombocytopenia [PET-MF]). JAK2V617F affected 562 patients (57.6% of PMF, 97% of PPV-MF, and 42.6% of PET-MF). Almost all patients with mCAs involving the JAK2 region (97.9%) were JAK2V617-positive. In PMF, JAK2V617F mutation status, allele burden, or identified mCAs were not associated with disease progression/relapse, nonrelapse mortality (NRM), or overall survival. Almost all PPV-MF were JAK2V617F-positive (97%), with no association between HCT outcomes and mutation allele burden or mCAs. In PET-MF, JAK2V617F high mutation allele burden (≥60%) was associated with excess risk of NRM, restricted to transplants received in the era of JAK inhibitors (2013-2016; hazard ratio = 7.65; 95% confidence interval = 2.10-27.82; P = .002). However, allele burden was not associated with post-HCT disease progression/relapse or survival. Our findings support the concept that HCT can mitigate the known negative effect of JAK2V617F in patients with MF, particularly for PMF and PPV-MF.

Cadmium facilitates the formation of large lipid droplets via PLCß2-DAG-DGKε-PA signal pathway in Leydig cells.

Cadmium (Cd) exposure damages the reproductive system. Lipid droplets (LDs) play an important role in steroid-producing cells to provide raw material for steroid hormone. We have found that the LDs of Leydig cells exposed to Cd are bigger than those of normal cells, but the effects on steroidogenesis and its underlying mechanism remains unclear. Using Isobaric tag for relative and absolute quantitation (iTARQ) proteomics, phosphodiesterase beta-2 (PLCß2) was identified as the most significantly up-regulated protein in immature Leydig cells (ILCs) and adult Leydig cells (ALCs) derived from male rats exposed to maternal Cd. Consistent with high expression of PLCß2, the size of LDs was increased in Leydig cells exposed to Cd, accompanied by reduction in cholesterol and progesterone (P4) levels. However, the high PLCß2 did not result in high diacylglycerol (DAG) level, because Cd exposure up-regulated diacylglycerol kinases ε (DGKε) to promote the conversion from DAG to phosphatidic acid (PA). Exogenous PA, which was consistent with the intracellular PA concentration induced by Cd, facilitated the formation of large LDs in R2C cells, followed by reduced P4 level in the culture medium. When PLCß2 expression was knocked down, the increased DGKε caused by Cd was reversed, and then the PA level was decreased to normal. As results, large LDs returned to normal size, and the level of total cholesterol was improved to restore steroidogenesis. The accumulation of PA regulated by PLCß2-DAG-DGKε signal pathway is responsible for the formation of large LDs and insufficient steroid hormone synthesis in Leydig cells exposed to Cd. These data highlight that LD is an important target organelle for Cd-induced steroid hormone deficiency in males.

The distribution and accumulation of the shortest telomeres in telomere biology disorders.

Individuals with telomere biology disorders (TBDs) have very short telomeres, high risk of bone marrow failure (BMF), and reduced survival. Using data from TBD patients, a mean leukocyte Southern blot telomere length (TL) of 5 kilobases (kb) was estimated as the 'telomere brink' at which human survival is markedly reduced. However, the shortest telomere, not the mean TL, signals replicative senescence. We used the Telomere Shortest Length Assay (TeSLA) to tally TL of all 46 chromosomes in blood-derived DNA and examined its relationship with TBDs. Patients (n = 18) had much shorter mean TL (TeSmTL) (2.54 ± 0.41 kb vs. 4.48 ± 0.52 kb, p < 0.0001) and more telomeres <3 kb than controls (n = 22) (70.43 ± 8.76% vs. 33.05 ± 6.93%, p < 0.0001). The proportion of ultrashort telomeres (<1.6 kb) was also higher in patients than controls (39.29 ± 10.69% vs. 10.40 ± 4.09%, p < 0.0001). TeS <1.6 kb was associated with severe (n = 11) compared with non-severe (n = 7) BMF (p = 0.027). Patients with multi-organ manifestations (n = 10) had more telomeres <1.6 kb than those with one affected organ system (n = 8) (p = 0.029). Findings suggest that TBD clinical manifestations are associated with a disproportionately higher number of haematopoietic cell telomeres reaching a telomere brink, whose length at the single telomere level is yet to be determined.

LINC00178 promotes cell growth, migration, and invasion in colorectal cancer in vitro and in vivo.

Background: This study aimed to determine the role of LINC00178 in colorectal cancer (CRC) cell invasion and migration by examining its expression in CRC cells and tissues. Methods: Cancer tissues and corresponding adjacent tissue specimens were collected from 45 patients who experienced radical CRC resection in the hospital from March to September 2021. The expression of LINC00178 was measured in both CRC cells and tissues and normal human colorectal mucosal cells using quantitative fluorescence polymerase chain reaction (QF-PCR). Cell Counting Kit-8 (CCK-8), clonogenic, and transwell assays were used to assess the impact of LINC00178 overexpression or knockdown on the CRC cells invasion and proliferation. In addition, the expression levels of vimentin, E-cadherin, and N-cadherin in CRC cells were determined after either LINC00178 knockdown or overexpression was performed using western blotting. Results: The experiments revealed that LINC00178 was over expressed in CRC cells and tissues. Over-expression of LINC00178 could significantly promote the propagation, clone formation, invasion, and transportation of CRC, whereas knockdown of LINC00178 had the opposite function. When LINC00178 was expressed at high levels, it suppressed the vimentin and N-cadherin expression and prevented the upregulation of E-cadherin. In vivo (nude mouse) studies showed that the over expression of LINC00178 could significantly promote the propagation in CRC cells. Conclusions: LINC00178 is overexpressed in CRC cells and tissues. In vivo and in vitro experiments showed that LINC00178 can significantly promote the propagation of CRC cells, so it may develop a potential biological site for targeted therapy of CRC patients.

Germline-somatic JAK2 interactions are associated with clonal expansion in myelofibrosis.

Myelofibrosis is a rare myeloproliferative neoplasm (MPN) with high risk for progression to acute myeloid leukemia. Our integrated genomic analysis of up to 933 myelofibrosis cases identifies 6 germline susceptibility loci, 4 of which overlap with previously identified MPN loci. Virtual karyotyping identifies high frequencies of mosaic chromosomal alterations (mCAs), with enrichment at myelofibrosis GWAS susceptibility loci and recurrently somatically mutated MPN genes (e.g., JAK2). We replicate prior MPN associations showing germline variation at the 9p24.1 risk haplotype confers elevated risk of acquiring JAK2V617F mutations, demonstrating with long-read sequencing that this relationship occurs in cis. We also describe recurrent 9p24.1 large mCAs that selectively retained JAK2V617F mutations. Germline variation associated with longer telomeres is associated with increased myelofibrosis risk. Myelofibrosis cases with high-frequency JAK2 mCAs have marked reductions in measured telomere length - suggesting a relationship between telomere biology and myelofibrosis clonal expansion. Our results advance understanding of the germline-somatic interaction at JAK2 and implicate mCAs involving JAK2 as strong promoters of clonal expansion of those mutated clones.

Genetic testing in severe aplastic anemia is required for optimal hematopoietic cell transplant outcomes.

Patients with severe aplastic anemia (SAA) can have an unrecognized inherited bone marrow failure syndrome (IBMFS) because of phenotypic heterogeneity. We curated germline genetic variants in 104 IBMFS-associated genes from exome sequencing performed on 732 patients who underwent hematopoietic cell transplant (HCT) between 1989 and 2015 for acquired SAA. Patients with pathogenic or likely pathogenic (P/LP) variants fitting known disease zygosity patterns were deemed unrecognized IBMFS. Carriers were defined as patients with a single P/LP variant in an autosomal recessive gene or females with an X-linked recessive P/LP variant. Cox proportional hazard models were used for survival analysis with follow-up until 2017. We identified 113 P/LP single-nucleotide variants or small insertions/deletions and 10 copy number variants across 42 genes in 121 patients. Ninety-one patients had 105 in silico predicted deleterious variants of uncertain significance (dVUS). Forty-eight patients (6.6%) had an unrecognized IBMFS (33% adults), and 73 (10%) were carriers. No survival difference between dVUS and acquired SAA was noted. Compared with acquired SAA (no P/LP variants), patients with unrecognized IBMFS, but not carriers, had worse survival after HCT (IBMFS hazard ratio [HR], 2.13; 95% confidence interval[CI], 1.40-3.24; P = .0004; carriers HR, 0.96; 95% CI, 0.62-1.50; P = .86). Results were similar in analyses restricted to patients receiving reduced-intensity conditioning (n = 448; HR IBMFS = 2.39; P = .01). The excess mortality risk in unrecognized IBMFS attributed to death from organ failure (HR = 4.88; P < .0001). Genetic testing should be part of the diagnostic evaluation for all patients with SAA to tailor therapeutic regimens. Carriers of a pathogenic variant in an IBMFS gene can follow HCT regimens for acquired SAA.

Differentiation and Maturation Effect of All-Trans Retinoic Acid on Cultured Fetal RPE and Stem Cell-Derived RPE Cells for Cell-Based Therapy.

PURPOSE: Clinical trials using fetal retinal pigment epithelium (fRPE), human embryonic stem cell (hESC)-derived RPE, or human induced pluripotent stem cell (hiPSC)-derived RPE for cell-based therapy for degenerative retinal diseases have been carried out. We investigated the culture-induced changes in passaged fRPE, hESC-RPE, and hiPSC-RPE cells and explored the differentiation and maturation effect of all-trans retinoic acid (ATRA) on cells for manufacturing and screening high-quality RPE cells for clinical transplantation. METHODS: RPE cell lines were set up and the culture-induced changes in subsequent passages caused by manipulating plating density, dissociation method, and repeated passaging were studied by a microscope, real-time quantitative PCR, western blot, and immunofluorescent assays. Gene and protein expression and functional characteristics of RPE cells incubated with ATRA were evaluated. RESULTS: Compared with fRPE, hESC-RPE, and hiPSC-RPE showed decreased gene and protein expression of RPE markers. RPE cells underwent mesenchymal changes showing increased expression of mesenchymal markers including a-SMA, N-cadherin, fibronectin and decreased expression of RPE markers including RPE65, E-cadherin, and ZO-1, as a subsequence of low plating density, inappropriate dissociated method, and repeated passaging. RPE cells treated by ATRA showed increased expression of RPE markers and increased expression of negative complement regulatory proteins (CRPs) and increased transepithelial resistance as well. CONCLUSIONS: Differences in protein and gene expression among three RPE types exist. ATRA can increase RPE markers, CRPs gene expression in fRPE, and stem cell-derived RPE. These can be used to guide the standard of screening RPE cells for clinical translational cell therapy.

CORO1A regulates lipoprotein uptake in Leydig cells exposed to cadmium.

Cadmium (Cd) is one of the most common environmental pollutants, which has a long biological half-life. Maternal Cd-exposure in the natural environment causes steroidogenesis defects resulting in spermatogenesis disorder in male offspring. For better understanding its underlying mechanism, we have employed iTRAQ to screen the differentially expressed protein and found that the expression of CORO1A and Cofilin 1 was up-regulated approximately 2 fold in Leydig cells of maternal Cd-exposure offspring. As the major source of steroid hormone, cholesterol is transported to cells via receptor-mediated endocytosis which relies on the remodel of cytoskeleton, then stores in lipid droplets (LDs). However, few studies have focused on the role of cytoskeleton in abnormal steroidogenesis. This study was performed to explore the role of CORO1A in androgen deficiency caused by Cd exposure and its involvement of low-density lipoprotein (LDL) uptake and effects on LDs. We found that Cd resulted in the up-regulation of CORO1A and Cofilin 1, and down-regulation of Profilin 1 in the testis of male offspring with maternal exposure. The structure of filamentous actin was broken, disordered and even crumpled up in Cd-treated R2C cells. F-actin disassembly led to a low uptake of LDL with a reduced number of LDs, followed by decreased total cholesterol and low progesterone production. When CORO1A was silenced, the expression of Cofilin 1 was down-regulated and Profilin 1 was up-regulated in Cd-treated R2C cells. The filamentous actin was rescued and the integrated cytoskeleton prompted LDL uptake, which resulted in the increased total cholesterol and high progesterone production. These findings highlight the crucial role of CORO1A as a cytoskeleton regulatory protein in steroidogenesis, which may help to better understand Cd-induced steroid hormone deficiency in children.

Prognostic impact of pre-transplant chromosomal aberrations in peripheral blood of patients undergoing unrelated donor hematopoietic cell transplant for acute myeloid leukemia.

To improve risk stratification and treatment decisions for patients with acute myeloid leukemia (AML) undergoing hematopoietic cell transplantation (HCT). We used SNP-array data from the DISCOVeRY-BMT study to detect chromosomal aberrations in pre-HCT peripheral blood (collected 2-4 weeks before the administration of conditioning regimen) from 1974 AML patients who received HCT between 2000 and 2011. All aberrations detected in ≥ 10 patients were tested for their association with overall survival (OS), separately by remission status, using the Kaplan-Meier estimator. Cox regression models were used for multivariable analyses. Follow-up was through January 2019. We identified 701 unique chromosomal aberrations in 285 patients (7% of 1438 in complete remission (CR) and 36% of 536 not in CR). Copy-neutral loss-of-heterozygosity (CNLOH) in chr17p in CR patients (3-year OS = 20% vs. 50%, with and without chr17p CNLOH, p = 0.0002), and chr13q in patients not in CR (3-year OS = 4% vs. 26%, with and without chr13q CNLOH, p < 0.0001) are risk factors for poor survival. Models adjusted for clinical factors showed approximately three-fold excess risk of post-HCT mortality with chr17p CNLOH in CR patients (hazard ratio, HR = 3.39, 95% confidence interval CI 1.74-6.60, p = 0.0003), or chr13q CNLOH in patients not in CR (HR = 2.68, 95% CI 1.75-4.09, p < 0.0001). The observed mortality was mostly driven by post-HCT relapse (HR = 2.47, 95% CI 1.01-6.02, p = 0.047 for chr17p CNLOH in CR patients, and HR = 2.58, 95% CI 1.63-4.08, p < 0.0001 for chr13q CNLOH in patients not in CR. Pre-transplant CNLOH in chr13q or chr17p predicts risk of poor outcomes after unrelated donor HCT in AML patients. A large prospective study is warranted to validate the results and evaluate novel strategies to improve survival in those patients.

Drug-Wide Association Study (DWAS): Challenges and Opportunities.

Cancer risk associations with commonly prescribed medications have been mainly evaluated in hypothesis-driven studies that focus on one drug at a time. Agnostic drug-wide association studies (DWAS) offer an alternative approach to simultaneously evaluate associations between a large number of drugs with one or more cancers using large-scale electronic health records. Although cancer DWAS approaches are promising, a number of challenges limit their applicability. This includes the high likelihood of false positivity; lack of biological considerations; and methodological shortcomings, such as inability to tightly control for confounders. As such, the value of DWAS is currently restricted to hypothesis generation with detected signals needing further evaluation. In this commentary, we discuss those challenges in more detail and summarize the approaches to overcome them by using published cancer DWAS studies, including the accompanied article by Støer and colleagues. Despite current concerns, DWAS future is filled with opportunities for developing innovative analytic methods and techniques that incorporate pharmacology, epidemiology, cancer biology, and genetics.See related article by Støer et al., p. 682.

Regulation of lipid droplets via the PLCß2-PKCα-ADRP pathway in granulosa cells exposed to cadmium.

In steroidogenic cells, steroids are synthesized de novo from cholesterol stored in lipid droplets (LDs). The size of LDs regulated by adipose differentiation-related protein (ADRP) is closely related to cholesterol ester hydrolysis. Many studies reported that cadmium (Cd) had dual effects on steroidogenesis in granulosa cells (GCs). However, the role of LD and its regulation in abnormal steroidogenesis caused by Cd exposure remain unknown. In current study, female rats were exposed to CdCl2 during gestation and lactation, and influence of such exposure was investigated in ovarian GCs of female offspring. The size of LDs was found much smaller than normal in GCs; ADRP was down-regulated and hormone-sensitive lipase (HSL) phosphorylation was increased, followed by up-regulation of steroidogenic acute regulatory protein (StAR) and cholesterol side-chain cleavage enzyme (CYP11A1); the expression of 1-phosphatidylinositol 4,5-bisphosphate phosphodiesterase beta-2 (PLCß2) and protein kinase C alpha type (PKCα) were both decreased accompanying the ADRP down-regulation. This series of events resulted in a high level of progesterone in serum. Similar results were demonstrated in GCs treated with 20 µM CdCl2 for 24 h in vitro. The protein level of ADRP was decreased after gene silencing of PLCß2/PKCα, and the knockdown of PLCß2/PKCα/ADRP led to micro-sized LD formation. We found that Cd exposure down-regulated ADRP by inhibiting the PLCß2-PKCα signaling pathway, reduced the size of LDs, and promoted HSL phosphorylation. StAR and CYP11A1 were both up-regulated following the hydrolysis of cholesterol ester, which led to a high production of progesterone. LD thereby is a target subcellular organelle for Cd to affect steroid hormone synthesis in ovarian GCs. These findings might help to uncover the mechanism of ovarian dysfunction and precocious puberty caused by Cd pollution.

Association of donor IFNL4 genotype and non-relapse mortality after unrelated donor myeloablative haematopoietic stem-cell transplantation for acute leukaemia: a retrospective cohort study.

BACKGROUND: The interferon lambda 4 gene (IFNL4) regulates immune responses by controlling the production of IFNλ4, a type III interferon. We hypothesised that IFNλ4 could play a role in infection clearance or alloreactivity in patients with acute leukaemia who received a myeloablative 10/10 HLA-matched haematopoietic stem-cell transplantation (HSCT). Therefore, we aimed to assess the association between recipient and donor IFNL4 genotype with post-HSCT survival outcomes in patients with acute leukaemia. METHODS: We did a two-stage retrospective cohort study using the Center for International Blood and Marrow Transplant Research (CIBMTR) repository and database, in which nearly all patients underwent the procedure in the USA. We included patients with acute myeloid leukaemia or acute lymphocytic leukaemia, who received a HSCT at any age from an unrelated 10/10 HLA-matched donor, with a myeloablative conditioning regimen, between Jan 1, 2000, and Dec 31, 2008, and had a pre-HSCT recipient or donor blood sample available. The discovery dataset included patients from an existing National Cancer Institute (NCI) cohort of the CIBMTR database, in which donor and recipient IFNL4 polymorphisms (rs368234815, rs12979860, and rs117648444) were genotyped with TaqMan assays. According to their genotype, donors and recipients were categorised into IFNL4-positive, if they had at least one copy of the allele that supports the production of IFNλ4, or IFNL4-null for the analyses. The findings were independently validated with patients from the DISCOVeRY-BMT cohort (validation dataset) with existing Illumina array genotype data. We also did a combined analysis using data from patients included in both the NCI and DISCOVeRY-BMT cohorts. FINDINGS: We assessed 404 patients (who had a HSCT from Jan 9, 2004, to Dec 26, 2008) in the discovery dataset and 1245 patients in the validation dataset (HSCT Jan 7, 2000, to Dec 26, 2008). The combined analysis included 1593 overlapping participants in both cohorts. Donor, but not recipient IFNL4-positive genotype was associated with increased risk of non-relapse mortality (HR 1·60, 95% CI 1·23-2·10; p=0·0005 in the discovery dataset; 1·22, 1·05-1·40; p=0·0072 in the validation dataset; and 1·27, 1·12-1·45; p=0·0001 in the combined dataset). Associations with post-HSCT overall survival were as follows: HR 1·24, 95% CI 1·02-1·51; p=0·034 in the discovery dataset; 1·10, 0·98-1·20; p=0·10 in the validation dataset; and 1·11, 1·02-1·22; p=0·018 in the combined dataset. INTERPRETATION: Prioritising HSCT donors with the IFNL4-null genotype might decrease non-relapse mortality and improve overall survival without substantially limiting the donor pool. If these findings are validated, IFNL4 genotype could be added to the donor selection algorithm. FUNDING: The National Cancer Institute Intramural Research Program. For full funding list see Acknowledgments.

Population Frequency of Fanconi Pathway Gene Variants and Their Association with Survival After Hematopoietic Cell Transplantation for Severe Aplastic Anemia.

Severe aplastic anemia (SAA) is most frequently immune-mediated; however, rare inherited bone marrow failure syndromes, such as Fanconi anemia (FA), may be causal and can present as aplastic anemia (AA). FA is primarily an autosomal recessive disorder caused by the presence of 2 pathogenic variants in a single FA/BRCA DNA repair pathway gene. Patients with SAA often undergo genetic testing during clinical evaluation that may identify single deleterious alleles in FA pathway genes. We quantified the rate of germline single deleterious alleles in 22 FA genes using both a general population database (3234 variants, 125,748 exomes) and in a cohort of patients with SAA undergoing hematopoietic cell transplantation (HCT) (21 variants in 730 patients). The variants were classified as deleterious using in silico tools (REVEL, MetaSVM, VEP) and database resources (ClinVar, LOVD-FA). We found similar rates of single deleterious alleles in FA genes in both groups (2.6% and 2.9%). The presence of a single deleterious variant in a gene for FA in SAA HCT recipients did not affect the overall survival after HCT (hazard ratio, 0.85; 95% CI, 0.37 to 1.95; P  = 0.71), or post-HCT cancer risk (P = 0.52). Our results demonstrate that the identification of a germline monoallelic deleterious variant in an FA gene in patients with idiopathic SAA does not influence the outcome of HCT. Our findings suggest that there is no need for special treatment considerations for patients with SAA and a single deleterious FA allele identified on genetic testing.

Pre-transplant short telomeres are associated with high mortality risk after unrelated donor haematopoietic cell transplant for severe aplastic anaemia.

Telomeres are essential for chromosomal stability and markers of biological age. We evaluated the effect of pre-transplant short (<10th percentile-for-age) or very short (<5th or <1st percentile-for-age) leucocyte telomere length on survival after unrelated donor haematopoietic cell transplantation (HCT) for acquired severe aplastic anaemia (SAA). Patient pre-transplant blood samples and clinical data were available at the Center for International Blood and Marrow Transplant Research. We used quantitative real time polymerase chain reaction to measure relative telomere length (RTL) in 490 SAA patients who received HCT between 1990 and 2013 (median age = 20 years). One hundred and twelve patients (22·86%) had pre-HCT RTL <10th percentile-for-age, with the majority below the 5th percentile (N = 80, 71·43%). RTL <10th percentile-for-age was associated with a higher risk of post-HCT mortality (hazard ratio [HR] = 1·78, 95% confidence interval [CI]=1·18-2·69, P = 0·006) compared with RTL ≥50th percentile; no survival differences were noted in longer RTL categories (P > 0·10). Time-dependent effects for post-HCT mortality were only observed in relation to very short RTL; HR comparing RTL <5th versus ≥5th percentile = 1·38, P = 0·15 for the first 12 months after HCT, and HR = 3·91, P < 0·0001, thereafter, P-heterogeneity = 0·008; the corresponding HRs for RTL <1st versus ≥1st percentile = 1·29, P = 0·41, and HR = 5·18, P < 0·0001, P-heterogeneity = 0·005. The study suggests a potential role for telomere length in risk stratification of SAA patients in regard to their HCT survival.

Diabetes, metformin and cancer risk in myotonic dystrophy type I.

Myotonic dystrophy type I (DM1) is an autosomal dominant multisystem disorder characterized by myotonia and muscle weakness. Type 2 diabetes (T2D) and cancer have been shown to be part of the DM1 phenotype. Metformin, a well-established agent for the management of T2D, is thought to have cancer-preventive effects in the general population. In our study, we aimed to assess the association between T2D, metformin use and the risk of cancer in DM1 patients. We identified a cohort of 913 DM1 patients and an age-, sex- and clinic-matched cohort of 12,318 DM1-free controls from the UK Clinical Practice Research Datalink, a large primary care records database. We used Cox regression models to assess cancer risk in T2D patients who were metformin users or nonusers compared to patients without T2D. Separate analyses were conducted for DM1 patients and controls. T2D was more prevalent in DM1 than in controls (8% vs. 3%, p < 0.0001). DM1 patients with T2D, compared to those without T2D, were more likely to develop cancer (hazard ratio [HR] = 3.60, 95% confidence interval [CI] = 1.18-10.97; p = 0.02), but not if they were treated with metformin (HR = 0.43, 95% CI = 0.06-3.35; p = 0.42). Among controls, we observed no significant associations between T2D and cancer risk in either users or nonusers of Metformin (HR = 1.28, 95% CI = 0.91-1.79; p = 0.16 and HR = 1.13, 95% CI = 0.72-1.79; p = 0.59, respectively). These results show an association between T2D and cancer risk in DM1 patients and may provide new insights into the possible benefits of Metformin use in DM1.

Relationship between plasma cell-free DNA (cfDNA) and prognosis of TACE for primary hepatocellular carcinoma.

BACKGROUND: Our study aims to investigate changes in cell-free DNA (cfDNA) concentration and integrity in primary hepatocellular carcinoma (PHC) patients before and after transcatheter arterial chemoembolization (TACE) treatment and their influence on the evaluation of prognosis of the disease. METHODS: A total of 84 PHC patients admitted to the Affiliated Hospital of Nanjing University of Chinese Medicine from December 2016 to December 2017 were included as the study group, while 55 healthy people served as the control group. Plasma cfDNA concentration and integrity were determined using qRT-PCR. The correlation between cfDNA concentration/integrity and clinical characteristics of PHC patients were analyzed. A ROC curve was used to investigate the sensitivity and specificity of cfDNA as detection indices. Univariate and multivariate analyses were used to analyze factors affecting recurrence in PHC patients and compare recurrence-free survival (RFS) of PHC patients with high cfDNA expression and low cfDNA expression. RESULTS: Plasma cfDNA concentration and integrity were significantly higher in PHC patients before TACE treatment than in healthy people and significantly lower after treatment than before (P<0.05). The cfDNA concentration was significantly correlated with tumor size, lymph node metastasis, TNM stage, and BCLC stage, while cfDNA integrity was significantly correlated with tumor size, TNM stage, and BCLC stage (P<0.05). ROC results showed that the area under the curve (AUC) value of cfDNA concentration was the largest, with an optimal cut-off of 10.51 ng/mL. Multivariate regression analysis for COX showed that the TNM stage, cfDNA concentration, and AFP were independent risk factors that affected PHC patients' survival. CONCLUSIONS: Plasma cfDNA concentration in PHC patients is more sensitive and specific than any other tumor marker. It is an independent risk factor for PHC patients treated with TACE. Therefore, it is hypothesized cfDNA is a potential biomarker for prognostic evaluation of PHC patients treated with TACE.