Publications by authors named "Anna Schuh"

129 Publications

Comparison of changes in corneal volume and corneal thickness after myopia correction between LASIK and SMILE.

PLoS One 2021 4;16(5):e0250700. Epub 2021 May 4.

Department of Ophthalmology, Ludwig-Maximilians-University, Munich, Germany.

Myopia is the most common refractive error. Surgical correction with laser is possible. LASIK and SMILE are the techniques currently most used. Aim of the study was to compare changes in corneal volume and thickness after the respective laser treatment. 104 eyes of 52 patients were matched based on refractive error into two equally sized groups, either treated with LASIK or SMILE. Measurements were obtained from the Scheimpflug camera (Pentacam) preoperatively and at 3 and 12 months postoperatively. 3 months postoperatively, the flapless SMILE procedure resulted in a significant overall greater loss of corneal volume (P < 0.01) and corneal thickness (P < 0.01) compared to LASIK. No significant difference was found when comparing the 3 to 12-months values in each group. Within the currently used ranges of refractive error correction, loss in central corneal thickness and corneal volume with SMILE is higher in comparison to LASIK. As greater loss in corneal volume and thickness might contribute to higher level of corneal instability maximum ranges of refractive error correction with SMILE should not supersede those set currently for LASIK until more long-term results on corneal ectasia are available for SMILE.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0250700PLOS
May 2021

TET2 drives 5hmc marking of GATA6 and epigenetically defines pancreatic ductal adenocarcinoma transcriptional subtypes.

Gastroenterology 2021 Apr 26. Epub 2021 Apr 26.

Department of Oncology, University of Oxford, OX3 7DQ. Electronic address:

Background And Aims: Pancreatic ductal adenocarcinoma (PDAC) is characterised by advanced disease stage at presentation, aggressive disease biology and resistance to therapy resulting in extremely poor five-year survival <10%. PDAC is classified into transcriptional subtypes with distinct survival characteristics, although how these arise is not known. Epigenetic deregulation, rather than genetics, has been proposed to underpin progression but exactly why is unclear and hindered by technical limitations of analysing clinical samples.

Methods: Genome-wide epigenetic mapping of DNA modifications 5-methylcytosine (5mc) and 5-hydroxymethylcytosine (5hmc) using oxidative bisulphite sequencing (oxBS) from formalin embedded sections. Bioinformatics using iCluster and mutational profiling to identify overlap with transcriptional signatures in FFPE from resected patients and confirmation in vivo.

Results: We find that aggressive squamous-like PDAC subtypes result from epigenetic inactivation of loci including GATA6 that promote differentiated classical-pancreatic subtypes. We show that squamous-like PDAC transcriptional subtypes are associated with greater loss of 5hmc due to reduced expression of the 5mc-hydroxylase TET2. Furthermore, we find that SMAD4 directly supports TET2 levels in classical-pancreatic tumors and loss of SMAD4 expression is associated reduced 5hmc, GATA6 and squamous-like tumors. Importantly, enhancing TET2 stability using Metformin and VitaminC/ascorbic acid (AA) restores 5hmc and GATA6 levels, reverting squamous-like tumor phenotypes and WNT-dependence in vitro and in vivo.

Conclusions: We identify epigenetic deregulation of pancreatic differentiation as an underpinning event behind the emergence of transcriptomic subtypes in PDAC. Our data shows that restoring epigenetic control increases biomarkers of classical-pancreatic tumors which are associated with improved therapeutic responses and survival.
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http://dx.doi.org/10.1053/j.gastro.2021.04.044DOI Listing
April 2021

Short and long-read genome sequencing methodologies for somatic variant detection; genomic analysis of a patient with diffuse large B-cell lymphoma.

Sci Rep 2021 Mar 19;11(1):6408. Epub 2021 Mar 19.

Wellcome Centre for Human Genetics, University of Oxford, Oxford, UK.

Recent advances in throughput and accuracy mean that the Oxford Nanopore Technologies PromethION platform is a now a viable solution for genome sequencing. Much of the validation of bioinformatic tools for this long-read data has focussed on calling germline variants (including structural variants). Somatic variants are outnumbered many-fold by germline variants and their detection is further complicated by the effects of tumour purity/subclonality. Here, we evaluate the extent to which Nanopore sequencing enables detection and analysis of somatic variation. We do this through sequencing tumour and germline genomes for a patient with diffuse B-cell lymphoma and comparing results with 150 bp short-read sequencing of the same samples. Calling germline single nucleotide variants (SNVs) from specific chromosomes of the long-read data achieved good specificity and sensitivity. However, results of somatic SNV calling highlight the need for the development of specialised joint calling algorithms. We find the comparative genome-wide performance of different tools varies significantly between structural variant types, and suggest long reads are especially advantageous for calling large somatic deletions and duplications. Finally, we highlight the utility of long reads for phasing clinically relevant variants, confirming that a somatic 1.6 Mb deletion and a p.(Arg249Met) mutation involving TP53 are oriented in trans.
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http://dx.doi.org/10.1038/s41598-021-85354-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7979876PMC
March 2021

Molecular response to imatinib in patients with chronic myeloid leukemia in Tanzania.

Blood Adv 2021 Mar;5(5):1403-1411

Department of Hematology and Blood Transfusion, Muhimbili University of Health and Allied Sciences, Dar es Salaam, Tanzania.

Imatinib is the mainstay of treatment of patients with chronic myeloid leukemia (CML) in Tanzania. Monitoring molecular response to therapy by real-time polymerase chain reaction at defined milestones is necessary for early detection of treatment failure. However, this assay is not routinely performed in Tanzania; therefore, the depth of molecular response among patients with CML is not known. A total of 158 patients with previously diagnosed CML who received imatinib treatment were recruited from January 2019 and followed up through October 2020 at Ocean Road Cancer Institute. Information was obtained at the time of diagnosis and follow-up. Blood samples were collected in EDTA tubes to measure the BCR/ABL ratio on the Gene Xpert system for molecular response determination. The median age of the 158 adult patients was 45 years (range, 18-86). By reference to established treatment milestones, only 37 (23.4%) achieved optimal molecular response. Signs of advanced-stage disease, in particular the need for red cell transfusions before diagnosis (adjusted odds ratio [AOR], 3.4; 95% CI, 1.32-9.17) and cytopenias (AOR, 2.26; 95% CI, 1.03-4.96) necessitating drug interruptions were statistically validated predictors of treatment failure on multivariate, multinomial logistic regression. Patient survival at the 22-month follow-up was lowest, with 78.6% (95% CI, 69.4-85.4) in the failure-to-respond category and highest in patients achieving optimal response 97.0% (95% CI, 80.9-99.6). In summary, the majority of patients with CML treated with imatinib in Tanzania do not obtain deep molecular response. This outcome can be attributed to late diagnosis, the development of cytopenias requiring multiple drug interruptions, and poor adherence to treatment.
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http://dx.doi.org/10.1182/bloodadvances.2020002973DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7948290PMC
March 2021

Effect of IPL in Patients with Meibomian Gland Dysfunction.

Klin Monbl Augenheilkd 2021 Feb 4. Epub 2021 Feb 4.

Department of Ophthalmology, Ludwig-Maximilians-University, München, Germany.

Purpose: To evaluate the effect of IPL (intense pulsed light) treatment in patients with meibomian gland dysfunction (MGD).

Methods: Clinical data of 25 patients with MGD who underwent IPL treatment at the department of ophthalmology of Ludwig-Maximilians-University between 2016 and 2018 were analyzed. Demographics, clinical history, examination findings (eyelid vascularization, meibomian gland findings, conjunctival redness, tear film break-up time [TFBUT], corneal staining (Oxford grading scale [OGS]), and subjective patients' findings (including ocular surface disease index [OSDI]) were collected from each visit (D1, D15, D45, D75).

Results: All included patients underwent three sessions of IPL treatment in both eyes (D1, D15, D45). There was a significant improvement after IPL treatment (D75) in TFBUT (p < 0.001), corneal staining (OGS) (p < 0.001), conjunctival redness (p < 0.001), lid margin edema (p < 0.001) and redness (p < 0.001), meibum quality (p < 0.001), lid margin telangiectasia (p = 0.005), meibomian gland obstruction (p = 0.001), and OSDI score (p = 0.004). Even after the first IPL session, significant improvements in TFBUT (p < 0.001), corneal staining (OGS p < 0.001), conjunctival redness (p < 0.022), lid margin edema (p < 0.001) and redness (p < 0.016), meibum quality (p = 0.014), and OSDI score (p < 0.013) were noted. There were no relevant negative side effects. Subgroup analysis for age, sex, duration or severity of disease, and associated diagnosis of rosacea showed no significant difference in effectiveness.

Conclusion: IPL is an effective and safe treatment for patients with MGD, which can be used as a supportive therapeutic option.
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http://dx.doi.org/10.1055/a-1333-3032DOI Listing
February 2021

Genome-wide association study identifies risk loci for progressive chronic lymphocytic leukemia.

Nat Commun 2021 01 28;12(1):665. Epub 2021 Jan 28.

Centre for Atherothrombosis and Metabolic Disease, Hull York Medical School, Hull, UK.

Prognostication in patients with chronic lymphocytic leukemia (CLL) is challenging due to heterogeneity in clinical course. We hypothesize that constitutional genetic variation affects disease progression and could aid prognostication. Pooling data from seven studies incorporating 842 cases identifies two genomic locations associated with time from diagnosis to treatment, including 10q26.13 (rs736456, hazard ratio (HR) = 1.78, 95% confidence interval (CI) = 1.47-2.15; P = 2.71 × 10) and 6p (rs3778076, HR = 1.99, 95% CI = 1.55-2.55; P = 5.08 × 10), which are particularly powerful prognostic markers in patients with early stage CLL otherwise characterized by low-risk features. Expression quantitative trait loci analysis identifies putative functional genes implicated in modulating B-cell receptor or innate immune responses, key pathways in CLL pathogenesis. In this work we identify rs736456 and rs3778076 as prognostic in CLL, demonstrating that disease progression is determined by constitutional genetic variation as well as known somatic drivers.
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http://dx.doi.org/10.1038/s41467-020-20822-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7843618PMC
January 2021

Refractive and Visual Outcome of Misaligned Toric Intraocular Lens After Operative Realignment.

Am J Ophthalmol 2021 04 10;224:150-157. Epub 2020 Dec 10.

Department of Ophthalmology, Goethe University, Frankfurt am Main, Frankfurt am Main, Germany; Department of Ophthalmology, Ludwig-Maximilians-University, Munich, München, Germany.

Purpose: The study was performed to evaluate the refractive and visual outcome of patients with misaligned toric intraocular lenses (IOLs) after operative realignment, with and without back-calculation of the toric axis after implantation of the IOL.

Design: Institutional, retrospective case-control study.

Methods: This is a retrospective case series of 39 patients who underwent a second operation to realign a misaligned toric IOL from August 2013 to December 2019 at the Department of Ophthalmology, Goethe University, Frankfurt, Germany. Ideal toric axis was calculated using the back-calculator astigmatismfix.com.

Results: The study consists of 39 treated eyes (20 [51%] right eyes). The toric IOLs showed a postoperative misalignment of 25.69 ± 26.06°. Postrotational, uncorrected distance visual acuity (UDVA) improved from 0.39 ± 0.29 logMAR to 0.27 ± 0.18 logMAR. Refractive outcome showed a reduction of residual sphere and cylinder. The postoperative UDVA when performing alignment to the preoperative calculated axis (51%) was 0.24 ± 0.16 logMAR with a cylinder of 0.90 ± 0.90 diopter (D). In the group with alignment to a back-calculated axis (49%), the UDVA was 0.32 ± 0.20 logMAR with a cylinder of 0.76 ± 0.72 D. High cylinder power IOLs (≥2 D) showed a higher decrease in residual cylinder when back-calculation was performed than low cylinder power IOLs (<2 D) (27% vs 9%). The mean spherical equivalent prediction error of the back-calculator was 0.54 ± 0.55 D.

Conclusion: Realignment of misaligned toric IOLs improves visual acuity and reduces residual refractive errors. Especially for high cylinder power IOLs, better refractive outcome can be seen when performing a back-calculation before realignment.
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http://dx.doi.org/10.1016/j.ajo.2020.11.024DOI Listing
April 2021

Twelve Cases of Hb Manitoba [α102(G9)Ser→Arg]: the Fluctuation in the Variant Expression.

Hemoglobin 2020 Nov 29;44(6):442-445. Epub 2020 Nov 29.

National Haemoglobinopathy Reference Laboratory, Oxford Radcliffe Hospitals National Health Service Trust, Oxford, Oxfordshire, UK.

Hb Manitoba [α102(G9)Ser→Arg] is a rare α chain variant with diverse ethnic origins. It is mildly unstable with an expression of around 10.0-14.2% in the heterozygous state in most literature. In this study, 12 cases of Hb Manitoba [11 cases carried Hb Manitoba II (: c.309C>A) and one case carried Hb Manitoba IV (: c.307A>C)] were detected during a wide-spectrum study of α chain variants in the UK. Fluctuation in variant expression from 6.9 to 15.2% of total Hb on high performance liquid chromatography (HPLC) would pose a diagnostic dilemma in routine laboratories. Focusing on the variant expression, the median of Hb Manitoba was around 11.5% of total Hb in three cases, apparently with normal hemoglobin (Hb), and normal red blood cell (RBC) indices. Two cases showed a higher expression (13.9 and 15.2%) and five cases showed a lower expression (6.9-9.9%). The common α-thalassemia (α-thal) -α (rightward) deletion coexisted with one case of increased Hb Manitoba expression. Iron (or other nutrient) deficiency was likely the cause of decreased Hb Manitoba percentage in this study. The α73(EF2)Val→Val (α2) (: c.222G>T) polymorphism is published for the first time and coexisted with two cases. The Cap +14 (C>G) (: c.-24C>G) polymorphism coexisted with another case in a heterozygous state. In conclusion, the fluctuation in variant expression can cause a diagnostic dilemma, especially in routine laboratories. Screening for the common -α deletion and iron deficiency is recommended when an α chain variant is suspected.
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http://dx.doi.org/10.1080/03630269.2020.1850473DOI Listing
November 2020

Genomic and transcriptomic correlates of Richter's transformation in Chronic Lymphocytic Leukemia.

Blood 2020 Nov 18. Epub 2020 Nov 18.

University of Oxford, Oxford, United Kingdom.

The transformation of chronic lymphocytic leukemia (CLL) to high-grade B-cell lymphoma is known as Richter's Syndrome (RS) and it is a rare event with dismal prognosis. In this study, we conducted whole genome sequencing (WGS) of paired circulating CLL (PB-CLL) and RS biopsies (tissue-RS) from 17 clinical trial (CHOP-O) patients. We found that tissue-RS was enriched for mutations in poor-risk CLL drivers and genes in the DNA damage response (DDR) pathway. In addition, we identified genomic aberrations not previously implicated in RS, including the protein tyrosine phosphatase receptor (PTPRD) and tumour necrosis factor receptor associated factor three (TRAF3). In the non-coding genome, we discovered AID-related and unrelated kataegis in tissue-RS affecting regulatory regions of key immune regulatory genes. These include BTG2, CXCR4, NFATC1, PAX5, NOTCH-1, SLC44A5, FCRL3, SELL, TNIP2 andTRIM13. Furthermore, differences between the global mutation signatures of pairs of PB-CLL and tissue-RS samples implicate DDR as the dominant mechanism driving transformation. Pathway-based clonal de-convolution analysis showed that genes in the MAPK and DDR pathways demonstrate high clonal expansion probability. Direct comparison of nodal-CLL and tissue-RS pairs from an independent cohort confirmed differential expression of the same pathways by RNA expression profiling. Our integrated analysis of WGS and RNA expression data significantly extends previous targeted approaches, which were limited by the lack of germline samples, and it facilitates the identification of novel genomic correlates implicated in RS transformation, which could be targeted therapeutically. Our results inform the future selection of investigative agents for a UK clinical platform study (NCT03899337).
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http://dx.doi.org/10.1182/blood.2020005650DOI Listing
November 2020

Comparison of variables measured with a Scheimpflug device for evaluation of progression and detection of keratoconus.

Sci Rep 2020 11 9;10(1):19308. Epub 2020 Nov 9.

Department of Ophthalmology, Ludwig-Maximilians University, Munich, Germany.

Keratoconus is a progressive ectatic corneal disorder, which can result in severe visual impairment. The new ABCD keratoconus classification system allows differentiated description of the disease. Aim of the study was to evaluate the components of this novel staging system (ARC, PRC, thinnest pachymetry) as well as topometric indices, deviation of normality indices, and other parameters in terms of repeatability and reliability. 317 eyes with keratoconus were examined twice with a Scheimpflug device (Pentacam, Oculus). Bland Altman analysis and intraclass correlations were carried out to evaluate the parameters repeatability and reliability. Apart from IHA (ICC = 0.730), all parameters showed excellent reliability (ICC > 0.900). ARC, PRC, thinnest pachymetry, Kmax, CKI, KI, Rmin, and Progression Avg were the best repeatable parameters with relative repeatability values < 2.5%. Other parameters performing well in terms of repeatability were IHD, ISV, IVA, and final D (RR < 13%). Regression analysis revealed consistently high repeatability along all stages of keratoconus for PRC, thinnest pachymetry, and CKI. All parameters of the ABCD staging system showed excellent reliability and repeatability, PRC and thinnest pachymetry even at all stages of keratoconus and can consequently be reliably used in the determination of keratoconus progression.
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http://dx.doi.org/10.1038/s41598-020-76020-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7652832PMC
November 2020

Fifteen Cases of Hb J-Meerut: The Rare Association with Hb E and/or : c.-24C>G (or ) Variants.

Hemoglobin 2020 Sep 14;44(5):364-367. Epub 2020 Sep 14.

National Haemoglobinopathy Reference Laboratory, Oxford Radcliffe Hospitals National Health Service Trust, Oxford, UK.

Hb J-Meerut [: c.362C>A (or )] is a rare, stable, nonpathogenic α-globin gene variant that peaks in the area between the P3 and A windows on high performance liquid chromatography (HPLC). Few cases from different ethnic origins have been published but the majority were Asian Indians. Coinheritance with other hemoglobin (Hb) variants are rarer and can change the Hb J-Meerut phenotype making a diagnostic dilemma. In this study, we have reported 15 cases of Hb J-Meerut, discovered during a wide spectrum study of α-globin chain variants in the UK. The diagnosis was confirmed by forward and reverse DNA sequencing of the α1- and α2-globin genes. The average of the Hb J-Meerut expression was 20.9% of total Hb and characterized by a retention time (RT) of 1.9 min. (on average) on HPLC. The median of isoelectric focusing (IEF) was 5.6 mm above Hb A. Among the 15 cases studied, one case coinherited the Hb E (: c.79G>A) mutation in heterozygosity and another case was associated with the Cap +14 (C>G) [: c.-24C>G (or )] variant. We noticed that the coinheritance of the Hb E mutation reduced the Hb J-Meerut expression with the formation of a hybrid peak missed on the HPLC chromatograph. We also noticed an increased expression of Hb J-Meerut in the case showing the coinheritance of the : c.-24C>G (or ) variant.
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http://dx.doi.org/10.1080/03630269.2020.1817755DOI Listing
September 2020

A statistical approach for tracking clonal dynamics in cancer using longitudinal next-generation sequencing data.

Bioinformatics 2021 04;37(2):147-154

Department of Oncology, University of Oxford, Oxford, OX3 7DQ, UK.

Motivation: Tumours are composed of distinct cancer cell populations (clones), which continuously adapt to their local micro-environment. Standard methods for clonal deconvolution seek to identify groups of mutations and estimate the prevalence of each group in the tumour, while considering its purity and copy number profile. These methods have been applied on cross-sectional data and on longitudinal data after discarding information on the timing of sample collection. Two key questions are how can we incorporate such information in our analyses and is there any benefit in doing so?

Results: We developed a clonal deconvolution method, which incorporates explicitly the temporal spacing of longitudinally sampled tumours. By merging a Dirichlet Process Mixture Model with Gaussian Process priors and using as input a sequence of several sparsely collected samples, our method can reconstruct the temporal profile of the abundance of any mutation cluster supported by the data as a continuous function of time. We benchmarked our method on whole genome, whole exome and targeted sequencing data from patients with chronic lymphocytic leukaemia, on liquid biopsy data from a patient with melanoma and on synthetic data and we found that incorporating information on the timing of tissue collection improves model performance, as long as data of sufficient volume and complexity are available for estimating free model parameters. Thus, our approach is particularly useful when collecting a relatively long sequence of tumour samples is feasible, as in liquid cancers (e.g. leukaemia) and liquid biopsies.

Availability And Implementation: The statistical methodology presented in this paper is freely available at github.com/dvav/clonosGP.

Supplementary Information: Supplementary data are available at Bioinformatics online.
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http://dx.doi.org/10.1093/bioinformatics/btaa672DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8055230PMC
April 2021

[Alliance of Young Doctors - Shaping the future of the healthcare system].

Ophthalmologe 2020 09;117(9):945-946

Augenklinik, Klinikum der Universität München, Campus Innenstadt, München, Deutschland.

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http://dx.doi.org/10.1007/s00347-020-01180-9DOI Listing
September 2020

Comparative analysis of somatic variant calling on matched FF and FFPE WGS samples.

BMC Med Genomics 2020 07 6;13(1):94. Epub 2020 Jul 6.

Department of Plant Biotechnology and Bioinformatics, Department of Information Technology, IDLab, imec, iGent Toren, Ghent, Belgium.

Background: Research grade Fresh Frozen (FF) DNA material is not yet routinely collected in clinical practice. Many hospitals, however, collect and store Formalin Fixed Paraffin Embedded (FFPE) tumor samples. Consequently, the sample size of whole genome cancer cohort studies could be increased tremendously by including FFPE samples, although the presence of artefacts might obfuscate the variant calling. To assess whether FFPE material can be used for cohort studies, we performed an in-depth comparison of somatic SNVs called on matching FF and FFPE Whole Genome Sequence (WGS) samples extracted from the same tumor.

Methods: Four variant callers (i.e. Strelka2, Mutect2, VarScan2 and Shimmer) were used to call somatic variants on matching FF and FFPE WGS samples from a metastatic prostate tumor. Using the variants identified by these callers, we developed a heuristic to maximize the overlap between the FF and its FFPE counterpart in terms of sensitivity and precision. The proposed variant calling approach was then validated on nine matched primary samples. Finally, we assessed what fraction of the discrepancy could be attributed to intra-tumor heterogeneity (ITH), by comparing the overlap in clonal and subclonal somatic variants.

Results: We first compared variants between an FF and an FFPE sample from a metastatic prostate tumor, showing that on average 50% of the calls in the FF are recovered in the FFPE sample, with notable differences between callers. Combining the variants of the different callers using a simple heuristic, increases both the precision and the sensitivity of the variant calling. Validating the heuristic on nine additional matched FF-FFPE samples, resulted in an average F1-score of 0.58 and an outperformance of any of the individual callers. In addition, we could show that part of the discrepancy between the FF and the FFPE samples can be attributed to ITH.

Conclusion: This study illustrates that when using the correct variant calling strategy, the majority of clonal SNVs can be recovered in an FFPE sample with high precision and sensitivity. These results suggest that somatic variants derived from WGS of FFPE material can be used in cohort studies.
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http://dx.doi.org/10.1186/s12920-020-00746-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7336445PMC
July 2020

A Wide Spectrum Study of α-Globin Chain Variants: Cases from the UK.

Hemoglobin 2020 May 29;44(3):195-200. Epub 2020 Jun 29.

National Haemoglobinopathy Reference Laboratory, Oxford Radcliffe Hospitals National Health Service Trust, Oxford, UK.

Over many years, cases of suspected α-globin chain variants were collected from different parts of the UK. The suspicion was based on the clinical picture, high performance liquid chromatography (HPLC) variant percentage, retention time (RT) and isoelectric focusing (IEF). DNA sequencing and the restriction enzyme EI were used for definitive diagnosis. One hundred and forty-eight variants were confirmed on one or both of the two α-globin genes (, ). These cases were identified as 46 different α-globin chain variants. The most common variants were Hb J-Meerut [: c.362C>A (or )] (10.1%) and Hb Q-India (: c.193G>C) (8.1%), followed by Hb J-Paris-I [: c.38C>A (or )] and Hb Manitoba II (: c.309C>A) (7.4% for each). Other α variants were detected at lower frequencies. Two novel alleles were also detected: Hb Walsgrave [α116(GH4)Glu→Val (: c.350A>T)] and Hb Coombe Park [α127(H10)Lys→Glu (: c.382A>G)]. The majority of the ethnic origin was Indian. The positive predictive value for α variant identification by HPLC-RT analysis was 65.9%, 41.9% by IEF, and using both RT and IEF, the value was 72.1%. The number of variants was higher in than in genes and in exons 1 and 2 than in exon 3. There was no clustering of mutations in consecutive codons. This study, the characterization of a wide spectrum of α-globin chain variants, can facilitate the presumptive diagnosis of these variants prior to screening by a panel of amplification refractory mutation system-polymerase chain reaction (ARMS-PCR), and a definitive diagnosis by DNA sequencing.
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http://dx.doi.org/10.1080/03630269.2020.1783288DOI Listing
May 2020

SAMHD1 Limits the Efficacy of Forodesine in Leukemia by Protecting Cells against the Cytotoxicity of dGTP.

Cell Rep 2020 05;31(6):107640

Medical Research Council Human Immunology Unit, Medical Research Council Weatherall Institute of Molecular Medicine, Radcliffe Department of Medicine, University of Oxford, Oxford OX3 9DS, UK. Electronic address:

The anti-leukemia agent forodesine causes cytotoxic overload of intracellular deoxyguanosine triphosphate (dGTP) but is efficacious only in a subset of patients. We report that SAMHD1, a phosphohydrolase degrading deoxyribonucleoside triphosphate (dNTP), protects cells against the effects of dNTP imbalances. SAMHD1-deficient cells induce intrinsic apoptosis upon provision of deoxyribonucleosides, particularly deoxyguanosine (dG). Moreover, dG and forodesine act synergistically to kill cells lacking SAMHD1. Using mass cytometry, we find that these compounds kill SAMHD1-deficient malignant cells in patients with chronic lymphocytic leukemia (CLL). Normal cells and CLL cells from patients without SAMHD1 mutation are unaffected. We therefore propose to use forodesine as a precision medicine for leukemia, stratifying patients by SAMHD1 genotype or expression.
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http://dx.doi.org/10.1016/j.celrep.2020.107640DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7225753PMC
May 2020

A five-year follow-up of untreated patients with chronic lymphocytic leukaemia treated with ofatumumab and chlorambucil: final analysis of the Complement 1 phase 3 trial.

Br J Haematol 2020 09 31;190(5):736-740. Epub 2020 Mar 31.

Department of Haematology, St James's University Hospital, Leeds, UK.

The Complement 1 trial investigated the efficacy and safety of ofatumumab + chlorambucil with chlorambucil monotherapy in patients with previously untreated chronic lymphocytic leukaemia (CLL). On long-term follow-up in the chemoimmunotherapy arm vs. the chemotherapy arm there was an estimated 12% (not significant) and 39% risk reduction in overall survival and progression-free survival, respectively. A high rate (61%) of treatment with next-line therapies in both the treatment arms may dilute any potential OS difference and confound the interpretation of the OS results. Addition of ofatumumab to chlorambucil demonstrated clinical benefit and tolerability as a frontline treatment option in patients unfit for fludarabine-containing therapy, with no new safety concerns.
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http://dx.doi.org/10.1111/bjh.16625DOI Listing
September 2020

Clinical significance of TP53, BIRC3, ATM and MAPK-ERK genes in chronic lymphocytic leukaemia: data from the randomised UK LRF CLL4 trial.

Leukemia 2020 07 3;34(7):1760-1774. Epub 2020 Feb 3.

Academic Unit of Cancer Sciences, Faculty of Medicine, University of Southampton, Southampton, UK.

Despite advances in chronic lymphocytic leukaemia (CLL) treatment, globally chemotherapy remains a central treatment modality, with chemotherapy trials representing an invaluable resource to explore disease-related/genetic features contributing to long-term outcomes. In 499 LRF CLL4 cases, a trial with >12 years follow-up, we employed targeted resequencing of 22 genes, identifying 623 mutations. After background mutation rate correction, 11/22 genes were recurrently mutated at frequencies between 3.6% (NFKBIE) and 24% (SF3B1). Mutations beyond Sanger resolution (<12% VAF) were observed in all genes, with KRAS mutations principally composed of these low VAF variants. Firstly, employing orthogonal approaches to confirm <12% VAF TP53 mutations, we assessed the clinical impact of TP53 clonal architecture. Whilst ≥ 12% VAF TP53mut cases were associated with reduced PFS and OS, we could not demonstrate a difference between <12% VAF TP53 mutations and either wild type or ≥12% VAF TP53mut cases. Secondly, we identified biallelic BIRC3 lesions (mutation and deletion) as an independent marker of inferior PFS and OS. Finally, we observed that mutated MAPK-ERK genes were independent markers of poor OS in multivariate survival analysis. In conclusion, our study supports using targeted resequencing of expanded gene panels to elucidate the prognostic impact of gene mutations.
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http://dx.doi.org/10.1038/s41375-020-0723-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7326706PMC
July 2020

A Massive Extradural Hematoma in Sickle Cell Disease and the Importance of Rapid Neuroimaging.

Case Rep Hematol 2019 18;2019:1742472. Epub 2019 Dec 18.

Department of Haematology and Blood Transfusion, Muhimbili University of Health and Allied Sciences, Dar-es-Salaam, Tanzania.

Sickle cell disease (SCD) is an inherited hemoglobinopathy leading to several serious organ complications and early death. It is mostly found in equatorial countries like Tanzania. Extradural hematoma (EDH) is a rare, but serious complication to SCD and may have debilitating consequences. Hitherto, there is no report of EDH in SCD where neuroimaging has been available before, during, and after such an event. Here, we describe a young female SCD patient who developed EDH that required surgical evacuation. She had made full recovery after three months. Neuroimaging performed two years prior to this event was unremarkable except for multiple small cerebral infarcts. On admission, neuroimaging revealed a subgaleal hematoma, possibly indicating disruption of the skull cortex due to increased hematopoiesis. Three months after evacuation of the hematoma, neuroimaging showed evidence of brain atrophy and the previously reported cerebral infarcts and multifocal bone infarction, but no vasculopathy. Possibly, disruption of the skull cortex with subsequent bleeding caused the EDH. As the differential diagnoses of neurological complications in SCD are many and some complications are reversible, neuroimaging should be performed without delay.
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http://dx.doi.org/10.1155/2019/1742472DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6935788PMC
December 2019

Acalabrutinib monotherapy in patients with relapsed/refractory chronic lymphocytic leukemia: updated phase 2 results.

Blood 2020 04;135(15):1204-1213

Weill Cornell Medicine, NewYork-Presbyterian Hospital, New York, NY.

Therapeutic targeting of Bruton tyrosine kinase (BTK) has dramatically improved survival outcomes for patients with chronic lymphocytic leukemia (CLL)/small lymphocytic lymphoma (SLL). Acalabrutinib is an oral, highly selective BTK inhibitor that allows for twice-daily dosing due to its selectivity. In this phase 1b/2 study, 134 patients with relapsed/refractory CLL or SLL (median age, 66 years [range, 42-85 years]; median prior therapies, 2 [range, 1-13]) received acalabrutinib 100 mg twice daily for a median of 41 months (range, 0.2-58 months). Median trough BTK occupancy at steady state was 97%. Most adverse events (AEs) were mild or moderate, and were most commonly diarrhea (52%) and headache (51%). Grade ≥3 AEs (occurring in ≥5% of patients) were neutropenia (14%), pneumonia (11%), hypertension (7%), anemia (7%), and diarrhea (5%). Atrial fibrillation and major bleeding AEs (all grades) occurred in 7% and 5% of patients, respectively. Most patients (56%) remain on treatment; the primary reasons for discontinuation were progressive disease (21%) and AEs (11%). The overall response rate, including partial response with lymphocytosis, with acalabrutinib was 94%; responses were similar regardless of genomic features (presence of del(11)(q22.3), del(17)(p13.1), complex karyotype, or immunoglobulin variable region heavy chain mutation status). Median duration of response and progression-free survival (PFS) have not been reached; the estimated 45-month PFS was 62% (95% confidence interval, 51% to 71%). BTK mutation was detected in 6 of 9 patients (67%) at relapse. This updated and expanded study confirms the efficacy, durability of response, and long-term safety of acalabrutinib, justifying its further investigation in previously untreated and treated patients with CLL/SLL. This trial was registered at www.clinicaltrials.gov as #NCT02029443.
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http://dx.doi.org/10.1182/blood.2018884940DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7146022PMC
April 2020

Distinct genetic changes reveal evolutionary history and heterogeneous molecular grade of DLBCL with MYC/BCL2 double-hit.

Leukemia 2020 05 16;34(5):1329-1341. Epub 2019 Dec 16.

Department of Pathology, University of Cambridge, Cambridge, UK.

Using a Burkitt lymphoma-like gene expression signature, we recently defined a high-risk molecular high-grade (MHG) group mainly within germinal centre B-cell like diffuse large B-cell lymphomas (GCB-DLBCL), which was enriched for MYC/BCL2 double-hit (MYC/BCL2-DH). The genetic basis underlying MHG-DLBCL and their aggressive clinical behaviour remain unknown. We investigated 697 cases of DLBCL, particularly those with MYC/BCL2-DH (n = 62) by targeted sequencing and gene expression profiling. We showed that DLBCL with MYC/BCL2-DH, and those with BCL2 translocation, harbour the characteristic mutation signatures that are associated with follicular lymphoma and its high-grade transformation. We identified frequent MYC hotspot mutations that affect the phosphorylation site (T58) and its adjacent amino acids, which are important for MYC protein degradation. These MYC mutations were seen in a subset of cases with MYC translocation, but predominantly in those of MHG. The mutations were more frequent in double-hit lymphomas with IG as the MYC translocation partner, and were associated with higher MYC protein expression and poor patient survival. DLBCL with MYC/BCL2-DH and those with BCL2 translocation alone are most likely derived from follicular lymphoma or its precursor lesion, and acquisition of MYC pathogenic mutations may augment MYC function, resulting in aggressive clinical behaviour.
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http://dx.doi.org/10.1038/s41375-019-0691-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7192846PMC
May 2020

Dual-specificity protein phosphatase DUSP4 regulates response to MEK inhibition in BRAF wild-type melanoma.

Br J Cancer 2020 02 16;122(4):506-516. Epub 2019 Dec 16.

Department of Oncology, Old Road Campus Research Building, University of Oxford, Oxford, UK.

Background: Aiming to improve treatment options for BRAF wild-type melanoma, we previously conducted the DOC-MEK study of docetaxel with MEK inhibitor (MEKi) selumetinib or placebo, revealing trends to prolongation of progression-free survival (hazard ratio 0.75, P = 0.130), and improved response rates (32% vs 14%, P = 0.059) with docetaxel plus selumetinib. NRAS status did not associate with outcome. Here, the aim was to identify novel biomarkers of response to MEKi.

Methods: A MEK 6 gene signature was quantified using NanoString and correlated with clinical outcomes. Two components of the gene signature were investigated by gene silencing in BRAF/NRAS wild-type melanoma cells.

Results: In melanomas of patients on the selumetinib but not the placebo arm, two gene signature components, dual-specificity protein phosphatase 4 (DUSP4) and ETS translocation variant 4 (ETV4), were expressed more highly in responders than non-responders. In vitro, ETV4 depletion inhibited cell survival but did not influence sensitivity to MEKi selumetinib or trametinib. In contrast, DUSP4-depleted cells showed enhanced cell survival and increased resistance to both selumetinib and trametinib.

Conclusions: ETV4 and DUSP4 associated with clinical response to docetaxel plus selumetinib. DUSP4 depletion induced MEKi resistance, suggesting that DUSP4 is not only a biomarker but also a mediator of MEKi sensitivity.

Clinical Trial Registration: DOC-MEK (EudraCT no: 2009-018153-23).
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http://dx.doi.org/10.1038/s41416-019-0673-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7028919PMC
February 2020

Idelalisib addition has neutral to beneficial effects on quality of life in bendamustine/rituximab-treated patients: results of a phase 3, randomized, controlled trial.

Health Qual Life Outcomes 2019 Nov 15;17(1):173. Epub 2019 Nov 15.

Memorial Sloan Kettering Cancer Center, New York, NY, USA.

Background: In a phase 3 randomized, double-blind, placebo-controlled trial, treatment with idelalisib, a phosphoinositol-3 kinase δ inhibitor, + bendamustine/rituximab improved progression-free survival (PFS) and overall survival (OS) in adult patients with relapsed/refractory chronic lymphocytic leukemia (R/R CLL). Here we report the results of health-related quality of life (HRQL) analyses from this study.

Methods: From June 15, 2012 to August 21, 2014, 416 patients with R/R CLL were enrolled; 207 patients were randomized to the idelalisib arm and 209 to the placebo arm. In the 416 patients randomized to receive bendamustine/rituximab and either oral idelalisib 150 mg twice-daily or placebo, HRQL was assessed at baseline and throughout the blinded part of the study using the Functional Assessment of Cancer Therapy-Leukemia (FACT-Leu) and EuroQoL Five-Dimension (EQ-5D) visual analogue scale (VAS) questionnaires. The assessments were performed at scheduled patient visits; every 4 weeks for the first 6 months from the initiation of treatment, then every 8 weeks for the next 6 months, and every 12 weeks thereafter until end of study. Least-squares mean changes from baseline were estimated using a mixed-effects model by including treatment, time, and treatment-by-time interaction, and stratification factors as fixed effects. Time to first symptom improvement was assessed by Kaplan-Meier analysis.

Results: In mixed-effects model analysis, idelalisib + bendamustine/rituximab treatment led to clinically meaningful improvements from baseline in leukemia-associated symptoms. Moreover, per Kaplan-Meier analysis, the proportion of patients with symptom improvement was higher and time to improvement was shorter among patients in the idelalisib-containing arm compared with those who did not receive idelalisib. The physical and social/family FACT-Leu subscale scores, along with the self-rated health assessed by EQ-VAS, showed improvement with idelalisib over placebo, but the difference did not reach statistical significance. The functional and emotional FACT-Leu subscale scores remained similar to placebo.

Conclusions: Addition of idelalisib to bendamustine/rituximab, apart from improving PFS and OS, had a neutral to beneficial impact on HRQL in patients with R/R CLL, particularly by reducing leukemia-specific disease symptoms.

Trial Registration: Clinicaltrials.gov NCT01569295. Registered April 3, 2012.
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http://dx.doi.org/10.1186/s12955-019-1232-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6858733PMC
November 2019

Clinical significance of DNA methylation in chronic lymphocytic leukemia patients: results from 3 UK clinical trials.

Blood Adv 2019 08;3(16):2474-2481

Cancer Genomics, Cancer Research UK Centre and Experimental Cancer Medicine Centre, Academic Unit of Cancer Sciences, University of Southampton, Southampton General Hospital, Southampton, United Kingdom.

Chronic lymphocytic leukemia patients with mutated immunoglobulin heavy-chain genes (IGHV-M), particularly those lacking poor-risk genomic lesions, often respond well to chemoimmunotherapy (CIT). DNA methylation profiling can subdivide early-stage patients into naive B-cell-like CLL (n-CLL), memory B-cell-like CLL (m-CLL), and intermediate CLL (i-CLL), with differing times to first treatment and overall survival. However, whether DNA methylation can identify patients destined to respond favorably to CIT has not been ascertained. We classified treatment-naive patients (n = 605) from 3 UK chemo and CIT clinical trials into the 3 epigenetic subgroups, using pyrosequencing and microarray analysis, and performed expansive survival analysis. The n-CLL, i-CLL, and m-CLL signatures were found in 80% (n = 245/305), 17% (53/305), and 2% (7/305) of IGHV-unmutated (IGHV-U) cases, respectively, and in 9%, (19/216), 50% (108/216), and 41% (89/216) of IGHV-M cases, respectively. Multivariate Cox proportional analysis identified m-CLL as an independent prognostic factor for overall survival (hazard ratio [HR], 0.46; 95% confidence interval [CI], 0.24-0.87; = .018) in CLL4, and for progression-free survival (HR, 0.25; 95% CI, 0.10-0.57; = .002) in ARCTIC and ADMIRE patients. The analysis of epigenetic subgroups in patients entered into 3 first-line UK CLL trials identifies m-CLL as an independent marker of prolonged survival and may aid in the identification of patients destined to demonstrate prolonged survival after CIT.
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http://dx.doi.org/10.1182/bloodadvances.2019000237DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6712529PMC
August 2019

The complete costs of genome sequencing: a microcosting study in cancer and rare diseases from a single center in the United Kingdom.

Genet Med 2020 01 30;22(1):85-94. Epub 2019 Jul 30.

Health Economics Research Centre, Nuffield Department of Population Health, University of Oxford, Oxford, UK.

Purpose: The translation of genome sequencing into routine health care has been slow, partly because of concerns about affordability. The aspirational cost of sequencing a genome is $1000, but there is little evidence to support this estimate. We estimate the cost of using genome sequencing in routine clinical care in patients with cancer or rare diseases.

Methods: We performed a microcosting study of Illumina-based genome sequencing in a UK National Health Service laboratory processing 399 samples/year. Cost data were collected for all steps in the sequencing pathway, including bioinformatics analysis and reporting of results. Sensitivity analysis identified key cost drivers.

Results: Genome sequencing costs £6841 per cancer case (comprising matched tumor and germline samples) and £7050 per rare disease case (three samples). The consumables used during sequencing are the most expensive component of testing (68-72% of the total cost). Equipment costs are higher for rare disease cases, whereas consumable and staff costs are slightly higher for cancer cases.

Conclusion: The cost of genome sequencing is underestimated if only sequencing costs are considered, and likely surpasses $1000/genome in a single laboratory. This aspirational sequencing cost will likely only be achieved if consumable costs are considerably reduced and sequencing is performed at scale.
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http://dx.doi.org/10.1038/s41436-019-0618-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6944636PMC
January 2020