Search

Abstract Whole genome sequencing (WGS) provides comprehensive, individualised cancer genomic information. However, routine tumour biopsies are formalin-fixed and paraffin-embedded (FFPE), damaging DNA, historically limiting their use in WGS. Here we analyse FFPE cancer WGS datasets from England’s 100,000 Genomes Project, comparing 578 FFPE samples with 11,014 fresh frozen (FF) samples across multiple tumour types. We use an approach that characterises rather than discards artefacts. We identify three artefactual signatures, including one known (SBS57) and two previously uncharacterised (SBS FFPE, ID FFPE), and develop an “FFPEImpact” score that quantifies sample artefacts. Despite inferior sequencing quality, FFPE-derived data identifies clinically-actionable variants, mutational signatures and permits algorithmic stratification. Matched FF/FFPE validation cohorts shows good concordance while acknowledging SBS, ID and copy-number artefacts. While FF-derived WGS data remains the gold standard, FFPE-samples can be used for WGS if required, using analytical advancements developed here, potentially democratising whole cancer genomics to many.

Background:. We aimed to investigate the effect of compression dressing on edema, ecchymosis, pain, and ocular surface irritation after ptosis surgery. Methods:. After ptosis correction [anterior levator reinsertion (and resection) (ALR), if necessary additional blepharoplasty], the eye was randomized for compression dressing or transparent eye shield. Edema and ecchymosis were scored on a four-point rating scale by a blinded observer 1 day (D1), 1 week (D7), and 8 weeks (D56) after surgery; the same was done for scar formation regarding redness and bulging at D7 and D56. Aesthetic outcome was ranked by patient and blinded observer using the Global Aesthetic Improvement Score at D1, D7, and D56. Postoperative pain was scored using a visual analogue scale (0 to 10) at D1. Impairment after surgery by dressing or eye shield was evaluated at D1. Results:. Ecchymosis, edema, scar formation, and aesthetic outcome ranked by the patient and blinded observer did not differ between the groups with compression dressing and eye shield at any day of follow-up (P > 0.05). Postoperative pain and impairment were the same in both groups (P > 0.05). One case of corneal erosion occurred in the group with compression dressing at D1 (P = 0.342). At D7, corneal staining was increased in the group without compression dressing (P = 0.930). Conclusions:. Compression dressing after ALR does not reduce ecchymosis, edema, or postoperative pain and has no effect on early scar formation or aesthetic results. To prevent corneal erosion caused by the dressing, it can be omitted after ALR without inferiority for the early postoperative results.

Abstract Background The capacity for invasive tissue biopsies followed by histopathology diagnosis in sub-Saharan Africa is severely limited. Consequently, many cancer patients are diagnosed late and outcomes are poor. Here, we propose to evaluate circulating tumour (ct) DNA analysis (“liquid biopsy”), a less invasive and faster approach to diagnose endemic EBV-driven lymphomas (EBVL) in East Africa. Methods We will evaluate the clinical utility of an already validated ctDNA test prospectively in a head-to-head comparison against histopathology. The primary endpoint is the time from presentation to the specialist centre to a final diagnosis of EBV- Lymphoma. Secondary endpoints include the sensitivity and specificity of liquid biopsy and health economic benefits over histopathology. One hundred forty-six patients will be recruited over 18 months. Patients will be eligible if they are 3–30 years of age and have provided written consent or assent as per IRB guidelines. Tissue and venous blood samples will be processed as per established protocols. Clinical data will be captured securely and in real-time into a REDCap database. The time from presentation to diagnosis will be documented. The sensitivity and specificity of the methods can be estimated within 5% error margin with 95% confidence level using 73 cases and 73 controls. Health-economic assessment will include micro-costing of ctDNA test and histopathology. All results will be reviewed in a multidisciplinary tumour board. Discussion The study evaluates the clinical utility of ctDNA in improving the speed of diagnostic pathways for EBVL in sub-Saharan Africa. Our results would provide proof-of-principle that ctDNA can be used as a diagnostic tool in areas without access to regular pathology, that transfer of the tool is feasible, and that it leads to an earlier and faster diagnosis. The potential clinical and economic impact of this proposal is thus significant. If successful, this study will provide appropriate, and cost-effective diagnostic tools that will promote earlier diagnosis of EBVL and potentially other cancers in countries with restricted healthcare resources. Trial registration Pan African Clinical Trials Registry: PACTR202204822312651 , registered on 14th-April-2022.

Burkitt lymphoma (BL) has a cure rate of around 95% when treated with chemo-immunotherapy that is standard of care in high-income countries (Minard-Colin et al., 2020, New England Journal of Medicine 382, 2207–2219), but currently, more than 50% of children and young adults with endemic BL (Epstein Barr virus driven BL) in sub-Saharan Africa (SSA) do not survive. Treatment for BL is largely free of charge, but there is limited access to reliable diagnostic services leading to significant delays and misdiagnoses. Innovations in histopathology such as whole slide imaging and the use of novel diagnostic approaches, in particular using circulating cell-free viral and/or lymphoma DNA (liquid biopsy), could increase access to timely and reliable diagnosis and improve outcomes in SSA.

Background: Telepathology utilizing high-throughput static whole slide image scanners is proposed to address the challenge of limited pathology services in resource-restricted settings. However, the prohibitive equipment costs and sophisticated technologies coupled with large amounts of space to set up the devices make it impractical for use in resource-limited settings. Herein, we aimed to address this challenge by validating a portable whole slide imaging (WSI) device against glass slide microscopy (GSM) using lymph node biopsies from suspected lymphoma cases from Sub-Saharan Africa. Material and methods: This was part of a multicenter prospective case–control head-to-head comparison study of liquid biopsy against conventional pathology. For the portable WSI scanner validation, the study pathologists evaluated 105 surgical lymph node specimens initially confirmed by gold-standard pathology between February and December 2021. The tissues were processed according to standard protocols for Hematoxylin and Eosin (H&E) and Immunohistochemistry (IHC) staining by well-trained histotechnicians, then digitalized the H& E and IHC slides at each center. The digital images were anonymized and uploaded to a HIPAA-compliant server by the histotechnicians. Three study pathologists independently accessed and reviewed the images after a 6-week washout. The agreement between diagnoses established on GSM and WSI across the pathologists was described and measured using Cohens’ kappa coefficient (κ). Results: On GSM, 65.5% (n=84) of specimens were lymphoma; 25% were classified as benign, while 9.5% were metastatic. Morphological quality assessment on GSM and WSI established that 79.8% and 53.6% of cases were of high quality, respectively. When diagnoses by GSM were compared to WSI, the overall concordance for various diagnostic categories was 93%, 100%, and 86% for lymphoma, metastases, and benign conditions respectively. The sensitivity and specificity of WSI for the detection of lymphoma were 95.2% and 85.7%, respectively, with an overall inter-observer agreement (κ) of 0.86; 95% CI (0.70–0.95). Conclusions: We demonstrate that mobile whole slide imaging (WSI) is non-inferior to conventional glass slide microscopy (GSM) for the primary diagnosis of malignant infiltration of lymph node specimens. Our results further provide proof of concept that mobile WSI can be adapted to resource-restricted settings for primary surgical pathology and would significantly improve patient outcomes.

Abstract Background Sickle cell disease (SCD) is an important cause of under-five mortality. Tanzania is the 5th country in the world with the highest births prevalence of SCD individuals. Significant advances in the neonatal diagnosis of SCD using rapid point-of-care testing have been made. However genetic confirmation is still required for positive cases, in uncertain cases, in multiply transfused patients, to resolve compound heterozygosity (Hb S/ β0 Thal or Hb S/ β+ thal) not uncommon in the coastal regions of East Africa and increasingly also for pre-marital counselling and potentially for future curative approaches such as gene therapy. The currently available DNA tests are prohibitively expensive. Here, we describe an easy-to-use, affordable and accurate β-globin sequencing approach that can be easily integrated within existing NBS for SCD and other haemoglobinopathies especially in Low- and Middle-income Countries. Aim To evaluate an affordable DNA technology for the diagnosis of Sickle cell disease and other haemoglobinopathies in a resource-limited setting. Methods Laboratory-based validation study was conducted by Muhimbili University of Health and Allied Sciences and the University of Oxford involving sequencing of the entire β -haemoglobin locus using the Oxford Nanopore MinION platform. A total number of 36 Dried blood spots and whole blood samples were subjected to conventional protein-based methods (isoelectric focusing, HPLC), and/or sequenced by the Sanger method as comparators. Results Sequencing results for SCD using the MinION were 100% concordant with those from the Sanger method. In addition, the long-read DNA sequencing method enabled the resolution of cases with unusual phenotypes which make up 1% of all children in Tanzania. The cost is £11/ sample for consumables, which is cheaper compared to other sequencing platforms. Conclusions This is the first report of a comprehensive single DNA assay as a definitive diagnostic test for SCD and other haemoglobinopathies. The test is fast, precise, accurate and affordable.

Abstract Studying anterior lens capsule cutting edge profiles from femtosecond laser-assisted capsulotomy procedures performed before and after lens fragmentation. Twenty eyes (10 patients) with age-related cataract underwent femtosecond laser-assisted surgery (FLACS) using the Ziemer Z8 platform. First step of laser surgery was either capsulotomy (group first) or fragmentation (group second). One eye of each patient was assigned randomly, the second eye treated with the different sequence of procedures. After anterior capsule removal, tissue was fixed in cacodylate-buffered solution and cutting-edge profiles were analysed using scanning electron microscopy (SEM). All cases had cataract grade 2 and 3 based on LOCS III grading. SEM analysis showed more smooth edges in the first group, especially in cases with pseudoexfoliation (P = 0.037); more tags and bridges and a significant number of staggered cutting patterns (7 out of 10 cases) in the second group. All cases evolved the same microgroves with “valleys and mountains “ as signs of the photodisruption process. Femtosecond laser capsulotomy should be performed before lens fragmentation minimizing the rate of cutting errors. Especially in eyes with advanced cataract, as intracapsular pressure may increase due to lens fragmentation without anterior capsular opening.

Background: Castleman’s Disease (CD) is a diverse group of non-neoplastic lymphoproliferative disorders characterized by lymphoid endothelial and follicular hyperplasia. Human Immunodeficiency Virus (HIV)-associated Human Herpes Virus type-8 (HHV-8) CD is a form of CD affecting HIV-positive individuals with concomitant HHV-8 infection as the aetiological driver. While the prevalence of HIV and HHV-8 is high in Sub-Saharan Africa (SSA), there are however very few reported cases of CD from the region. This might reflect a high rate of misdiagnosis and under-reporting of HIV-associated HHV-8 CD in limited-resource settings. If diagnosed early and precisely, the HIV-associated HHV-8 CD can be successfully treated with chemotherapy and/or single-agent targeted therapy (rituximab), regimens that are increasingly becoming available. Case presentation: We highlight a rare case of HHV-8 associated Multicentric Castleman Disease (MCD) in an HIV-positive individual who presented with lymphadenopathy, haemolytic anaemia, monoclonal gammopathy and atypical hyaline vascular variant and showed good early response with single-agent rituximab. Conclusions: A high index of suspicion is needed to diagnose CD, especially in regions where infectious diseases are common and share clinicopathological features with malignant diseases. Early diagnosis is the key to appropriate management, with a potential cure for the disease.

Limited data exists to show the correlation of (tumour protein 53) TP53 mutation detected by Next generation sequencing (NGS) and the presence/absence of deletions of 17p13 detected by FISH. The study which is the largest series to date includes 2332 CLL patients referred for analysis of del(17p) by FISH and TP53 mutations by NGS before treatment. Using a 10% variant allele frequency (VAF) threshold, cases were segregated into high burden mutations (≥10%) and low burden mutations (

Abstract 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.

The clinical course of chronic lymphocytic leukaemia (CLL) is variable and difficult to predict. Here, the authors conduct a genome wide association study meta-analysis for time to first treatment in CLL patients and report two loci associating with progressive disease.

Abstract 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.

Abstract 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.

Abstract: 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; P = .018) in CLL4, and for progression-free survival (HR, 0.25; 95% CI, 0.10-0.57; P = .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.

Abstract Background Transformation of chronic lymphocytic leukaemia (CLL) to diffuse large B-cell lymphoma (DLCBL) type Richter’s syndrome (RS) carries a dismal prognosis. Standard-of-care chemoimmunotherapy for de novo RS is inadequate with median survival of less than one year. Patients are frequently elderly or have co-morbidities limiting dose-intense chemotherapy. Treatment of relapsed/refractory (R/R) RS and RS emerging after CLL-directed therapy represent urgent unmet clinical needs. Agents targeting Bruton’s tyrosine kinase (BTK) deliver improved outcomes for patients with high-risk CLL and expand effective treatments to frailer patients. Acalabrutinib is an oral, second-generation BTK inhibitor with a favourable toxicity profile and demonstrated activity in CLL and B-cell lymphomas. Combination of acalabrutinib with standard-of-care CHOP-R chemoimmunotherapy offers a sound rationale to test in a prospective trial for de novo RS. Methods The prospective multicentre STELLAR study is designed in two elements, consisting of a randomised study to evaluate the safety and activity of CHOP-R chemoimmunotherapy in combination with acalabrutinib in newly diagnosed RS and single-arm studies of novel agents for other RS patient cohorts. Eligible patients with newly diagnosed DLBCL-type RS are randomised between six cycles of CHOP-R therapy and six cycles CHOP-R plus acalabrutinib, followed by acalabrutinib maintenance. The primary endpoint of the randomised component is progression free survival (PFS). Cohort 1 enrols RS patients with progressive disease following chemoimmunotherapy for acalabrutinib monotherapy. Patients with RS diagnosed while on ibrutinib may enrol in Cohort 2, a single-arm study of CHOP-R plus acalabrutinib. The primary endpoint for the single-arm studies is overall response rate (ORR). Secondary endpoints for all cohorts are overall survival (OS), quality of life and proportion of patients proceeding to stem cell transplantation. The study will be accompanied by exploratory analysis of the mutational landscape of RS and the relationship between dynamic changes in sequential circulating tumour DNA samples and clinical outcomes. Discussion The STELLAR randomised trial evaluates the role of CHOP-R plus acalabrutinib in newly diagnosed RS patients. The single-arm platform studies enable the incorporation of promising novel therapies into the protocol. The STELLAR study has potential to identify novel biomarkers of treatment response in this high-risk malignancy. Trial registration EudraCT: 2017–004401-40, registered on the 31-Oct-2017. IRSCTN: https://www.isrctn.com/ISRCTN52839057, registered on the 04-Mar-2019. ClinicalTrials.gov: NCT03899337, registered on 02-April-2019.

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.

Abstract Background One of the functions of the reformed Cancer Drugs Fund in England is as a managed access fund, providing conditional funding for cancer drugs where there is uncertainty in the economic case, and where that uncertainty can be addressed by data collection during two years’ use in the NHS. Our study characterises likely sources of such uncertainty, through a review of recent NICE Technology Appraisals. Methods Discussions of uncertainty in NICE Appraisal Committees were extracted from published Single Technology Appraisals of cancer drugs, 2014–2016, and categorised inductively. The location of the comments within the structured Appraisal document was used as a proxy for the degree of concern shown by the Committee. Results Twenty-nine appraisals were analysed, of which 23 (79%) were recommended for funding. Six main sources of uncertainty were identified. Immaturity of survival data, and issues relating to comparators, were common sources of uncertainty regardless of degree of concern. Uncertainties relating to quality of life, and the patient population in the trial, were discussed frequently but rarely occurred in the more uncertain appraisals. Concerns with trial design, and cost uncertainty, were less common, but a high proportion contributed to the most uncertain appraisals. Funding decisions were not driven by uncertainty in the evidence base, but by the expected cost per QALY relative to acceptance thresholds, and the resultant level of uncertainty in the decision. Conclusions The reformed CDF is an improvement on its predecessor. However the main types of uncertainty seen in recent cancer appraisals will not readily be resolved solely by 2 years’ RWD collection in the reformed CDF; where there are no ongoing trials to provide longer-term data, randomised trials rather than RWD may be needed to fully resolve questions of relative efficacy. Other types of uncertainty, and concerns with generalisability, may be more amenable to the RWD approach, and it is these that we expect to be the focus of data collection arrangements in the reformed CDF.

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.

Chronic lymphocytic leukemia is associated with a highly heterogeneous disease course in terms of clinical outcomes and responses to chemoimmunotherapy. This heterogeneity is partly due to genetic aberrations identified in chronic lymphocytic leukemia cells such as mutations of TP53 and/or deletions in chromosome 17p [del(17p)], resulting in loss of one TP53 allele. These aberrations are associated with markedly decreased survival and predict impaired response to chemoimmunotherapy thus being among the strongest predictive markers guiding treatment decisions in chronic lymphocytic leukemia. Clinical trials demonstrate the importance of accurately testing for TP53 aberrations [both del(17p) and TP53 mutations] before each line of treatment to allow for appropriate treatment decisions that can optimize patients’ outcomes. The current report reviews the diagnostic methods to detect TP53 disruption better, the role of TP53 aberrations in treatment decisions and current therapies available for patients with chronic lymphocytic leukemia carrying these abnormalities. The standardization in sequencing technologies for accurate identification of TP53 mutations and the importance of continued evaluation of TP53 aberrations throughout initial and subsequent lines of therapy remain unmet clinical needs as new therapeutic alternatives become available.

We performed an observational study on the efficacy of ben-damustine and rituximab (BR) as first salvage regimen in chronic lymphocytic leukemia (CLL). In an intention-to-treat analysis including 237 patients, the median progression-free survival (PFS) was 25 months. The presence of del(17p), unmutated IGHV and advanced stage were associated with a shorter PFS at multivariate analysis. The median time-to-next treatment was 31.3 months. Front-line treatment with a chemoimmunotherapy regimen was the only predictive factor for a shorter time to next treatment at multivariate analysis. The median overall survival (OS) was 74.5 months. Advanced disease stage (i.e. Rai stage III-IV or Binet stage C) and resistant disease were the only parameters significantly associated with a shorter OS. Grade 3-5 infections were recorded in 6.3% of patients. A matched-adjusted indirect comparison with ibrutinib given second-line within Named Patient Programs in the United Kingdom and in Italy was carried out with OS as objective end point. When restricting the analysis to patients with intact 17p who had received chemoimmunotherapy in first line, there was no difference in OS between patients treated with ibrutinib (63% alive at 36 months) and patients treated with BR (74.4% alive at 36 months). BR is an efficacious first salvage regimen in CLL in a real-life population, including the elderly and unfit patients. BR and ibrutinib may be equally effective in terms of OS when used as first salvage treatment in patients without 17p deletion.

MABLE investigated the efficacy and safety of rituximab plus bendamustine or rituximab plus chlorambucil in fludarabine-ineligible patients with chronic lymphocytic leukemia. Patients received rituximab plus bendamustine or rituximab plus chlorambucil every four weeks for six cycles. Rituximab plus chlorambucil-treated patients without a complete response after Cycle 6 received chlorambucil monotherapy for at least six additional cycles or until complete response. The primary endpoint was complete response rate (confirmed by bone marrow biopsy) after Cycle 6 in first-line patients. Secondary endpoints included progression-free survival, overall survival, minimal residual disease, and safety. Overall, 357 patients were randomized (rituximab plus bendamustine, n=178; rituximab plus chlorambucil, n=179; intent-to-treat population), including 241 first-line patients (n=121 and n=120, respectively); 355 patients received treatment (n=177 and n=178, respectively; safety population). In first-line patients, complete response rate after Cycle 6 (rituximab plus bendamustine, 24%; rituximab plus chlorambucil, 9%; P=0.002) and median progression-free survival (rituximab plus bendamustine, 40 months; rituximab plus chlorambucil, 30 months; P=0.003) were higher with rituximab plus bendamustine than rituximab plus chlorambucil. Overall response rate and overall survival were not different. In first-line patients with a complete response, minimal residual disease-negativity was higher with rituximab plus bendamustine than rituximab plus chlorambucil (66% vs. 36%). Overall adverse event incidence was similar (rituximab plus bendamustine, 98%; rituximab plus chlorambucil, 97%). Rituximab plus bendamustine may be a valuable first-line option for fludarabine-ineligible patients with chronic lymphocytic leukemia.

Acute chest syndrome (ACS) is a life-threatening complication of sickle cell disease (SCD) with blood transfusion an integral part in its management. Red cell exchange (RCE) transfusion is usually regarded as preferable to top-up transfusion, because it reduces the proportion of Hemoglobin (Hb) S while at the same time avoiding circulatory overload. Despite its obvious benefits, RCE is underutilized, particularly in low-resource settings which may be due to scarcity of blood products and of expertise in carrying out exchange transfusion. We report on a young woman with SCD with severe ACS who responded promptly and dramatically to a RCE of only 0.95 L (instead of the recommended 1.4 L) and had in the end an HbS level of 48% (instead of the recommended level below 30%). Limited RCE resulted in significant clinical improvement. We suggest that limited RCE may be of benefit than no RCE in SCD patients with ACS, particularly in settings where RCE is not available.

BackgroundSingle gene tests to predict whether cancers respond to specific targeted therapies are performed increasingly often. Advances in sequencing technology, collectively referred to as next generation sequencing (NGS), mean the entire cancer genome or parts of it can now be sequenced at speed with increased depth and sensitivity. However, translation of NGS into routine cancer care has been slow. Healthcare stakeholders are unclear about the clinical utility of NGS and are concerned it could be an expensive addition to cancer diagnostics, rather than an affordable alternative to single gene testing.Methods and findingsWe validated a 46-gene hotspot cancer panel assay allowing multiple gene testing from small diagnostic biopsies. From 1 January 2013 to 31 December 2013, solid tumour samples (including non-small-cell lung carcinoma [NSCLC], colorectal carcinoma, and melanoma) were sequenced in the context of the UK National Health Service from 351 consecutively submitted prospective cases for which treating clinicians thought the patient had potential to benefit from more extensive genetic analysis. Following histological assessment, tumour-rich regions of formalin-fixed paraffin-embedded (FFPE) sections underwent macrodissection, DNA extraction, NGS, and analysis using a pipeline centred on Torrent Suite software. With a median turnaround time of seven working days, an integrated clinical report was produced indicating the variants detected, including those with potential diagnostic, prognostic, therapeutic, or clinical trial entry implications. Accompanying phenotypic data were collected, and a detailed cost analysis of the panel compared with single gene testing was undertaken to assess affordability for routine patient care. Panel sequencing was successful for 97% (342/351) of tumour samples in the prospective cohort and showed 100% concordance with known mutations (detected using cobas assays). At least one mutation was identified in 87% (296/342) of tumours. A locally actionable mutation (i.e., available targeted treatment or clinical trial) was identified in 122/351 patients (35%). Forty patients received targeted treatment, in 22/40 (55%) cases solely due to use of the panel. Examination of published data on the potential efficacy of targeted therapies showed theoretically actionable mutations (i.e., mutations for which targeted treatment was potentially appropriate) in 66% (71/107) and 39% (41/105) of melanoma and NSCLC patients, respectively. At a cost of £339 (US$449) per patient, the panel was less expensive locally than performing more than two or three single gene tests. Study limitations include the use of FFPE samples, which do not always provide high-quality DNA, and the use of "real world" data: submission of cases for sequencing did not always follow clinical guidelines, meaning that when mutations were detected, patients were not always eligible for targeted treatments on clinical grounds.ConclusionsThis study demonstrates that more extensive tumour sequencing can identify mutations that could improve clinical decision-making in routine cancer care, potentially improving patient outcomes, at an affordable level for healthcare providers.

Use of circulating tumour DNA (ctDNA) as a liquid biopsy has been proposed for potential identification and monitoring of solid tumours. We investigate a next-generation sequencing approach for mutation detection in ctDNA in two related studies using a targeted panel. The first study was retrospective, using blood samples taken from melanoma patients at diverse timepoints before or after treatment, aiming to evaluate correlation between mutations identified in biopsy and ctDNA, and to acquire a first impression of influencing factors. We found good concordance between ctDNA and tumour mutations of melanoma patients when blood samples were collected within one year of biopsy or before treatment. In contrast, when ctDNA was sequenced after targeted treatment in melanoma, mutations were no longer found in 9 out of 10 patients, suggesting the method might be useful for detecting treatment response. Building on these findings, we focused the second study on ctDNA obtained before biopsy in lung patients, i.e. when a tentative diagnosis of lung cancer had been made, but no treatment had started. The main objective of this prospective study was to evaluate use of ctDNA in diagnosis, investigating the concordance of biopsy and ctDNA-derived mutation detection. Here we also found positive correlation between diagnostic lung biopsy results and pre-biopsy ctDNA sequencing, providing support for using ctDNA as a cost-effective, non-invasive solution when the tumour is inaccessible or when biopsy poses significant risk to the patient.

We report the largest retrospective, phase IV non-interventional, observational study of ofatumumab therapy in heavily pre-treated patients with poor-prognosis chronic lymphocytic leukemia. Total number of patients was 103; median age was 65 years (range 39–85). Median number of prior lines of therapy was 4 (range 1–13), including, in most cases, rituximab-, fludarabine- and alemtuzumab-based regimens; 13 patients had been allografted. Of 113 adverse events, 28 (29%) were considered to be directly related to ofatumumab. Grade 3–4 toxicities included neutropenia (10%), thrombocytopenia (5%), anemia (3%), pneumonia (17%), and fever (3%). Two heavily pre-treated patients developed progressive multifocal leukoencephalopathy. On an intention-to-treat analysis, the overall response rate was 22% (3 complete response, 1 incomplete complete response). Median progression-free and overall survival times were 5 and 11 months, respectively. This study confirms in a daily-life setting the feasibility and acceptable toxicity of ofatumumab treatment in advanced chronic lymphocytic leukemia. The complete response rate, however, was low. Therefore, treatment with ofatumumab should be moved to earlier phases of the disease. Ideally, this should be done in combination with other agents, as recently approved for ofatumumab plus chlorambucil as front-line treatment for patients unfit for fludarabine. This study is registered at clinicaltrials.gov identifier:01453062.

Interstitial deletion of chromosome 5q is the most common chromosomal abnormality in myelodysplastic syndromes. The catalogue of genes involved in the molecular pathogenesis of myelodysplastic syndromes is rapidly expanding and next-generation sequencing technology allows detection of these mutations at great depth. Here we describe the design, validation and application of a targeted next-generation sequencing approach to simultaneously screen 25 genes mutated in myeloid malignancies. We used this method alongside single nucleotide polymorphism-array technology to characterize the mutational and cytogenetic profile of 43 cases of early or advanced del(5q) myelodysplastic syndromes. A total of 29 mutations were detected in our cohort. Overall, 45% of early and 66.7% of advanced cases had at least one mutation. Genes with the highest mutation frequency among advanced cases were TP53 and ASXL1 (25% of patients each). These showed a lower mutation frequency in cases of 5q- syndrome (4.5% and 13.6%, respectively), suggesting a role in disease progression in del(5q) myelodysplastic syndromes. Fifty-two percent of mutations identified were in genes involved in epigenetic regulation (ASXL1, TET2, DNMT3A and JAK2). Six mutations had allele frequencies

We investigated whether mutations in the KLF1 gene are associated with increased Hb F levels in ethnically diverse patients referred to our laboratory for hemoglobinopathy investigation. Functionally effective KLF1 mutations were identified in 11 out of 131 adult samples with an elevated Hb F level (1.5–25.0%). Eleven different mutations were identified, 9 of which were previously unreported. KLF1 mutations were not identified in a matched cohort of 121 samples with normal Hb F levels (

Mutations in the C-terminal region of nucleophosmin in acute myeloid leukemia (AML) result in aberrant cytoplasmic nucleophosmin (cNPM) in leukemic blast cells which is detectable by immunocytochemistry in bone marrow trephine (BMT) biopsy sections. We tested whether cNPM is detectable by immunocytochemistry in air-dried smears of AML with nucleophosmin1 (NPM1) mutations. An immunoalkaline phosphatase method was developed using the OCI-AML3 cell line, known to have mutated NPM1, and assessed on blood and marrow smears of 60 AML cases. NPM was detectable in all blast cell nucleoli and cNPM in 21 of 31 of NPM1 mutated and 15 of 29 wild-type cases. Paired air-dried smears and BMT biopsies from the same case (mutated and wild-type) gave discrepancies in cNPM expression and there was no correlation in 10 of 22 cases. Due to the high false positive and negative rates for cNPM in cell smears, this method should not be used as a surrogate for NPM1 mutations in AML.