Marta Cabezón

Co-existence of JAK2 V617F and CALR mutations in primary myelofibrosis

In December 2013 mutations were described in the calreticulin (CALR) gene in 67–71% and 56–88% of patients with JAK2 V617F and MPL negative essential thrombocythemia (ET) and primary myelofibrosis (PMF), respectively [1,2]. Since this discovery, not only have CALR mutations been recommended to be included in the diagnostic algorithm for myeloproliferative neoplasms [3], but also CALR exon 9 mutations have been recognized to have clinical utility, as patients with these mutations have a better outcome than JAK2 V617F positive patients [4,5]. CALR mutations have also been reported to be mutually exclusive from JAK2 V617F or MPL mutations [1,2], so the majority of retrospective studies do not test for CALR mutations in JAK2 V617F or MPL positive patients. Recently, some series have described patients harboring mutations in both JAK2 V617F and CALR genes [5–7], but the true frequency of patients with “double-positive” disease is unknown, as mutations in JAK2 V617F, MPL and CALR are not routinely studied in all patients. For this reason, the objective of this study was to establish the true frequency of patients with double-positive PMF. Peripheral blood or bone marrow samples were obtained from 73 patients (45 males, median age 69 years) diagnosed with PMF according to the World Health Organization (WHO) classification [8]. Written informed consent for sample collection was received from all participants. DNA was extracted from blood or bone marrow samples collected in ethylenediaminetetraacetic acid anticoagulant using a QiaAmp DNA Blood Mini Kit (Qiagen, Hilden, Germany) and diluted with double-distilled water. In compliance with the Declaration of Helsinki this study was approved by the Institutional Review Board of the Hospital Germans Trias i Pujol. In all patients JAK2 V617F, MPL and CALR mutations were studied. To detect the presence of JAK2 V617F mutation, an allele-specific polymerase chain reaction (PCR) with TaqMan probes was used. We employed Sanger sequencing to detect MPL exon 10 mutations. Moreover we screened for insertions or deletions in the CALR gene with 6-carboxyfluorescein (6-FAM) labeled primers spanning exon 9 as previously described [1]. We confirmed and described the CALR mutation type with Sanger sequencing. A total of 46 out of 73 (63%) patients were JAK2 V617F positive, and among the 27 JAK2 V617F negative patients, five had MPL mutations (18.5%). Twelve patients out of 73 (16.5%) had a CALR mutation (eight type 1, one type 2 and three different from the 36 described). One patient harbored both JAK2 V617F and CALR mutations (c.1142_1144del) (Figure 1), the other 11 patients being JAK2 V617F and MPL negative. The biological implication of this mutation is unknown, as it implies the deletion of one amino acid but the reading frame does not change, and the somatic nature of this alteration could not be confirmed in constitutional DNA as the patient died of acute myocardial infarction in October 2011. In our series, 85% of patients carried a JAK2 V617F, MPL or CALR mutation, and a single patient had JAK2 V617F and CALR double-positive disease, representing 1.4% of the cohort studied. The patient with double-positive disease was an 86-yearold male with hemoglobin of 142 g/L, white blood cell count 11.7  109/L, platelet count 1170  109/L and a history of rectorrhagia. A diagnosis of PMF was made on the basis of the clinical characteristics, morphology and JAK2 V617F mutation status (allele burden of 60.58%). To date, and taking into account the present study, only four patients have been described (two with ET and two with PMF) with concurrent JAK2 V617F and CALR exon 9 mutations [5–7]; however, this percentage could be underLeukemia & Lymphoma, October 2015; 56(10): 2973–2974

Usefulness of IGH/TCR PCR studies in lymphoproliferative disorders with inconclusive clonality by flow cytometry.

In up to 5–15% of studies of lymphoproliferative disorders (LPD), flow cytometry (FCM) or immunomorphologic methods cannot discriminate malignant from reactive processes. The aim of this work was to determine the usefulness of PCR for solving these diagnostic uncertainties. We analyzed IGH and TCRγ genes by PCR in 106 samples with inconclusive FCM results. A clonal result was registered in 36/106 studies, with a LPD being confirmed in 27 (75%) of these cases. Specifically, 9/9 IGH clonal and 16/25 TCRγ clonal results were finally diagnosed with LPD. Additionally, two clonal TCRγ samples with suspicion of undefined LPD were finally diagnosed with T LPD. Although polyclonal results were obtained in 47 of the cases studied (38 IGH and nine TCRγ), hematologic neoplasms were diagnosed in 4/38 IGH polyclonal and in 1/9 TCRγ polyclonal studies. There were also 14 PCR polyclonal results (four IGH, 10 TCRγ), albeit nonconclusive. Of these, 2/4 were eventually diagnosed with B‐cell lymphoma and 3/10 with T‐cell LPD. In eight IGH samples, the results of PCR techniques were noninformative but in 3/8 cases a B lymphoma was finally confirmed. We concluded that PCR is a useful technique to identify LPD when FCM is inconclusive. A PCR clonal B result is indicative of malignancy but IGH polyclonal and nonconclusive results do not exclude lymphoid neoplasms. Interpretation of T‐cell clonality should be based on all the available clinical and analytical data.

Targeted deep sequencing improves outcome stratification in chronic myelomonocytic leukemia with low risk cytogenetic features

Clonal cytogenetic abnormalities are found in 20-30% of patients with chronic myelomonocytic leukemia (CMML), while gene mutations are present in >90% of cases. Patients with low risk cytogenetic features account for 80% of CMML cases and often fall into the low risk categories of CMML prognostic scoring systems, but the outcome differs considerably among them. We performed targeted deep sequencing of 83 myeloid-related genes in 56 CMML patients with low risk cytogenetic features or uninformative conventional cytogenetics (CC) at diagnosis, with the aim to identify the genetic characteristics of patients with a more aggressive disease. Targeted sequencing was also performed in a subset of these patients at time of acute myeloid leukemia (AML) transformation. Overall, 98% of patients harbored at least one mutation. Mutations in cell signaling genes were acquired at time of AML progression. Mutations in ASXL1, EZH2 and NRAS correlated with higher risk features and shorter overall survival (OS) and progression free survival (PFS). Patients with SRSF2 mutations associated with poorer OS, while absence of TET2 mutations (TET2wt) was predictive of shorter PFS. A decrease in OS and PFS was observed as the number of adverse risk gene mutations (ASXL1, EZH2, NRAS and SRSF2) increased. On multivariate analyses, CMML-specific scoring system (CPSS) and presence of adverse risk gene mutations remained significant for OS, while CPSS and TET2wt were predictive of PFS. These results confirm that mutation analysis can add prognostic value to patients with CMML and low risk cytogenetic features or uninformative CC.

Frequency of ABL gene mutations in chronic myeloid leukemia patients resistant to imatinib and results of treatment switch to second-generation tyrosine kinase inhibitors

BACKGROUND AND OBJECTIVES Tyrosine kinase inhibitors (TKI) have improved the management of patients with chronic myeloid leukemia (CML). However, a significant proportion of patients do not achieve the optimal response or are resistant to TKI. ABL kinase domain mutations have been extensively implicated in the pathogenesis of TKI resistance. Treatment with second-generation TKI has produced high rates of hematologic and cytogenetic responses in mutated ABL patients. The aim of this study was to determine the type and frequency of ABL mutations in patients who were resistant to imatinib or had lost the response, and to analyze the effect of second-generation TKI on their outcome. PATIENTS AND METHODS The presence of ABL mutations in 45 CML patients resistant to imatinib was evaluated by direct sequencing and was correlated with the results of the cytogenetic study (performed in 39 cases). The outcome of these patients after therapy with nilotinib or dasatinib was analyzed. RESULTS ABL mutations were detected in 14 out of 45 resistant patients. Patients with clonal cytogenetic evolution tended to develop mutations more frequently than those without clonal evolution. Nine out of the 15 patients with ABL mutation responded to a treatment switch to nilotinib (n=4), dasatinib (n=2), interferon (n=1) or hematopoietic stem cell transplantation (n=2). CONCLUSION The frequency of ABL mutations in CML patients resistant to imatinib is high and is more frequent among those with clonal cytogenetic evolution. The change to second-generation TKI can overcome imatinib resistance in most of the mutated patients.

The role of T-cell phenotype and T-cell receptor rearrangement in the diagnosis of T-cell malignancies

We read with interest the article by Flammiger et al. [1] on the study of a series of patients with aberrant T-cell phenotype of unclear significance by flow cytometry (FC). They divided their cases into mature T-cell malignancies (TCM) and reactive (non-TCM) cases, focusing on the phenotypes that pose a diagnostic challenge, having excluded those cases whose clinical evolution and further testing did not allow for a clear diagnosis, as well as those cases with obviously aberrant phenotypes. After analyzing their FC reports they argue that an aberrant T-cell phenotype of unclear significance is of little help in the diagnosis of TCM. They only found a correlation with a loss of expression of CD7, more frequent and marked in TCM, but also present in up to 30% of the non-TCM cases (which, notably, included B-cell malignancies in 50% of cases). The authors conclude that, in those cases without an obviously aberrant phenotype, FC can only be a part of a much larger set of diagnostic tools, which include cytogenetics, the study of T-cell receptor (TCR) rearrangements, clinical course and histological examination whenever possible. However, it is well established that TCR clonality is often detected in reactive cases or simply with aging [2–8]. It has been our experience that, indeed, T-cell FC analysis often shows abnormal markers that ultimately do not reflect an underlying TCM [3]. Thus, we set out to analyze the role of T-cell phenotype and TCR rearrangements in the diagnosis of TCM. From October 2007 to December 2014, there were 452 TCR rearrangement studies performed at our institution. After exclusion of all patients who had previously been tested for TCR clonality and those not simultaneously studied by FC, 120 cases were left and were the object of this study. Samples tested were primarily peripheral blood but also bone marrow, lymph node biopsies, lymph node fine needle aspiration and pleural or ascitic exudates. Demographic, clinical and diagnostic data were collected and, based on the FC results, four groups were established: (1) those with an obviously aberrant phenotype, including T-cell expression of CD30, CD10 or CD1a, loss of CD26, expression of gammadelta chains and CD4  CD8 co-expression ( 5% of the lymphocytic population); (2) those with an aberrant T-cell phenotype of unclear significance, as described by Flammiger et al. [1]. In our series, those abnormalities included diminished expression of CD7, CD5 or alpha-beta chains and expression of CD56 or CD57 by T-cells (Table I); (3) those with an abnormal CD4/CD8 ratio, defined as CD4/CD8  3 or CD4/CD8  1; and (4) those with a normal T-cell phenotype in whom TCR rearrangement testing was ordered to study absolute lymphocytosis or lymphopenia. Concerning diagnosis, we divided TCM into aggressive T-cell lymphomas (TCL), i.e. those high-grade lymphomas that required chemotherapy, including Sézary syndrome (SS), all diagnosed by histological tissue examination, and indolent TCM, which in our series were limited to T-cell large granular lymphocyte leukemias (TLGL), diagnosed by peripheral blood smear and according to WHO criteria. We diagnosed as reactive those cases that were clearly related to a non-hematological disorder as well as those that eventually resolved, even without a specific diagnosis. Cases with unclear clinical course and diagnosis were excluded and labeled as unclassified There were 20 patients in group 1, 17 (85%) of them with a clonal TCR, 23 patients in group 2, 15 (65%) of them with a clonal TCR, 47 patients in group 3, 18 (38%) of them with a clonal TCR and 30 patients in group 4, 6 (20%) of them with a clonal TCR (p  0.001). Table I shows for each of the phenotype groups the number of TCM, non-T-cell hematological malignancies (mostly B-cell lymphomas), non-malignant hematological disorders (mostly bone marrow aplasia) and reactive cases. Of note, not only do clearly aberrant phenotypes almost always reflect a hematological disorder (mostly TCL, but also TLGL or B-cell malignancies), but TCL and SS were mostly found in the group with clearly aberrant phenotypes (9/11 and 3/6, respectively). However, TLGL were found in all four groups. Furthermore, aberrant phenotypes

Highly variable mutational profile of ASXL1 in myelofibrosis

Somatic mutations in ASXL1 seem to have a negative prognostic impact in patients with several myeloid neoplasms, including myelofibrosis (MF). The aim of this work was to determine the prevalence and profile of ASXL1 mutations in MF.

A thirty-five nucleotides BCR-ABL1 insertion mutation of controversial significance confers resistance to imatinib in a patient with chronic myeloid leukemia (CML)

Tyrosine kinase inhibitors (TKI) have improved the management of patients with chronic myeloid leukemia (CML). However, a significant proportion of patients does not achieve the optimal response or are resistant to TKI. ABL1 kinase domain mutations have been extensively implicated in the pathogenesis of TKI resistance. Although deletion or insertion of nucleotides in BCR-ABL1 has rarely been described, we identified a CML patient with an already described 35 nucleotides insertion (BCR-ABL1(35INS)) of controversial significance, that confers resistance to imatinib but sensitivity to dasatinib.

Impact of mutational studies on the diagnosis and the outcome of high-risk myelodysplastic syndromes and secondary acute myeloid leukemia patients treated with 5-azacytidine

Myelodysplastic syndromes (MDS) are stem cell disorders caused by various gene abnormalities. We performed targeted deep sequencing in 39 patients with high-risk MDS and secondary acute myeloid leukemia (sAML) at diagnosis and follow-up (response and/or relapse), with the aim to define their mutational status, to establish if specific mutations are biomarkers of response to 5-azacytidine (AZA) and/or may have impact on survival. Overall, 95% of patients harbored at least one mutation. TP53, DNMT3A and SRSF2 were the most frequently altered genes. Mutations in TP53 correlated with higher risk features and shorter overall survival (OS) and progression free survival (PFS) in univariate analysis. Patients with SRSF2 mutations were associated with better OS and PFS. Response rate was 55%; but we could not correlate the presence of TET2 and TP53 mutations with AZA response. Patients with sAML presented more variations than patients with high-risk MDS, and usually at relapse the number of mutations increased, supporting the idea that in advanced stages of the disease there is a greater genomic complexity. These results confirm that mutation analysis can add prognostic value to high-risk MDS and sAML patients, not only at diagnosis but also at follow-up.

DNA methylation profile in chronic myelomonocytic leukemia associates with distinct clinical, biological and genetic features

ABSTRACT Chromosomal abnormalities are detected in 20–30% of patients with chronic myelomonocytic leukemia (CMML) and correlate with prognosis. On the mutation level, disruptive alterations are particularly frequent in chromatin regulatory genes. However, little is known about the consequential alterations in the epigenetic marking of the genome. Here, we report the analysis of genomic DNA methylation patterns of 64 CMML patients and 10 healthy controls, using a DNA methylation microarray focused on promoter regions. Differential methylation analysis between patients and controls allowed us to identify abnormalities in DNA methylation, including hypermethylation of specific genes and large genome regions with aberrant DNA methylation. Unsupervised hierarchical cluster analysis identified two main clusters that associated with the clinical, biological, and genetic features of patients. Group 1 was enriched in patients with adverse clinical and biological characteristics and poorer overall and progression-free survival. In addition, significant differences in DNA methylation were observed between patients with low risk and intermediate/high risk karyotypes and between TET2 mutant and wild type patients. Taken together, our results demonstrate that altered DNA methylation patterns reflect the CMML disease state and allow to identify patient groups with distinct clinical features.

CD34 expression and the outcome of nucleophosmin 1-mutated acute myeloid leukemia.

CD34 positivity has been considered as an adverse prognostic factor in acute myeloid leukemia (AML). Although nucleophosmin 1-mutated (NPM1m) AML is usually CD34 negative, this marker may be expressed at diagnosis or acquired at relapse in a variable number of cases. Our objective was to ascertain if CD34 expression has any influence on the general outcome of this form of acute leukemia. Analysis of clinical outcome (complete remissions, relapses, disease-free survival, and overall survival) was performed depending on the degree of expression of CD34 determined by flow cytometry, in 67 adult patients with NPM1m AML. CD34 expression did not have any influence on the variables analyzed whatever the percentage of blasts expressing this marker. In contrast to other forms of AML, CD34 expression is not an unfavorable prognostic factor in NPM1m AML, neither at diagnosis nor at relapse.