David Oscier

Inactivating mutations of the histone methyltransferase gene EZH2 in myeloid disorders

Abnormalities of chromosome 7q are common in myeloid malignancies, but no specific target genes have yet been identified. Here, we describe the finding of homozygous EZH2 mutations in 9 of 12 individuals with 7q acquired uniparental disomy. Screening of a total of 614 individuals with myeloid disorders revealed 49 monoallelic or biallelic EZH2 mutations in 42 individuals; the mutations were found most commonly in those with myelodysplastic/myeloproliferative neoplasms (27 out of 219 individuals, or 12%) and in those with myelofibrosis (4 out of 30 individuals, or 13%). EZH2 encodes the catalytic subunit of the polycomb repressive complex 2 (PRC2), a highly conserved histone H3 lysine 27 (H3K27) methyltransferase that influences stem cell renewal by epigenetic repression of genes involved in cell fate decisions. EZH2 has oncogenic activity, and its overexpression has previously been causally linked to differentiation blocks in epithelial tumors. Notably, the mutations we identified resulted in premature chain termination or direct abrogation of histone methyltransferase activity, suggesting that EZH2 acts as a tumor suppressor for myeloid malignancies.

Assessment of fludarabine plus cyclophosphamide for patients with chronic lymphocytic leukaemia (the LRF CLL4 Trial): a randomised controlled trial

BACKGROUND Previous studies of patients with chronic lymphocytic leukaemia reported high response rates to fludarabine combined with cyclophosphamide. We aimed to establish whether this treatment combination provided greater survival benefit than did chlorambucil or fludarabine. METHODS 777 patients with chronic lymphocytic leukaemia requiring treatment were randomly assigned to fludarabine (n=194) or fludarabine plus cyclophosphamide (196) for six courses, or chlorambucil (387) for 12 courses. The primary endpoint was overall survival, with secondary endpoints of response rates, progression-free survival, toxic effects, and quality of life. Analysis was by intention to treat. This study is registered as an International Standard Randomised Controlled Trial, number NCT 58585610. FINDINGS There was no significant difference in overall survival between patients given fludarabine plus cyclophosphamide, fludarabine, or chlorambucil. Complete and overall response rates were better with fludarabine plus cyclophosphamide than with fludarabine (complete response rate 38%vs 15%, respectively; overall response rate 94%vs 80%, respectively; p<0.0001 for both comparisons), which were in turn better than with chlorambucil (complete response rate 7%, overall response rate 72%; p=0.006 and 0.04, respectively). Progression-free survival at 5 years was significantly better with fludarabine plus cyclophosphamide (36%) than with fludarabine (10%) or chlorambucil (10%; p<0.00005). Fludarabine plus cyclophosphamide was the best combination for all ages, including patients older than 70 years, and in prognostic groups defined by immunoglobulin heavy chain gene (V(H)) mutation status and cytogenetics, which were tested in 533 and 579 cases, respectively. Patients had more neutropenia and days in hospital with fludarabine plus cyclophosphamide, or fludarabine, than with chlorambucil. There was less haemolytic anaemia with fludarabine plus cyclophosphamide (5%) than with fludarabine (11%) or chlorambucil (12%). Quality of life was better for responders, but preliminary analyses showed no significant difference between treatments. A meta-analysis of these data and those of two published phase III trials showed a consistent benefit for the fludarabine plus cyclophosphamide regimen in terms of progression-free survival. INTERPRETATION Fludarabine plus cyclophosphamide should now become the standard treatment for chronic lymphocytic leukaemia and the basis for new protocols that incorporate monoclonal antibodies.

Prognostic Subgroups in B-Cell Chronic Lymphocytic Leukemia Defined by Specific Chromosomal Abnormalities

BACKGROUND AND METHODS Specific chromosomal abnormalities have been shown to affect the overall survival of patients with acute leukemia, but the possibility that specific chromosomal defects may influence the course of B-cell chronic lymphocytic leukemia (CLL) is controversial. We assessed this possibility as follows: blood mononuclear cells from 433 patients with B-cell CLL in five European centers were cultured with B-cell mitogens, and banded metaphases were studied. RESULTS Three hundred ninety-one patients could be evaluated cytogenetically, and 218 had clonal chromosomal changes. The most common abnormalities were trisomy 12 (n = 67) and structural abnormalities of chromosome 13 (n = 51; most involving the site of the retinoblastoma gene) and of chromosome 14 (n = 41). Patients with a normal karyotype had a median overall survival of more than 15 years, in contrast to 7.7 years for patients with clonal changes. Patients with single abnormalities (n = 113) did better than those with complex karyotypes (P less than 0.001). Patients with abnormalities involving chromosome 14q had poorer survival than those with aberrations of chromosome 13q (P less than 0.05). Among patients with single abnormalities, those with trisomy 12 alone had poorer survival than patients with single aberrations of chromosome 13q (P = 0.01); the latter had the same survival as those with a normal karyotype. A high percentage of cells in metaphase with chromosomal abnormalities, indicating highly proliferative leukemic cells, was associated with poor survival (P less than 0.001). Cox proportional-hazards analysis identified age, sex, the percentage of cells in metaphase with chromosomal abnormalities, and the clinical stage of the disease (Binet classification system) as independent prognostic variables. CONCLUSIONS Chromosomal analysis provides prognostic information about overall survival in addition to that supplied by clinical data in patients with B-cell CLL.

ZAP-70 expression and prognosis in chronic lymphocytic leukaemia

BACKGROUND Chronic lymphocytic leukaemia (CLL) is a heterogeneous disease; many patients never need treatment, whereas some have poor outcomes. New treatments, which can induce complete remissions, allow patients with poor outlook to be treated while they are still asymptomatic. Whether or not the IgVH gene is mutated is the best predictor of clinical outcome, but this assay is unsuited to the routine laboratory. The gene coding for ZAP-70, a tyrosine kinase protein normally expressed in T and NK cells, has been shown by gene-expression profiling to be differentially expressed between patients with mutated and unmutated IgVH genes. We assessed whether ZAP-70 could be used as a prognostic marker in CLL. METHODS We developed a flow cytometry assay for ZAP-70 protein expression and investigated its concordance with ZAP-70 mRNA expression, IgVH gene mutational status, and clinical outcome in 167 patients with CLL. FINDINGS We showed high concordance between ZAP-70 protein expression and IgVH gene mutations. 108 patients (65%) had mutated IgVH genes and were ZAP-70 negative; 46 (28%) had unmutated IgVH genes and were ZAP-70 positive. Findings were discordant in 13 patients: six (4%) had mutated IgVH genes but were ZAP-70 positive, and seven (4%) had unmutated IgVH genes and were ZAP-70 negative. Expression of mRNA showed 97% concordance with ZAP-70 protein. Median survival was 24.4 years (95% CI 15.1-33.8) in ZAP-70 negative patients and 9.3 years (7.0-11.5) in those who were ZAP-70 positive (hazard ratio 5.5, 2.8-.8). INTERPRETATION ZAP-70 protein, which can be measured by flow cytometry in the general laboratory, is a reliable prognostic marker in CLL, equivalent to that of IgVH gene mutational status.

Myelodysplastic syndromes: a scoring system with prognostic significance

Summary 141 patients with MDS were classified according to the FAB criteria and followed up for a period of 4–192 months. It was recognized that patients with RAEBT had a uniformly poor prognosis. However, there was a wide variation in survival among the other subgroups. A score of 1 was assigned to each of the following presenting haematological features: bone marrow blasts ≤ 5%, platelets ≤ 100 × 109/1, neutrophils ≤ 2.5 × 109/1 and Hb ≤ 10.0 g/dl. Therefore the score for each patient ranged between 0 and 4. There were no statistically significant differences between those patients who scored 0 or 1, or between those who scored 2 and 3. Therefore patients were put into three groups: Group A (score 0 or 1), Group B (score 2 or 3), Group C (score 4). The differences in survival between each of the three groups are highly significant (P<0.00001). This system further separates patients with RA, RAS, RAEB into good and bad prognostic groups.

Analysis of the chronic lymphocytic leukemia coding genome: role of NOTCH1 mutational activation

Next generation sequencing and copy number analysis provide insights into the complexity of the CLL coding genome, and reveal an association between NOTCH1 mutational activation and poor prognosis.

Eradication of Minimal Residual Disease in B-Cell Chronic Lymphocytic Leukemia After Alemtuzumab Therapy Is Associated With Prolonged Survival

PURPOSE To test whether eradication of minimal residual disease (MRD) in B-cell chronic lymphocytic leukemia (CLL) by alemtuzumab is associated with a prolongation of treatment-free and overall survival. PATIENTS AND METHODS Ninety-one previously treated patients with CLL (74 men and 17 women; median age, 58 years [range, 32 to 75 years]; 44 were refractory to purine analogs) received a median of 9 weeks of alemtuzumab treatment between 1996 and 2003. Regular bone marrow assessments by MRD flow cytometry were performed with the aim of eradicating detectable MRD (< 1 CLL cell in 10(5) normal cells). RESULTS Responses according to National Cancer Institute-sponsored working group response criteria were complete remission (CR) in 32 patients (36%), partial remission (PR) in 17 patients (19%), and no response (NR) in 42 patients (46%). Twenty-two (50%) of 44 purine analog-refractory patients responded to alemtuzumab. Detectable CLL was eradicated from the blood and marrow in 18 patients (20%). Median survival was significantly longer in MRD-negative patients compared with those achieving an MRD-positive CR, PR, or NR. Patients achieving an MRD-negative CR had a longer treatment-free survival than patients with MRD-positive CRs, PR, or NR: MRD-negative CRs, not reached; MRD-positive CRs, 20 months; PRs, 13 months; NR, 6 months (P < .0001). Overall survival for the 18 patients with MRD-negative remissions was 84% at 60 months. Eight (47%) of the MRD-negative patients converted to MRD positivity at a median of 28 months. CONCLUSION MRD-negative remission in CLL is achievable with alemtuzumab, leading to an improved overall and treatment-free survival.

Frequent CBL mutations associated with 11q acquired uniparental disomy in myeloproliferative neoplasms

Recent evidence has demonstrated that acquired uniparental disomy (aUPD) is a novel mechanism by which pathogenetic mutations in cancer may be reduced to homozygosity. To help identify novel mutations in myeloproliferative neoplasms (MPNs), we performed a genome-wide single nucleotide polymorphism (SNP) screen to identify aUPD in 58 patients with atypical chronic myeloid leukemia (aCML; n = 30), JAK2 mutation-negative myelofibrosis (MF; n = 18), or JAK2 mutation-negative polycythemia vera (PV; n = 10). Stretches of homozygous, copy neutral SNP calls greater than 20Mb were seen in 10 (33%) aCML and 1 (6%) MF, but were absent in PV. In total, 7 different chromosomes were involved with 7q and 11q each affected in 10% of aCML cases. CBL mutations were identified in all 3 cases with 11q aUPD and analysis of 574 additional MPNs revealed a total of 27 CBL variants in 26 patients with aCML, myelofibrosis or chronic myelomonocytic leukemia. Most variants were missense substitutions in the RING or linker domains that abrogated CBL ubiquitin ligase activity and conferred a proliferative advantage to 32D cells overexpressing FLT3. We conclude that acquired, transforming CBL mutations are a novel and widespread pathogenetic abnormality in morphologically related, clinically aggressive MPNs.

JAK2 haplotype is a major risk factor for the development of myeloproliferative neoplasms.

Chronic myeloproliferative neoplasms (MPNs) are a group of related conditions characterized by the overproduction of cells from one or more myeloid lineages. More than 95% of cases of polycythemia vera, and roughly half of essential thrombocythemia and primary myelofibrosis acquire a unique somatic 1849G>T JAK2 mutation (encoding V617F) that is believed to be a critical driver of excess proliferation. We report here that JAK2V617F-associated disease is strongly associated with a specific constitutional JAK2 haplotype, designated 46/1, in all three disease entities compared to healthy controls (polycythemia vera, n = 192, P = 2.9 × 10−16; essential thrombocythemia, n = 78, P = 8.2 × 10−9 and myelofibrosis, n = 41, P = 8.0 × 10−5). Furthermore, JAK2V617F specifically arises on the 46/1 allele in most cases. The 46/1 JAK2 haplotype thus predisposes to the development of JAK2V617F-associated MPNs (OR = 3.7; 95% CI = 3.1–4.3) and provides a model whereby a constitutional genetic factor is associated with an increased risk of acquiring a specific somatic mutation.

Trisomy 12 defines a group of CLL with atypical morphology: correlation between cytogenetic, clinical and laboratory features in 544 patients

We have analysed the clinical and laboratory features in 544 patients with chronic lymphocytic leukaemia (CLL) with available cytogenetics and fluorescence in‐situ hybridization (FISH) analysis for trisomy 12 in half of them, to examine the correlation between chromosome abnormalities and clinical or laboratory parameters. Five chromosome groups were defined: (1) trisomy 12 (18%), detected as the sole abnormality or associated with other changes; (2) del(13)(q12–14) (7%); (3) other abnormal karyotypes (20%); (4) normal karyotype (41%); and (5) no divisions (14%). There were no differences in the age distribution between the five groups. Clinical stages (Binet) were: A (74%), B (12%) and C (14%). Stage A was common in cases with del(13q)(82%), normal (84%) and other abnormal karyotypes (74%), whereas it was less common in trisomy 12 cases (64%) and those with no divisions (48%). Typical CLL morphology was found in 83% of cases; 10% had more than 10% prolymphocytes (CLL/PL) and 7% had other atypical features. CLL with trisomy 12 was the only group with a high frequency of either CLL/PL (31%) or atypical morphology (24%). Atypical morphology and CLL/PL were even more frequent when trisomy 12 was associated with other chromosomal abnormalities (70% v 46%). The incidence of cases with CLL/PL and other atypical morphology was significantly lower in the other chromosome groups (P<0.001. There were no differences in immunophenotype among the various groups except for a higher frequency of stronger Smlg and FMC7 expression in cases with trisomy 12, particularly those with CLL/PL and other atypical morphology. Our findings confirm that trisomy 12 defines a subgroup of CLL with more frequent atypical morphology, including CLL/PL, stronger SmIg and FMC7 expression, more advanced stages (B and C in 18%) and possibly worse prognosis.