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The clinical outcome of chronic myeloid leukaemia patients has vastly improved since the introduction of tyrosine kinase inhibitor treatment, with a significant proportion of patients able to achieve treatment-free remission. However, studies have shown that patients with the e13a2 transcript were less likely to achieve major molecular response compared to those with e14a2 transcripts. Most quantitative polymerase chain reaction (PCR) assays for detection of the BCR-ABL1 fusion gene do not differentiate between the two transcripts and we therefore hypothesised that technical bias linked to the qPCR assay could partially explain the discrepancy in outcomes. We designed an e14a2-specific assay and identified no difference in results compared to an e13a2 standard assay. We then demonstrated that the commercial e14a2 standards were causing a significant overestimation of the e13a2 transcripts. Finally, we reviewed patient management after the qPCR values were corrected, using our new evaluation. We concluded that despite statistically significant differences in qPCR results, there was no impact on patient management or outcome. We conclude that, at least in our institution, it would be inappropriate to perform separate assays for patients with e13a2 or e14a2.

Purpose Approximately 1–2% of chronic myeloid leukemia (CML) patients harbor atypical BCR-ABL1 transcripts that cannot be monitored by real-time quantitative PCR (RT-qPCR) using standard methodologies. Within the European Treatment and Outcome Study (EUTOS) for CML we established and validated robust RT-qPCR methods for these patients. Methods BCR-ABL1 transcripts were amplified and sequenced to characterize the underlying fusion. Residual disease monitoring was carried out by RT-qPCR with specific primers and probes using serial dilutions of appropriate BCR-ABL1 and GUSB plasmid DNA calibrators. Results were expressed as log reduction of the BCR-ABL1/GUSB ratio relative to the patient-specific baseline value and evaluated as an individual molecular response (IMR). Results In total, 330 blood samples (2–34 per patient, median 8) from 33 CML patients (19 male, median age 62 years) were analyzed. Patients expressed seven different atypical BCR-ABL1 transcripts (e1a2, n = 6; e6a2, n = 1; e8a2, n = 2; e13a3, n = 4; e14a3, n = 6; e13a3/e14a3, n = 2; e19a2, n = 12). Most patients (61%) responded well to TKI therapy and achieved an IMR of at least one log reduction 3 months after diagnosis. Four patients relapsed with a significant increase of BCR-ABL1/GUSB ratios. Conclusions Characterization of atypical BCR-ABL1 transcripts is essential for adequate patient monitoring and to avoid false-negative results. The results cannot be expressed on the International Scale (IS) and thus the common molecular milestones and guidelines for treatment are difficult to apply. We, therefore, suggest reporting IMR levels in these cases as a time-dependent log reduction of BCR-ABL1 transcript levels compared to baseline prior to therapy.

Formalin is the principal tissue fixative used worldwide for clinical and research purposes. Despite optimal preservation of morphology, its preservation of DNA and RNA is poor. As clinical diagnostics increasingly incorporates molecular-based analysis, the requirement for maintaining nucleic acid quality is of increasing importance. Here we assess an alternative non-formalin-based tissue fixation method, PAXgene Tissue system, with the aim of better preserving nucleic acids, while maintaining the quality of the tissue to be used for vital existing diagnostic techniques. In this study, these criteria are assessed in a clinically representative setting. In total, 203 paired PAXgene Tissue and formalin-fixed samples were obtained. Blind-scored haematoxylin and eosin (H&E) sections showed comparable and acceptable staining. Immunohistochemistry (IHC) staining was suboptimal using existing protocols but improved with minor method adjustment and optimisation. Quality of DNA and RNA was significantly improved by PAXgene tissue fixation [RIN 2.8 versus 3.8 (p

The demonstration of clonality is important in differentiating non-Hodgkin lymphomas (NHL) from reactive lymphoid conditions that can have similar morphological characteristics. For B-cell NHL, thi...

Solid pseudopapillary neoplasms of the pancreas are rare neoplasms that have been shown to harbor recurrent somatic pathogenic variants in the beta-catenin gene, CTNNB1. Here, we used targeted next generation sequencing to analyze these tumors for other associated mutations. Six cases of solid pseudopapillary neoplasms were studied. DNA extracted from formalin-fixed paraffin embedded tissue blocks was analyzed using the Ion Torrent platform, with the 50-gene Ampliseq Cancer Hotspot Panel v2 (CHPv2), with further variant validation performed by Sanger sequencing. Four tumors (67%) were confirmed to harbor mutations within CTNNB1, two with c.109T > G p.(Ser37Ala) and two with c.94G > A p.(Asp32Asn). One case showed a frameshift deletion in the Adenomatous Polyposis Coli gene, APC c.3964delG p.(Glu1322Lysfs*93) with a variant allele frequency of 42.6%. Sanger sequencing on non-tumoral tissue confirmed the variant was somatic. The patient with the APC mutation developed metastasis and died. In addition to the four cases harboring CTNNB1 variants, we found a case characterized by poor outcome, showing a rare frameshift deletion in the APC gene. Since the APC product interacts with beta-catenin, APC variants may, in addition to CTNNB1, contribute to the pathogenesis of solid pseudopapillary neoplasms via the Wnt signaling pathway.

AimsTargeted therapies for non-small cell lung carcinoma (NSCLC) rely on the detection of specific genomic lesions, such as mutations within the epidermal growth factor receptor (EGFR) gene. The Biocartis Idylla platform and single-use EGFR mutation test cartridge is CE-IVD for use with formalin-fixed paraffin embedded (FFPE) tumour material, but can also function off-scope using extracted DNA as input material. This can expand the utility of the platform and potentially conserve valuable tissue.MethodsWe sought to evaluate the performance of this system to detect known EGFR mutations using extracted DNA at different input levels. 130 next generation sequencing-characterised NSCLC cases possessing EGFR mutations that were theoretically detectable by the Idylla system were selected. Replicate analyses were performed using the Idylla EGFR test with up to three different DNA input levels (20 ng, 50 ng and 250 ng).ResultsConsidering only variants within the test manufacturer’s specified scope, the Idylla EGFR test generated concordant findings for 90.77% of cases at 20 ng DNA input, 98.46% at 50 ng input and 100% at 250 ng input. Analyses with discordant findings all generated control quantification cycle (CQ) values greater than 23. Very low CQ values were associated with EGFR gene amplification.ConclusionsThe Idylla EGFR Mutation Test can be used at least as well with pre-extracted DNA than with direct FFPE input. In cases with control CQ >23, reanalysis with an increased DNA input should ideally be undertaken. If this is not possible, the risk of false negative calls may be mitigated by manual review of the quantitative PCR data and/or by reflexing to alternative analysis options.

Primary breast cancer treatment relying on surgery with the use neo-adjuvant therapies has long been established treatment. Side effects from these and varying efficacy has lead to the search for novel therapies that may result in improved results. The use of Photodynamic therapy as a novel neo-adjuvant treatment alone and in combination with agents cytotoxic to breast cancer cells was investigated.

There are no validated molecular biomarkers to identify newly-diagnosed individuals with chronic-phase chronic myeloid leukemia likely to respond poorly to imatinib and who might benefit from first-line treatment with a more potent second-generation tyrosine kinase inhibitor. Our inability to predict these ‘high-risk’ individuals reflects the poorly understood heterogeneity of the disease. To investigate the potential of genetic variants in epigenetic modifiers as biomarkers at diagnosis, we used Ion Torrent next-generation sequencing of 71 candidate genes for predicting response to tyrosine kinase inhibitors and probability of disease progression. A total of 124 subjects with newly-diagnosed chronic-phase chronic myeloid leukemia began with imatinib (n=62) or second-generation tyrosine kinase inhibitors (n=62) and were classified as responders or non-responders based on the BCRABL1 transcript levels within the first year and the European LeukemiaNet criteria for failure. Somatic variants affecting 21 genes (e.g. ASXL1, IKZF1, DNMT3A, CREBBP) were detected in 30% of subjects, most of whom were non-responders (41% non-responders, 18% responders to imatinib, 38% non-responders, 25% responders to second-generation tyrosine kinase inhibitors). The presence of variants predicted the rate of achieving a major molecular response, event-free survival, progression-free survival and chronic myeloid leukemia-related survival in the imatinib but not the second-generation tyrosine kinase inhibitors cohort. Rare germline variants had no prognostic significance irrespective of treatment while some pre-leukemia variants suggest a multi-step development of chronic myeloid leukemia. Our data suggest that identification of somatic variants at diagnosis facilitates stratification into imatinib responders/non-responders, thereby allowing earlier use of second-generation tyrosine kinase inhibitors, which, in turn, may overcome the negative impact of such variants on disease progression.

// Tamara M.H. Gall 1 , Gareth Gerrard 2, 3 , Adam E. Frampton 1 , Leandro Castellano 1 , Raida Ahmad 4 , Nagy Habib 1 , Duncan Spalding 1 , Madhava Pai 1 , Letizia Foroni 4 and Long R. Jiao 1 1 Department of Surgery and Cancer, Imperial College, Hammersmith Hospital Campus, London W12 0HS, United Kingdom 2 Faculty of Medicine, Imperial College, Hammersmith Hospital Campus, London W12 0HS, United Kingdom 3 Current address: Sarah Cannon Molecular Diagnostics, HCA Healthcare UK, London WC1E 6JA, United Kingdom 4 Department of Histopathology, Imperial College, Hammersmith Hospital Campus, London W12 0HS, United Kingdom Correspondence to: Long R. Jiao, email: l.jiao@imperial.ac.uk Keywords: PDAC; ion torrent; next-generation sequencing; nanostring; cell-free DNA Received: December 02, 2018 Accepted: December 12, 2018 Published: January 22, 2019 ABSTRACT Objective: Pancreatic ductal adenocarcinoma (PDAC) is an aggressive tumour associated with poor 5-year survival. We aimed to determine factors which differentiate short and long-term survivors and identify a prognostic biomarker. Methods: Over a ten-year period, patients with resected PDAC who developed disease recurrence within 12 months (Group I) and those who had no disease recurrence for 24 months (Group II) were identified. Clinicopathological data was analysed. Ion Torrent high-throughput sequencing on DNA extracted from FFPE tumour samples was used to identify mutations. Additionally, peripheral blood samples were analysed for variants in cell-free DNA, circulating tumour cells (CTCs), and microRNAs. Results: Multivariable analysis of clinicopathological factors showed that a positive medial resection margin was significantly associated with short disease-free survival ( p = 0.007). Group I patients ( n = 21) had a higher frequency of the KRAS mutant mean variant allele (16.93% ± 11.04) compared to those in Group II ( n = 13; 7.55% ± 5.76, p = 0.0078). Group I patients also trended towards having a KRAS c.35G>A p.Gly12Asp mutation in addition to variants in other genes, such as TP53 , CDKN2A , and SMAD4 . Mutational status of cell-free DNA, and number of CTCs, was not found to be useful in this study. A circulating miRNA (hsa-miR-548ah-5p) was found to be significantly differentially expressed. Conclusions: Medial resection margin status and the frequency of KRAS mutation in the tumour tissue are independent prognostic indicators for resectable PDAC. Circulating miRNA hsa-miR-548ah-5p has the potential to be used as a prognostic biomarker.

Solid pseudopapillary neoplasms of the pancreas are rare neoplasms that have been shown to harbor recurrent somatic pathogenic variants in the beta-catenin gene, CTNNB1. Here, we used targeted next generation sequencing to analyze these tumors for other associated mutations. Six cases of solid pseudopapillary neoplasms were studied. DNA extracted from formalin-fixed paraffin embedded tissue blocks was analyzed using the Ion Torrent platform, with the 50-gene Ampliseq Cancer Hotspot Panel v2 (CHPv2), with further variant validation performed by Sanger sequencing. Four tumors (67%) were confirmed to harbor mutations within CTNNB1, two with c.109T G p.(Ser37Ala) and two with c.94G A p.(Asp32Asn). One case showed a frameshift deletion in the Adenomatous Polyposis Coli gene, APC c.3964delG p.(Glu1322Lysfs*93) with a variant allele frequency of 42.6%. Sanger sequencing on non-tumoral tissue confirmed the variant was somatic. The patient with the APC mutation developed metastasis and died. In addition to the four cases harboring CTNNB1 variants, we found a case characterized by poor outcome, showing a rare frameshift deletion in the APC gene. Since the APC product interacts with beta-catenin, APC variants may, in addition to CTNNB1, contribute to the pathogenesis of solid pseudopapillary neoplasms via the Wnt signaling pathway.

The tyrosine kinase inhibitor (TKI) imatinib has revolutionized the management of chronic myeloid leukaemia (CML). However, around 25% of patients fail to sustain an adequate response. We sought to identify gene-expression biomarkers that could be used to predict imatinib response. The expression of 29 genes, previously implicated in CML pathogenesis, were measured by TaqMan Low Density Array in 73 CML patient samples. Patients were divided into low and high expression for each gene and imatinib failure (IF), probability of achieving CCyR, progression free survival and CML related OS were compared by Kaplan-Meier and log-rank. Results were validated in a second cohort of 56 patients, with a further technical validation using custom gene-expression assays in a conventional RT-qPCR in a sub-cohort of 37 patients. Patients with low PTCH1 expression showed a worse clinical response for all variables in all cohorts. PTCH1 was the most significant predictor in the multivariate analysis compared with Sokal, age and EUTOS. PTCH1 expression assay showed the adequate sensitivity, specificity and predictive values to predict for IF. Given the different treatments available for CML, measuring PTCH1 expression at diagnosis may help establish who will benefit best from imatinib and who is better selected for second generation TKI.

The Philadelphia chromosome positive arm of the UKALLXII/ECOG2993 study for adult acute lymphoblastic leukemia (ALL) enrolled 266 patients between 1993 and 2003 (pre-imatinib cohort). In 2003 imatinib was introduced as a single-agent course following induction (N = 86, late imatinib). In 2005 imatinib was added to the second phase of induction (N = 89, early imatinib). The complete remission (CR) rate was 92% in the imatinib cohort vs 82% in the preimatinib cohort (P = .004). At 4 years, the overall survival (OS) of all patients in the imatinib cohort was 38% vs 22% in the preimatinib cohort (P = .003). The magnitude of the difference between the preimatinib and imatinib cohorts in event-free survival (EFS), OS, and relapse-free survival (RFS) seen in univariate analysis was even greater in the multivariate analysis. In the preimatinib cohort, 31% of those starting treatment achieved hematopoietic stem cell transplant (alloHSCT) compared with 46% in the imatinib cohort. A Cox multivariate analysis taking alloHSCT into account showed a modest additional benefit to imatinib (hazard ratio for EFS = 0.64, 95% confidence interval 0.44-0.93, P = .02), but no significant benefit for OS and RFS. Adding imatinib to standard therapy improves CR rate and long-term OS for adults with ALL. A proportion of the OS benefit derives from the fact that imatinib facilitates alloHSCT. This trial was registered at clinicaltrials.gov as NCT00002514.

Ikaros and Foxp1 are transcription factors that play key roles in normal lymphopoiesis and lymphoid malignancies. We describe a novel physical and functional interaction between the proteins, which requires the central zinc finger domain of Ikaros. The Ikaros-Foxp1 interaction is abolished by deletion of this region, which corresponds to the IK6 isoform that is commonly associated with high-risk acute lymphoblastic leukemia (ALL). We also identify the Gpr132 gene, which encodes the orphan G protein-coupled receptor G2A, as a novel target for Foxp1. Increased expression of Foxp1 enhanced Gpr132 transcription and caused cell cycle changes, including G2 arrest. Co-expression of wild-type Ikaros, but not IK6, displaced Foxp1 binding from the Gpr132 gene, reversed the increase in Gpr132 expression and inhibited G2 arrest. Analysis of primary ALL samples revealed a significant increase in GPR132 expression in IKZF1-deleted BCR-ABL negative patients, suggesting that levels of wild-type Ikaros may influence the regulation of G2A in B-ALL. Our results reveal a novel effect of Ikaros haploinsufficiency on Foxp1 functioning, and identify G2A as a potential modulator of the cell cycle in Ikaros-deleted B-ALL.

Background: Minimal Residual Disease (MRD) monitoring in BCR-ABL+ acute lymphoblastic leukaemia (ALL) is a valuable tool in the management of Philadelphia positive adult ALL due to the markedly poor prognosis engendered by this leukemia subtype. As part of the UKALLXII trial we received samples from 104 de novo adult ALL patients, 26 of which (25%) were found to be BCR-ABL+. BM and PB received at presentation and during chemotherapy or post SCT were tested for MRD as part of a modified UKALLXII trial protocol which aimed at assessing the efficacy of combined chemotherapy and Imatinib treatment and SCT. Aims: 1) To establish which sample source offers the highest level of sensitivity; 2) To determine if differences in disease level is associated with the BCR-Abl rearrangement exhibited. Methods: BM and/or PB samples were received at presentation and at monthly follow-up points and were tested by quantitative real-time PCR (QRT-PCR) using a Roche LightCycler 1.3 with the SYBR-green fluorescent detection system. The BCR-ABL transcript levels were normalised as a ratio against Abl levels. Operating procedures were adapted from the recommendations of the European Study Group for MRD in ALL. Results: 138 samples (65 PB, 73 BM) were analysed from 26 BCR-ABL+ patients: 15M, 11F; median age 43y (range 17y to 58y). 17 (65%) were found to be minor and nine (35%) were major. Ten patients have received Imatinib and nine have undergone transplant (7 allo-SCT, 2 autograft). 2 patients have relapsed and 2 have died (1 following relapse). Paired t-tests between PB and BM sample BCR-Abl transcript levels show that BM offers a significantly higher level of sensitivity with a median BCR-ABL level of 2.93 x 10−4 against 1.8 x 10−4 for PB (p= 0.0056, n=46). Baseline ABL levels show the converse (1.49 x 104 BM against 1.77 x 104 PB, p= 0.016, n=46) suggesting that the generally greater quantity of material associated with PB samples may result in higher quality RNA. BCR-Abl levels between major and minor patients show that patients exhibiting the major form have significantly higher levels of disease in both PB (median 3.1 x 10−2 major (n= 24) against 3.3x10−4 in patients exhibiting minor BCR-Abl (n=41), (p< 0.0001) and BM samples (median 2.0 x 10−2 major (n= 28) against 3.3 x 10−4 minor (n=45), (p= 0.0009). Paired sequential analysis for 11 patients with 2 or more follow-up samples confirms this observation (median 3.5 x 10−2 major (n=4) against 3.5 x 10−4 minor (n=7), (p= 0.008). Conclusions: In the QRT-PCR MRD monitoring of adult ALL BM samples offer a small but significantly higher level of sensitivity than PB samples. Patients exhibiting the major BCR-ABL rearrangement have significantly higher levels of disease than patients with the minor form. These data describing differential disease status within the BCR-ABL+ subgroup and variance due to sample type may be important in providing guidelines for patient management.

Malignant B-cells from a high proportion of chronic lymphocytic leukaemia (B-CLL) patients over express the multidrug resistance (MDR) -1 gene encoded transmembrane efflux pump P-glycoprotein (P-gp). Inhibition of glucosylceramide synthesis has been shown to correlate with the expression and function of P-gp and sensitise cells to cytotoxic agents. We analysed the ability of glucosylceramide synthetase (GCS) inhibitors N-butyl-deoxygalactonojirimycin (OGB-1, 500μM) and N-nonyl-deoxygalactonojirimycin (OGB-2, 100μM) to sensitise B-CLL cells to conventional cytotoxic drugs 2-chlorodeoxyadenosine (CdA), chlorambucil (Chl) and fludarabine (FdR) using the in vitro cytotoxicity MTT assay. The effect on P-gp activity was also analysed using the calcein-AM accumulation assay and the results expressed as multidrug activity factor (MAF), where a MAF of >10 in the presence of a P-gp inhibitor denotes P-gp functional activity. GCS inhibitors were cultured with B-CLL cells for 24-48h before the assays were performed. The P-gp negative cell line CEM-CCRF had no MAF activity with an IC50 for vincristine (a known P-gp substrate) of 10ug/ml to 55.5ng/ml in the presence of OGB-2. In peripheral blood mononuclear cells from 3 normal volunteers (all P-gp +ve), the mean MAF value for Z.3HCL was 23.86 and for OGB-2 was 16.2. In 9/13 B-CLL samples there was P-gp functional activity in the presence of Z.3HCL with a mean MAF value of 22.15 (range 11.27–37.3). P-gp was over expressed in10/13 B-CLL samples. However, when available samples from this cohort were assessed with OGB-1 (n=4) and OGB-2 (n=13) the MAF value was

Molecular monitoring of BCR-ABL1 expression in chronic myeloid leukaemia (CML) is well established. As the International Scale (IS) normalised BCR-ABL1/ABL1 ratio at 3 months post-treatment is now an important milestone in patients’ treatment schedule, the reliable and reproducible measurement of BCR-ABL1 levels is therefore paramount. IS conversion factors (CF) are established via sample exchange and yearly ratification with an external reference laboratory. Since any change to an established IS CF could lead to discontinuity in longitudinal results, we wished to add an internal verification step as an additional safeguard. We used the Cepheid GeneXpert qPCR and IS calibrated Xpert BCR-ABL Monitor cartridge system, parallel to our in-house pipeline on 50 CML samples, over the period of one week to verify the CF for those samples and compare it to the externally provided CF. The median non-IS in-house BCR-ABL1/ABL1 values were significantly different than that from the IS GeneXpert, but they became non-significant when adjusted to CF provided by the CXM and by the current external CF, validating it. These metrics can help decide to accept or reject an updated CF value, especially where a significant change in CF might lead to a discontinuity in ongoing patient monitoring.

Ikaros and Foxp1 are transcription factors that play key roles in normal lymphopoiesis and lymphoid malignancies. We describe a novel physical and functional interaction between the proteins, which requires the central zinc finger domain of Ikaros. The Ikaros-Foxp1 interaction is abolished by deletion of this region, which corresponds to the IK6 isoform that is commonly associated with high-risk acute lymphoblastic leukemia (ALL). We also identify the Gpr132 gene, which encodes the orphan G protein-coupled receptor G2A, as a novel target for Foxp1. Increased expression of Foxp1 enhanced Gpr132 transcription and caused cell cycle changes, including G2 arrest. Co-expression of wild-type Ikaros, but not IK6, displaced Foxp1 binding from the Gpr132 gene, reversed the increase in Gpr132 expression and inhibited G2 arrest. Analysis of primary ALL samples revealed a significant increase in GPR132 expression in IKZF1-deleted BCR-ABL negative patients, suggesting that levels of wild-type Ikaros may influence the regulation of G2A in B-ALL. Our results reveal a novel effect of Ikaros haploinsufficiency on Foxp1 functioning, and identify G2A as a potential modulator of the cell cycle in Ikaros-deleted B-ALL.

Molecular monitoring of chronic myeloid leukemia patients using robust BCR-ABL1 tests standardized to the International Scale (IS) is key to proper disease management, especially when treatment cessation is considered. Most laboratories currently use a time-consuming sample exchange process with reference laboratories for IS calibration. A World Health Organization (WHO) BCR-ABL1 reference panel was developed (MR 1 -MR 4), but access to the material is limited. In this study, we describe the development of the first cell-based secondary reference panel that is traceable to and faithfully replicates the WHO panel, with an additional MR 4.5 level. The secondary panel was calibrated to IS using digital PCR with ABL1, BCR and GUSB as reference genes and evaluated by 44 laboratories worldwide. Interestingly, we found that >40% of BCR-ABL1 assays showed signs of inadequate optimization such as poor linearity and suboptimal PCR efficiency. Nonetheless, when optimized sample inputs were used, >60% demonstrated satisfactory IS accuracy, precision and/or MR 4.5 sensitivity, and 58% obtained IS conversion factors from the secondary reference concordant with their current values. Correlation analysis indicated no significant alterations in %BCR-ABL1 results caused by different assay configurations. More assays achieved good precision and/or sensitivity than IS accuracy, indicating the need for better IS calibration mechanisms., 0, SCOPUS: ar.j, info:eu-repo/semantics/published

Molecular genetic assays for the detection of the JAK2 V617F (c.1849G>T) and other pathogenetic mutations within JAK2 exon 12 and MPL exon 10 are part of the routine diagnostic workup for patients presenting with erythrocytosis, thrombocytosis or otherwise suspected to have a myeloproliferative neoplasm. A wide choice of techniques are available for the detection of these mutations, leading to potential difficulties for clinical laboratories in deciding upon the most appropriate assay, which can lead to problems with inter-laboratory standardization. Here, we discuss the most important issues for a clinical diagnostic laboratory in choosing a technique, particularly for detection of the JAK2 V617F mutation at diagnosis. The JAK2 V617F detection assay should be both specific and sensitive enough to detect a mutant allele burden as low as 1-3%. Indeed, the use of sensitive assays increases the detection rate of the JAK2 V617F mutation within myeloproliferative neoplasms. Given their diagnostic relevance, it is also beneficial and relatively straightforward to screen JAK2 V617F negative patients for JAK2 exon 12 mutations (in the case of erythrocytosis) or MPL exon 10 mutations (thrombocytosis or myelofibrosis) using appropriate assays. Molecular results should be considered in the context of clinical findings and other haematological or laboratory results. © 2012 Blackwell Publishing Ltd.

Purpose We studied BCR-ABL1 transcript levels in patients with chronic myeloid leukemia in chronic phase (CML-CP) at 3, 6, and 12 months after starting imatinib to identify molecular milestones that would predict for overall survival (OS) and other outcomes more reliably than serial marrow cytogenetics. Patients and Methods We analyzed 282 patients with CML-CP who received imatinib 400 mg/d as first-line therapy followed by dasatinib or nilotinib if treatment with imatinib failed. We used a receiver operating characteristic curve to identify the cutoffs in transcript levels at 3, 6, and 12 months that would best predict patient outcome. We validated our findings in an independent cohort of 95 patients treated elsewhere. Results Patients with transcript levels of more than 9.84% (n = 68) at 3 months had significantly lower 8-year probabilities of OS (56.9% v 93.3%; P < .001), progression-free survival, cumulative incidence of complete cytogenetic response, and complete molecular response than those with higher transcript levels. Similarly, transcript levels of more than 1.67% (n = 87) at 6 months and more than 0.53% (n = 93) at 12 months identified high-risk patients. However, transcript levels at 3 months were the most strongly predictive for the various outcomes. When we compared OS for the groups defined molecularly at 6 and 12 months with the usual cytogenetic milestones, categorization by transcript numbers was the only independent predictor for OS (relative risk, 0.207; P < .001 and relative risk, 0.158; P < .001, respectively). Conclusion A single measurement of BCR-ABL1 transcripts performed at 3 months is the best way to identify patients destined to fare poorly, thereby allowing early clinical intervention.

We studied the relation between adherence to imatinib measured with microelectronic monitoring systems and the probabilities of losing a complete cytogenetic response (CCyR) and of imatinib failure in 87 CCyR chronic myeloid leukemia patients receiving long-term therapy. We included in our analysis the most relevant prognostic factors described to date. On multivariate analysis, the adherence rate and having failed to achieve a major molecular response were the only independent predictors for loss of CCyR and discontinuation of imatinib therapy. The 23 patients with an adherence rate less than or equal to 85% had a higher probability of losing their CCyR at 2 years (26.8% vs 1.5%, P = .0002) and a lower probability of remaining on imatinib (64.5% vs 90.6%, P = .006) than the 64 patients with an adherence rate more than 85%. In summary, we have shown that poor adherence is the principal factor contributing to the loss of cytogenetic responses and treatment failure in patients on long-term therapy.

Molecular testing for the BCR-ABL1 fusion gene by real time quantitative polymerase chain reaction (RT-qPCR) is the most sensitive routine approach for monitoring the response to therapy of patients with chronic myeloid leukaemia. In the context of tyrosine kinase inhibitor (TKI) therapy, the technique is most appropriate for patients who have achieved complete cytogenetic remission and can be used to define specific therapeutic milestones. To achieve this effectively, standardization of the laboratory procedures and the interpretation of results are essential. We present here consensus best practice guidelines for RT-qPCR testing, data interpretation and reporting that have been drawn up and agreed by a consortium of 21 testing laboratories in the United Kingdom and Ireland in accordance with the procedures of the UK Clinical Molecular Genetics Society.

Activation of the EVI-1 oncogene has been reported in acute myeloid leukemia, chronic myeloid leukemia (CML) in blast crisis, and less commonly, in chronic-phase CML patients. We screened an unselected cohort of 75 chronic-phase CML patients who had failed imatinib for expression of EVI-1 and sought a correlation with subsequent outcome on the second-generation tyrosine kinase inhibitors dasatinib (n = 61) or nilotinib (n = 14). The 8 patients (10.7%) who expressed EVI-1 transcripts detectable by real-time polymerase chain reaction had significantly lower event-free survival, progression-free survival, and overall survival than patients with undetectable transcript. The predictive value of EVI-1 expression was validated in an independent cohort. In a multivariate analysis, EVI-1 expression status and the best cytogenetic response obtained on imatinib were the only independent predictors for overall survival, progression-free survival, and event-free survival. Our data suggest that screening for EVI-1 expression at the time of imatinib failure may predict for response to second-line TKI therapy and consequently aid clinical management.

Background We have previously shown that DNA based digital PCR (dPCR) is a more sensitive approach for monitoring patients with CML in deep molecular remission compared to the 3 alternatives: qPCR, RT-qPCR and RT-dPCR. In this study we compared dPCR and RT-qPCR for their ability to predict molecular relapse in the context of national study of de-escalation of TKI dose followed by cessation of therapy (DESTINY). Methods The DESTINY study recruited 174 patients in major (or deeper) molecular remission, stratified according to whether their molecular remission status at study entry was MR3 or ≥MR4. The standard dose of TKI (imatinib, dasatinib and nilotinib) was halved at study entry and stopped 12 months later provided the patient remained in ≥ MR3. Material from diagnosis, required to clone the patient specific breakpoint for dPCR, was available only in a limited subset of patients. We cloned the breakpoints in 30 patients who reflected the range of outcomes of the study: 4 relapsed within the first 12 months of de-escalation, 12 relapsed 12-24 months from study entry (0-12 months from stopping TKI) and one relapsed 26 months from study entry (14 months from stopping therapy). All 30 patients were in ≥MR4 at study entry, of whom 7, 13 and 10 patients were in MR4, MR4.5 and MR5, respectively. Relapse was defined as loss of MR3. Fusion specific assays were designed and validated for all patients and dPCR was performed using the RainDrop platform (BioRad®). Multiple negative samples were used to establish the true positive quantification threshold per assay, including a pooled DNA samples from healthy controls and from other CML patients. Approximately 80,000 cells were included per reaction. RT-qPCR was performed according to standard protocols and all results expressed on the international scale (IS). Samples were collected at a minimum of 3 specified intervals, study entry, time of stopping TKI (month 12), month 24 and/or at the time of relapse. The log rank test was used to predict the risk of relapse and the Spearman correlation coefficient to compare the sensitivity of the two methods. Results At trial entry, patients were in MR4, MR4.5 or MR5 as defined by RT-qPCR: the depth of remission was not associated with the risk of relapse (log rank test p = 0.18 and 0.07, 0.19, 0.59 for MR4 vs MR4.5, MR4 vs MR5 and MR4.5 vs MR5, respectively). However, dPCR was positive in only 5 of the 30 patients of whom 3/5 (60%) relapsed compared to 14/25 (56%) who were dPCR negative (p=0.03). At 12 months neither the depth of remission by RT-qPCR nor the detection of malignant cells by dPCR, predicted subsequent relapse. In order to account for the fact that both methods were detecting different types of target molecules, the results were treated as a binary outcome (target detected/target not detected). 54 samples (49%) were positive by both methods, 6 (5%) were negative by both, 47 (42%) were positive only by RT-qPCR and 4 (4%) were positive only by dPCR. There was no difference in the predictive value of relapse when both methods were positive or negative. The majority of target molecules were not detected by dPCR when RT-qPCR showed transcript numbers ≤ 7 suggesting the possibility of false positivity and that dPCR is more specific in detecting true positive residual disease. Target molecules were detected by dPCR in all samples defined as > MR3 by RT-qPCR: absolute numbers of target molecules were understandably lower by dPCR (detecting cells) than by RT-qPCR (detecting transcripts). Conclusion Both methods can detect truly negative samples, however, dPCR is more specific in excluding false positivity. Neither dPCR nor RT-qPCR was capable of predicting relapse at the time of study entry. The ability of dPCR to predict loss of MR3 while off-therapy however requires the monitoring of serial time points leading to relapse before a final conclusion could be made. The question that remains posed here is the clinical validity of early residual disease detection given that, in the case of relapse (defined by ≥MR3 by RT-qPCR), all patients regain molecular remission after the TKI is reintroduced as it has been shown by all the STOP clinical trials. Disclosures Milojkovic: Incyte: Honoraria, Speakers Bureau; Novartis: Honoraria, Speakers Bureau; Pfizer: Honoraria, Speakers Bureau; BMS: Honoraria, Speakers Bureau. Clark:Pfizer: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; Bristol Myers Squibb: Consultancy, Research Funding; Ariad/Incyte: Consultancy. Apperley:Novartis: Honoraria, Research Funding, Speakers Bureau; BMS: Honoraria, Speakers Bureau; Incyte: Honoraria, Speakers Bureau; Pfizer: Honoraria, Speakers Bureau.

Analysis of BCR–ABL1 tyrosine kinase domain mutational spectra in primitive chronic myeloid leukemia cells suggests a unique mutator phenotype

The molecular basis of the progression of chronic myeloid leukaemia from the chronic stage to the acute phase is poorly understood. Now, work in mouse models of chronic myeloid leukaemia shows that this progression is controlled by the cell fate regulator Musashi2, which in turn regulates Numb, Notch and p53 to block cellular differentiation. Musashi2 expression can be increased by aberrant transcription factors found in leukaemia and is observed during cancer progression in human patients with leukaemia, where it is associated with poorer prognosis. This raises the possibility that modulating Musashi–Numb associated signalling may serve as a new approach to therapies against this disease. Chronic myelogenous leukaemia (CML) can progress from a chronic to an acute phase. These authors show in mouse models that leukaemia progression is controlled by the cell-fate regulator Musashi2, which in turn regulates Numb, Notch and p53 to block cellular differentiation. Musashi2 expression can be increased by aberrant transcription factors found in leukaemia, is observed during cancer progression in human CML patients and is associated with poorer prognosis. Chronic myelogenous leukaemia (CML) can progress from a slow growing chronic phase to an aggressive blast crisis phase1, but the molecular basis of this transition remains poorly understood. Here we have used mouse models of CML2,3 to show that disease progression is regulated by the Musashi–Numb signalling axis4,5. Specifically, we find that the chronic phase is marked by high levels of Numb expression whereas the blast crisis phase has low levels of Numb expression, and that ectopic expression of Numb promotes differentiation and impairs advanced-phase disease in vivo. As a possible explanation for the decreased levels of Numb in the blast crisis phase, we show that NUP98–HOXA9, an oncogene associated with blast crisis CML6,7, can trigger expression of the RNA-binding protein Musashi2 (Msi2), which in turn represses Numb. Notably, loss of Msi2 restores Numb expression and significantly impairs the development and propagation of blast crisis CML in vitro and in vivo. Finally we show that Msi2 expression is not only highly upregulated during human CML progression but is also an early indicator of poorer prognosis. These data show that the Musashi–Numb pathway can control the differentiation of CML cells, and raise the possibility that targeting this pathway may provide a new strategy for the therapy of aggressive leukaemias.

The predictive value of molecular minimal residual disease (MRD) monitoring using polymerase chain reaction amplification of clone-specific immunoglobulin or T-cell Receptor rearrangements was analysed in 161 patients with non T-lineage Philadelphia-negative acute lymphoblastic leukaemia (ALL) participating in the UK arm of the international ALL trial UKALL XII/Eastern Cooperative Oncology Group (ECOG) 2993. MRD positivity (> or =10(-4)) in patients treated with chemotherapy alone was associated with significantly shorter relapse-free survival (RFS) at several time-points during the first year of therapy. MRD status best discriminated outcome after phase 2 induction, when the relative risk of relapse was 8.95 (2.85-28.09)-fold higher in MRD-positive (> or =10(-4)) patients and the 5-year RFS 15% [95% confidence interval (CI) 0-40%] compared to 71% (56-85%) in MRD-negative (

Recent studies indicate that 40% of chronic myeloid leukemia patients who achieve sustained undetectable BCR-ABL1 transcripts on tyrosine kinase inhibitor therapy remain disease-free after drug discontinuation. In contrast, 60% experience return of detectable disease and have to restart treatment, thus highlighting the need for an improved method of identifying patients with the lowest likelihood of relapse. Here we describe the validation of a personalized DNA-based digital PCR (dPCR) approach for quantifying very low levels of residual disease, which involves the rapid identification of t(9;22) fusion junctions using targeted next-generation sequencing coupled with the use of a dPCR platform. t(9;22) genomic breakpoints were successfully mapped in samples from 32 of 32 patients with early stage disease. Disease quantification by DNA-based dPCR was performed using the Fluidigm BioMark platform on 46 follow-up samples from 6 of the 32 patients, including 36 samples that were in deep molecular remission. dPCR detected persistent disease in 81% of molecular-remission samples, outperforming both RT-dPCR (25%) and DNA-based quantitative PCR (19%). We conclude that dPCR for BCR-ABL1 DNA is the most sensitive available method of residual-disease detection in chronic myeloid leukemia and may prove useful in the management of tyrosine kinase inhibitor withdrawal.

Serial quantification of BCR-ABL1 mRNA is an important therapeutic indicator in chronic myeloid leukaemia, but there is a substantial variation in results reported by different laboratories. To improve comparability, an internationally accepted plasmid certified reference material (CRM) was developed according to ISO Guide 34:2009. Fragments of BCR-ABL1 (e14a2 mRNA fusion), BCR and GUSB transcripts were amplified and cloned into pUC18 to yield plasmid pIRMM0099. Six different linearised plasmid solutions were produced with the following copy number concentrations, assigned by digital PCR, and expanded uncertainties: 1.08±0.13 × 10 6, 1.08±0.11 × 10 5, 1.03±0.10 × 10 4, 1.02±0.09 × 10 3, 1.04±0.10 × 10 2 and 10.0±1.5 copies/μl. The certification of the material for the number of specific DNA fragments per plasmid, copy number concentration of the plasmid solutions and the assessment of inter-unit heterogeneity and stability were performed according to ISO Guide 35:2006. Two suitability studies performed by 63 BCR-ABL1 testing laboratories demonstrated that this set of 6 plasmid CRMs can help to standardise a number of measured transcripts of e14a2 BCR-ABL1 and three control genes (ABL1, BCR and GUSB). The set of six plasmid CRMs is distributed worldwide by the Institute for Reference Materials and Measurements (Belgium) and its authorised distributors (https://ec.europa.eu/jrc/en/reference-materials/catalogue/; CRM code ERM-AD623a-f)., 0, SCOPUS: ar.j, info:eu-repo/semantics/published

Diamond-Blackfan anemia (DBA) is a disorder characterized by a selective defect in erythropoiesis. Delineation of the precise defect is hampered by a lack of markers that define cells giving rise to erythroid burst- and erythroid colony-forming unit (BFU-E and CFU-E) colonies, the clonogenic assays that quantify early and late erythroid progenitor (EEP and LEP) potential, respectively. By combining flow cytometry, cell-sorting, and single-cell clonogenic assays, we identified Lin(-)CD34(+)CD38(+)CD45RA(-)CD123(-)CD71(+)CD41a(-)CD105(-)CD36(-) bone marrow cells as EEP giving rise to BFU-E, and Lin(-)CD34(+/-)CD38(+)CD45RA(-)CD123(-)CD71(+)CD41a(-)CD105(+)CD36(+) cells as LEP giving rise to CFU-E, in a hierarchical fashion. We then applied these definitions to DBA and identified that, compared with controls, frequency, and clonogenicity of DBA, EEP and LEP are significantly decreased in transfusion-dependent but restored in corticosteroid-responsive patients. Thus, both quantitative and qualitative defects in erythroid progenitor (EP) contribute to defective erythropoiesis in DBA. Prospective isolation of defined EPs will facilitate more incisive study of normal and aberrant erythropoiesis.

The cytogenetic picture in multiple myeloma (MM) is highly complex, from which non-random numerical and structural chromosomal changes have been identified. Specifically, translocations involving the immunoglobulin heavy chain gene (IGH) at 14q32 and either monosomy or deletions of chromosome 13 have been reported in a significant number of patients from both cytogenetic and interphase fluorescence in situ hybridization (FISH) studies. Importantly, these abnormalities of chromosome 13 have recently been associated with a poor prognosis. In view of the highly complex nature of the karyotypes in MM patients, interphase FISH results may be difficult to interpret. In this study, cytogenetics and/or interphase FISH were carried out on bone marrow samples or purified plasma cells from 37 MM patients. Abnormal karyotypes, characterized by multiplex FISH (M-FISH) were found in 11 patients, all of which were highly complex. Interphase FISH revealed translocations involving the IGH locus in 16 (43%) patients. The IGH/cyclin D1 (CCND1) gene fusion characteristic of the translocation, t(11;14)(q13;q32), was seen in 12 (32%) of these patients and other rearrangements of IGH in four (11%) patients. Fourteen patients had additional copies of chromosome 11. Twenty patients (54%) had 13q14 deletions, 10 of whom also had t(11;14) or another IGH translocation. By comparing cytogenetic and FISH results, this study has revealed that significant chromosomal abnormalities might be hidden within highly complex karyotypes. Therefore, extreme caution is required in the interpretation of interphase FISH results in MM, particularly in relation to certain abnormalities, such as 13q14 deletions, which have an impact on prognosis.

Systemic monoclonal immunoglobulin light chain amyloidosis (AL) is associated with clonal plasma cell dyscrasias that are often subtle and non-proliferating. AL shares numerical chromosomal changes with multiple myeloma (MM) and monoclonal gammopathy of undetermined significance (MGUS). Illegitimate translocations involving the immunoglobulin heavy chain gene (IGH ) at 14q32 and deletions of the long arm of chromosome 13, [del(13q)], commonly occur in MM, MGUS and plasma cell leukaemia. In AL IGH rearrangements have been identified but, to date, there are no reports of del(13q). In this study of 32 patients with AL, 24 with systemic and eight with localized disease, translocations involving IGH and del(13q) were found using dual-colour interphase fluorescence in situ hybridization (FISH). IGH translocations were observed in 11 patients (37% overall and in 46% with systemic disease), of which nine had the IGH/CCND1 fusion from t(11;14)(q13;q32). Two showed IGH translocations other than the t(11;14) or t(4;14)(p16;q32). In one of these patients a breakpoint within the constant region of IGH between Calpha1 and Calpha2 was indicated. In the second a deletion covering Calpha1 and Calpha2 accompanied the translocation. Ten patients (27% overall and 33% of those with systemic disease) showed del(13q). The gain or loss of IGH and CCND1 signals provided evidence of numerical chromosomal changes in three patients.

Molecular monitoring of minimal residual disease (MRD) using quantitative real-time PCR (qPCR) to measure BCR-ABL1 transcript levels has become an essential tool in management of patients with chronic myeloid leukaemia (CML). The assay shows superior specificity and sensitivity compared with conventional morphology, cytogenetic, and fluorescent in situ hybridization. In an attempt to increase sample throughput and assay accuracy, we have developed a duplex assay that measures both BCR-ABL1 target and endogenous control transcripts in the same reaction with no loss of sensitivity compared with the conventional simplex test.

The tyrosine kinase inhibitor (TKI) imatinib has revolutionized the management of chronic myeloid leukaemia (CML). However, around 25% of patients fail to sustain an adequate response. We sought to identify gene-expression biomarkers that could be used to predict imatinib response. The expression of 29 genes, previously implicated in CML pathogenesis, were measured by TaqMan Low Density Array in 73 CML patient samples. Patients were divided into low and high expression for each gene and imatinib failure (IF), probability of achieving CCyR, progression free survival and CML related OS were compared by Kaplan-Meier and log-rank. Results were validated in a second cohort of 56 patients, with a further technical validation using custom gene-expression assays in a conventional RT-qPCR in a sub-cohort of 37 patients. Patients with low PTCH1 expression showed a worse clinical response for all variables in all cohorts. PTCH1 was the most significant predictor in the multivariate analysis compared with Sokal, age and EUTOS. PTCH1 expression assay showed the adequate sensitivity, specificity and predictive values to predict for IF. Given the different treatments available for CML, measuring PTCH1 expression at diagnosis may help establish who will benefit best from imatinib and who is better selected for second generation TKI.

The BCR-ABL1 fusion gene, the causative lesion of chronic myeloid leukemia (CML) in >95 % of newly presenting patients, offers both a therapeutic and diagnostic target. Reverse-transcription quantitative polymerase chain reaction technology (RT-qPCR), utilizing primer-probe combinations directed to exons flanking the breakpoint junctional region, offers very high levels of both specificity and sensitivity, in a scalable, robust, and cost-effective assay.

Introduction Despite significant progress in the understanding of the molecular pathogenesis of myeloproliferative neoplasms (MPN) and the identification of high molecular risk (HMR) genes (i.e. ASXL1, EZH2, IDH1 and IDH2 genes), the mechanisms by which different cell types predominate in the different disease subtypes and their implications for prognosis remain uncertain. Given the recently described association of senescence and fibrosis in a number of pathologies by Menoz-Espin et al, we hypothesized that genes implicated in oncogene-induced senescence (OIS) and senescence associated secretory phenotype (SASP) may contribute to the pathogenesis of these neoplastic bone marrow disorders that frequently show evidence of fibrosis. Specifically, we were interested in the gene expression levels in different disease subtypes, at a cell-type level, and whether these patterns of differential expression were distinct from the transforming JAK-STAT pathway and the HMR genes. Aim To elucidate the role of OIS and SASP genes in the pathogenesis of MPN subtypes by determining the differential expression of the genes in specific cell types in patients with MPN. Methods We performed gene expression profiling on normal controls (NC) and patients with MPN who were diagnosed with essential thrombocythemia (ET), polycythemia vera (PV) and primary myelofibrosis (PMF) according to the 2008 WHO diagnostic criteria. Two cohorts of patients, the patient and validation cohorts, from 3 tertiary-level hospitals were recruited prospectively over 3 years. Peripheral blood samples were taken and sorted into polymorphonuclear cells (PMN), mononuclear cells (MNC) and T cells. RNA was extracted from each cell population. Gene expression profiling of the human transcriptome was performed using microarray and RNA sequencing on the patient and validation cohorts respectively. Gene expression analyses (GEA) were performed on 4 sets of genes derived from publicly available or custom derived gene set enrichment analysis: 92 OIS genes, 88 SASP genes (Gil et al), 4 HMR genes, and 126 genes associated with JAK-STAT pathway. Gene expression levels for each cell type in each disease were compared with NC to obtain the differential expression of the genes. RNA-seq analysis of samples from the validation cohort was used to validate the microarray results from the patient cohort. Results Twenty-eight patients (10 ET, 11 PV and 7 PMF) and 11 NC were recruited into the patient cohort. Twelve patients (4 ET, 4 PV and 4 PMF) and 4 NC were recruited into the validation cohort. After combination of the microarray and RNA-seq datasets, GEA of the OIS genes revealed the differential expressions of MCTP1 and SULT1B1 genes by PMN in PV but of none in PMF. In contrast, the BEX1 gene was identified as differentially expressed by MNC in PMF but none in PV. GEA of the SASP genes revealed differential expression of THBS1 gene by MNC in PMF but of none in PV. None of the SASP genes were differentially expressed by PMN in either PV or PMF. No differentially expressed genes were identified by PMN or MNC in ET, or by T cells in any of the diseases. Notably, GEA of the HMR genes and genes associated with the JAK-STAT pathways did not show any differential expression in any disease subtype by any cell type. Conclusions We have found strikingly distinct patterns of differential expression of senescence associated genes by PMN (in PV) and MNC (in PMF). These results provide a novel insight into the mechanisms underlying the different phenotype of the MPN subtypes and also to the cells responsible for mediating the differences. The lack of differential expression of OIS and SASP genes in ET may reflect the milder clinical phenotype of the disease. Although mutations in the HMR genes are associated with poor prognosis in PMF, the lack of differential expression in these genes and genes associated with the JAK-STAT pathway is in keeping with their mutated status and suggests that they give rise to the disease phenotypes via altering downstream expression of genes associated in other pathways such as the senescence pathways studied here. Further studies are warranted to investigate the role of these genes and the pathways involved in senescence at a cell-type specific level in order to gain further insight into how they can potentially give rise to the various disease phenotypes in MPN and unmask potential therapeutic targets. Disclosures Aitman: Illumina: Honoraria.

Introduction The discoveries of JAK2 V617F and CALR mutations have significantly improved our understanding of MPN and its subtypes. Somatic JAK2 V617F mutation is present in almost all patients with polycythemia vera (PV) and in 50% to 60% of those with essential thrombocythemia (ET) or primary myelofibrosis (PMF).Somatic CALR mutations are generally absent in PV but their frequencies are approximately 20-25% in ET and PMF. Initial reports suggested that these variants were mutually exclusive. However subsequently co-occurrence has been reported and it is suggested but not yet confirmed that this may confer a distinct phenotype. The role of germline variation is unknown. We report here the first occurrence of a germline CALR variant in association with a somatic JAK2 V617F mutation. Case report A 73-year old Malay gentleman was noted to have an abnormal cardiac rhythm and function during admission for herniorrhaphy. On further questioning, he reported a 2 month history of reduced cognitive function, short term memory loss, occasional urinary incontinence and involuntary movements. Splenomegaly was noted and blood test showed Hb 205 g/L, WCC 33.5 x 109/L and platelet 304 x 109/L. Cardiac investigations revealed an anteroseptal myocardial infarction requiring an angioplasty and stent insertion. Targeted Ion Torrent high-throughput sequencing and fragment analysis showed a somatic JAK2 V617F mutation and a germline 3bp in-frame deletion c.1213_1215delGAG (p.Glu405del) in exon 9 of CALR. Discussion Although somatic CALR exon 9 mutations are mostly found in patients with ET and PMF who are JAK2-negative, coexistence of somatic JAK2 V617F and CALR exon 9 alterations have been reported previously, mostly in patients with ET. To the best of our knowledge, this is the first case of a patient with PV presenting with a somatic JAK2 V617F mutation and a germline CALR variant, Glu405del, comprising a 3 bp in-frame deletion in exon 9. According to existing databases, this CALR variant is a previously undescribed germline variant not found in the 60,000 exomes on the ExAC database; but one instance of an overlapping p.Glu405_Asp408del was reported (frequency of 2 x 10-5). Although it is at present of unknown clinical significance, it is predicted by in silico modeling as being likely pathogenic: MutationTaster predicted both splice-site and protein feature changes, including loss of an endoplasmic reticulum export motif. Unlike the "classical" CALR-positive patients who present at a younger age with a higher platelet count, this patient presented incidentally at the age of 73 with a normal platelet count but a rather severe clinical phenotype with significant thrombosis requiring surgical intervention. Further studies are required to examine in detail the molecular signatures in this group of patients and whether they represent a distinct group of patients with clinical phenotypes which are different from the "classical" cases. Disclosures Aitman: Illumina: Honoraria. Laffan:CSL: Other: Travel support; Octapharma: Speakers Bureau.

Approximately 2‐3% of CML patients (pts) have variant BCR‐ABL fusions transcripts that cannot be monitored by RQ‐PCR using standard methodologies. Most rare variants are accounted for by seven variant fusions (e1a2/3, b2/3a3, e6a2, e8a2, e19a2). Within the European Treatment and Outcome Study (EUTOS) we sought to establish and validate robust RQ-PCR methods for each of these abnormalities. RNA was extracted from leukocytes obtained from 20 mL peripheral blood. Samples were received either locally or by mail and spent between 1 to 3 days in transit. Multiplex reverse transcription polymerase chain reaction (RT-PCR) experiments were performed to identify BCR-ABL1 transcripts (Cross et al., Leukemia 1994). In all cases, gel electrophoresis showed an unusual band. PCR products were then sequenced to characterize BCR-ABL1 rearrangements. Monitoring of residual disease was carried out by RQ-PCR with specific forward and/or reverse primers and probes using a serial dilution of individual BCR-ABL1 or GUSB plasmid DNA calibrators. GUSB transcripts were quantified as internal control and results were expressed as the BCR-ABL1/GUSB ratio with no conversion according to the international scale (IS) as it is recommended for only typical major (M) BCR-ABL1 and cannot be applied to other transcripts in CML. 43 pts from 8 prospective studies and pts outside of studies were investigated. A total of 435 samples (1-72 per patient, median 8) were analyzed. Pts expressed 6 different atypical BCR-ABL1 transcripts (e19a2, n=18; e1a2, n=10; b2a3, n=5; b3a3, n=5; b2a3&b3a3, n=2, e8a2, n=3). At the start of the observation period, ratios BCR-ABL1/GUSB ranged between 0.01% and 179.1% (median 13.75%). Follow-up results were determined in comparison to the starting level and expressed as relative log reduction. 9 pts were in deep molecular remission considering a minimum GUSB transcript level of 20,000/reaction. During the observation period, six pts relapsed with a significant increase of BCR-ABL1/GUSB ratios. We conclude that very few pts are diagnosed with unusual BCR-ABL1 transcripts, but their characterization is important for proper assessment of treatment response in affected patients, and to avoid false negative assessment of residual disease status. Based on the individual PCR strategies, results cannot be expressed with precision on the International Scale and thus the common molecular milestones and triggers for treatment discontinuation are difficult to apply. We suggest that residual disease in these cases should be assessed primarily by considering trends in BCR-ABL/reference gene ratios over time. Disclosures Saussele: NOVARTIS PHARMA: Consultancy, Honoraria; BMS: Honoraria. Cross:Novartis: Consultancy, Honoraria, Research Funding, Speakers Bureau.

Background: While chronic myeloid leukaemia (CML) originates from a single genetic aberration (BCR-ABL1) remarkably heterogeneity characterises treatment response and outcome. Most CML patients respond well to tyrosine kinase inhibitors (TKI), particularly 2nd generation (2G) TKI but a significant minority shows resistance and a proportion experience progression. At diagnosis there are currently no biomarkers for patients at higher risk of progression who could be treated with more effective treatment or be selected for BMT at an early stage of therapy. Such biomarkers may also provide useful prognostic information in addition to the most valid biomarker to date, the BCR-ABL1 IS ratio during the first 3-6-12 months of TKI therapy. Aims: The aim of our study is to analyse a panel of mutations in epigenetic modifiers in pre-treatment CML-CP using Ion Torrent next-generation sequencing (IONT-NGS) to assess the prognostic value of potential mutations as novel biomarkers of response to 1st and 2G TKI and risk of progression to advanced phase disease. Methods: 100 samples from untreated patients with newly diagnosed CML-CP were included in the study, 62 from patients treated frontline with imatinib (IM) and 38 with a 2G-TKI (31 dasatinib, 4 nilotinib, 3 bosutinib). The patients were classified as TKI responders (R) (34 IM, 22 2G-TKI) or non-responders (NR) (28 IM, 16 2G-TKI) based on BCR-ABL1 IS ratio at 3 months. DNA was extracted from CD34+ cells isolated from diagnostic samples, while DNA from T cells was used as constitutional non-leukemic control to exclude confounding germline mutations. Samples from healthy donors (n=14) and CML blast crisis (BC) (n=5) patients were used as negative and positive controls, respectively. We used a custom panel covering the coding region of 71 epigenetic enzymes. After sequencing data processing was performed excluding variants of low quality, common in the general population with minor allele frequency (maf) >1% or present in the healthy controls, we analysed the genomic data and integrated them with annotated clinical data. Results: After using a variant reduction pipeline, 164 non-synonymous variants that affected protein function were identified: 52 somatic and 112 germline. The somatic mutations (including missense, nonsense, frameshift insertions and splice site variants) were confined to 30 genes, with ASXL1, IKZF1, CREBBP beingthe most frequently mutated (n=9, 7 and 4 respectively). The mutations were detected in 34/100 (34%) CML-CP patients (19/62 IM and 15/38 2G-TKI), in higher proportion in NR (19/44, 43%) compared to R (15/56, 27%; p=0.027). We next correlated the presence of mutations with overall survival (OS), TKI failure free survival (TFFS) and progression free survival (PFS). IM patients carrying somatic mutations demonstrated a poorer response to IM [HR=2.1 (1-4.4 95% CI), p=0.05] and were more likely to progress to advanced phase [HR=3.1 (1-9.4 95% CI), p=0.03] (Figure 1). Nonsense mutations in particular (in ASXL1, IKZF1, DNMT3A, EP300) were found in 4 IM NR vs 1 R and their presence led to poor OS [HR= 6.1 (1.6-23 95% CI), p=0.002] and PFS [HR= 5.4 (1.4-21 95% CI), p=0.006]. As these were observed in 5 patients, further testing is required to corroborate this initial observation. Multivariate analysis revealed that both increased Sokal score and occurrence of somatic mutations negatively influenced outcome: somatic mutations detected in 6/24 low and in 8/13 high Sokal IM patients were associated with worse OS and PFS compared to unmutated patients with the same Sokal score. Among 38 patients treated with 2G-TKI, neither somatic mutations (including nonsense variants) nor combination of somatic mutations with Sokal score had any influence on OS, TFFS, PFS, neither did the presence of germline mutations in either IM or 2G-TKI patients. Summary/Conclusion: Somatic mutations identified using IONT-NGS on 71 epigenetic modifiers potentially predict 1st generation (IM) patient poor survival, drug failure and progression to advance phase disease. However, the more effective therapeutic effect of 2G-TKI seems to overcome the poor prognostic influence of such mutations though further validation on larger cohort of patients may be required to validate preliminary data. Our results suggest that occurrence of somatic mutations at diagnosis have the potential to identify patients who would benefit from upfront treatment with 2G-TKI. Disclosures Apperley: Incyte: Speakers Bureau; Novartis: Honoraria, Speakers Bureau; Ariad: Honoraria, Speakers Bureau; Pfizer: Honoraria, Speakers Bureau; Bristol Myers Squibb: Honoraria, Speakers Bureau.

Diamond-Blackfan anaemia (DBA) is caused by inactivating mutations in ribosomal protein (RP) genes, with mutations in 13 of the 80 RP genes accounting for 50-60% of cases. The remaining 40-50% cases may harbour mutations in one of the remaining RP genes, but the very low frequencies render conventional genetic screening as challenging. We, therefore, applied custom enrichment technology combined with high-throughput sequencing to screen all 80 RP genes. Using this approach, we identified and validated inactivating mutations in 15/17 (88%) DBA patients. Target enrichment combined with high-throughput sequencing is a robust and improved methodology for the genetic diagnosis of DBA.

Approximately one-third of patients with chronic myeloid leukaemia will fail to achieve or maintain responses to imatinib. Changes in solute carrier family 22 (organic cation transporter), member 1 (SLC22A1, also termed OCT1), the main transporter for imatinib, have been proposed as a possible predictive factor. We analysed SLC22A1 mRNA levels and single nucleotide polymorphisms (SNPs) located in exon 7 in 153 diagnostic whole blood samples from two patient cohorts. The level of SLC22A1 expression did not significantly correlate with imatinib failure or achievement of molecular remission. The SNP 408V>M (g.1222G>A) was present in 65% of patients and was associated in all cases with an eight base-pair insertion (8+ allele) at the 3′ end of exon 7. The latter generates an alternative splice site, leading to a premature stop codon. M420del was found in 33% of patients and never in cis with 8+ (the 3 allele). Significantly longer times to 1% and 0 1% molecular responses (by quantitative reverse transcription polymerase chain reaction) were seen in patients with 8+8+ or 8+N compared to those with the remaining four genotypes (N = no insertion or deletion). Patients lacking 8+ and 3 (NN, 18%) showed the best outcomes overall. Thus, while SLC22A1 expression does not appear to affect response, alterations in its splicing or amino acid sequence may do so. Refereed/Peer-reviewed

Several groups have shown that that the BCR-ABL1 transcript level measured at 3 or 6 months after starting treatment with tyrosine kinase inhibitors strongly predicts clinical outcomes for patients with chronic myeloid leukemia. In this work, we asked whether the prognostic value of the 3-month transcript level could be improved by combining the 3- and 6-month results. We classified patients treated with imatinib and patients treated with dasatinib according to their transcript levels at 3 months and 6 months. The patients who met the 3-month landmark but failed the 6-month one had outcomes identical to those of patients who met both landmarks, whereas the patients who failed the first landmark but met the second one had prognoses similar to those who failed both landmarks. In summary, early intervention strategies can be based robustly just on the transcript level at 3 months. This trial was registered at www.clinicaltrials.gov as # NCT01460693.

Background The past decade has witnessed a significant progress in the understanding of the molecular pathogenesis of myeloproliferative neoplasms (MPN). A large number of genes have now been implicated in the pathogenesis of MPN but their relative importance, the mechanisms by which they cause different cell types to predominate and their implications for prognosis remain unknown. Aim The aim of this study was to perform targeted next generation sequencing on different cell types directed at a panel of mutations shown to be associated with MPN in order to identify driver mutations and novel variants, to determine their relative importance to pathogenesis and to predict survival in patients with MPN. Methods We performed targeted sequencing on normal controls (NC) and patients with MPN from 3 different races (Malay, Chinese and Indian) in Malaysia who were diagnosed with essential thrombocythemia (ET), polycythemia vera (PV) and primary myelofibrosis (PMF) according to the 2008 WHO diagnostic criteria for MPN. Patients from 3 tertiary-level hospitals were recruited prospectively over 3 years and informed consents were obtained. Peripheral blood samples were taken and sorted into polymorphonuclear cells (PMNs), mononuclear cells (MNCs) and T cells. DNA was extracted from the different cell population and high-throughput sequencing was performed using an Ion Torrent PGM and AmpliSeq targeting platform, with a panel of 26 MPN-related genes. Results Twenty-nine patients (11 ET, 11 PV, 7 PMF) and 3 NC were recruited into the study. Ten of 29 (34.5%) and 9 of 29 (31%) patients were Malay and Chinese men respectively. Twenty-four of 29 (82.8%) patients harbored the JAK2V617F mutation which was detected in 7 (63.6%) ET, 11 (100%) PV and 6 (85.7%) PMF patients. The allelic frequency of JAK2V617F mutation was highest in PMF. 11 of the 24 JAK2V617F positive patients (2 ET, 5 PV and 4 PMF) also harbored potentially pathogenic variants involving the ASXL1, CALR, CBL, CHEK2, DNMT3A, TET2, U2AF1, RUNX1 , SF3B1 and TP53 genes. In patients who were found to have both JAK2V617F mutation and a TET2 variant, the allelic frequencies of the TET2 variantswere significantly lower than the JAK2V617F mutations. Conversely, in 1 ET patient with high TET2 allelic frequency, JAK2V617F mutation was not detected. Several novel variants involving the CHEK2, DNMT3A, RUNX1, SF3B1 and TET2 genes were identified in the study. All the novel variants were somatic (detected predominantly in the PMNs) except CHEK2 which was detected in all 3 cell types (PMNs, MNCs and T cells) at approximately 50% allele frequency. A similar spectrum of mutations was found in PMNs and MNCs, usually at a lower frequency in MNCs. Somatic mutations were absent or detected at very low frequencies in T cells. One of the 7 JAK2V617F positive ET patients harbored 4 somatic variants in CHEK2, SF3B1, TET2 and TP53 genes, with the highest allelic frequencies in the PMNs for 2 variants and MNCs for the other 2 variants. Three of the 4 JAK2V617F negative ET patients had CALR, MPL exon 10, SH2B3 and TET2 variants. One of the 3 ET patients with CALR and TET2 variant presented with a very high platelet count of 2251 x 109/L. One PV patient who harbored both JAK2V617F and CALR mutations presented with high haemoglobin and white cell counts. The CALR mutation in this patient was thought to be germline as it was detected in all cell types at approximately 50% allelic frequencies. The 2 PV patients who harbored both JAK2V617F and DNMT3A mutations presented with more severe phenotype with leukocytosis, splenomegaly of >15cm, constitutional and vasomotor symptoms requiring treatment with Hydroxycarbamide. The 3 PMF patients with JAK2V617F mutation in combination with ASXL1, DNMT3A, RUNX1, TET2 and U2AF1 variants also presented with severe clinical phenotypes, 2 of whom died within 2 years from diagnosis. Conclusion JAK2V617F is the commonest somatic mutation detected in MPN patients in Malaysia and is predominantly found in the PMNs. Nearly half of the JAK2V617F positive patients also harbor other potentially pathogenic variants. Several novel somatic and one germline variants were found in this study. Patients who harbor the JAK2V617F in combination with ASXL1, DNMT3A, RUNX1, TET2 and U2AF1 putatively pathogenic variants were found to have more severe clinical phenotypes with very poor prognosis. Further studies are required to explore cell type differences, prognosis and clonal evolution in MPN. Disclosures Aitman: Illumina: Honoraria.