Silvia Marce

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.

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.