PDF file - 442K, Functional Analysis of the ATM-p53-p21 pathway in CLL4 trial

Key Points Cladribine is regarded as the first treatment of choice for symptomatic hairy cell leukemia. This large international study reports a complete response in 72% of cases and a continuous complete response in 20% of patients.

This chapter presents case studies related to T-cell prolymphocytic leukaemia (T-PLL). A first patient, an 82-year-old woman, was admitted for investigation of progressive fatigue. She was found to have widespread small-volume lymphadenopathy and splenomegaly. The blood film showed medium to large cells with oval, bean-shaped and sometimes clefted nuclei, condensed chromatin, frequent nucleoli and basophilic cytoplasm with occasional tufts and blebs. Flow cytometry showed these cells to have a CD4+, CD7+, CD5+, CD2+ and uniform CD26+ immunophenotype, in keeping with a diagnosis of T-PLL. A second patient, a 33-year-old man, presented with progressive erythema and swelling of the skin of his face and torso. The chapter also contains a series of images to illustrate key features to recognize when undertaking a morphological review and show how they can be integrated with supplementary information to reach a final diagnosis. This is followed by a related multiple choice question with the preferred answers.

Causes of death, in particular deaths due to infection, have not been widely studied in randomised trials in chronic lymphocytic leukaemia. With long-term follow-up (median 13 years) we examined the cause of death in 600/777 patients in the LRF CLL4 trial. Blood samples, taken at randomisation from 499 patients, were available for identifying gene mutations. Infection was a cause of death in 258 patients (43%). Patients dying of infection were more likely than those who died of other causes to have received ≥2 lines of treatment (194/258 [75%] versus 231/342 [68%], P = 0.04) and to have died in the winter months (149/258 [58%] versus 166/342 [49%], P = 0.03), respectively. In patients with mutation data, the factors significantly associated with death from infection versus all other deaths were 11q deletion (47/162 [29%] versus 40/209 [19%], P = 0.03) and mutations of the BRAF, FBXW7, NRAS and XPO1 genes. Death was caused by an infection in 46/67 assessable patients (69%) who had a mutation of one or more of these four genes versus only 129/333 patients (39%) without any of these mutations (odds ratio: 3.46 [95% CI 1.98–6.07] P < 0.0001). Careful management of infection risk, including prophylaxis against infection, may be important in patients who carry these mutations.

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

Abstract We studied the clinical and laboratory characteristics of 71 patients with hairy cell leukemia to ascertain whether prognostic factors important for the therapy of the disease existed. Gra...

Background - Although there has been a revolution in the treatment of chronic lymphocytic leukemia (CLL), the challenge remains to identify the right drugs for the right patients. It is widely accepted that CIT, including the 'gold standard' fludarabine, cyclophosphamide and rituximab (FCR), is contraindicated for patients with TP53 disruption and, more recently, unmutated IGHV genes. Also patients with short, dysfunctional telomeres were shown to have inferior outcomes when treated with FCR-based regimens. To date, a role for CD49d in this setting has not been established. Aims - Here we evaluated the ability of telomere lenght (TL) and CD49d to cooperate with IGHV gene status to predict progression-free survival (PFS) in patients treated with FCR-based regimens in the frontline setting in three UK trials, ARCTIC, ADMIRE and CLL4. Methods - The study included a discovery cohort of 245 CLL treated with FCR/FCR-like regimens according to the two UK trials ARCTIC and ADMIRE. As there was no significant difference in PFS between the three arms of the study (P = 0.97), analysis was performed on the combined cohort. The median follow-up was 77.5 months with 157 progressions and 76 deaths. Twenty-nine patients were TP53 deleted and/or mutated, with shorter PFS compared to cases without TP53 disruption (final cohort, 216 TP53 wild-type CLL). The validation cohort was composed of 119 CLL samples derived from patients randomised to receive fludarabine, cyclophosphamide (FC) from the UK CLL4 trial. The median follow-up was 67.2 months with 99 progressions and 77 deaths. Fifteen CLL were TP53 mutated/deleted, with shorter PFS compared to cases without TP53 disruption (final cohort, 104 TP53 wild-type CLL). TL was measured using the high-throughput STELA assay and patients were bifurcated into two groups with either short telomeres inside the fusogenic range (TL-IFR) or long telomeres outside the fusogenic range (TL-OFR). CD49d was measured by flow cytometry and dichotomized as CD49dpos and CD49dneg based on the established 30% cut-off. For IGHV gene status, the 2% cutoff was used to split patients in mutated (IGHV-M) and ummutated (IGHV-UM). Results - In the 216 CLL with wild-type TP53 status from the ARCTIC/ADMIRE trials, CD49d expression was a predictor of PFS (P=0.02; HR=1.46 [1.03-2.06]). In keeping with previous reports, patients with IGHV-UM genes (P<.0001; HR=2.53 [1.79-3.58]) or TL-IFR (P=0.0002; HR=1.97 [1.30-2.98]) showed also significantly shorter PFS. This data was used as a starting point for a risk-stratification algorithm (Figure 1A). IGHV-UM cases could not be further dissected by TL or CD49d expression (P=0.76 log-rank test), their 8-year PFS being just 19.0%; (HR=5.58 [3.70-8.42]). In contrast, the IGHV-M group could be stratified by TL and CD49d expression (P<0.001 log-rank test). In particular, 13/84 (15.5%) of IGHV-M CLL with TL-IFR showed a median PFS of 3.0 years with a 8-year PFS of 15.4% (HR=6.45 [1.84-22.58]), similar to IGHV-UM cases (P = 0.19). Patients with IGHV-M genes and TL-OFR could be further stratified by CD49d expression into categories with different 8-year PFS: 43.1% in IGHV-M/TL-OFR/CD49dpos cases (HR=2.52 [1.08-5.89]), and 75.5% in the IGHV-M/TL-OFR/CD49dneg reference group (Table 1). Figure 1B shows the overlaid Kaplan-Meier curves for this hierarchical stratification. The proposed algorithm was then evaluated in the UK CLL4 trial. In keeping with the known inferiority of FC when compared with FCR, the 8-year PFS was just 5.2% in the IGHV-UM subset, irrespective of TL or CD49d expression. In concordance with the discovery cohort, the IGHV-M subset with TL-IFR had a similar median PFS to IGHV-UM cases (P=0.84). Furthermore, CD49d expression was able to stratify cases with TL-OFR; in particular, 18/104 (17.3%) were IGHV-M/TL-OFR/CD49dneg CLL which had a 8-year PFS of 77.8% (summary in Table 1 and Figure 1C). Conclusion - Our analysis shows that only IGHV-M/TL-OFR/ CD49dneg patients may benefit from CIT; this group represented just 56/321 cases (17.4%) of the combined cohort, suggesting that approximately 82% of patients should be considered for alternative therapies. Incorporation of this algorithm into clinical trial design and real-world practice would enable rational, risk-adapted, clinical management with the aim of treating all CLL patients with the optimal therapeutic regimen in the frontline setting. Disclosures Norris: TeloNostiX Ltd: Current equity holder in private company, Patents & Royalties. Hillmen:Gilead: Other: Financial or material support, Research Funding; Alexion: Consultancy, Research Funding, Speakers Bureau; Apellis: Consultancy, Research Funding, Speakers Bureau; AstraZeneca: Consultancy, Speakers Bureau; Acerta: Other: Financial or material support; Roche: Consultancy, Other: Financial or material support, Research Funding, Speakers Bureau; AbbVie: Consultancy, Other: Financial or material support, Research Funding, Speakers Bureau; Pharmacyclics: Other: Financial or material support, Research Funding; Janssen: Consultancy, Other: Financial or material support, Research Funding, Speakers Bureau. Rawstron:BD Biosciences (Intrasure): Patents & Royalties. Baird:TeloNostiX Ltd: Current equity holder in private company, Patents & Royalties. Fegan:TeloNostiX Ltd: Current equity holder in private company, Patents & Royalties. Pepper:TeloNostiX Ltd: Current equity holder in private company, Patents & Royalties.

This is an historical account of the randomised trials in chronic lymphocytic leukaemia in the UK between the years 1978 to 2004, describing their gestation, the treatments used and the main lessons learnt. Those lessons include: (1) how best to use chlorambucil, which was the first effective treatment for CLL; (2) the significant difference in survival between the sexes; and (3) the value of prognostic markers, both morphological and molecular, which continue to be relevant to current practice.

Overview, Daniel Catovsky cytochemistry in the classification of leukemias, Georges Flandrin, Marie-Therese Daniel, Alistair Crockard bone marrow histology, Reiner Bartl, Bertha Frisch, Rolf Burkhardt ultrastructure of the leukemic cell, Janine Breton-Gorius the ultrastructure of lymphoid cells, Estela Matutes, Daniele Robinson monoclonal antibodies in the diagnosis of acute leukemia, George Janossy, D.Campana immunocytochemical labelling of leukemia samples with monoclonal antibodies by the APAAP procedure, David Y.Mason, Wendy N.Erber terminal deoxynucleotidyl transferase, adenosine deaminase and purine nucleoside phosphorylase - diagnostic tools and chemotherapeutic targets, Mary Sue Colman, Lela K.Riley, Jennifer K.Morrow lymphocyte functional assays, Frank Miedema the clonal culture in vitro of human myeloid cells, Donald Metcalf cytogenetics of acute leukemia, Dorothy L.Williams cytogenetic abnormalities of mature T-cell malignancies, Vasantha Brito-Babapulle immunoglobin and T-cell receptor gene analysis for the investigation of lymphoproliferative disorders, L.Foroni, P.Mason, L.Luzzatto the analysis of molecular changes in leukemia, Bryan D.Young, Trivadi S.Ganesan, Lynne Hiorns.

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.

This chapter deals with the basic demographics and the presenting features of CLL and how to assess patients once a diagnosis has been made. CLL is predominantly a disease of the elderly with a preponderance of male patients. New evidence suggests that there are variations in gender incidence according to the clinical status of patients, with a higher male:female ratio in the groups with worse prognosis. Full blood counts and a physical examination are the basis of the existing staging systems of Rai and Binet. Establishing the patient’s clinical stage sets the scene for the frequency of follow-up, the possible need for therapy and the need for cytogenetic and molecular investigations. Examination of blood films is still important, to identify the presence of prolymphocytes and to consider alternative diagnostic possibilities. In addition, biochemical tests such as beta-2 microglobulin and lactate dehydrogenase are a valuable part of the prognostic evaluation. Patients may need support to deal with the psychological and quality of life issues arising from their disease.

Genome-wide association studies have provided evidence for inherited genetic predisposition to chronic lymphocytic leukemia (CLL). To gain insight into the mechanisms underlying CLL risk we analyze chromatin accessibility, active regulatory elements marked by H3K27ac, and DNA methylation at 42 risk loci in up to 486 primary CLLs. We identify that risk loci are significantly enriched for active chromatin in CLL with evidence of being CLL-specific or differentially regulated in normal B-cell development. We then use in situ promoter capture Hi-C, in conjunction with gene expression data to reveal likely target genes of the risk loci. Candidate target genes are enriched for pathways related to B-cell development such as MYC and BCL2 signalling. At 14 loci the analysis highlights 63 variants as the probable functional basis of CLL risk. By integrating genetic and epigenetic information our analysis reveals novel insights into the relationship between inherited predisposition and the regulatory chromatin landscape of CLL.

Grafting of diethylphophonate-based ILs onto γ-Al2O3 powder in solvothermal condition was achieved on mesoporous γ-alumina powder and membrane (A = organic spacer).

The clustering of different types of B-cell malignancies in families raises the possibility of shared aetiology. To examine this, we performed cross-trait linkage disequilibrium (LD)-score regression of multiple myeloma (MM) and chronic lymphocytic leukaemia (CLL) genome-wide association study (GWAS) data sets, totalling 11,734 cases and 29,468 controls. A significant genetic correlation between these two B-cell malignancies was shown ( R g = 0.4, P = 0.0046). Furthermore, four of the 45 known CLL risk loci were shown to associate with MM risk and five of the 23 known MM risk loci associate with CLL risk. By integrating eQTL, Hi-C and ChIP-seq data, we show that these pleiotropic risk loci are enriched for B-cell regulatory elements and implicate B-cell developmental genes. These data identify shared biological pathways influencing the development of CLL and, MM and further our understanding of the aetiological basis of these B-cell malignancies.

Even in the era of the targeted therapies, there remains clinical value in exploring the impact of disease characteristics in chemo-immunotherapy (CIT) trials, namely to identify features that contribute most to long-term outcomes, thereby pinpointing patients destined to benefit from these therapies. One such feature is the CLL DNA methylome, that recapitulates normal B cell maturation, with IGHV mutated (M-CLL) and unmutated CLL (U-CLL) retaining an imprint of the DNA methylation signature of memory (m-CLL) and naive B cells (n-CLL), respectively, with a third intermediate epigenetic subgroup (i-CLL) with borderline mutation status. The pyrosequencing analysis of 5 CpG sites can divide CLL into these three subgroups (Queiros 2015, Leukemia 29:598-605), each with different clinico-biological features.

The previous edition of the consensus guidelines of the International Workshop on Chronic Lymphocytic Leukemia (iwCLL), published in 2008, has found broad acceptance by physicians and investigators caring for patients with CLL. Recent advances including the discovery of the genomic landscape of the disease, the development of genetic tests with prognostic relevance, and the detection of minimal residual disease (MRD), coupled with the increased availability of novel targeted agents with impressive efficacy, prompted an international panel to provide updated evidence- and expert opinion-based recommendations. These recommendations include a revised version of the iwCLL response criteria, an update on the use of MRD status for clinical evaluation, and recommendations regarding the assessment and prophylaxis of viral diseases during management of CLL.