H3 G34-mutant high-grade gliomas: integrated clinical, imaging and pathological characterisation of a single-centre case series.

BACKGROUND: 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. METHODS: 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. RESULTS: 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%) was the prevalent location, with a mixed cystic-nodular appearance (10/12) and presence of vascular flow voids coursing through/being encased by the mass (8/12), and all tumours showed cortical invasion. Nine patients had subtotal resection limited by functional margins, two patients underwent supra-total resection, and one patient had biopsy only. 5-ALA was administered to 6 patients, all of whom showed positive fluorescence. Histologically, the tumours showed a marked heterogeneity and aggressive spread along pre-existing brain structures and leptomeninges. In addition to the diagnostic H3 G34R/V mutation, pathogenic variants in TP53 and ATRX genes were found in most cases. Potential targetable mutations in PDGFRA and PIK3CA genes were detected in five cases. The MGMT promoter was highly methylated in half of the samples. Methylation profiling was a useful diagnostic tool and highlighted recurrent structural chromosome abnormalities, such as PDGFRA amplification, CDKN2A/B deletion, PTEN loss and various copy number changes in the cyclin D-CDK4/Rb pathway. Radiochemotherapy was the most common adjuvant treatment (9/12), and the average survival was 19.3 months. CONCLUSIONS: H3 G34R-mutant hemispheric glioma is a distinct entity with characteristic imaging and pathological features. Genomic landscaping of individual tumours can offer an opportunity to adapt individual therapies and improve patient management.

Assessment of the iliocapsularis muscle on magnetic resonance imaging.

INTRODUCTION: Morphologic features of Iliocapsularis (IC) may aid clinical decision-making in the symptomatic hip. The relationship between IC muscle size and underlying hip pathology is emerging; however, research is limited in the imaging literature. The purpose of this study was to determine the reliability and reproducibility of measurements of the IC muscle and its MRI appearances. It also looked for any association between IC dimension and axial levels, side, gender and bony features of hip instability. METHODS: Retrospective study of 37 MRI scans were assessed by four observers. MRI axial T1 images were used to define the IC anatomy, measure the IC and rectus femoris at the femoral head centre (FHC) and adjacent levels and calculate the iliocapsularis-to-rectus femoris (IR) ratio. Measurements were repeated at least 2 weeks later. Radiographic assessment of the lateral centre edge angle, acetabular index and femoral neck-shaft angle were also conducted. RESULTS: The IC was always present, but was well-defined in only 4% of cases with fair agreement. The intraclass correlation coefficient for reliability and reproducibility was the highest for IC width 0.94 (0.91-0.96). No significant correlation was identified between the IR ratio and radiographic parameters. CONCLUSION: Iliocapsularis is visible and reliably measured on MRI despite observers considering the muscle to be not well-defined. Despite gender differences in muscle size, the IR ratio was unchanged. There is a significant difference in the IR ratio above and below the FHC; therefore, clinicians need to be aware of how this may impact the clinical use when utilising the IR ratio.

A Sri Lankan girl with a new genetic variant in the PKLR gene causing pyruvate kinase deficiency: a case report.

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.

Improving the laboratory diagnosis of pyruvate kinase deficiency.

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.

TLR9 expression in chronic lymphocytic leukemia identifies a promigratory subpopulation and novel therapeutic target.

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, ß2-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.

Validity of whole genomes sequencing results in neoplasms in precision medicine.

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.

A Plea for the Newborn Diagnosis of Hb S-Hereditary Persistence of Fetal Hemoglobin.

The gene for hereditary persistence of fetal hemoglobin (HPFH) in the Caribbean is much more common than previously estimated. To avoid labeling persons with the benign syndrome Hb S (HBB: c.20A>T)/HPFH as a disease and wasting scarce resources, parental studies are recommended when newborn screening reveals a pattern consistent with an SS phenotype.

Detection of Hb Rothschild HBB: c.[112T>A or 112T>C], Through High Index of Suspicion on Abnormal Pulse Oximetry.

We describe a case with a low oxygen affinity hemoglobin (Hb) variant who presented with cyanosis in the absence of cardiopulmonary disease. The patient, a 27-year-old pregnant female (P1G2), complained of a productive cough and bluish discoloration of the lips that started 3 days prior to seeking attention. She had no previous episodes and has generally been in good health. A positive family history of cyanosis was obtained in one sibling. Systematic examination, notably the cardiorespiratory system, revealed no abnormalities. The arterial Hb oxygen saturation (SpO2) on pulse oximetry was 81.0% and Hb separation studies revealed an Hb variant identified as Hb Rothschild [ß37(C3)Trp→Arg] (HBB: c.[112 T>A or 112 T>C]) by gene sequencing. The amino acid substitution (Trp→Arg) is an important contact point at the α1ß2 interface and favors a T-quaternary state of the Hb tetramer. This leads to a low oxygen affinity state, which results in premature release of oxygen and drop in oxygen saturation. In the absence of cardiopulmonary disease, a decreased oxygen saturation reading, with or without cyanosis, should arouse suspicion for a possible dysHb.

Loss of Major DNase I Hypersensitive Sites in Duplicated ß-globin Gene Cluster Incompletely Silences HBB Gene Expression.

We report an infant with sickle cell disease phenotype by biochemical analysis whose ß-globin gene (HBB) sequencing showed sickle cell mutation (HBBS ) heterozygosity. The proband has a unique head-to-tail duplication of the ß-globin gene cluster having wild-type (HBBA ) and HBBS alleles inherited from her father; constituting her HBBS /HBBS -HBBA genotype. Further analyses revealed that proband's duplicated ß-globin gene cluster (∼650 kb) encompassing HBBA does not include the immediate upstream locus control region (LCR) or 3' DNase I hypersensitivity (HS) element. The LCR interacts with ß-globin gene cluster involving long range DNA interactions mediated by various transcription factors to drive the regulation of globin genes expression. However, a low level of HBBA transcript was clearly detected by digital PCR. In this patient, the observed transcription from the duplicated, distally displaced HBBA cluster demonstrates that the loss of LCR and flanking 3'HS sites do not lead to complete silencing of HBB transcription.

Genomic DNA "finger-printing" in diagnostic virology to clarify discrepant HIV results.

BACKGROUND: We describe two case studies in the antenatal setting where human immunodeficiency virus (HIV) antibody results were discrepant in serial sera. Root cause analysis including genomic DNA fingerprinting was performed. OBJECTIVE: To analyze serial sera with HIV antibody discrepant results using genomic DNA "finger-printing" to determine whether the sera were from the same or different individuals. STUDY DESIGN: In case study 1, two serial sera with HIV antibody discrepant results, supposedly from a pregnant lady were subjected to root cause analysis. In case study 2, four serial sera 'from' a pregnant lady were subjected to root cause analysis. All sera were analyzed forensically using genomic DNA (gDNA) fingerprinting. In the case study 1, public health look-back exercise was done prior to forensic analysis unlike case study 2, when it was planned for later. RESULTS: Case study 1 gDNA results showed that both sera had identical genotypes and sex indicating that these were from the same individual and that the problem was within automated serum separator pipette syringe. Case study 2 gDNA analysis showed that the 3 of 4 sera were from three different unrelated females indicating an intentional sample mix-up. CONCLUSIONS: Forensic gDNA "finger-printing" in diagnostic virology changed the direction of the investigations; one pointing to a laboratory issue and the other to an intentional deception by the patient. We recommend considering gDNA fingerprinting in any sample discrepancy investigation prior to any public health investigation. This is the first reported use of gDNA analysis in diagnostic virology.

Next generation sequencing identifies a novel rearrangement in the HBB cluster permitting to-the-base characterization.

Genetic testing for hemoglobinopathies is required for prenatal diagnosis, understanding complex cases where multiple pathogenic variants may be present or investigating cases of unexplained anemia. Characterization of disease causing variants that range from single base changes to large rearrangements may require several different labor-intensive methodologies. Multiplex ligation probe amplification analysis is the current method used to detect indels, but the technique does not characterize the breakpoints or detect balanced translocations. Here, we describe a next-generation sequencing (NGS) method that is able to identify and characterize a novel rearrangement of the HBB cluster responsible for ÎµÎ³Î´ß thalassemia in an English family. The structural variant involved a 59.0 kb inversion encompassing HBG2 exon 3, HBG1, HBD, HBB, and OR51V1, juxtaposed by a deletion of 122.6 kb including 82 bp of the inverted sequence, HBG2 exon 1 and 2, HBE, and the ß-locus control region. Identification of reads spanning the breakpoints provided to-the-base resolution of the rearrangement, subsequently confirmed by gap-PCR and Sanger sequence analysis. The same rearrangement, termed Inv-Del English V ÎµÎ³Î´ß thalassemia (HbVar 2935), was identified in two other unrelated English individuals with a similar hematological phenotype. Our NGS approach should be applicable as a diagnostic tool for other disorders.