Histone mutations (H3 K27M, H3 G34R/V) are molecular features defining subtypes of paediatric-type diffuse high-grade gliomas (HGG) (diffuse midline glioma (DMG), H3 K27-altered, diffuse hemispheric glioma (DHG), H3 G34-mutant). The WHO classification recognises in exceptional cases, these mutations co-occur. We report one such case of a 2-year-old female presenting with neurological symptoms; MRI imaging identified a brainstem lesion which was biopsied. Histology showed diffusely infiltrating pleomorphic astrocytes, multinucleated cells, and conspicuous mitotic activity; the diagnosis was DMG, H3 K27-altered (immunohistochemistry: H3K27me3 loss, H3K27M positivity). DNA methylation profiling (Illumina EPIC BeadArrays, brain tumour classifier (MNP v12.5 R package)) classified the tumour as ‘DMG, H3 K27-altered’ (calibrated score = 0.99). Further molecular studies (whole exome, whole genome sequencing) revealed concurrent H3.1 K27M and G34R mutations (clonal, in the same reads) of H3C3, FGF11 and PIK3CA somatic variants, and a pathogenic germline NBN variant. The RNAseq profile clustered with H3K27M-mutant tumours. A patient-derived cell culture was established enabling unbiased in vitro drug screening; no selective sensitivities were identified. Chromatin immunoprecipitation assays with sequencing (ChIP-seq; H3K27ac, H3K27me3, H3K36me3, RNApol2 marks) showed features in keeping with DMG H3 K27M-mutant tumours (H3K27ac loci including OLIG2, IRX1/2, PKDCC). The patient was treated with adjuvant radiotherapy, but progressed and passed away 13 months post-diagnosis. This case is an exceptionally rare, complex variant of histone-mutant paediatric HGG, illustrating that the H3.1 K27M mutation demonstrates a dominance over the molecular and clinical profiles compared to G34R, and highlights the importance of broad molecular profiling to identify such examples for further study.

Abstract BACKGROUND Histone mutations (H3 K27M, H3 G34R/V) define subtypes of paediatric-type diffuse high-grade gliomas (HGG) (diffuse midline glioma (DMG), H3 K27-altered, diffuse hemispheric glioma (DHG), H3 G34-mutant). The WHO classification recognises exceptional cases where these mutations co-occur. We report one such case of a 2-year-old female presenting with neurological symptoms and lethargy. METHODS MRI imaging identified a brainstem lesion; a stereotactic needle biopsy was undertaken, followed by standard diagnostic neuropathology workflows including histology review, panel immunohistochemistry, DNA methylation profiling (Illumina EPIC BeadArrays, brain tumour classifier (MNP v12.5 R package)) and WES/WGS. Patient-derived in vitro cell cultures were established allowing further characterisation using RNAseq and chromatin immunoprecipitation assays with sequencing (ChIP-seq). RESULTS Histology showed diffusely infiltrating pleomorphic astrocytes, multinucleated cells, and conspicuous mitotic activity; the diagnosis was DMG, H3 K27-altered (immunohistochemistry: H3K27me3 loss, H3K27M positivity). DNA methylation profiling classified the tumour as ‘DMG, H3 K27-altered’ (calibrated score=0.99). WES/WGS revealed concurrent H3.1 K27M and G34R mutations (clonal, in the same reads) of HIST1H3C (H3C3), somatic variants in FGF11 and PIK3CA, and a pathogenic germline NBN variant. The RNAseq profile of the primary tumour and cell cultures clustered with H3 K27M-mutant tumours. Unbiased in vitro drug screening showed no selective sensitivities. ChIP-seq (H3K27ac, H3K27me3, H3K36me3, RNApol2 marks) showed features in keeping with DMG H3 K27M-mutant tumours (H3K27ac loci including OLIG2, IRX1/2, PKDCC). The patient was treated with adjuvant radiotherapy and everolimus, but progressed and passed away 13 months post-diagnosis. CONCLUSION This case is an exceptionally rare, complex variant of histone-mutant paediatric HGG, and the first reported occurrence in HIST1H3C (H3C3). It illustrates that in cis, the H3.1 K27M mutation demonstrates a dominance over molecular and clinical profiles compared to G34R, and highlights the importance of broad molecular profiling to identify such examples for further study.

The spleen is prone to both physical damage and functional impairment, which can be difficult to detect before catastrophic complications occur. Currently available tests of splenic function are laborious, user-dependent and unreliable, so there is an unmet need for a reliable test offered routinely in diagnostic laboratories. In this study, we have assessed a simple flow cytometry-based method measuring high mannose glycans (HMGs) on erythrocytes, which has previously been proposed as a potential test of splenic function. We developed the test as a diagnostic assay using blood from a range of control and potentially hyposplenic samples, including people with sickle cell disease. HMG expression correlated well with manual pit counting, an established method for assessing splenic function (r = 0.6). A threshold of >36% difference compared to the mean of control samples was used to define hyposplenism. At this threshold, the test is 93% sensitive and 100% specific for detecting splenic dysfunction. The test was highly reproducible and stable in blood samples of up to 4 days old. This test is non-invasive with quantitative data output and requires significantly less operator time than other available techniques, making it a robust new clinical assay for determining splenic function.

Diffuse hemispheric glioma, H3 G34-mutant, is a novel paediatric tumour type in the fifth edition of the WHO classification of CNS tumours associated with an invariably poor outcome. We present a comprehensive clinical, imaging and pathological review of this entity. Patients with confirmed H3 G34R-mutant high-grade glioma were included in a single-centre retrospective cohort study and examined for clinical, radiological and histo-molecular data. Twelve patients were enrolled in the study — 7 males/5 females; the mean age was 17.5 years (10–57 years). Most patients presented with signs of raised intracranial pressure (8/12). The frontal lobe (60

Abstract AIMS Whole genome sequencing is currently being offered to paediatric, teenage and young adult neuro-oncology patients and this was being managed by a genomics practitioner who works cross-site. We found that some patients are being discharged from the hospital without 1 component of the test or the consent. Our aim is to create a solid pathway in which fresh tissue, blood sample and record of discussion are taken at a certain stage in their hospital admission. METHOD A poster was created to remind the theatre team that a fresh tumour tissue and EDTA blood sample are the required samples to proceed with whole genome sequencing. Consent training is being provided to all clinical nurse specialists who look after CNS tumours in order to secure the record of discussion at any point in their hospital appointment. After several shadowing and doing the consenting process with supervision, they are expected to be able to be independent in doing this. RESULTS The clinical nurse specialist team have consented 24 out of 37 paediatric, teenage and young adult neuro- oncology patients that were operated since July 2022. Almost all of them have had their bloods taken intra- operatively with some taken whilst still an in-patient. CONCLUSIONS Whole genome sequencing will soon be offered to all neuro-oncology patients and the entire team will need to be equipped in securing the record of discussion in order for this test to proceed and this will be led by the clinical nurse specialist team.

METHODOLOGY The British Society for Haematology (BSH) produces Good Practice Papers to recommend good practice in areas where there is a limited evidence base but for which a degree of consensus or uniformity is likely to be beneficial to patient care. The Grading of Recommendations Assessment, Development and Evaluation (GRADE) nomenclature was used to evaluate levels of evidence and to assess the strength of recommendations. The GRADE criteria can be found at http://www.gradeworkinggroup.org. This Good Practice Paper was produced as a collaboration with the European Hematology Association (EHA) compiled according to the BSH process at http://scanmail.trustwave.com/?c=8248&d=68DV3b1jbPPsVn. This guideline group included UK-based medical experts representing the BSH and members of the EHA Red Cell and Iron Scientific Working Group (SWG). Literature review details MEDLINE, EMBASE and PubMED were searched systematically for publications in English from 2000 to 2019 using the following key words. 'NGS' and 'next-generation sequencing' or 'high throughput sequencing' AND 'haemolytic anaemia' or 'DBA' or 'Diamond Blackfan anaemia' or 'CDA' or 'congenital dyserythropoietic anaemia' or 'sideroblastic anaemia' or 'HS' or 'hereditary spherocytosis' or 'red cell membrane disorders' or 'red cell enzyme disorders' or 'PK deficiency' or 'PKD'. References from relevant publications were also searched. Conference abstracts were included if deemed to be of particular relevance. Review of the manuscript Review of the manuscript was performed by the BSH Guidelines Committee General Haematology Task Force, the BSH Guidelines Committee and the General Haematology sounding board of the BSH. It was also on the members section of the BSH website for comment. It has also been reviewed by members of the EHA Red Cell and Iron SWG and the EHA Guidelines Executive Committee. INTRODUCTION The use of next-generation sequencing (NGS) in the diagnosis of rare inherited anaemias is increasingly common, as evidenced by a growing number of publications describing its clinical utility.1–6 Excluding disorders of globin synthesis, rare anaemias include Diamond-Blackfan anaemia (DBA), congenital dyserythropoietic anaemias (CDA), congenital sideroblastic anaemias (CSA), and disorders of red cell membrane and enzymes. Other forms of genetic anaemias can also be considered while establishing NGS panels, in particular genetic syndromes, where anaemia comprises one of the constellation of symptoms. Table 1 briefly summarises the key aspects of these conditions. Table 1. - Key Aspects of the Rare Anaemias Not Due to Disorders of Haemoglobin Synthesis DBA CDA Sideroblastic Anaemia Red Cell Membrane/Cation Leaking and Enzyme Disorders Age at presentation Usually 2–3 mo of age or 98%/90%, which is higher than for t-NGS. Although these are often mild conditions, they can result in significant morbidity including foetal anaemia, kernicterus and transfusion dependence, and genetic counselling is useful, particularly in families who wish to avoid further affected pregnancies. It is particularly important to be certain of the precise diagnosis before performing splenectomy for presumed HS, to avoid ill-advised splenectomy in dehydrated hereditary stomatocytosis as this procedure is accompanied by a greatly increased risk of thromboembolic disease.15 Phenotypically these conditions can be very similar unless some assessment of red cell hydration is performed, such as osmotic gradient ektacytometry or osmotic fragility measurement. In general, genetic diagnosis should be confirmed before recommending splenectomy in HS, and this will typically involve analysis using an NGS panel. Additionally, documenting genetic variants will eventually lead to some genotype–phenotype correlations.16,17 This is the case with pyruvate kinase (PK) deficiency, where response to the new drug AG-348 depends on whether the mutations are missense or not.18 For some conditions, NGS is far superior to Sanger sequencing of specific genes, due to the phenotypic variability and the unreliability of phenotypic tests such as enzyme assays for rare enzymopathies, making it difficult to target genes precisely, particularly when the patient is transfusion dependent. Because of frequent misdiagnosis of 'dyserythropoiesis' in some haemolytic anaemias,1,6 genetic analysis should always be used to confirm a 'CDA'. One condition where genetic analysis is particularly useful is dehydrated hereditary stomatocytosis (xerocytosis) due to autosomal dominant mutations in the gene Piezo-type mechanosensitive ion channel component 1 (PIEZO1), a mechanosensitive calcium channel. Patients with this condition are probably at high risk of developing post-splenectomy thrombosis and splenectomy in these cases is generally contraindicated.15,19 This condition is difficult to diagnose and can be associated with only occasional stomatocytes on the blood film; genetic diagnosis should usually be performed before splenectomy when there is a possibility that the diagnosis could be dehydrated hereditary stomatocytosis; this will include most cases of presumed HS. Finally, NGS-based genetic testing is useful for the identification of complex modes of inheritance that are recognised to account for at least 4% of diagnosed Mendelian conditions.20 Recommendations NGS should only be used in cases where acquired causes are thought to be very unlikely (IA) Appropriate consent should be obtained (IA) Globin gene abnormalities should be considered and investigated appropriately before NGS is carried out, including haemoglobin analysis and sequencing of individual globin genes, depending on the genetic distribution that is already known in the local population. Specific consideration should be given to globin gene CNVs, with use of Gap-PCR and MLPA as appropriate (IIB) Conditions that should be tested on the panel include DBA, CDA, CSA, suspected red cell enzyme deficiencies and red cell membrane disorders (IIB) Genetic analysis should be used to confirm conditions when there is diagnostic uncertainty (IIB) Genetic analysis should be performed before undertaking splenectomy for inherited haemolytic anaemias or other irreversible procedures such as bone marrow transplantation, where the genetic variant should be excluded from a potential stem cell sibling donor (IIB) Question 2: At which point in the diagnostic pathway should NGS be used? The use of NGS will partly depend on each country or hospital system's technical and reimbursement characteristics. The traditional investigative pathway is to take a history and examination, full blood count, reticulocyte count, and haemolytic markers, before selecting specialised tests (enzyme assays, osmotic gradient ektacytometry, eosin-5-maleimide [EMA] test, erythrocyte adenosine deaminase [eADA], etc.). In some cases, this may lead to a bone marrow biopsy or aspiration, with genetic analysis being kept at the end of the pathway. In other places, genetic analysis may occur much earlier in the pathway.21 The advantages are that this may lead to a more rapid diagnosis, may be cost effective in reducing delay in diagnosis (at the expense of a higher cost upfront) and may (in some conditions) preclude the need for a bone marrow biopsy. Figure 2 shows examples of aspects of the history and examination that should be sought when evaluating the patient, as well as standard blood tests. The requirement for specialised tests, bone marrow aspiration and biopsy, and genetic analysis and the order in which they are requested, will differ between services, but in time, genetic analysis is likely to be carried out earlier in the pathway, with specialised functional analysis used to confirm the genetic diagnosis.Figure 2.: Clinical and laboratory assessment of the patient with a suspected diagnosis of inherited anaemia. These are indicative only and not exhaustive. eADA, erythrocyte adenosine deaminase; EMA, eosin-5′-maleimide test; FBC, full blood count; HPLC, high performance liquid chromatography; LDH, lactate dehydrogenase; LFTs, liver function tests; retics, reticulocytes; U&Es, urea and electrolytes.Recommendations NGS should primarily be used once the phenotype has been characterised. In particular, it should be established whether the patient has haemolysis, ineffective erythropoiesis, dyserythropoiesis, or bone marrow failure, as this may direct the analysis of the variants identified (IC) Clinical-grade NGS should ensure that variants are reported with reference to the phenotype of the patient (a sample request form detailing minimal phenotypic information can be found in Suppl. Figure S1) (IC) If further investigations are required to confirm the diagnosis (eg, family studies, RNA studies, specialist haematological tests directed by the variant identified), these can be recommended on the genetics report (IC) Question 3: What are the important considerations in choosing the most appropriate NGS method and which quality criteria must be met? Most panels are currently carried out as t-NGS, although some diagnostic laboratories carry out target enrichment across thousands of regions, then analyse the variants among genes that have been grouped together into virtual panels. As some countries move towards conducting all genetic analysis in the form of WGS, virtual panels will be increasingly used. The choice of using t-NGS over virtual panels is mostly due to availability, cost and turnaround time. Although cost-per-base may be lower for WGS, this requires a capital investment beyond the scope of most diagnostic laboratories. However, a major disadvantage of using t-NGS is that if any new genes are found to be associated with a known phenotype, adding a gene to the panel requires complete redesign and revalidation. This time-consuming and expensive process limits updating t-NGS panels to about once a year. WGS is also better suited for determination of CNVs, a common genetic cause of a number of inherited anaemias, with alpha globin gene deletions remaining a particular challenge for all technologies. New bioinformatic protocols to improve CNV assessment from targeted panels are improving their detection across modalities. Bait capture and unique molecular indexed amplicon methods may be combined with bioinformatic algorithms to determine the breakpoint mapping from short reads.22 As the selected method will depend on many factors, it is critical that a laboratory is aware of the limitations of the technique, and that additional steps are taken to either overcome some of these limitations (eg, gap-filling by Sanger Sequencing) or that the report produced is explicitly clear on the limitations of the analysis. This may require suggesting alternative methods (eg, MLPA) to address CNVs that may not be detected reliably by t-NGS. The availability of complementary diagnostic tools such as erythrocyte morphology, red cell and reticulocyte indices, EMA dye binding or osmotic gradient ektacytometry for red cell membrane disorders, may allow a phenotypic confirmation of the diagnosis in the absence of a definitive genetic diagnosis. Recommendations The NGS method should be chosen based on local resources and required turnaround time (IC) Depending on the method chosen, the laboratory should be aware of the limitations and either reduce these (MLPA, gap-fill) or make it clear in the report what has not been tested (IC) Question 4: What criteria should be used for reporting NGS variants identified? Once variants have been identified and graded for pathogenicity, a multidisciplinary team meeting (MDT) is carried out, where variants are discussed in the context of the clinical presentation and a final report is written. In cases of an established pathogenic variant that fits with the phenotype, a report can be issued by the clinical scientists in the absence of an MDT meeting. The ACMG guidelines must be followed for pathogenicity of single-nucleotide variants (5 classes)—pathogenic (class 5) and likely pathogenic (class 4) variants related to the clinical suspicion should be included in the report. Recommendations Variant types to include in the final report (IIB): 1. Pathogenic/likely pathogenic variants related to the clinical suspicion Variants to which a pathogenic role can be attributed with certainty, including: known variants in genes already associated with phenotype/disease novel variants in genes already associated with the phenotype/disease that have a clear causative role (eg, loss-of-function of a known gene that is associated with disease with a mechanism of haploinsufficiency), and fits with the pattern of inheritance, if available. 2. Pathogenic variants unrelated to the clinical suspicion Variants with a well-known pathogenic role not related to clinical suspicion, including: causative variants in genes already associated with a phenotype but different from the suspected disease (reverse phenotyping) incidental findings (eg, carrier state for other condition), which should be reported only if consent explicitly signed for this as per the ACMG guidelines. 3. Variants with unknown clinical and functional role (VUSs) that could provide a diagnosis pending further investigation or evidence These variants can be identified in: genes related to the suspected phenotype, which can be included in the final report. However, it should be made clear that the variant is a VUS and that without functional or family studies one cannot be sure that this variant is involved in the pathogenicity of the condition. genes not related to the clinical suspicion, which should not be included in the report. In general, it is not recommended that intronic/splice (noncanonical) and 5′ and 3′ variants are reported unless substantial functional data is available. Some laboratories may report a recessive disorder where one pathogenic mutation (classes 4 or 5) has been found together with a VUS. Family studies are strongly recommended, and the report must make clear that there is no definite pathogenicity associated with the second variant. This also includes circumstances where 2 very rare VUSs are identified in a gene(s) implicated in the phenotype, and family studies indicate they are in trans and functional data supports this gene as being causative. Variants of uncertain significance or variants that would suggest a novel complex mode of inheritance can form the basis of research studies, with the caveat that this almost universally requires a different form of consent to that obtained for diagnostic testing. Question 5: How should variants be stored and shared between laboratories? The sharing of variants between laboratories plays a very important role in ensuring high-quality data, high-diagnostic rates, and cost efficiency. However, this is often much more difficult to achieve than might be imagined, with issues such as data storage and the practicalities of sharing variants being significant obstacles. One of the prerequisites for variant sharing is that participating laboratories use the same system for variant classification. Sharing of variants is difficult because ideally the information to be shared includes the clinical phenotype, how the pathogenicity was assessed including individual components of the overall score, and knowledge of the other variants found in the same patient. A potentially pathogenic variant where a different definitive genetic cause has also been found in the patient means the first one is less likely to be pathogenic. However, the more variants are shared, the more identifiable the data are, raising the possibility that individuals may be identified according to specific haplotypes. Other obstacles to routine variant sharing across laboratories include practical technical reasons (not everyone shares and stores data in the same way) and time (the need to keep the database up to date and curated, with someone to take responsibility for any discrepancies). Variant sharing is also predicated upon using the same nomenclature (eg, Human Genome Variation Society [HGVS]) and reporting against the same transcripts. In cases of multiple transcripts, the specific 'disease transcript' must be used, but this is not always known. Laboratories should make reasonable efforts to ensure that the transcript they are using is expressed in erythroid cells. LRG (Locus Reference Genomic) may be useful in this assessment: https://www.lrg-sequence.org. Recommendation Laboratories should share variants with other laboratories analysing the same genes (IIC) Laboratories should ensure they are using commonly used transcripts which have been shown to be expressed in erythroid cells (IIC) Ethical and legal issues in sharing variants between laboratories, often located in different countries should be clearly reported and discussed (IIC) Question 6: What criteria are essential for a laboratory to be able to offer clinical-grade NGS? For a laboratory to offer clinical-grade NGS, a number of parameters must be met, which relate to the laboratory itself, the panel design, the analytical pathway and the report. Patient consent This will depend on the legal framework of each country. However

Background Erythrocyte pyruvate kinase is expressed under the control of the PKLR gene located on chromosome 1q21. Pyruvate kinase catalyzes the final steps of the glycolytic pathway and creates 50% of the red cell total adenosine triphosphate. Pyruvate kinase deficiency is the commonest glycolytic defect causing congenital non-spherocytic hemolytic anemia inherited in an autosomal recessive trait in which homozygotes and compound heterozygotes are common. Over 200 mutations have been described in patients with pyruvate kinase deficiency. This case report identifies a new pathogenic variant in PKLR gene detected in a patient with severe pyruvate kinase deficiency. Case presentation A Sri Lankan Sinhalese girl who developed neonatal anemia and jaundice within 24 hours of birth with mild hepatomegaly. She was from a nonconsanguineous marriage and had two siblings who had no hematological disorders. She had repeated admissions due to similar illnesses and at the age of 8 years was found to have pyruvate kinase deficiency associated with a novel homozygous pathogenic variant c.507+1delG in the PKLR gene. Conclusions A novel genetic variant in PKLR gene, consistent with pyruvate kinase deficiency, was detected in a Sri Lankan girl. This genetic variant may be specific to the Asian population and requires further studies.

Chronic lymphocytic leukemia (CLL) remains incurable despite B-cell receptor–targeted inhibitors revolutionizing treatment. This suggests that other signaling molecules are involved in disease escape mechanisms and resistance. Toll-like receptor 9 (TLR9) is a promising candidate that is activated by unmethylated cytosine guanine dinucleotide–DNA. Here, we show that plasma from patients with CLL contains significantly more unmethylated DNA than plasma from healthy control subjects (P < .0001) and that cell-free DNA levels correlate with the prognostic markers CD38, β2-microglobulin, and lymphocyte doubling time. Furthermore, elevated cell-free DNA was associated with shorter time to first treatment (hazard ratio, 4.0; P = .003). We also show that TLR9 expression was associated with in vitro CLL cell migration (P < .001), and intracellular endosomal TLR9 strongly correlated with aberrant surface expression (sTLR9; r = 0.9). In addition, lymph node–derived CLL cells exhibited increased sTLR9 (P = .016), and RNA-sequencing of paired sTLR9hi and sTLR9lo CLL cells revealed differential transcription of genes involved in TLR signaling, adhesion, motility, and inflammation in sTLR9hi cells. Mechanistically, a TLR9 agonist, ODN2006, promoted CLL cell migration (P < .001) that was mediated by p65 NF-κB and STAT3 transcription factor activation. Importantly, autologous plasma induced the same effects, which were reversed by a TLR9 antagonist. Furthermore, high TLR9 expression promoted engraftment and rapid disease progression in a NOD/Shi-scid/IL-2Rγnull mouse xenograft model. Finally, we showed that dual targeting of TLR9 and Bruton's tyrosine kinase (BTK) was strongly synergistic (median combination index, 0.2 at half maximal effective dose), which highlights the distinct role for TLR9 signaling in CLL and the potential for combined targeting of TLR9 and BTK as a more effective treatment strategy in this incurable disease.

Pyruvate kinase (PK) deficiency is an autosomal recessive disease caused by mutations in the PKLR gene, which reduce erythrocyte PK enzyme activity and result in decreased energy synthesis in red cells, causing haemolytic anaemia. Historically, the investigation into pyruvate kinase deficiency (PKD) has been led by a red cell enzyme assay determining PK enzyme activity per unit of haemoglobin. For our laboratory, the reference range was set by Beutler et al. in 1977 when the test was first established. The introduction of genetic testing permitted the creation of reference sample datasets, with positive controls having two pathogenic variants causing disease. This permitted re-assessment of the enzyme assay's sensitivity and specificity, and was used to reassess the reference range of the enzyme assay. Using sequenced samples, we have devised an enzyme assay, DNA testing workflow, which minimises false negative/positive results and improves the diagnostic efficiency. This combined enzyme-DNA testing strategy should improve the diagnostic accuracy whilst limiting the number of expensive DNA tests. During this evaluation, 10 novel genetic variants were identified and are described.

Objective To compare the whole genomes sequencing (WGS) results in the 100K Genomes project with the results of routine molecular diagnostics in precision medicine. Materials and methods We analysed 374 cancers including a high tumour mutational burden (TMB-high) subgroup, defined as >10 non-synonymous single nucleotide variations per megabase. Colon cancers were evaluated for microsatellite instability (MSI), mismatch repair (MMR) genes and NRAS, KRAS and BRAF mutations using routine molecular diagnostics. Fluorescence in-situ hybridisation/immunohistochemistry was used to evaluate the Her2Neu status in breast cancers. Results There was high correlation between WGS and routine diagnostic testing results irrespective of TMB status in colon cancers. Her2Neu status was discordant in 3 out of the 5 TMB-high breast cancers (p=0.049). The presence of ductal carcinoma in-situ correlated significantly with discordance (p=0.04). There were 3 (5%) discordant colorectal cases, all in the KRAS gene, 2 of which were from the non-invasive adenomatous component (p=0.0058). Of the 374 cases we identified 24 tumours with a TMB >10; comprising (colorectal carcinomas (CRCs) n=16, breast carcinomas n=5, bladder urothelial cell cancers n=3). Of the 16 TMB-high colorectal adenocarcinomas, 13 had MSI-high status. The same 13 had defective MMR protein expression. TMB-high colorectal cancers had 100% concordant results between WGS and NGS testing for KRAS, BRAF and NRAS (16/16). Conclusion The microsatellite and mutational status of colorectal cancers evaluated by WGS seem to correlate well with the routine diagnostic testing if it is ensured that the invasive component is sequenced. Evaluation of WGS results need to be carefully correlated with histomorphology, as tumour heterogeneity/contamination with pre-malignant components needs to be taken into account.

Non-invasive prenatal testing (NIPT) to date is used in the clinic primarily to detect foetal aneuploidy. Few studies so far have focused on the detection of monogenic autosomal recessive disorders where mother and foetus carry the same mutation. In particular, NIPT is currently not available for the detection of Sickle Cell Anaemia (SCA), the most common monogenic disorder world-wide and the most common indication for invasive prenatal testing in high-income countries. Here, we report the clinical validation of a novel diagnostic approach that combines ultra-sensitive amplicon-based sequencing of cell-free DNA (cfDNA) with internal controls and bias factor correction to calculate the probability for the presence of allelic imbalance from maternal plasma without prior knowledge of the paternal genotype. Identification of the foetal genotype was determined using a hierarchical probabilistic model based on the relative number of reads from the sequencing, along with the foetal fraction. NIPT was performed on a cohort of 57 patients, all of whom had previously undergone invasive prenatal testing so that the foetal genotype was known. Overall, NIPT demonstrated 100% sensitivity and negative predictive value for foetal fractions higher than 0.5%, and 100% specificity and positive predictive value for foetal fractions higher than or equal to 4%. Our methodology can be used as a safe, non-invasive screening tool in any clinical scenarios where early prenatal diagnosis of SCA or other recessive disorders is important.

We evaluated deficiency of adenosine deaminase 2 (DADA2) in a non-consanguineous family with four children (Fig 1A). Both parents and their son are unaffected. The father has panlymphopenia but is clinically well. The eldest sibling, 4 years older than the proband, had bilateral renal dysplasia and died following a myocardial infarction aged 8 years. The proband, a 45-year-old woman, presented with recurrent upper respiratory tract infections at the age of 13 years. Subsequently, she developed persistent moderate neutropenia. From the age of 24 years, she had recurrent episodes of fever with no identifiable pathogens. Aged 44 years, she was diagnosed with panhypogammaglobulinaemia, neutropenia, panlymphopenia, mild thrombocytopenia and mild bone marrow hypocellularity. She declined immunoglobulin replacement as she had minimal infections. She responded to granulocyte colony-stimulating factor (GCSF) treatment for neutropenia. The proband's sister, who is 7 years younger, presented with shingles at the age of 11 years. She experienced recurrent upper respiratory tract infections and was diagnosed with moderate neutropenia aged 20 years. She had recurrent bouts of fever with persistent severe neutropenia and a mildly hypocellular bone marrow aspirate from the age of 35 years. Occasionally, Escherichia coli was cultured and she was treated with intravenous antibiotics. Unlike the proband, she had no response to GCSF but received antibiotic, antifungal and antiviral prophylaxis. Aged 36 years, her IgA and IgM levels were low with panlymphopenia. She had poor responses to pneumococcal vaccination. At the age of 38 years, she commenced immunoglobulin replacement due to ongoing infections despite anti-microbial prophylaxis. Salient clinical features and laboratory findings of the patients are summarised in Table 1. Chickenpox URTIs (no organism isolated) Chickenpox Shingles URTIs (no organism isolated) Lower GI (Escherichia coli) Immunoglobulin replacement Antibiotic, antiviral and antifungal prophylaxis We undertook whole exome sequencing of all family members except for the deceased sibling. Analysis of the data considered a recessive inheritance model to identify homozygous or compound heterozygous genotypes. Systematic assessment of bioinformatical predictions, published literature, gene annotation and the clinical and immunological phenotype identified a compound heterozygous mutation in the Adenosine Deaminase 2 gene (ADA2) that encodes the protein enzyme ADA2. Deficiency of ADA2 (DADA2) is an autosomal recessive disorder that typically causes systemic vasculopathy and early-onset recurrent stroke, but is also known to cause hypogammaglobulinemia (Meyts & Aksentijevich, 2018). ADA2 has an isoform, ADA1, the deficiency of which causes severe combined immunodeficiency (SCID). ADA2 is released extracellularly by myeloid cells and binds directly to widely expressed transmembrane adenosine receptors. ADA2 catalyses the conversion of adenosine to inosine and appears to have a growth factor-like function (Zavialov et al, 2010a). Sanger sequencing (Fig 1B) confirmed that the affected sisters carried compound heterozygous mutations at positions 506 (exon 2) and 1057 (exon 6) of the coding sequence of ADA2 (c.506G>A, c.1057T>C), resulting respectively in amino acid substitutions p.Arg169Gln and p.Tyr353His in ADA2 (NM_001282225.1). The unaffected family members were heterozygous carriers for either of the two missense variants. The predicted p.Arg169Gln protein change is in the putative protein binding domain of ADA2 and has been previously associated with DADA2 (Table SI). The previously unreported variant at amino acid position 353, in the catalytic domain of ADA2, replacing the highly conserved tyrosine with histidine (Fig 1C), was predicted to be a loss of function mutation. The combined annotation dependent depletion score for both variants demonstrated that they were highly deleterious (Table SI). Plasma samples form the family showed that ADA2 enzyme activity was lacking in the affected sisters compared to healthy controls, indicating DADA2 (Fig 1D). The plasma of unaffected family members had measurable ADA2 activity but this was significantly lower than the healthy controls, suggesting that each of the two described variants in isolation reduced ADA2 enzyme activity in plasma. Thus, both alleles of ADA2 need to be expressed normally to sustain normal ADA2 enzyme activity, but reduction, as opposed to absence of such activity, is not sufficient to cause a clinical phenotype. Additionally, it was demonstrated that the p.Arg169Gln variant did not affect ADA2 mRNA expression but all ADA2 protein was retained intracellularly (Navon Elkan et al, 2014). This indicates that p.Arg169Gln affects secretion of ADA2, its stability or both. Although neutropenia has previously been observed in DADA2 (Zhou et al, 2014; Cipe et al, 2018), we have identified a novel genetic variant c.1057T>C; p.(Tyr353His) in the catalytic domain of ADA2. Based on crystallographic analyses of human ADA2 protein structure (Zavialov et al, 2010a), residue 356 is crucial for zinc ion binding, which is a cofactor for ADA2 activity, and residue 359 is part of the enzyme's active site. This provides a potential structural basis for impairment of ADA2 activity by the substitution at residue 353. How impaired activity of ADA2 results in neutropenia remains unclear. ADA2 has been demonstrated to have a role in embryogenesis (Iijima et al, 2008) and cell differentiation (Zavialov et al, 2010b). Therefore, it could possibly be involved in haematopoietic stem cell differentiation into neutrophils. The growth factor-like function has been shown to be dependent on ADA2's catalytic activity in fruit flies and frogs (Zurovec et al, 2002; Iijima et al, 2008), while in humans the two functions are independent (Zavialov et al, 2010b). Alternatively, ADA2 has been shown to upregulate myeloperoxidase (Caorsi et al, 2016), which delays the apoptosis of neutrophils (El Kebir & Filep, 2013). Put together, neutropenia in our patients could be a result of either decreased neutrophil production, increased cell death or a combination of both. We have thus reported a family with DADA2 presenting with neutropenia and/or lymphopenia with evidence of bone marrow failure and fevers but without the typical cardinal manifestations of the syndrome. DADA2 should be considered in cases of otherwise unexplained cytopenia, especially when associated with panhypogammaglobulinaemia and bone marrow hypocellularity, irrespective of the age of the patient at presentation. The mechanism(s) with which ADA2 mutations lead to the immunological and clinical manifestations of DADA2 are not clear. Elaboration of the pathophysiology of this condition could enhance our understanding of immune cell differentiation and function, and unveil new therapeutic opportunities for these and other patients with immune-mediated inflammatory diseases. The authors would like to acknowledge the contribution of the following colleagues to the clinical care of patients described in this work: Patrick Gordon, Roopen Arya, Dorothea Grosse-Kreul and Elzbieta Bialas, as well as the co-operation of the patients and their families. RRG and KS contributed equally to the study. RRG, ZA, GJM, JM and MAAI handled clinical aspects of the study. KS and MAAI designed research aspects of the study. RRG, KS, FS, BC and LF generated data. MAS and MAAI performed bioinformatics analyses for identification of the mutations. RRG and MAAI analyzed clinical data. KS and MAAI analyzed research data. KS prepared figures. KS, RRG and MAAI prepared manuscript. All authors edited the manuscript. None of the authors have any conflict of interest to declare. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

Background: Autoimmune hemolytic anemia (AIHA) is clinically heterogeneous, from chronic compensated to abrupt hemolysis. Together with the rate of antibody mediated erythrocyte destruction, bone marrow reticulocyte compensation is a recently recognized determinant of outcome. Erythropoietin (EPO) has been anecdotally used in AIHA to ameliorate bone marrow response, but only one systematic series has been published and predictors of response are not knownAims: To evaluate EPO efficacy and its predictors in a cohort of AIHA patients Methods: Data on primary and secondary AIHA cases who had received EPO either alone or concomitantly to other therapies were retrospectively collected using a preformed survey. Efficacy was evaluated at 15 and 30 days, and then at 3,6 and 12 months; Hb response was considered partial (PR, ≥2 g/dL Hb increase or >10 g/dL) or complete (CR, >12 g/dL) and hemolytic markers (LDH, reticulocytes) were registered Results: 29 AIHA cases followed from June 2007 to February 2019 at 7 centers in Italy, France, Norway, Austria, and UK were included in the study. Main AIHA types (warm, cold, mixed, and DAT negative) were present, and 3 cases were secondary to a lymphoproliferative disorder (not active and without specific treatment at the moment of the study). Patients’ characteristics are shown in table 1: at diagnosis 74% of cases presented with severe anemia and 95% displayed inadequate reticulocytosis (i.e. bone marrow responsiveness index<121). Bone marrow evaluation at diagnosis (N = 16) showed hypercellularity with dyserythropoiesis in 7 cases, and reticulin fibrosis in 3; a lymphoid infiltrate was found in 13 patients (T-cell in 4, B-cell in 7, mixed in 2), greater than 10% in the 3 secondary cases only. All patients had received at least one previous therapy, and the majority (69%) started EPO because of non-response to ongoing treatment (steroids 15, immunosuppressor 4, sutimlimab 1). Six patients had received rituximab during the 3 months before EPO start (median 1 month, range 0–5). At EPO initiation, 21% of cases displayed severe anemia, 73% had inadequate reticulocytosis, and 89% (of 18 tested) showed inappropriately low endogenous EPO levels. Patients were treated for a median of 7 months and responses were observed in about 70% of cases at month+1 and +3 (table1), with a median Hb and reticulocyte increase of 21.5 (2–48) g/L (p < 0.001) and 25(0–220)x109/L at month+1; and 29 (0–66) g/L (p < 0.001) and 49 (0–195)x109/L at month+3, respectively. Notably, 64% of patients responded as soon as at day+15; this finding supports an activity of EPO although recent or concomitant treatments may have contributed. At last follow up, 13 cases had discontinued EPO: 6 for long standing CR and 7 because of NR (3 with hemolytic flares). We observed an association of response to EPO and primary AIHA (73 vs 33% in secondary), inadequate reticulocytosis (76 vs 50% with adequate reticulocytosis), and not-warm (85 vs 50% in warm cases) not transfusion dependent cases (76 vs 50% transfusion dependent), although the small number did not allow statistical significance Summary/Conclusion: Use of EPO is effective in about 70% of AIHA patients unresponsive to ongoing/previous treatments, particularly in cases with inadequate reticulocytosis. Although preliminary, these data advise EPO use to stimulate bone marrow compensatory response

Sideroblastic anemias (SA), both hereditary and acquired, are characterized by ring sideroblasts (RS), which are bone marrow erythroid precursors (erythroblasts) with iron loaded mitochondria visualized as a perinuclear ring by Perl’s stain. Acquired SA, myelodysplastic syndrome with RS (MDS-RS),