Raquel Longarón

Increased ALK Gene Copy Number and Amplification are Frequent in Non-small Cell Lung Cancer

Introduction: Translocation of the anaplastic lymphoma kinase (ALK) gene is involved in the tumorigenesis of a subset of non-small cell lung carcinomas (NSCLCs) and identifies patients sensitive to ALK inhibitors. ALK copy number changes and amplification, which plays an oncogenic role in tumors such as neuroblastoma, are poorly characterized in NSCLC. We aimed to study the prevalence of ALK copy number changes and their correlation to ALK protein expression, epidermal growth factor receptor (EGFR) status, and clinicopathological data in patients with NSCLC. Methods: ALK status was evaluated by fluorescence in situ hybridization (FISH). Specimens with ALK translocation were studied for echinoderm microtubule-associated protein-like 4 (EML4), KIF5B, and TFG status. ALK expression was assessed by immunohistochemistry. EGFR gene and protein status were evaluated in adenocarcinomas. Survival analysis was performed. Results: One hundred seven NSCLC cases were evaluated. There were two cases of EML4-ALK translocation and one with an atypical translocation of ALK. Both cases of EML4-ALK translocation had ALK protein expression, whereas in the rest, ALK was undetected. Eleven cases (10%) exhibited ALK amplification and 68 (63%) copy number gains. There was an association between ALK amplification and EGFR FISH positivity (p < 0.0001) but not with prognosis. In conclusion, EML4-ALK translocation is a rare event in NSCLC. Conclusion: The study reveals a significant frequency of ALK amplification and its association with EGFR FISH positivity in lung adenocarcinomas. Based on these findings, a potential role of ALK amplification in the response to ALK inhibitors alone or combined with EGFR inhibitors in NSCLC merits further studies.

Modulation of JAK2 V617F allele burden dynamics by hydroxycarbamide in polycythaemia vera and essential thrombocythaemia patients

The modulation of JAK2 V617F allele burden dynamics was prospectively analysed in 47 patients (26 polycythaemia vera [PV] and 21 essential thrombocythaemia [ET]) treated with first‐line hydroxyurea (HU) and compared with the JAK2 V617F dynamics of a control group of 45 PV and ET patients. A partial molecular response (PMR), according to European Leukaemia Net criteria, was observed in 27/47 (57%) patients. Median time to PMR was 14 months (3–66) with a probability of PMR at 3 years of 57%. A significant decrease in JAK2 V617F allele load was observed at 36 months both in PV and ET patients, being the reduction in PV higher than in ET patients (P = 0·01). A haematocrit ≥0·45 L/L was associated with a higher probability of attaining a PMR (HR:3·4; 95%CI:1·02–11·6, P = 0·04). Control group showed a slight increase of JAK2 V617F allele burden over time. The reduction in the mutated allele load comparing treated patients versus controls was highly significant both in PV and ET, demonstrating a clear effect of HU on the JAK2 V617F allele burden. In conclusion, first‐line HU can attain PMR in more than 50% of newly diagnosed PV and ET patients, with a continuous decrease of the JAK2 V617F allele burden in PV patients during treatment.

JAK2V617F monitoring in polycythemia vera and essential thrombocythemia: clinical usefulness for predicting myelofibrotic transformation and thrombotic events.

The JAK2V617F allele burden has been identified as a risk factor for vascular events and myelofibrotic transformation in polycythemia vera (PV) and essential thrombocythemia (ET). However, all previous studies have evaluated a single time point JAK2V617F measurement. Therefore, the frequency and the clinical significance of changes in the JAK2V617F mutant load occurring during the disease evolution remain unknown. In the present study, JAK2V617F monitoring was performed during the follow‐up of 347 patients (PV = 163, ET = 184). According to their JAK2V617F evolutionary patterns, patients were stratified as stable < 50% (n = 261), stable ≥50% (n = 52), progressive increase (n = 24) and unexplained decrease (n = 10). After a 2,453 person‐years follow‐up, a total of 59 thrombotic events, 16 major hemorrhages, and 27 cases of myelofibrotic transformations were registered. At multivariate analyses, patients with a persistently high (≥50%) or unsteady JAK2V617F load during follow‐up had an increased risk of myelofibrotic transformation (Incidence rate ratio [IRR]: 20.7, 95% CI: 6.5–65.4; P < 0.001) and a trend for a higher incidence of thrombosis (IRR: 1.7, 1–3.3; P = 0.05) than patients with a stable allele burden below 50%. In conclusion, JAK2V617F monitoring could be useful in patients with PV and ET for predicting diseases complications, especially myelofibrotic transformation. Am. J. Hematol. 89:517–523, 2014.

miR-203 and miR-221 regulate SOCS1 and SOCS3 in essential thrombocythemia

The biological basis of essential thrombocythemia (ET) patients lacking known mutations is still unknown. MicroRNAs (miRNA) regulate hematopoietic differentiation and are deregulated in several hematopoietic malignancies. However, miRNA expression in ET patients has been poorly explored. We performed miRNA profiling in platelets from 19 ET patients and 10 healthy controls. Hierarchical cluster analysis showed two well-separated clusters between patients and controls, indicating that ET platelets had a characteristic 70-miRNA signature (P<0.0001), 68 of which were downregulated. According to the mutational status, three differentially expressed miRNAs, miR-15a (P=0.045), miR-150 (P=0.001) and miR-519a (P=0.036), were identified. A 40-miRNA signature was identified characterizing JAK2V617F-positive ET patients. Eight genes, whose interaction with the miRNAs could activate the JAK/STAT pathway were identified. An inverse correlation was observed between miRNAs expression and their target genes for SOCS1 and miR-221, SOCS3 and miR-221, SOCS3 and miR-203, and PTPN11 and miR-23a. All three miRNAs were upregulated in JAK2V617F-negative ET patients. SOCS1 and SOCS3 were validated as targets of miR-221 and miR-203, respectively. In summary, our study shows that platelets from JAK2V617F-negative ET patients harbor a specific miRNA signature that can participate in the modulation of the JAK/STAT pathway through regulation of key genes as SOCS1 and SOCS3.

Dynamics of JAK2 V617F allele burden of CD34+ haematopoietic progenitor cells in patients treated with ruxolitinib

Ruxolitinib has demonstrated splenomegaly reduction and symptom relief in patients with polycythaemia vera (PV) and myelofibrosis (MF) (Harrison et al, 2012; Verstovsek et al, 2012, 2014; Vannucchi et al, 2015) with limited effect on granulocytic JAK2 V617F mutant load (Harrison et al, 2012; Vannucchi et al, 2015). However, the effect of ruxolitinib on the JAK2 V617F allelle burden at the progenitor level is still unknown. We have assessed the effect of ruxolitinib on haematopoietic stem cells (HSCs, CD34 CD38 ), haematopoietic progenitor cells (HPCs, CD34 CD38) and granulocytes from seven patients with JAK2 V617F myeloproliferative neoplasms (PV, n = 4; secondary MF, n = 3). HPCs and HSCs were isolated from peripheral blood in all patients. Isolation of cell subpopulations and quantification of JAK2 V617F allele burden was performed in circulating HSCs, HPCs and granulocytes before starting ruxolitinib and after 6 and 12 months of therapy as previously described (Angona et al, 2015). Additional mutations in TET2 (whole coding sequence), DNMT3A (exon 23), TP53 (exons 2–10), ASXL1 (exon 12), SF3B1 (exons 14 and 15), SRSF2 (exon 1) and U2AF1 (exons 2 and 6–7) genes were studied in granulocytes by Sanger sequencing or pirosequencing using a Next Generation Sequencing (NGS) 454 GS Junior platform (Roche Applied Science, Mannhion, Germany). Mutations detected were also analysed by NGS in DNA extracted from granulocytes, HSCs and HPCs after 6 and 12 months of therapy. The study was approved by the Ethics Committee and informed consent was obtained according to the Declaration of Helsinki. Ruxolitinib was indicated as second-line treatment in all seven patients. Palpable splenomegaly was present in six patients before starting ruxolitinib with a reduction during the treatment in all of them. Most patients had marked improvement in symptoms. Two patients discontinued ruxolitinib after 6 months of treatment, due to severe anaemia (n = 1) and transformation to acute leukaemia (n = 1). The quantification of JAK2 V617F allele burden in HSCs, HPCs and granulocytes during ruxolitinib therapy is shown in Fig 1. In HSCs, JAK2 V617F allele burden remained stable in five patients (>50% and <50% in three and two patients, respectively), decreased from 54% to 27% and duplicated from 22% to 47% in one patient each (Fig 1A). A reduction of JAK2 V617F allele burden in HPCs was observed after 6 months of therapy in three patients but this reduction was not confirmed at 1 year (Fig 1B). The granulocytic JAK2 V617F allele burden remained stable in all but one patient (Patient 2) in whom a progressive increase, from 37% at baseline to 87% at 12 months, was observed (Fig 1C). One patient (Patient 4) showed additional mutations in TET2 (p.R1179 fs and p.E1483X). The mutational burden of both TET2 mutations remained stable in HSCs and granulocytes during ruxolitinib treatment (Fig 2). The allele burden of TET2 p.R1179 fs remained stable in HPCs whereas TET2 p.E1483X presented a mild progressive increase from 13% to 33 9%. To the best of our knowledge, this is the first study evaluating the effect of ruxolitinib on circulating HSCs and HPCs. Our results showed that ruxolitinib had a minimal impact on the mutant JAK2 V617F allele burden in PV and secondary MF patients, both in granulocytes and CD34 cell subpopulations, suggesting a minimal inhibition of the bone marrow JAK2 V617F-mutated cells. It is noteworthy that the six patients with palpable splenomegaly prior to therapy experienced a reduction of the spleen size during treatment in contrast with the absence of JAK2 V617F allele burden variation in bone marrow progenitor cells in all patients. This could be explained by differences in the microenvironment of the bone marrow and the spleen, with bone marrow progenitors being less sensitive to this treatment in contrast to those stem cells present in the spleen. In this regard, Wang et al (2014) recently showed that AZD1480, a JAK1/2/3 inhibitor, may induce apoptosis in myeloproliferative neoplasm progenitor cells in the spleens of MF patients with a subsequent reduction in splenomegaly. It has been reported that the presence of mutations in additional genes other than JAK2 V617F may play a role in the transformation of chronic phase PV and MF to acute leukaemia (Swierczek et al, 2011; Brecqueville et al, 2012). In our series, one PV patient harboured two mutations in the TET2 gene, without significant mutational burden variation in any of the cell populations during ruxolitinib treatment. In this sense, if ruxolitinib has an effect on HSCs, its role might be different depend on whether JAK2 V617F and TET2 mutations coexist in the same clone or are present in two independent clones. In the first situation, we would expect that ruxolitinib, by inhibiting JAK2, also had some effect on TET2 mutational load. By contrast, if the two mutations are present in different clones, JAK2 inhibition

Trombocitemia esencial: características iniciales y factores de riesgo de supervivencia y trombosis en una serie de 214 pacientes

BACKGROUND AND OBJECTIVE Two prognostic models to predict overall survival and thrombosis-free survival have been proposed: International Prognostic Score for Essential Thrombocythemia (IPSET) and IPSET-Thrombosis, respectively, based on age, leukocytes count, history of previous thrombosis, the presence of cardiovascular risk factors and the JAK2 mutational status. The aim of the present study was to assess the clinical and biological characteristics at diagnosis and during evolution in essential thrombocythemia (ET) patients as well as the factors associated with survival and thrombosis and the usefulness of these new prognostic models. PATIENTS AND METHODS We have evaluated the clinical data and the mutation status of JAK2, MPL and calreticulin of 214 ET patients diagnosed in a single center between 1985 and 2012, classified according to classical risk stratification, IPSET and IPSET-Thrombosis. RESULTS With a median follow-up of 6.9 years, overall survival was not associated with any variable by multivariate analysis. Thrombotic history and leukocytes>10×10(9)/l were associated with thrombosis-free survival (TFS). In our series, IPSET prognostic systems of survival and thrombosis did not provide more clinically relevant information regarding the classic risk of thrombosis stratification. CONCLUSION Thrombotic history and leukocytosis>10×10(9)/l were significantly associated with lower TFS, while the prognostic IPSET-Thrombosis system did not provide more information than classical thrombotic risk assessment.

Molecular characterisation of triple negative essential thrombocythaemia patients by platelet analysis and targeted sequencing.

Molecular characterisation of triple negative essential thrombocythaemia patients by platelet analysis and targeted sequencing

Hematopoietic clonal dominance, stem cell mutations, and evolutionary pattern of JAK2V617F allele burden in polycythemia vera

Clonal dominance is characteristic of patients with post‐polycythemia vera myelofibrosis (post‐PV MF), whereas patients in chronic phase usually display polyclonal hematopoiesis. The aim of this work was to study the mutational burden of JAK2V617F at the progenitor level in patients with PV and correlate it with the evolutive phase of the disease and the presence of mutations in genes different to JAK2V617F.

Characterization of CD34+ hematopoietic progenitor cells in JAK2V617F and CALR-mutated myeloproliferative neoplasms

Mutations in JAK2 or CALR are observed in patients with myeloproliferative neoplasms (MPN). To get further insight in the dynamics of the mutant clone, we assessed the mutant allele burden in hematopoietic stem cells (HSCs), hematopoietic progenitor cells (HPCs) and granulocytes from 138 patients [51 polycythemia vera (PV), 58 essential thrombocythemia (ET) and 29 myelofibrosis (MF)]. CALR-mutated ET patients harbored a higher mutant load at progenitor level than JAK2V617F-positive ET (HSCs: 39.9% vs 7.5% p<0.001, HPCs: 32.7% vs 7.7% p<0.001). Moreover, HSCs of CALR-mutated ET patients showed a similar mutational load than patients with CALR-mutated MF (39.9% vs 48.2%, p=0.17). Regarding JAK2V617F MPN, PV and ET patients showed a low mutational burden at progenitor level whereas in the myelofibrotic phase the dominance of the mutated clone was a constant finding. In conclusion, the size of the mutated clone in chronic phase MPN is different according to genotype with CALR-mutated ET showing a pattern similar to that observed in MF.

11P Prognostic impact of MET mutations in exon 14 and copy number alterations in a series of NSCLC patients

Background: Mutations in the exon 14 of the MET gene affecting RNA splice acceptor and donor sites, which lead to exon skipping, have been recently associated to responsiveness to crizotinib. Up to date, patient selection for Met inhibitors has been made according to MET amplification/high polysomy and/or protein overexpression. We have investigated the prevalence of MET exon 14 mutations and their correlation with gene copy number alterations (CNAs) in NSCLC patients. Methods: We tested 132 lung adenocarcinomas and adenosquamous carcinoma samples for mutations in MET exon 14 by Sanger sequencing and for MET gene CNAs by FISH (Abbott Molecular). We collected clinical data together with EGFR and KRASmutational status and ALK, ROS1 and RET rearrangements. Results: MET alterations were found in 23 patients (17.5%): 3 exon 14 skipping mutations (2.3%), 8 gene amplifications (6.1%), and 12 high polysomy cases (9.1%). Among the 11 MET altered cases (3 mutated and 8 amplified): 8 patients were male, with a median age of 64.5 years (range: 46–91), current or former smokers (50 pack/years, range: 30–80), and all were diagnosed in an advanced stage disease (III and IV). None of the 3 mutated cases had concurrent MET CNAs. One MET mutated case presented also a KRAS p.G12C substitution while the other two were wild type for EGFR (exons 18 to 21) and KRAS (exon 2). Interestingly, patients with MET alterations have a shorter overall survival (p < 0.001). Conclusions: We have identified alterations of the MET gene (mutations in exon 14 and CNAs) in 8.4% of NSCLC patients. Our data support the usefulness of prospective screening of MET exon 14 mutations and gene amplifications due to their prognostic and predictive role. These mutations define a new subset of NSCLC patients that should be analyzed independently of the MET gene copy number status. Legal entity responsible for the study: Institut Hospital del Mar d’Investigacions Mediques (IMIM), Barcelona, Spain Funding: Institut Hospital del Mar d’Investigacions Mediques (IMIM), Barcelona, Spain Disclosure: All authors have declared no conflicts of interest.