A novel, somatic, transforming mutation in the extracellular domain of Epidermal Growth Factor Receptor identified in myeloproliferative neoplasm.

We describe a novel ERBB1/EGFR somatic mutation (p. C329R; c.985 T > C) identified in a patient with JAK2V617F Polycythaemia Vera (PV). This substitution affects a conserved cysteine residue in EGFR domain 2 and leads to the formation of a ligand-independent covalent receptor dimer, associated with increased transforming potential. Aberrant signalling from the EGFRC329R receptor is cell type-dependent and in the TF1.8 erythroid cell line expression of this mutant suppresses EPO-induced differentiation. Clonal analysis shows that the dominant JAK2V617F-positive clone in this PV patient harbors EGFRC329R, thus this mutation may contribute to clonal expansion. Somatic mutations affecting other ERBB and related receptor tyrosine kinases are observed in myeloproliferative neoplasms (MPN), and we show elevated EGFR levels in MPN samples, consistent with previous reports. Thus activation of this group of receptors, via multiple mechanisms, may contribute to clonal growth and survival of the JAK2V617F disease clone in MPN.

BCR-ABL1 kinase domain mutations may persist at very low levels for many years and lead to subsequent TKI resistance.

BACKGROUND: BCR-ABL1 mutation analysis is recommended for chronic myeloid leukaemia patients. However, mutations may become undetectable after changing therapy, and it is unknown whether they have been eradicated. METHODS: We examined longitudinal data of patients with imatinib-resistant mutations, which became undetectable by Sanger sequencing to determine whether mutations could reappear, and the related circumstances. RESULTS: Identical imatinib- and nilotinib-resistant mutations reappeared following further therapy changes in five patients, and was associated with subsequent nilotinib resistance in four. CONCLUSION: The data suggest that some BCR-ABL1 mutations may persist at undetectable levels for many years after changing therapy, and can be reselected and confer resistance to subsequent inhibitors.

Early molecular and cytogenetic response is predictive for long-term progression-free and overall survival in chronic myeloid leukemia (CML).

In the face of competing first-line treatment options for CML, early prediction of prognosis on imatinib is desirable to assure favorable survival or otherwise consider the use of a second-generation tyrosine kinase inhibitor (TKI). A total of 1303 newly diagnosed imatinib-treated patients (pts) were investigated to correlate molecular and cytogenetic response at 3 and 6 months with progression-free and overall survival (PFS, OS). The persistence of BCR-ABL transcript levels >10% according to the international scale (BCR-ABL(IS)) at 3 months separated a high-risk group (28% of pts; 5-year OS: 87%) from a group with >1-10% BCR-ABL(IS) (41% of pts; 5-year OS: 94%; P=0.012) and from a group with ≤1% BCR-ABL(IS) (31% of pts; 5-year OS: 97%; P=0.004). Cytogenetics identified high-risk pts by >35% Philadelphia chromosome-positive metaphases (Ph+, 27% of pts; 5-year OS: 87%) compared with ≤35% Ph+ (73% of pts; 5-year OS: 95%; P=0.036). At 6 months, >1% BCR-ABL(IS) (37% of pts; 5-year OS: 89%) was associated with inferior survival compared with ≤1% (63% of pts; 5-year OS: 97%; P<0.001) and correspondingly >0% Ph+ (34% of pts; 5-year OS: 91%) compared with 0% Ph+ (66% of pts; 5-year OS: 97%; P=0.015). Treatment optimization is recommended for pts missing these landmarks.

Establishment of a standardized multiplex assay with the analytical performance required for quantitative measurement of BCR-ABL1 on the international reporting scale.

Accurate and standardized methods for the quantitative measurement of BCR-ABL1 are a prerequisite for monitoring of treatment response in t(9;22)-positive leukemia. Here, we describe a novel multiplex assay system based on the proven TaqMan and Armored RNA technologies and optimized for sensitive detection of three BCR-ABL1 fusion transcripts and ABL1 in a single reaction. Analytical experiments confirmed the absence of significant competition between the simultaneous amplification reactions and established the sensitivity, linearity and precision of the assay. Comparative studies with 115 clinical specimens resulted in high qualitative and quantitative agreement with independent singleplex laboratory-developed tests routinely used in clinical testing. Direct comparison with a reference laboratory calibrated to the international scale (IS) demonstrated minimal analytical bias between methods and an overall accuracy and precision within the performance range required for quantitative measurement of BCR-ABL1 on the IS. We conclude that detection of e1a2, b2a2, b3a2 and ABL1 can be achieved in a multiplex assay format compatible with IS reporting. Further clinical validation of the assay could improve the operational efficiency of clinical laboratories, increase their adherence to current recommendations for b2a2/b3a2 reporting on the IS and provide for the first time an opportunity to standardize e1a2-monitoring results.

Patients with chronic myeloid leukemia who maintain a complete molecular response after stopping imatinib treatment have evidence of persistent leukemia by DNA PCR.

Around 40-50% of patients with chronic myeloid leukemia (CML) who achieve a stable complete molecular response (CMR; undetectable breakpoint cluster region-Abelson leukemia gene human homolog 1 (BCR-ABL1) mRNA) on imatinib can stop therapy and remain in CMR, at least for several years. This raises the possibility that imatinib therapy may not need to be continued indefinitely in some CML patients. Two possible explanations for this observation are (1) CML has been eradicated or (2) residual leukemic cells fail to proliferate despite the absence of ongoing kinase inhibition. We used a highly sensitive patient-specific nested quantitative PCR to look for evidence of genomic BCR-ABL1 DNA in patients who sustained CMR after stopping imatinib therapy. Seven of eight patients who sustained CMR off therapy had BCR-ABL1 DNA detected at least once after stopping imatinib, but none has relapsed (follow-up 12-41 months). BCR-ABL1 DNA levels increased in all of the 10 patients who lost CMR soon after imatinib cessation, whereas serial testing of patients in sustained CMR showed a stable level of BCR-ABL1 DNA. This more sensitive assay for BCR-ABL1 provides evidence that even patients who maintain a CMR after stopping imatinib may harbor residual leukemia. A search for intrinsic or extrinsic (for example, immunological) causes for this drug-free leukemic suppression is now indicated.

Sensitive detection and quantification of minimal residual disease in chronic myeloid leukaemia using nested quantitative PCR for BCR-ABL DNA.

Increasing numbers of patients with chronic myeloid leukaemia (CML) treated with tyrosine kinase inhibitors achieve undetectable levels of BCR-ABL mRNA using sensitive quantitative real-time reverse transcriptase PCR (RT-qPCR) methods and a method to measure minimal residual disease (MRD) in patients with low levels could be of value. Following isolation and sequencing of the patient-specific BCR-ABL breakpoint, a DNA-based nested qPCR assay was established, and MRD was measured by this method and one-round RT-qPCR in 38 samples from 24 patients with CML. Mixing experiments using patient DNA in normal DNA indicated that DNA qPCR could detect BCR-ABL sequences at a limit of approximately 10⁻6. In 22 samples in which MRD was detectable by both methods, comparison of the results of DNA qPCR with the results obtained on the same sample by RT-qPCR showed good correlation. In another 16 samples, BCR-ABL mRNA was not detectable by RT-qPCR. In 8 of the 16 samples, BCR-ABL DNA was detected at levels ranging from 1.1 × 10⁻5 up to 2.8 × 10⁻4 and in the remaining eight samples BCR-ABL was not detected by either method. In one patient, who had stopped imatinib, an almost 1000-fold rise in MRD, to 5.2 × 10⁻4 was observed in sequential samples. Nested DNA qPCR was more sensitive than one-round RT-qPCR and could be used for the monitoring of patients with CML with very low levels of MRD.

Harmonization of molecular monitoring of CML therapy in Europe.

The high efficacy of the standard treatment of chronic myeloid leukemia (CML) with imatinib has prompted the need for accurate methods to monitor response at levels below the landmark of complete cytogenetic remission. Quantification of BCR-ABL transcripts has proven to be the most sensitive method available, and has shown prognostic impact with regard to progression-free survival. Until recently, variations in methods used to quantify BCR-ABL made it difficult to compare results between laboratories. An international program is now underway to harmonize the reporting of results according to an international scale (IS). In this review, we consider the background to the IS and the progress that has been made to date, with a particular focus on ongoing harmonization efforts in Europe. We provide recommendations for the propagation of the IS by national or regional laboratory networks.

Dasatinib-associated major molecular responses in patients with chronic myeloid leukemia in chronic phase following imatinib failure: response dynamics and predictive value.

Dasatinib is a highly potent BCR-ABL inhibitor that has shown durable efficacy in patients with chronic phase (CP) chronic myeloid leukemia (CML) after resistance, suboptimal response, or intolerance to prior imatinib. In patients with CML, BCR-ABL transcript measurement is the most sensitive method for assessing minimal residual disease. Here, molecular responses were analyzed in 1067 patients with CML-CP treated with dasatinib during phase II/III trials. After 3, 6, 12, and 24 months of follow-up, a major molecular response (MMR) was achieved by 12, 22, 35, and 40% of patients, respectively. The 24-month MMR rate was 34% in patients with resistance or suboptimal response to imatinib (n=829) and 63% in imatinib-intolerant patients (n=238). Among patients who had achieved a complete cytogenetic response (CCyR), 72% also achieved MMR. Responses with dasatinib 100 mg once daily were similar to other doses. In landmark analyses, 24-month progression-free survival was higher in patients who had achieved MMR or CCyR at 12 months than in those without MMR or CCyR at 12 months. MMR at 12 months was associated with a longer duration of CCyR. Overall, this analysis shows that dasatinib treatment results in high MMR rates in patients with CML-CP after imatinib failure.

Six-year follow-up of patients receiving imatinib for the first-line treatment of chronic myeloid leukemia.

Imatinib mesylate is considered standard of care for first-line treatment of chronic phase chronic myeloid leukemia (CML-CP). In the phase III, randomized, open-label International Randomized Study of Interferon vs STI571 (IRIS) trial, previously untreated CML-CP patients were randomized to imatinib (n=553) or interferon-alpha (IFN) plus cytarabine (n=553). This 6-year update focuses on patients randomized to receive imatinib as first-line therapy for newly diagnosed CML-CP. During the sixth year of study treatment, there were no reports of disease progression to accelerated phase (AP) or blast crisis (BC). The toxicity profile was unchanged. The cumulative best complete cytogenetic response (CCyR) rate was 82%; 63% of all patients randomized to receive imatinib and still on study treatment showed CCyR at last assessment. The estimated event-free survival at 6 years was 83%, and the estimated rate of freedom from progression to AP and BC was 93%. The estimated overall survival was 88% -- or 95% when only CML-related deaths were considered. This 6-year update of IRIS underscores the efficacy and safety of imatinib as first-line therapy for patients with CML.

Characterization of a reference material for BCR-ABL (M-BCR) mRNA quantitation by real-time amplification assays: towards new standards for gene expression measurements.

Monitoring of BCR-ABL transcripts has become established practice in the management of chronic myeloid leukemia. However, nucleic acid amplification techniques are prone to variations which limit the reliability of real-time quantitative PCR (RQ-PCR) for clinical decision making, highlighting the need for standardization of assays and reporting of minimal residual disease (MRD) data. We evaluated a lyophilized preparation of a leukemic cell line (K562) as a potential quality control reagent. This was found to be relatively stable, yielding comparable respective levels of ABL, GUS and BCR-ABL transcripts as determined by RQ-PCR before and after accelerated degradation experiments as well as following 5 years storage at -20 degrees C. Vials of freeze-dried cells were sent at ambient temperature to 22 laboratories on four continents, with RQ-PCR analyses detecting BCR-ABL transcripts at levels comparable to those observed in primary patient samples. Our results suggest that freeze-dried cells can be used as quality control reagents with a range of analytical instrumentations and could enable the development of urgently needed international standards simulating clinically relevant levels of MRD.

Rationale for the recommendations for harmonizing current methodology for detecting BCR-ABL transcripts in patients with chronic myeloid leukaemia.

Molecular monitoring for patients with chronic myeloid leukaemia (CML) has become an important practice in the era of imatinib therapy. For successful widespread introduction into the mainstream patient monitoring schedule, many procedural aspects of the complex real-time quantitative polymerase chain reaction (RQ-PCR) technique for measuring BCR-ABL transcripts require optimization. Recommendations for harmonizing the differing methodologies have recently been proposed. These recommendations were designed to maximize reliability of analysis for clinical decision making and proposed the adoption of an International Scale of measurement. The purpose of this review is to present the evidence and supporting data for specific recommendations. These recommendations include use of the same source of cells, either blood or marrow, for analysis; for validation of equal PCR amplification efficiencies of cDNA and standards when using a plasmid to construct standard curves and for ensuring ongoing high-level performance by undertaking a quality assurance programme. Clinicians must know the measurement reliability of an RQ-PCR assay to be able to determine the significance of a change in BCR-ABL level. An assay with poor precision limits the clinical usefulness of results. International harmonization should establish RQ-PCR measurement of BCR-ABL as the best method for monitoring treatment response for patients with CML.

Limited clinical value of regular bone marrow cytogenetic analysis in imatinib-treated chronic phase CML patients monitored by RQ-PCR for BCR-ABL.

Real-time quantitative polymerase chain reaction (PCR) for BCR-ABL mRNA in the peripheral blood (RQ-PCR) provides an accurate and reliable measure of response to therapy in chronic myeloid leukaemia (CML). We wanted to determine in what circumstances additional clinically relevant information was provided by simultaneous cytogenetic analysis in RQ-PCR monitored patients receiving imatinib treatment. We analysed 828 simultaneous RQ-PCR and bone marrow cytogenetic analyses from 183 patients with chronic phase CML with a median follow-up of 20 months. Cytogenetic progression was defined as Philadelphia (Ph)-positive clonal evolution, loss of complete cytogenetic response or an increase of > or = 20% Ph-positive cells. Cytogenetic progression occurred in 24/183 (13%) patients. At the time of cytogenetic progression, none of the 24 patients had a major molecular response (MMR; > or = 3-log reduction in BCR-ABL from standardised baseline). There were 320 RQ-PCR results from 95 patients indicating MMR. No abnormality was detected in any of the corresponding cytogenetic analyses. A policy of regular RQ-PCR monitoring with cytogenetic analysis targetted only to patients who have not achieved, or have lost MMR would represent a rational approach to monitoring and spare most patients the discomfort of multiple marrow aspirates. This approach depends upon availability of an accurate, reproducible RQ-PCR assay with ongoing quality assurance.

Imatinib produces significantly superior molecular responses compared to interferon alfa plus cytarabine in patients with newly diagnosed chronic myeloid leukemia in chronic phase.

We analyzed molecular responses in 55 newly diagnosed chronic-phase chronic myeloid leukemia (CML) patients enrolled in a phase 3 study (the IRIS trial) comparing imatinib to interferon-alfa plus cytarabine (IFN+AraC). BCR-ABL/BCR% levels were measured by real-time quantitative RT-PCR and were significantly lower for the imatinib-treated patients at all time points up to 18 months, P<0.0001. The median levels for imatinib-treated patients continued to decrease and had not reached a plateau by 24 months. A total of 24 IFN+AraC-treated patients crossed over to imatinib. Once imatinib commenced, the median BCR-ABL/BCR% levels in these patients were not significantly different to those on first-line imatinib for the equivalent number of months. The incidence of progression in imatinib-treated patients, defined by hematologic, cytogenetic or quantitative PCR criteria, was significantly higher in the patients who failed to achieve a 1 log reduction by 3 months or a 2 log reduction by 6 months, P=0.002. A total of 49 patients were screened for BCR-ABL kinase domain mutations. Mutations were detected in two imatinib-treated patients who crossed over from IFN+AraC and both lost their imatinib response. In conclusion, first-line imatinib-treated patients had profound reductions in BCR-ABL/BCR%, which significantly exceeded those of IFN+AraC-treated patients and early measurements were predictive of subsequent response.

Successful peripheral blood stem cell mobilisation with filgrastim in patients with chronic myeloid leukaemia achieving complete cytogenetic response with imatinib, without increasing disease burden as measured by quantitative real-time PCR.

Imatinib mesylate (Glivec) is a selective inhibitor of bcr-abl tyrosine kinase, the product of the Philadelphia chromosome, which is the hallmark of chronic myeloid leukaemia (CML). With imatinib, complete cytogenetic response (CCR) can be achieved in over 70% of newly diagnosed patients with CML. However, the optimal long-term management of patients who achieve CCR after imatinib is unknown. With longer follow-up, it is anticipated that some patients are likely to progress and become candidates for autologous transplantation. We studied filgrastim (r-metHuG-CSF) mobilisation of peripheral blood stem cells (PBSC) in 32 patients who have achieved CCR with imatinib. Our data demonstrate that (1) the target CD34(+) cell yields of >/=2.0 x 10(6)/kg were attained with filgrastim 10 microg/kg/day, in 9/18 (50%) of patients during uninterrupted imatinib therapy, and in 10/14 (70%) when imatinib was temporarily withheld. The median CD34(+) cell yield per aphaeresis was 0.70 x 10(6)/kg (range 0.14-2.18) and 2.90 x 10(6)/kg (range 0.15-8.71) in the two groups, respectively (P&<0.005). (2) The cell yields did not correlate with the duration of imatinib administration. (3) There was no impact of the mobilisation procedure on the level of leukaemia as measured by serial blood bcr-abl levels using real-time quantitative PCR with either protocol. (4) bcr-abl remained detectable at low levels in the harvests in most but not all patients. In conclusion, filgrastim can safely be used to mobilise PBSC in patients who have achieved CCR with imatinib, but CD34(+) cell yields are significantly improved when imatinib is temporarily withheld.

A novel BCR-ABL transcript (e8a2) with the insertion of an inverted sequence of ABL intron 1b in a patient with Philadelphia-positive chronic myeloid leukaemia.

An atypical BCR-ABL transcript in a patient with Philadelphia (Ph)-positive chronic myeloid leukaemia (CML) was detected using a long polymerase chain reaction technique. Sequence analysis revealed a joining of BCR exon e8 and ABL exon a2, with a 55-base pair (bp) insert of an inverted sequence of ABL intron 1b between them, giving rise to an in frame e8a2 BCR-ABL transcript. This is the first report of a BCR-ABL transcript with the insertion of an inverted sequence. The long PCR technique is a useful screen for atypical BCR-ABL transcripts not detected by standard techniques.