Identification of Somatic Mutations in Primary Cutaneous Diffuse Large B-Cell Lymphoma, Leg Type by Massive Parallel Sequencing.

To determine whether the mutational profile of primary cutaneous diffuse large B-cell lymphoma, leg type (PCLBCL-LT) is unique by comparison with other diffuse large B-cell lymphoma subtypes, we analyzed a total cohort of 20 PCLBCL-LT patients by using next-generation sequencing with a lymphoma panel designed for diffuse large B-cell lymphoma. We also analyzed 12 pairs of tumor and control DNA samples by whole-exome sequencing, which led us to perform resequencing of three selected genes not included in the lymphoma panel: TBL1XR1, KLHL6, and IKZF3. Our study clearly identifies an original mutational landscape of PCLBCL-LT with a very restricted set of highly recurrent mutations (>40%) involving MYD88 (p.L265P variant), PIM1, and CD79B. Other genes involved in B-cell signaling, NF-κB activation, or DNA modeling were found altered, notably TBL1XR1 (33%), MYC (26%) CREBBP (26%), and IRF4 (21%) or HIST1H1E (41%). MYD88L265P variant was associated with copy number variations or copy neutral loss of heterozygosity in 60% of patients. The most frequent genetic losses involved CDKN2A/2B, TNFAIP3/A20, PRDM1, TCF3, and CIITA. Together, these results show that PCLBCL-LT exhibits a unique mutational landscape, combining highly recurrent hotspot mutations in genes involved in NF-kB and B-cell signaling pathways, which provides a rationale for using selective inhibitors of the B-cell receptor.

[Comparison between the centrifugation on MPA C10 (Roche Diagnostics) and the centrifugation according recommendations of GEHT (Groupe d'étude de l'hémostase et de la thrombose) for the daily hemostasis assays]. / Comparaison de la centrifugation sur MPA C10 (Roche Diagnostics) et de la centrifugation selon les préconisations du Groupe d'étude de l'hémostase et de la thrombose pour les analyses d'hémostase du quotidien.

Actually, many laboratories tend to acquire pre analytic automates to prepare specimens for analysis. For haemostasis, these pre analytical modules are not always in agreement with the recommendations from the Groupe d'étude de l'hémostase et de la thrombose (GEHT). For example in the MPA C10 module (Roche Diagnostics) the speed of centrifugation was not rather fast compared with the GEHT recommandations. Then, to be able to use this automate for routine coagulation assays, we compared results of Quick time, activated partial prothombin time, fibrinogen, factor II, factor V, factor VII, factor X and antithrombin levels and unfractioned heparin anti-Xa activity measurement after MPA (1,885 g - 999 sec) or GEHT (2,500 g - 900 sec) protocol of centrifugation. First, we verified platelet counts: in 82% of specimens, the platelet counts were under 10.10(9)/L after centrifugation on MPA module. Moreover, a good correlation was observed in all comparisons. Then we concluded the MPA C10 module was usable for routine coagulation tests.

Decreased procoagulant phospholipids in patients treated by vitamin K antagonists.

INTRODUCTION: The stimulation of cells by thrombin is associated with the release of microparticles (MPs) from cell membranes. These MPs can express procoagulant activity. As vitamin K antagonists (VKA) decrease the generation of thrombin, we compared plasma procoagulant phospholipids (PPL) levels in patients with a previous history of venous thrombosis who were being treated with VKA and compared them with an untreated group. MATERIALS AND METHODS: Plasma PPL were measured using a factor Xa-based coagulation assay. sGPV, a marker of platelet activation by thrombin, was measured by ELISA. Platelet MPs were also evaluated using standard flow cytometric techniques. Ninety-six VKA-treated patients and 80 patients not undergoing VKA therapy were tested and the results compared. RESULTS: PPL activity was significantly reduced (p<0.0001) in VKA-treated patients compared with the untreated group. PPL were correlated with platelet and white blood cell count and with sGPV levels in the untreated group, but not in VKA-treated patients. PPL were correlated with fibrinogen levels in both groups, but not with C-reactive protein. Polymorphonuclear neutrophils (PMN) were significantly lower (p=0.01) in VKA-treated patients than in untreated patients. CONCLUSION: The difference between PPL levels in VKA-treated patients and patients without treatment could be related to the decrease in PMN count. It remains to be established if this decrease of PPL is directly related to the capacity of activated PMN to generate MPs, or indirectly by reducing the amount of pro-inflammatory cytokines or reactive oxygen species produced by PMN.

Combination of cellular population data and CytoDiff analyses for the diagnosis of lymphocytosis.

BACKGROUND: Differentials with moderate lymphocytosis are common in hematology laboratories and it is important in these cases to discriminate monoclonal from reactive lymphocytosis (RL). Blood smear reflex examination is dependent of the expertise of a cytologist, time-consuming and not always informative. Therefore, rapid and easy orientation parameters are clearly needed to discriminate malignant from RL. METHODS: The differential performed by the Beckman-Coulter analyzers is based on the determination of three parameters (volume, conductivity and scatter of the cell subpopulations) called cellular population data (CPD). This study evaluated CPD in 332 patients with a typical B-chronic lymphocytic leukemia (B-CLL), 90 patients with other B-lymphoproliferative diseases (OLPD) and 55 patients with a proven RL, and established a discriminating protocol to identify these pathologies. Secondly, this approach was evaluated in a prospective study including 102 patients with lymphocyte counts above 3.5 × 10(9)/L and in each case the diagnosis suggested by CPD was compared with conventional flow cytometry (FC) analysis and that obtained using CytoDiff reagent, a combination of six antibodies/five colors which performs a rapid WBC differential by FC. RESULTS: Lymphocyte anisocytosis was observed for malignant and RL. A low lymphocyte volume identifies monoclonal B-cell lymphocytosis and classical B-CLL. CytoDiff analysis is helpful when lymphocyte volume is in the normal range. A ratio B-Ly/total Ly count >0.32 is suggestive of a B-malignancy, whereas a non-cytotoxic T-lymphocyte count above 2.43 × 10(9)/L suggests RL. CONCLUSIONS: The analysis of CPD in combination with CytoDiff analysis shows promise for the rapid and accurate identification of lymphocyte pathologies in routine practice.

Plasma thrombomodulin activity, tissue factor activity and high levels of circulating procoagulant phospholipid as prognostic factors for acute myocardial infarction.

Several studies have indicated an association between haemostatic markers and acute myocardial infarction, but few or no studies refer to their activity. We studied plasma levels of 10 coagulation factors (fibrinogen, protein C, protein S, von Willebrand factor, D-dimers, factor VIIa, free tissue factor pathway inhibitor, tissue-type plasminogen activator, plasminogen activator inhibitor-1, thrombomodulin) and using new specific assays analysed the activity of plasma tissue factor (TFa), thrombomodulin (TMa), and procoagulant phospholipid in 46 consecutive patients with acute myocardial infarction at the time of hospital admission, and compared them with 34 healthy normal volunteers. Plasma levels of TFa, TMa, and procoagulant phospholipid were significantly higher in cases than in control patients (P < 0.001). In addition the ratio of TFa/free tissue factor pathway inhibitor was higher in patients than in controls, whereas the tissue-type plasminogen activator (t-PA)/plasminogen activator inhibitor-1 ratio was lower in patients. Interestingly, patients with an unfavourable outcome during a 2-month follow-up had higher levels of TFa, TMa, procoagulant phospholipid, a higher ratio of TFa/free tissue factor pathway inhibitor and a lower ratio of t-PA/plasminogen activator inhibitor-1 than patients who recovered. The combination of these different parameters reveals an increase in procoagulant activity as well as impaired fibrinolytic activity during the acute phase of an acute myocardial infarction. The association of the level of the activity of these three factors may provide a new tool to assess the prognosis of acute myocardial infarction. Further studies are needed to support our findings and to elucidate the clinical interest of measuring these factors.

Multicenter study of ZAP-70 expression in patients with B-cell chronic lymphocytic leukemia using an optimized flow cytometry method.

BACKGROUND: Flow cytometry allows specific assessment of the expression of ZAP-70, a promising new prognostic factor in B-cell chronic lymphocytic leukemia (B-CLL), but suffers from a lack of multicenter standardization. DESIGN AND METHODS: An optimized method for direct detection of ZAP-70 in flow cytometry was tested in a multicenter fashion. Adapted for frozen cells, this method includes a normalization step by addition of B cells from a pool of peripheral blood mononuclear cells collected from normal donors. ZAP-70 expression levels were assessed for 153 patients with typical B-cell chronic lymphocytic leukemia chronic lymphocytic leukemia. Results were expressed as the ratio of ZAP-70 mean fluorescence intensity between B-CLL cells and normal B cells. RESULTS: The statistically optimized cut-off of ZAP-70 positivity was a ratio of 1.4. Concordance between ZAP-70 and CD38 expression was 67%. Concordance between the mutational status of IgVH genes and ZAP-70 or CD38 expression was 87% and 65%, respectively. ZAP-70 was significantly expressed in 28%, 54% and 61% of patients with Binet stages A, B and C B-cell chronic lymphocytic leukemia, respectively (p=0.008). The absence of ZAP-70 expression was associated with isolated del(13q14), a cytogenetic abnormality with a good prognosis, while most patients with the del(17p13) poor prognosis cytogenetic marker expressed ZAP-70 (p<10(-5)). ZAP-70 expression was not related to the other poor prognosis cytogenetic abnormality del(11q22.3) nor to trisomy 12. CONCLUSIONS: This new technique provides highly reliable results well correlated with the mutational status of IgVH genes, CD38 expression, Binet stage and cytogenetic abnormalities. This robust discriminative technique appears of particular interest for routine diagnosis and assessment of ZAP-70 expression in large, prospective, multicenter therapeutic trials.

Long-term incubation with IL-4 and IL-10 oppositely modifies procoagulant activity of monocytes and modulates the surface expression of tissue factor and tissue factor pathway inhibitor.

Monocytes can be induced to express both tissue factor (TF) and its inhibitor, TF pathway inhibitor-1 (TFPI-1). A short incubation (<6 h) with interleukin (IL)-4 and IL-10, two potent deactivators of monocyte functions, has been shown to modulate the synthesis and expression of TF by monocytes activated by lipopolysaccharide, but the consequences of longer incubations (up to 96 h) on both TF and TFPI-1 are unknown. The results of this study showed that adherent monocytes in culture spontaneously expressed TF and TFPI and that prolonged incubation with IL-10 induced a time- and dose-dependent decrease of monocyte TF synthesis, and an accumulation of TF/TFPI-1 complexes at the moncyte surface, suggesting a decreased clearance of these complexes. In contrast, IL-4 induced a time- and dose-dependent increase in TF synthesis, which remained intracytoplasmic, as shown by confocal microscopy. Surprisingly, TF:antigen (Ag) was decreased at the monocyte surface, but the procoagulant activity (PCA) of IL-4-treated monocytes was increased, as a result of more pronounced decrease of TFPI-1:Ag expression than that of TF. In conclusion, prolonged incubation with IL-4 and IL-10 oppositely modified PCA of cultured monocytes, and altered TF and TFPI trafficking and clearance. These data explain the respective deleterious or benefit effects of IL-4 or IL-10 in atherothrombosis.

Usefulness of cutaneous T-cell clonality analysis for the diagnosis of cutaneous T-cell lymphoma in patients with erythroderma.

CONTEXT: Demonstration of a dominant T-cell clone in skin biopsy specimens by a molecular assay constitutes an additional diagnostic criterion to differentiate cutaneous T-cell lymphomas (CTCLs) from inflammatory dermatoses. OBJECTIVE: To determine which patients, depending on their clinical presentations, could most benefit from a cutaneous T-cell clonality analysis in addition to histopathologic analysis for the diagnosis of CTCL. DESIGN: Comparison of sensitivity and specificity of histopathologic analysis and a combination of this method and the detection of a T-cell receptor gamma chain gene rearrangement by polymerase chain reaction denaturing gradient gel electrophoresis performed on skin biopsy specimens obtained at initial presentation. PATIENTS: One hundred forty consecutive patients were classified into 4 groups, depending on their clinical presentation: (1) eczematous patches suggestive of early-stage mycosis fungoides (MF) (IA and IB of the TNM classification) (n = 42); (2) plaques, nodules, or tumors that arise on or are associated with plaques suggestive of late-stage MF (IIB and III of the TNM classification) (n = 16); (3) erythroderma (n = 50); and (4) nodules or tumors that arise in normal skin, suggestive of non-MF CTCL (n = 32). RESULTS: When compared with histopathologic examination, the addition of clonality analysis increased the sensitivity of CTCL diagnosis in all groups of patients except those with cutaneous lesions suggestive of late-stage MF, because the diagnosis was made based on histopathologic analysis alone in 100% of these cases. The main increase in sensitivity of CTCL diagnosis was observed in patients with erythroderma: 62% with histopathologic analysis alone to 87% with the combination of both methods (P = .04). Diagnostic specificity of molecular assays decreased from 100% to 76% (P = .01) in patients with patch lesions and from 100% to 70% (P = .04) in patients with nodules that occurred in normal skin due to the detection of a T-cell clone in 6 patients with follicular mucinosis without a histologic pattern of MF and in 5 of 20 cases of T-cell pseudolymphoma (25%), respectively. In contrast, a T-cell clone was not detected in the 34 patients with erythroderma of inflammatory origin. CONCLUSION: Polymerase chain reaction analysis of cutaneous T-cell clonality could be useful for the diagnosis of CTCL in patients who present with erythroderma.

Clonal T cells in the blood of patients with systemic sclerosis.

BACKGROUND: It has been suggested that clonal T cells may play a critical role in the pathogenesis of systemic sclerosis. OBSERVATIONS: A monoclonal population of T cells was found in blood samples from 13 (34%) of 38 consecutive patients with a definite diagnosis of systemic sclerosis who were prospectively examined by T-cell receptor gamma gene rearrangement using polymerase chain reaction analysis and denaturating gradient gel electrophoresis. In the healthy control group, the same type of examination revealed a monoclonal population of T cells in the blood samples from only 3 healthy subjects (4%)(odds ratio, 12.28; 95% confidence interval, 2.76-54.64; P = .001). Patients who had a circulating clonal population of T cells were older than those who did not (67 years vs 48 years; P = .04). There was a marked relationship between systemic sclerosis subtypes and the presence of a circulating clonal population of T cells. Twelve (43%) of 28 patients with limited cutaneous sclerosis exhibited a circulating clonal population of T cells, whereas only 1 (10%) of the 10 patients with diffuse cutanous sclerosis had evidence of T-cell clonality (P<.01). CONCLUSIONS: Clonally expanded T cells were more commonly detected in patients with limited cutaneous sclerosis than in those with diffuse cutaneous sclerosis, which is also in accordance with a possible role of clonal T cells in patients with limited cutaneous sclerosis.

Value of interphase FISH for the diagnosis of t(11:14)(q13;q32) on skin lesions of mantle cell lymphoma.

The diagnosis of skin lesions of mantle cell lymphoma (MCL) may be difficult at the onset of the disease. We observed 2 patients with papules of the trunk and 1 with diffuse infiltration of the trunk and the face and 2 subcutaneous nodules. Skin samples showed diffuse infiltration of the dermis (n = 1) or perivascular infiltration (n = 2). The infiltrate corresponded to centrocytic cells (n = 2) or pleomorphic blastoid cells (n = 1) with a B-cell phenotype: CD3-, CD5+ (2/3), CD20+, CD23-, and CD43+. In only 1 case was cyclin D1 immunoreactivity detected, and the t(11;l4)(q13;q32) breakpoint was amplified from both lymph node and skin DNA. Competitive reverse transcriptase-polymerase chain reaction was not contributive for skin specimens. In all 3 cases, interphase fluorescence in situ hybridization (FISH) demonstrated t(11;14) fusion signals either on paraffin sections or on fresh frozen touch preparations of skin biopsies. The recognition of skin lesions of MCL from other B-cell infiltrates can be established by interphase FISH.