Supplementary Data Figure S1. Determination of IgLV3-21 positivity by real-time PCR. Supplementary Data Figure S2: CLL-OS analysis including only CLL-related deaths. Supplementary Data Figure S3. Validation of the poor prognosis associated with IgLV3-21 usage in population C. Supplementary Data Figure S4. Validation of the poor prognosis of IgLV3-21 usage in population D. Supplementary Data Figure S5: prognostic impact of IgLV3-21 usage in the different CLL-IPI subgroups. Supplementary Data Figure S6. Immunogenetic features of IgLV3-21 patients. Supplementary Data Figure S7. CXCR4 surface expression is downregulated in IgLV3-21 patients.

Hb J-Paris-I [HBA2: c.38C>A (or HBA1)] is a stable fast-moving hemoglobin (Hb) that elutes in the P3 window on high performance liquid chromatography (HPLC). The mutation can happen on either the alpha 1- or alpha 2-globin gene. Codon 12 changes from GCC to GAC to replace the alanine amino acid with aspartic acid. This change is external with no clinical significance. The elution in the P3 wave on HPLC can interfere with the glycated Hb assay by HPLC. In this study, data of 11 cases of Hb J-Paris-I were thoroughly presented. The majority of the cases were of Indian ethnicity. The mean value of Hb J-Paris-I on HPLC was 26.7 +/- 2.0%. The retention time (RT) was 1.75 +/- 0.03 min. The isoelectric focusing (IEF) mean value was -5.6 (range -6.1 to -4.9). Hb A(2) was consistently reduced to 1.8 +/- 0.3%. A fraction of 0.8% corresponding to the Hb A(2)-J-Paris-I (alpha(J-Paris-I)(2)delta(2)) is likely to be concealed within the A(0) peak of Hb A on HPLC. Interestingly, two cases were associated with two different polymorphisms [HBA2: c.-24C>G or Cap +14 (C>G) and HBA2: c.*136A>G polymorphism] without apparent effect on the variant expression.

Hb Winnipeg [α75(EF4)Asp→Tyr (α2); HBA2: c.226G>T (or HBA1)] is a stable α-globin chain variant described in a few articles. The majority of reported cases in older articles were clustered in Canada. It can occur on both α1- and α2-globin genes and in different populations. In this study, eight cases of Hb Winnipeg were characterized by DNA sequencing during a wide-spectrum study of suspected α-globin gene variants collected in the United Kingdom. All cases detected peaked in the S window between 4.4 and 4.54 min. on high performance liquid chromatography (HPLC). The isoelectric focusing (IEF) averaged at 6.21 below Hb A. All the mutations were detected on the α1-globin gene except in one case. The ethnic origin of the majority of the patients was Canadian. Only one case was associated with the common polymorphism HBA2: c.-24C>G (or HBA1) [Cap +14 (C>G)] on both α-globin genes without any apparent effect on the variant expression. All cases were detected in a heterozygous state. Hb Winnipeg expression was consistently lower than the theoretical value for α chain variants, ranging between 11.8 and 15.8% of total hemoglobin (Hb). This study gave more details about Hb Winnipeg that may help in presumptive diagnosis, especially in routine laboratories.

Hb J-Meerut [HBA2: c.362C>A (orHBA1)] is a rare, stable, nonpathogenic alpha-globin gene variant that peaks in the area between the P3 and A(0) 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 alpha-globin chain variants in the UK. The diagnosis was confirmed by forward and reverse DNA sequencing of the alpha 1- and alpha 2-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 (HBB: c.79G>A) mutation in heterozygosity and another case was associated with the Cap +14 (C>G) [HBA1: c.-24C>G (orHBA2)] 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 theHBA2: c.-24C>G (orHBA1) variant.

Hb Manitoba [alpha 102(G9)Ser -> Arg] is a rare alpha 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 (HBA1: c.309C>A) and one case carried Hb Manitoba IV (HBA1: c.307A>C)] were detected during a wide-spectrum study of alpha 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 alpha-thalassemia (alpha-thal) -alpha(3.7) (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 alpha 73(EF2)Val -> Val (alpha 2) (HBA2: c.222G>T) polymorphism is published for the first time and coexisted with two cases. The Cap +14 (C>G) (HBA2: 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 -alpha(3.7) deletion and iron deficiency is recommended when an alpha chain variant is suspected.

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 EaeI were used for definitive diagnosis. One hundred and forty-eight variants were confirmed on one or both of the two α-globin genes (HBA2, HBA1). These cases were identified as 46 different α-globin chain variants. The most common variants were Hb J-Meerut [HBA2: c.362C>A (or HBA1)] (10.1%) and Hb Q-India (HBA1: c.193G>C) (8.1%), followed by Hb J-Paris-I [HBA2: c.38C>A (or HBA1)] and Hb Manitoba II (HBA1: 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 (HBA2: c.350A>T)] and Hb Coombe Park [α127(H10)Lys→Glu (HBA2: 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 HBA1 than in HBA2 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.

Chronic lymphocytic leukaemia (CLL) consists of two biologically and clinically distinct subtypes defined by the abundance of somatic hypermutation (SHM) affecting the Ig variable heavy-chain locus (IgHV). The molecular mechanisms underlying these subtypes are incompletely understood. Here, we present a comprehensive whole-genome sequencing analysis of somatically acquired genetic events from 46 CLL patients, including a systematic comparison of coding and non-coding single-nucleotide variants, copy number variants and structural variants, regions of kataegis and mutation signatures between IgHVmut and IgHVunmut subtypes. We demonstrate that one-quarter of non-coding mutations in regions of kataegis outside the Ig loci are located in genes relevant to CLL. We show that non-coding mutations in ATM may negatively impact on ATM expression and find non-coding and regulatory region mutations in TCL1A, and in IgHVunmut CLL in IKZF3, SAMHD1,PAX5 and BIRC3. Finally, we show that IgHVunmut CLL is dominated by coding mutations in driver genes and an aging signature, whereas IgHVmut CLL has a high incidence of promoter and enhancer mutations caused by aberrant activation-induced cytidine deaminase activity. Taken together, our data support the hypothesis that differences in clinical outcome and biological characteristics between the two subgroups might reflect differences in mutation distribution, incidence and distinct underlying mutagenic mechanisms.

Chronic lymphocytic leukaemia (CLL) is characterised by considerable clinical and biological heterogeneity, with specific recurrent genomic alterations, including TP53 mutations, deletions of chromosome 17p, and IgHV mutational status, impacting on response to chemo-immunotherapy and targeted agents. Consequently, diagnostic screening for these predictive biomarkers is recommended in both national and international clinical guidelines. Current conventional methods, including fluorescent in-situ hybridisation and Sanger sequencing, exhibit shortcomings in terms of cost, speed and sensitivity, and even second-generation sequencing methods encounter technical limitations imposed by short-read lengths and bio-informatics analysis. The MinION platform from Oxford Nanopore Technologies generates exceptionally long (1-100kbp) read lengths in a short period of time and at low cost, making it a good candidate for diagnostic testing. In this paper, we present a nanopore-based CLL-specific screening assay, to simultaneously screen for both TP53 mutations and del17p13.1, as well as determining the IgHV mutation status for a single patient in one sequencing run. We sequenced 11 CLL patients and were able to generate a full diagnostic dataset for all. We identified somatic SNVs and indels in the coding region of TP53 , and demonstrate that, following error correction of the data, we could accurately define the somatically hypermutated IgHV region in all patients. We also demonstrated the ability of the MinION platform to detect large-scale genomic deletions through low-coverage whole-genome sequencing. We conclude that nanopore sequencing has the potential to provide accurate, low-cost and rapid diagnostic information, which could be applied to other cancer types.

Purpose of ReviewThe usual abundance of fresh cells and high-quality DNA derived from bone marrow aspirate and peripheral blood mean haematological malignancies are at the forefront of the application of genomics to malignancy. This review evaluates where genomics is routinely used in clinical care and where opportunities for further application exist.Recent FindingsThe 2016 revision of the WHO classification of tumours of haematopoietic and lymphoid tissues increased the number of disease entities defined by, or whose diagnosis was strongly supported by, a specific genetic change. Increasingly combinations of mutations rather than individual lesions are being used to genomically classify heterogeneous disorders to inform prognosis and direct treatment. Furthermore, the role of different genetic aberrations as markers of measurable residual disease is being evaluated in clinical trials to allow intensification/de-intensification of treatment as appropriate and early detection of relapse.SummaryImplementation of broader sequencing technologies such as whole exome/genome sequencing coupled with continuing developments in genomic technology to improve turn-around-times are likely to further reinforce the centrality of genomics in the management of haematological malignancies.

AbstractPurpose: Unmutated (UM) immunoglobulin heavy chain variable region (IgHV) status or IgHV3-21 gene usage is associated with poor prognosis in chronic lymphocytic leukemia (CLL) patients. Interestingly, IgHV3-21 is often co-expressed with light chain IgLV3-21, which is potentially able to trigger cell-autonomous BCR-mediated signaling. However, this light chain has never been characterized independently of the heavy chain IgHV3-21.Experimental Design: We performed total RNA sequencing in 32 patients and investigated IgLV3-21 prognostic impact in terms of treatment-free survival (TFS) and overall survival (OS) in 3 other independent cohorts for a total of 813 patients. IgLV3-21 presence was tested by real-time PCR and confirmed by Sanger sequencing.Results: Using total RNA sequencing to characterize 32 patients with high-risk CLL, we found a high frequency (28%) of IgLV3-21 rearrangements. Gene set enrichment analysis revealed that these patients express higher levels of genes responsible for ribosome biogenesis and translation initiation (P < 0.0001) as well as MYC target genes (P = 0.0003). Patients with IgLV3-21 rearrangements displayed a significantly shorter TFS and OS (P < 0.05), particularly those with IgHV mutation. In each of the three independent validation cohorts, we showed that IgLV3-21 rearrangements—similar to UM IgHV status—conferred poor prognosis compared with mutated IgHV (P < 0.0001). Importantly, we confirmed by multivariate analysis that this was independent of IgHV mutational status or subset #2 stereotyped receptor (P < 0.0001).Conclusions: We have demonstrated for the first time that a light chain can affect CLL prognosis and that IgLV3-21 light chain usage defines a new subgroup of CLL patients with poor prognosis. Clin Cancer Res; 24(20); 5048–57. ©2018 AACR.

Abstract Introduction Chronic Lymphocytic Leukaemia (CLL) is the most prevalent leukaemia in the Western world and characterised by clinical heterogeneity. IgHV mutation status, mutations in the TP53 gene and deletions of the p-arm of chromosome 17 are currently used to predict an individual patient's response to therapy and give an indication as to their long-term prognosis. Current clinical guidelines recommend screening patients prior to initial, and any subsequent, treatment. Routine clinical laboratory practices for CLL involve three separate assays, each of which are time-consuming and require significant investment in equipment. Nanopore sequencing offers a rapid, low-cost alternative, generating a full prognostic dataset on a single platform. In addition, Nanopore sequencing also promises low failure rates on degraded material such as FFPE and excellent detection of structural variants due to long read length of sequencing. Importantly, Nanopore technology does not require expensive equipment, is low-maintenance and ideal for patient-near testing, making it an attractive DNA sequencing device for low-to-middle-income countries. Methods Eleven untreated CLL samples were selected for the analysis, harbouring both mutated (n=5) and unmutated (n=6) IgHV genes, seven TP53 mutations (five missense, one stop gain and one frameshift) and two del(17p) events. Primers were designed to amplify all exons of TP53, along with the IgHV locus, and each primer included universal tails for individual sample barcoding. The resulting PCR amplicons were prepared for sequencing using a ligation sequencing kit (SQK-LSK108, Oxford Nanopore Technologies, Oxford, UK). All IgHV libraries were pooled and sequenced on one R9.4 flowcell, with the TP53 libraries pooled and sequenced on a second R9.4 flowcell. Whole genome libraries were prepared from 400ng genomic DNA for each sample using a rapid sequencing kit (SQK-RAD004, Oxford Nanopore Technologies, Oxford, UK), and each sample sequenced on individual flowcells on a MinION mk1b instrument (Oxford Nanopore Technologies, Oxford, UK). We developed a bespoke bioinformatics pipeline to detect copy-number changes, TP53 mutations and IgHV mutation status from the Nanopore sequencing data. Results were compared to short-read sequencing data obtained earlier by targeted deep sequencing (MiSeq, Illumina Inc, San Diego, CA, USA) and whole genome sequencing (HiSeq 2500, Illumina Inc, San Diego CA, USA). Results Following basecalling and adaptor trimming, the raw data were submitted to the IMGT database. In the absence of error correction, it was possible to identify the correct VH family for each sample; however the germline homology was not sufficient to differentiate between IgHVmut and IgHVunmut CLL cases. Following bio-informatic error correction and consensus building, the percentage to germline homology was the same as that obtained from short-read sequencing and nanopore sequencing also called the same productive rearrangements in all cases. A total of 77 TP53 variants were identified, including 68 in non-coding regions, and three synonymous SNVs. The remaining 6 were predicted to be functional variants (eight missense and two stop-gains) and had all been identified in early MiSeq targeted sequencing. However, the frameshift mutation was not called by the analysis pipeline, although it is present in the aligned reads. Using the low-coverage WGS data, we were able to identify del(17p) events, of 19Mb and 20Mb length, in both patients with high confidence. Conclusions Here we demonstrate that characterization of the IgHV locus in CLL cases is possible using the MinION platform, provided sufficient downstream analysis, including error correction, is applied. Furthermore, somatic SNVs in TP53 can be identified, although similar to second generation sequencing, variant calling of small insertions and deletions is more problematic. Identification of del(17p) is possible from low-coverage WGS on the MinION and is inexpensive. Our data demonstrates that Nanopore sequencing can be a viable, patient-near, low-cost alternative to established screening methods, with the potential of diagnostic implementation in resource-poor regions of the world. Disclosures Schuh: Giles, Roche, Janssen, AbbVie: Honoraria.

The 100 000 Genome Project aims to develop a diagnostics platform by introducing whole genome sequencing (WGS) into clinical practice. Samples from patients with chronic lymphocytic leukaemia were subjected to WGS. WGS detection of single nucleotide variants and insertion/deletions were validated by targeted next generation sequencing showing high concordance (96·3%), also for detection of sub-clonal variants and low-frequency TP53 variants. Copy number alteration detection was verified by fluorescent in situ hybridisation and genome-wide single nucleotide polymorphism array (concordances of 86·7% and 92·9%, respectively), confirming adequate sensitivity by WGS. Our results confirm that WGS can provide comprehensive genomic characterisation for clinical trials, drug discovery and, ultimately, precision medicine.

The current influx of economic migrants and asylum seekers from countries with a high prevalence of haemoglobinopathies creates new challenges for health care systems and diagnostic laboratories. The migration of carriers introduces new and novel haemoglobinopathy mutations to the diagnostic repertoire of a laboratory, often creating new pressures to improve and update the carrier screening technology and diagnostic scope. For antenatal screening programmes, the marriage of partners from different ethnic groups can lead to the risk of compound heterozygote children being born novel mutation combinations, creating problems in the provision of accurate advice regarding the expected phenotype of the thalassaemia or haemoglobinopathy disorder. In the UK, the impact of immigration required the National Haemoglobinopathy Reference laboratory to change the strategy and techniques used for the molecular diagnosis of thalassaemia and the haemoglobinopathies. In 2005, due to the increasingly large range of β-thalassaemia mutations that needed to be diagnosed, the laboratory switched from a three-step screening procedure using ARMS-PCR to a simpler but more expensive one-step strategy of DNA sequencing of the beta and alpha globin genes for all referrals. After ten years of employing this strategy, a further 57 novel thalassaemia and haemoglobionpopthy alleles were discovered (11 new β-chain variants, 15 α-chain variants, 19 β-thalassaemia mutations and 12 α+-thalassaemia mutations), increasing further the extremely heterogeneous spectrum of globin gene mutations in the UK population.

Disease relapse is the major cause of treatment failure after allogeneic stem cell transplantation (allo-SCT) in acute myeloid leukemia (AML). To identify AML-associated genes prognostic of AML relapse post-allo-SCT, we resequenced 35 genes in 113 adults at diagnosis, 49 of whom relapsed. Two hundred sixty-two mutations were detected in 102/113 (90%) patients. An increased risk of relapse was observed in patients with mutations in WT1 (P = .018), DNMT3A (P = .045), FLT3 ITD (P = .071), and TP53 (P = .06), whereas mutations in IDH1 were associated with a reduced risk of disease relapse (P = .018). In 29 patients, we additionally compared mutational profiles in bone marrow at diagnosis and relapse to study changes in clonal structure at relapse. In 13/29 patients, mutational profiles altered at relapse. In 9 patients, mutations present at relapse were not detected at diagnosis. In 15 patients, additional available pre-allo-SCT samples demonstrated that mutations identified posttransplant but not at diagnosis were detectable immediately prior to transplant in 2 of 15 patients. Taken together, these observations, if confirmed in larger studies, have the potential to inform the design of novel strategies to reduce posttransplant relapse highlighting the potential importance of post-allo-SCT interventions with a broad antitumor specificity in contrast to targeted therapies based on mutational profile at diagnosis.

The immunoglobulin heavy-chain variable region gene (IgHV) mutational status is considered the gold standard of prognostication in chronic lymphocytic leukemia (CLL) and is currently determined by Sanger sequencing that allows the analysis of the major clone. Using next-generation sequencing (NGS), we sequenced the IgHV gene from two independent cohorts: (A) 270 consecutive patient samples obtained at diagnosis and (B) 227 patients from the UK ARCTIC-AdMIRe clinical trials. Using complementary DNA from purified CD19+CD5+ cells, we demonstrate the presence of multiple rearrangements in independent experiments and showed that 24.4% of CLL patients express multiple productive clonally unrelated IgHV rearrangements. On the basis of IgHV-NGS subclonal profiles, we defined five different categories: patients with (a) multiple hypermutated (M) clones, (b) 1 M clone, (c) a mix of M-unmutated (UM) clones, (d) 1 UM clone and (e) multiple UM clones. In population A, IgHV-NGS classification stratified patients into five different subgroups with median treatment-free survival (TFS) of >280(a), 131(b), 94(c), 29(d), 15(e) months (P<0.0001) and a median OS of >397(a), 292(b), 196(c), 137(d) and 100(e) months (P<0.0001). In population B, the poor prognosis of multiple UM patients was confirmed with a median TFS of 2 months (P=0.0038). In conclusion, IgHV-NGS highlighted one quarter of CLL patients with multiple productive IgHV subclones and improves disease stratification and raises important questions concerning the pre-leukemic cellular origin of CLL.

Although TP53, NOTCH1, and SF3B1 mutations may impair prognosis of patients with chronic lymphocytic leukemia (CLL) receiving frontline therapy, the impact of these mutations or any other, alone or in combination, remains unclear at relapse. The genome of 114 relapsed/refractory patients included in prospective trials was screened using targeted next-generation sequencing of the TP53, SF3B1, ATM, NOTCH1, XPO1, SAMHD1, MED12, BIRC3, and MYD88 genes. We performed clustering according to both number and combinations of recurrent gene mutations. The number of genes affected by mutation was ≥ 2, 1, and 0 in 43 (38%), 49 (43%), and 22 (19%) respectively. Recurrent combinations of ≥ 2 mutations of TP53, SF3B1, and ATM were found in 22 (19%) patients. This multiple-hit profile was associated with a median progression-free survival of 12 months compared with 22.5 months in the remaining patients (P = .003). Concurrent gene mutations are frequent in patients with relapsed/refractory CLL and are associated with worse outcome.