Summary
Sequential measurement of BCR‐ABL1 mRNA levels by reverse transcription quantitative polymerase chain reaction (RT‐qPCR) is embedded in the management of patients with chronic myeloid ...leukaemia (CML), and has played an important role in the remarkable improvement in patient outcomes seen in this disease. As a provider of external quality assessment (EQA) in this area, UK NEQAS for Leucocyte Immunophenotyping (UKNEQAS LI) has a unique perspective on the changing face of BCR‐ABL1 testing in CML. To assess the impact of technical standardisation and the development of the International Scale (IS) upon the accuracy of BCR‐ABL1 testing, we reviewed EQA trial data from 2007 to 2015. Comparison of participant results identified considerable variability at both high and low levels of disease, including therapeutically important decision points; however, results converted to the IS showed less variability compared to unconverted data sets. We also found that different methods of converting to the IS produce consistently different median results within UKNEQAS LI IS data sets. This data suggests that whilst the development of the IS has improved the comparability of results between centres, there is still the need for further improvement in the processes of converting raw results to the IS in order to fully realise the benefits of molecular monitoring of CML.
Aberrant activation of tyrosine kinases, caused by either mutation or gene fusion, is of major importance for the development of many hematologic malignancies, particularly myeloproliferative ...neoplasms. We hypothesized that hitherto unrecognized, cytogenetically cryptic tyrosine kinase fusions may be common in non-classical or atypical myeloproliferative neoplasms and related myelodysplastic/myeloproliferative neoplasms.
To detect genomic copy number changes associated with such fusions, we performed a systematic search in 68 patients using custom designed, targeted, high-resolution array comparative genomic hybridization. Arrays contained 44,000 oligonucleotide probes that targeted 500 genes including all 90 tyrosine kinases plus downstream tyrosine kinase signaling components, other translocation targets, transcription factors, and other factors known to be important for myelopoiesis.
No abnormalities involving tyrosine kinases were detected; however, nine cytogenetically cryptic copy number imbalances were detected in seven patients, including hemizygous deletions of RUNX1 or CEBPA in two cases with atypical chronic myeloid leukemia. Mutation analysis of the remaining alleles revealed non-mutated RUNX1 and a frameshift insertion within CEBPA. A further mutation screen of 187 patients with myelodysplastic/myeloproliferative neoplasms identified RUNX1 mutations in 27 (14%) and CEBPA mutations in seven (4%) patients. Analysis of other transcription factors known to be frequently mutated in acute myeloid leukemia revealed NPM1 mutations in six (3%) and WT1 mutations in two (1%) patients with myelodysplastic/myeloproliferative neoplasms. Univariate analysis indicated that patients with mutations had a shorter overall survival (28 versus 44 months, P=0.019) compared with patients without mutations, with the prognosis for cases with CEBPA, NPM1 or WT1 mutations being particularly poor.
We conclude that mutations of transcription and other nuclear factors are frequent in myelodysplastic/myeloproliferative neoplasms and are generally mutually exclusive. CEBPA, NPM1 or WT1 mutations may be associated with a poor prognosis, an observation that will need to be confirmed by detailed prospective studies.
We sought to evaluate the efficacy of the purine analogue cladribine in 79 patients with advanced systemic mastocytosis (AdvSM) using data from the ‘German Registry on Disorders of Eosinophils and ...Mast Cells (GREM)’. The overall response rate according to modified Valent criteria (46 evaluable patients) for first- (1L) and second-line (2L) cladribine treatment was 41% (12/29) and 35% (6/17,
P
= 0.690), respectively, and the median overall survival (OS, all patients evaluable) was 1.9 years (
n
= 48) and 1.2 years (
n
= 31;
P
= 0.311). Univariate and multivariable analyses of baseline and on-treatment parameters identified diagnosis of mast cell leukemia (hazard ratio HR 3.5, 95% confidence interval CI, 1.3–9.1,
P
= 0.012), eosinophilia ≥ 1.5 × 10
9
/L (HR 2.9 CI 1.4–6.2,
P
= 0.006) and < 3 cycles of cladribine (HR 0.4 CI 0.2–0.8,
P
= 0.008) as independent adverse prognostic parameters for OS. There was no impact of other laboratory (anemia, thrombocytopenia, serum tryptase) or genetic markers (mutations in
SRSF2
,
ASXL1
or
RUNX1
) on OS. In consequence, none of the recently established prognostic scoring systems (MARS, IPSM, MAPS or GPSM) was predictive for OS. Modified Valent criteria were superior to a single factor-based response assessment (HR 2.9 CI 1.3–6.6,
P
= 0.026). In conclusion, cladribine is effective in 1L and 2L treatment of AdvSM. Mast cell leukemia, eosinophilia, application of < 3 cycles and a lack of response are adverse prognostic markers.
The FIP1L1-PDGFRA fusion gene is a recurrent molecular lesion in eosinophilia-associated myeloproliferative disorders, predicting a favorable response to imatinib mesylate. To investigate its ...prevalence, 376 patients with persistent unexplained hypereosinophilia were screened by the United Kingdom reference laboratory, revealing 40 positive cases (11%). To determine response kinetics following imatinib, real-time quantitative–polymerase chain reaction (RQ-PCR) assays were developed and evaluated in samples accrued from across the European LeukemiaNet. The FIP1L1-PDGFRA fusion transcript was detected at a sensitivity of 1 in 105 in serial dilution of the EOL-1 cell line. Normalized FIP1L1-PDGFRA transcript levels in patient samples prior to imatinib varied by almost 3 logs. Serial monitoring was undertaken in patients with a high level of FIP1L1-PDGFRA expression prior to initiation of imatinib (100 mg/d-400 mg/d). Overall, 11 of 11 evaluable patients achieved at least a 3-log reduction in FIP1L1-PDGFRA fusion transcripts relative to the pretreatment level within 12 months, with achievement of molecular remission in 9 of 11 (assay sensitivities 1 in 103-105). In 2 patients, withdrawal of imatinib was followed by a rapid rise in FIP1L1-PDGFRA transcript levels. Overall, these data are consistent with the exquisite sensitivity of the FIP1L1-PDGFRα fusion to imatinib, as compared with BCR-ABL, and underline the importance of RQ-PCR monitoring to guide management using molecularly targeted therapies.
Summary
This study evaluated the efficacy and safety of imatinib in chronic eosinophilic leukaemia (CEL, n = 23) and hypereosinophilic syndrome (HES, n = 13). In CEL with FIP1L1‐PDGFRA (n = 16) or ...various PDGFRB fusion genes (n = 5), complete haematological remission (CHR) was achieved in 95% (20/21) after 3 months. Complete molecular remission (CMR) was seen in 75% (12/16) of cases with FIP1L1‐PDGFRA positive CEL by 6 months, and in 87% (13/15) after 12 months. CMR was achieved in three of five PDGFRB fusion positive patients after 3, 9 and 18 months respectively. All patients are currently on imatinib (100 mg; n = 13, 400 mg; n = 8) and no molecular relapse has yet been observed (median 26·7 months; range, 6·9–39·9). Imatinib was less effective in HES and CEL without known molecular aberration (n = 15); CHR was observed in 40% (6/15) of patients, two patients relapsed after 4·8 and 24·5 months. Three patients died due to imatinib‐resistant progressive CEL (n = 2) or myocardial infarction (n = 1) unrelated to study treatment. Overall, imatinib was well tolerated with a low incidence of grade III/IV toxicities. These data confirmed the long‐term efficacy of imatinib for PDGFR‐rearranged CEL patients, and also showed that a minority of HES cases without known molecular aberrations may benefit from imatinib.
EZH2, a component of the polycomb repressive complex 2, catalyses the trimethylation of histone H3 lysine 27, a chromatin mark associated with transcriptional repression. EZH2 loss-of-function ...mutations are seen in myeloid neoplasms and are associated with an adverse prognosis. Missense mutations in the SET/CXC domain abrogate catalytic activity as assessed by in vitro histone methylation assays, but missense mutations clustering in the conserved DI and DII regions retain activity. To understand the role of DI and DII mutations, we initially developed a cell-based histone methylation assay to test activity in a cellular context. Murine induced pluripotent stem cells lacking EZH2 were transiently transfected with wild type or mutant EZH2 (n = 15) and any resulting histone methylation was measured by flow cytometry. All DI mutations (n = 5) resulted in complete or partial loss of methylation activity whilst 5/6 DII mutations retained activity. Next, we assessed the possibility of splicing abnormalities induced by exon 8 mutations (encoding DII) using RT-PCR from primary patient samples and mini-gene assays. Exon 8 mutations resulted in skipping of exon 8 and an out-of-frame transcript. We have therefore shown that mutations within regions encoding EZH2 domains DI and DII are pathogenic by loss of function and exon skipping, respectively.
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