IntroductionZAP-70 protein is a 70-kDa member of the Syk family of protein tyrosine kinases that was first identified as a crucial element for proximal signaling from the T-cell receptor. 1 Similar to Syk protein after the B-cell receptor (BCR) stimulation, ZAP-70 is recruited to the phosphorylated immunoreceptor tyrosine activation motifs of the -and CD3-chains present in the T-cell receptor, where it subsequently becomes phosphorylated and initiates several signaling cascades. 2 Expression of ZAP-70 protein was considered to be restricted to T lymphocytes and natural killer cells. However, it has also been found expressed in normal B-cell precursors and in some subsets of activated B cells. [3][4][5][6] Among B cell-derived malignancies, ZAP-70 is mainly expressed in chronic lymphocytic leukemia (CLL; 37%-57% of cases), 7-9 B-acute lymphoblastic leukemia (B-ALL; 56%-59% of cases) 4,10 and Burkitt lymphoma (8%-31% of cases). 11,12 An increased ZAP-70 expression in CLL has been associated with particular adverse biologic features, such as the presence of unmutated IgHV genes 7 or high CD38 expression, 13 and correlates with a poor clinical outcome. [7][8][9] In the same vein, ZAP-70 expression in B-ALL correlates with a short survival as well. 10 Although ZAP-70 expression in B-cell malignancies has an adverse prognostic influence, its role in the biology of the tumoral B cell is not fully defined. In this regard, the expression of ZAP-70 protein in CLL cells has been related to an enhanced BCR signaling. [13][14][15][16][17] In addition, increased ZAP-70 expression has been associated with increased migration capabilities of CLL cells toward different chemokines, such as CXCL12, CCL19, and CCL21, [18][19][20] and with increased signaling and survival on CXCL12 treatment. 18,21 However, whether these increased migrative capabilities are a direct effect of ZAP-70 expression or a mere reflection of the distinct biology features of ZAP-70-expressing cells needs to be further investigated.To ascertain the direct implication of ZAP-70 in B-cell signaling and migration, we analyzed the phenotypic effects of ectopic ZAP-70 expression in a B-cell system and studied the expression of adhesion molecules and chemokine receptors in CLL primary cells with high or low ZAP-70 expression within the same patient. Herein, we report that IgM, but not IgD, stimulation mobilizes and activates ZAP-70, which in turn enhances BCR-induced ERK1/2 and Akt phosphorylation and delays IgM and CD79b internalization. Moreover, we show that ZAP-70 induces the expression of the chemokine receptor CCR7 via ERK1/2 activation, thus directly enhancing the capacity of signaling and migration of the ZAP-70-expressing B cells toward CCL21. Finally, we show that CLL cells with higher ZAP-70 expression within the same patient have a different expression profile of adhesion molecules and chemokine receptors and enhanced migration capacity toward CCL21. Methods Cell lines and primary cellsThe Burkitt lymphoma B-cell lines Raji and Ramos were obtained from ...

Combining single-cell cytometry datasets increases the analytical flexibility and the statistical power of data analyses. However, in many cases the full potential of co-analyses is not reached due to technical variance between data from different experimental batches. Here, we present cyCombine, a method to robustly integrate cytometry data from different batches, experiments, or even different experimental techniques, such as CITE-seq, flow cytometry, and mass cytometry. We demonstrate that cyCombine maintains the biological variance and the structure of the data, while minimizing the technical variance between datasets. cyCombine does not require technical replicates across datasets, and computation time scales linearly with the number of cells, allowing for integration of massive datasets. Robust, accurate, and scalable integration of cytometry data enables integration of multiple datasets for primary data analyses and the validation of results using public datasets.

SummaryThis prospective multi-institutional phase II study was designed to assess the efficacy and safety of dose-adjusted EPOCH (etoposide, prednisone, vincristine, cyclophosphamide and doxorubicin) plus rituximab (DA-EPOCH-R) in untreated patients with poor prognosis large B-cell lymphomas. Eighty-one patients diagnosed with diffuse large B-cell lymphoma (DLBCL, n = 68), primary mediastinal DLBCL (n = 6) and follicular lymphoma Grade 3b (n = 7), with an age-adjusted International Prognostic Index >1, were eligible for analysis. Median age was 60 years (range: 21-77). Sixty-five patients (80Á2%) achieved complete response. After a median follow-up time of 64 months, 10-year event-free survival and overall survival (OS) were 47Á8% and 63Á6%, respectively. None of the studied clinical and biological characteristics were associated with poorer outcome. Interestingly, patients with BCL6 rearrangement achieved a 10-year OS of 100%, while patients with BCL2 rearrangement exhibited a poorer outcome compared to activated B-cell tumours and germinal centre B-cell without BCL2 rearranged tumours. Results achieved with DA-EPOCH-R showed a good long-term outcome and a tolerable toxicity profile in high-risk large B cell lymphoma patients. Outcome was not affected by tumour cell proliferation or by cell of origin, highlighting the requirement of new biological markers for patient subclassification of high-risk DLBCL patients.