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In splenic marginal zone lymphoma (SMZL), specific and functional Toll-like Receptor (TLR) patterns have been recently described, suggesting their involvement in tumoral proliferation. Splenic diffuse red pulp lymphoma with villous lymphocytes (SDRPL) is close to but distinct from SMZL, justifying here the comparison of TLR patterns and functionality in both entities. Distinct TLR profiles were observed in both lymphoma subtypes. SDRPL B cells showed higher expression of TLR7 and to a lesser degree TLR9, in comparison to SMZL B cells. In both entities, TLR7 and TLR9 pathways appeared functional, as shown by IL-6 production upon TLR7 and TLR9 agonists stimulations. Interestingly, circulating SDRPL, but not SMZL B cells, constitutively expressed CD86. In addition, stimulation with both TLR7 and TLR9 agonists significantly increased CD80 expression in circulating SDRPL but not SMZL B cells. Finally, TLR7 and TLR9 stimulations had no impact on proliferation and apoptosis of SMZL or SDRPL B cells. In conclusion, SMZL and SDRPL may derive from different splenic memory B cells with specific immunological features that can be used as diagnosis markers in the peripheral blood., Competing Interests: CONFLICTS OF INTEREST The authors declare no conflicts of interest.

Splenic diffuse red pulp lymphoma is an indolent small B-cell lymphoma recognized as a provisional entity in the World Health Organization 2008 classification. Its precise relationship to other related splenic B-cell lymphomas with frequent leukemic involvement or other lymphoproliferative disorders remains undetermined. We performed whole-exome sequencing to explore the genetic landscape of ten cases of splenic diffuse red pulp lymphoma using paired tumor and normal samples. A selection of 109 somatic mutations was then evaluated in a cohort including 42 samples of splenic diffuse red pulp lymphoma and compared to those identified in 46 samples of splenic marginal zone lymphoma and eight samples of hairy-cell leukemia. Recurrent mutations or losses in BCOR (the gene encoding the BCL6 corepressor) - frameshift (n=3), nonsense (n=2), splicing site (n=1), and copy number loss (n=4) - were identified in 10/42 samples of splenic diffuse red pulp lymphoma (24%), whereas only one frameshift mutation was identified in 46 cases of splenic marginal zone lymphoma (2%). Inversely, KLF2 , TNFAIP3 and MYD88 , common mutations in splenic marginal zone lymphoma, were rare (one KLF2 mutant in 42 samples; 2%) or absent ( TNFAIP3 and MYD88 ) in splenic diffuse red pulp lymphoma. These findings define an original genetic profile of splenic diffuse red pulp lymphoma and suggest that the mechanisms of pathogenesis of this lymphoma are distinct from those of splenic marginal zone lymphoma and hairy-cell leukemia., (Copyright© 2017 Ferrata Storti Foundation.)

Background: Altered Toll-like receptor (TLR) expression levels and/or mutations in its signaling pathway (such as MyD88 mutation) contribute to the pathogenesis of lymphoproliferative disorders (LPD). CD180 is an orphan member of the TLR family that modulates the signaling of several TLRs, but only limited studies have evaluated its expression by flow cytometry (FCM) in LPD., Methods: Using a multiparameter FCM approach, we have assessed CD180 mean fluorescence intensity (MFI) in lymph nodes (LNs) and peripheral blood (PB) samples obtained from patients with follicular lymphoma (FL; LN/PB, n = 44/n = 15), chronic lymphocytic leukemia (CLL, n = 26/n = 21), mantle cell lymphoma (MCL, n = 13/n = 17), and marginal zone lymphoma (MZL, n = 16/n = 12). Specimens from non-tumoral PB and LN (n = 8/n = 12) were used as controls., Results: In the LN specimens, FL and control B-cells showed similar CD180 expression (MFI = 1,049 vs. 1,381, P > 0.05; Mann-Whitney U-test). This level was markedly lower in the other LPDs, MCL (MFI = 396, P < 0.05), or CLL (MFI = 502 P < 0.05), and similar to MZL (MFI = 858, P > 0.05). However, the CD180 expression of FL B-cells assessed in PB was dim and/or negative, in the same range as MCL and CLL (FL MFI = 453, MCL MFI = 305, CLL MFI = 420, P > 0.05) but lower than in MZL (MFI = 895, P < 0.05). Therefore, these results suggest a modulation of CD180 expression by neoplastic FL B-cells based on the anatomical compartment., Conclusion: These FCM data confirm the usefulness of CD180 in the accurate diagnosis of LPDs and emphasize the need to interpret this marker according to the origin of the sample. © 2015 Clinical Cytometry Society., (© 2015 International Clinical Cytometry Society.)

New B-cell receptor-targeted therapies such as ibrutinib, a Bruton tyrosine kinase inhibitor, are now proposed for lymphoid pathologies. The putative benefits of its combination with glucocorticoids were evaluated here. We compared the effects of dexamethasone (DXM), ibrutinib and their in vitro combination on proliferation and metabolic stress markers in stimulated normal B-lymphocytes and in malignant lymphocytes from chronic lymphocytic leukemia (CLL) patients. In both cellular models, cell cycle progression was globally inhibited by DXM and/or ibrutinib. This inhibition was significantly amplified by DXM addition to ibrutinib and was related to a significant decrease in the expression of the cell cycle regulatory proteins CDK4 and cyclin E. Apoptosis increased especially with DXM/ibrutinib combination and was associated with a significant decrease in Mcl-1 expression. Treatment effects on metabolic stress were evaluated by DNA damage recognition after 53BP1 foci labeling. The percentage of cells with more than five 53BP1 foci decreased significantly with ibrutinib in normal and CLL lymphocytes. This decrease was strongly reinforced, in CLL, by DXM addition. Our data indicated that, in vitro, DXM potentiated antiproliferative effects of ibrutinib and decreased DNA damage in lymphoid B-cells. Thus their combination may be proposed for CLL treatment., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

We report five chronic myeloid leukaemia (CML) patients in whom we identified and characterized undescribed BCR-ABL1 fusion transcripts. We investigated the precise features of the molecular rearrangements and the minimal residual disease follow-up for these five patients. Three resulted from new rearrangements between the BCR and ABL1 sequences (the breakpoints being located within BCR exon 13 in two cases and within BCR exon 18 in one case). The other two cases revealed a complex e8-[ins]-a2 fusion transcript involving a third partner gene, PRDM12 and SPECC1L, respectively. Moreover, single nucleotide polymorphism-array analysis performed in the latter two cases showed copy number alterations shared by the two patients, thus identifying genes that were deleted during rearrangement and suggesting their potential role in CML pathogenesis. Interestingly, we highlight that the prognosis of alterations, such as the presence of an e8a2 transcript or the deletion of various genes, which have been controversial, may be definitively erased by the introduction of tyrosine kinase inhibitors (TKIs)., (© 2015 Wiley Periodicals, Inc.)

Background: The rigorous cytological review by manual or automatic microscopic analysis is critical in the detection of circulating neoplastic cells, since their morphology as well as their count contributes to the diagnosis and prognosis of many diseases. However, the cytological analysis is not always obvious and requires trained and competent cytologist. In this context, the alarms and/or parameters generated by hematology analyzer could be particularly informative to alert the operators., Methods: Blood samples from patients with Sezary syndrome (n = 9) were studied with Sysmex XN-1000 analyzer, and compared to patients with benign or tumoral skin lesions (n = 47) and patients with chronic lymphoproliferative B-cell diseases (n = 51) used as control., Results: In present series, the value of structural lymphoid parameters (LyX and LyZ) and the alarm Blast/Abn Lympho were statistically higher in Sezary cases than in control cases. In addition, the value of LyX was associated to the count of circulating Sezary cells and value of LyZ to the presence of large Sezary cells, both parameters described as prognostic factors., Conclusion: The combination of alarm Blast/Abn Lympho and structural parameters (Ly-X/Ly-Z/Ly-Y) may allow to define rule of blood slide review to screen circulating Sezary cells, and give promising results in B-cell diseases., (© 2014 Wiley Periodicals, Inc.)

Recent studies demonstrate that early molecular response to tyrosine-kinase inhibitors is strongly predictive of outcome in chronic myeloid leukemia patients and that early response landmarks may identify patients at higher risk for transformation who would benefit from an early switch to second-line therapy. In this study, we evaluated the ability of the control gene GUS to identify relevant thresholds for known therapeutic decision levels (BCR-ABL1/ABL1IS  = 10% and 0.1%). We then defined the most relevant cut-offs for early molecular response markers (transcript level at 3 months, halving time and log reduction between diagnosis and 3 months of treatment) using GUS or ABL1. We demonstrated that, although both control genes could be used (in an equivalent way) to accurately assess early molecular response, the BCR-ABL1/GUS level at diagnosis is impacted by the higher GUS copy number over-expressed in CML cells, thus negatively impacting its ability to completely replace ABL1 at diagnosis. Furthermore, we pointed out, for the first time, that it would be helpful to monitor BCR-ABL1 levels at an earlier time point than that currently performed, in order to assess response to first-line tyrosine-kinase inhibitors and consider a potential switch of therapy as early as possible. We evaluated this optimal time point as being 19 days after the start of treatment in our cohort.

Introduction: As most hematology cell analyzers, the different parameters of Sysmex XE-5000™ are little informative in the qualitative analysis of lymphoid cells, and especially when the lymphocyte count is below 4 × 10(9) /L (i.e., 'normal'). The aim of our study was to investigate whether some parameters and/or 'flags' not routinely provided by this analyzer, but reachable by operator could be reliable to define rules of slide review in absence of common qualitative and quantitative alarms particularly in case of 'normal' lymphocyte count., Methods: Blood samples from 13 mantle cell lymphoma fully annotated cases, and 180 control specimens were studied with Sysmex XE-5000™ analyzer. All cases did not present any anomalies in common quantitative and structural parameters., Results: Using the method of area under the curve and ROC curve analysis, we described a novel threshold of alarm VAL&#95;ABN LYMPH (≥40 instead of 100 defined by Sysmex), as well as a pertinent LyX threshold (≥89). The combination of these thresholds allowed defining a rule of slide review in context of 'normal' lymphocyte count., Conclusion: Among the parameters provided by the Sysmex XE-5000™ analyzer, the combination of the alarm VAL&#95;ABN LYMPH and the LyX value, routinely available on a simple blood analysis appears particularly informative to trigger slide review in a context of 'normal' lymphocyte count with a good sensitivity (85%) to detect circulating lymphoma cells and with <1% of false positive results., (© 2012 John Wiley & Sons Ltd.)

Somatic mutations in the NPM1 gene, which encodes for nucleophosmin, have been reported to be the most frequent genetic abnormalities found in acute myeloid leukaemia (AML). Their identification and quantification remain crucial for the patients' residual disease monitoring. We investigated a new method that could represent a novel reliable alternative to sequencing for its identification. This method was based on high-resolution melting analysis in order to detect mutated patients and on an allele-specific oligonucleotide real-time quantitative polymerase chain reaction (ASO-RQ-PCR) for the identification and quantification of the transcripts carrying NPM1 mutations (NPM1m). Few patients carrying known NPM1m enabled us to set up a table with the different primers' ΔCT values, identifying a profile for each mutation type. We then analysed a series of 337 AML patients' samples for NPM1 mutational status characterization and confirmed the ASO-RQ-PCR results by direct sequencing. We identified some mutations in 86 samples, and the results were fully correlated in 100% of the 36 sequenced samples. We also detected other rare NPM1m in two samples, that we confirmed by direct sequencing. This highly specific method provides a novel quick, useful, and costless tool, easy to use in routine practice.

The translocation t(14;18) and its t(2;18) and t(18,22) variants, which involve the BCL2 genetic hallmark for follicular lymphoma (FL), have been reported in several cases of chronic B-cell lymphoproliferative disease (CLPD) and frequently in chronic lymphocytic leukaemia (CLL). We describe here the clinical, morphological, immunological, cytogenetic and molecular findings from 37 cases of t(14;18)-positive CLPD, identified from our series of non-FL B-cell neoplasms (n=993) that were routinely analysed in peripheral blood by conventional cytogenetics analyses. The FL diagnosis was excluded by morphology and immunology (the samples were CD10 negative in all cases). The BCL2 translocations were observed in 22 CLL cases, including 7 monoclonal B-cell lymphocytosis (MBL) cases re-classified according to the new International Workshop on CLL criteria, six small lymphocytic lymphoma (SLL) cases, 1 splenic marginal zone lymphoma (SMZL) case and eight cases of unclassifiable CLPD with overlapping CLL/MZL features. In the CLL cases, the IGH/BCL2 fusion was remarkably associated with trisomy 12 (13/22) and mutated IGHV status (20/21) and did not affect the outcome. Moreover, most of these CLLs harboured a low mutation load of BCL6 gene and unmutated FAS (CD95) loci, which points to a post-germinal-centre cellular origin., (© 2012 Blackwell Publishing Ltd.)

Introduction: Immunohaematology examinations are usually prescribed preoperatively according to more or less standardized protocols. We wanted to assess the relevance of these protocols on the basis of factual data: an overview of the rate of transfusions carried out as part of surgery within the HCL in 2009., Study Design: The list of patients operated in 2009 in the HCL (IPOP by Cristalnet) has been combined with the list of patients transfused in the same time period (CTS server, Inlog). The percentage of patients transfused during the stay, and the percentage of patients transfused on the day of the intervention itself were determined for each type of surgery. The study focused on 13,571 patients affected by 44 surgeries., Patients and Methods: Six hundred and thirty-three patients were transfused, 45% of them the day of the intervention. The risk of needing to carry out a transfusion depends on the risk to the patient and surgery. For example, the total hip arthroplasty transfusion risk is 11.9% when it's programmed against 37.8% in emergency surgery. The transfusion risk of knee arthroscopies, osteosynthesis of wrist fracture, carpal canal surgeries and of appendectomies, thyroidectomies, herna repair surgeries are below 0.5%. The transfusion risk of colectomy is 18.1%. Thus, new recommendations for good clinical practices on the relevance of settled surgery-preoperative immunohematologic exams can be established., Conclusion: The emergency degree of the transfusion must be taken into account for such recommendation. Each hospital should perform its own cartography to justify its own protocols., (Copyright © 2011 Société française d'anesthésie et de réanimation (Sfar). Published by Elsevier SAS. All rights reserved.)

It has been recently shown that DNA methyl transferase overexpression is correlated with unfavourable prognosis in human malignancies while methylation deregulation remains a hallmark that defines acute myeloid leukemia (AML). The oncogenic transcription factor EVI1 is involved in methylation deregulation and its overexpression plays a major role for predicting an adverse outcome. Moreover, the identification of DNMT3A mutations in AML patients has recently been described as a poor prognostic indicator. In order to clarify relationship between these key actors in methylation mechanisms and their potential impact on patient outcomes, we analysed 195 de novo AML patients for the expression of DNMT3A, 3B (and its non-catalytic variant 3B(NC)) and their correlations with the outcome and the expression of other common prognostic genetic biomarkers (EVI1, NPM1, FLT3ITD/TKD and MLL) in adult AML. The overexpression of DNMT3B/3B(NC) is (i) significantly correlated with a shorter overall survival, and (ii) inversely significantly correlated with event-free survival and DNMT3A expression level. Moreover, multivariate analysis showed that a high expression level of DNMT3B/3B(NC) is statistically a significant independent poor prognostic indicator. This study represents the first report showing that the overexpression of DNMT3B/3B(NC) is an independent predictor of poor survival in AML. Its quantification should be implemented to the genetic profile used to stratify patients for therapeutical strategies and should be useful to identify patients who may benefit from therapy based on demethylating agents.

Angioimmunoblastic T-cell lymphoma is immunologically defined by the expression of CD10 and the follicular helper T cell (T(FH)) markers such as CXCL13, programmed death-1 (PD-1) and inducible T-cell costimulator (ICOS). This T(FH) profile has been mainly reported by immunohistochemistry. Here, using multiparametric flow cytometry, the relevance of ICOS and PD-1 to angioimmunoblastic T-cell lymphoma diagnosis was evaluated in lymph node (n=15) as well as in peripheral blood (n=13) among a series of 28 angioimmunoblastic T-cell lymphoma cases, in addition to the CD10 expression (available in 26 lymph node and 15 peripheral blood specimens). In this series, CD10 expression was present in 23/26 (88%) lymph node and in 12/15 (80%) peripheral blood cases and ICOS in 13/15 (87%) lymph node and in 6/13 (47%) peripheral blood cases, whereas neither significant CD10 nor ICOS T cells were identified in the control group (lymph nodes with reactive hyperplasia=10, peripheral blood of healthy donors=15). PD-1 expression was less informative as observed in both angioimmunoblastic T-cell lymphoma and control cases. The multiparametric approach allowed us to confirm the frequent blood dissemination in angioimmunoblastic T-cell lymphoma and to show that circulating neoplastic T cells correspond more often to a CD10-positive subset than to an ICOS-positive subset. Consequently, if ICOS constitutes an additional feature for the diagnosis of angioimmunoblastic T-cell lymphoma, it appears less sensitive than CD10 expression for the detection of circulating neoplastic T cells.

The p16(INK4a) protein regulates cell cycle progression mainly by inhibiting the activity of G1-phase cyclin-dependent kinases (CDKs) 4 and 6, the subsequent retinoblastoma protein (pRb) phosphorylation and E2F transcription factor release. The p16(INK4a) protein can also repress the activity of other transcription factors, such as c-myc, nuclear factor-kappaB and c-Jun/AP1. Here, we report that, in two p16(-/-), pRb(WT) and p53(WT) cell lines (MCF7 and U87), p16(INK4a) overexpression induces a dramatic decrease in CDK1 protein expression. In response to p16(INK4a), the decreased rate of CDK1 protein synthesis, its unchanged protein half-life, unreduced CDK1 mRNA steady-state levels and mRNA half-life allow us to hypothesize that p16(INK4a) could regulate CDK1 expression at the post-transcriptional level. This CDK1 downregulation is mediated by the 3'-untranslated region (3'UTR) of CDK1 mRNA as shown by translational inhibition in luciferase assays and is associated with a modified expression balance of microRNAs (miRNAs) that potentially regulate CDK1, analyzed by TaqMan Human microRNA Array. The p16(INK4a)-induced expression of two miRNAs (miR-410 and miR-650 chosen as an example) in MCF7 cells is confirmed by individual reverse transcription-qPCR. Furthermore, we show the interaction of miR-410 or miR-650 with CDK1-3'UTR by luciferase assays. Endogenous CDK1 expression decreases upon both miRNA overexpression and increases with their simultaneous inhibition. The induction of miR-410, but not miR-650 could be related to the pRb/E2F pathway. These results demonstrate the post-transcriptional inhibition of CDK1 by p16(INK4a). We suggest that p16(INK4a) may regulate gene expression by modifying the functional equilibrium of transcription factors and consequently the expression balance of miRNAs.

'Splenic red pulp lymphoma with numerous basophilic villous lymphocytes' (SRPL), recently described, is characterized by clinical, morphologic, immunologic, cytogenetic and molecular features distinct from SMZL/SLVL and HCL. In particular, the intensity of CD11c staining (expressed as fluorescence intensity -RFI-) in SRPL is significantly different from the RFI in SMZL/SLVL and HCL. Moreover the use of a scoring system based on the expression of CD11c, CD22, CD76, CD38 and CD27 appears to improve the differential diagnosis between SRPL and SMZL/SLVL and emphasizes that SRPL is an entity closed to but distinct from SMZL/SLVL., (Copyright © 2010 John Wiley & Sons, Ltd.)

This phase I/II study was designed to demonstrate the tolerance and the efficacy of a combination of pegylated interferon-α 2a to Imatinib mesylate (IM) 600mg daily in cytogenetically IM-resistant but in CHR chronic phase CML patients. The combination was generally well tolerated in the 15 evaluable patients. A significant reduction of the Ph1(+) BM metaphases was observed in these poor prognosis patients, with 2 long-term CCyR including 2 MMR. After a median follow-up of 43 months, 93% of patients are alive. The addition of PegIFNα2a to IM600 is feasible, and able to overcome resistance within this context., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

This report aims to more accurately define the frequency of the involvement of SRC Family Kinases (SFKs) in imatinib- and dasatinib-resistant CML patients. Clinical samples were analysed during in vivo treatment. We confirmed the high frequency of SFKs involvement in Tyrosine kinase inhibitor-resistant CML (52% of the cases) and even further in progressive disease and blast crises (60% of the cases). The SFKs deregulation is also observed in patients harboring BCR-ABL mutations. In T315I and F317L mutated patients, CML-resistance appears to be promoted by SFKs kinase protein reactivation once the BCR-ABL mutated clone has decreased on Omacetaxine., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

Purpose: The onset of a BCR-ABLT315I mutation during the course of chronic myelogenous leukemia (CML) on tyrosine kinase inhibitors (TKIs) usually results in poor survival, and therapeutic options remain few in the absence of any allogeneic donor., Patients and Methods: We have investigated the affect of subcutaneous omacetaxine (OMA, or homo-harringtonine) cycles on unmutated and T315I-mutated BCR-ABL transcripts in a series of 8 TKI-resistant chronic-phase CML patients and we have addressed the question of whether the administration of OMA could resensitize patients to TKIs. Patients were regularly monitored for total disease burden and for BCR-ABLT315I transcripts using a new quantitative sensitive technique (sensitivity threshold, 0.05%), for up to 27 cycles of OMA., Results: Overall, patients demonstrated hematologic, cytogenetic, or molecular improvement. An initial rapid decline and a sustained disappearance of T315I-mutated transcripts were observed in 50% of patients, after a median of 10.5 cycles (range, 3-27 cycles) of OMA. As the unmutated leukemic burden reduction was modest, 2 patients were submitted to nilotinib after 9 months of sustained BCR-ABLT315I transcripts negativity on OMA and mutated transcripts remained undetectable after a median follow-up of 12 months on nilotinib challenge., Conclusion: We suggest that OMA (ie, a non-targeted therapy) might provide a better disease control allowing the disappearance of the mutated clone probably elicited by the clone deselection after TKI release, and/or a preferential activity of OMA on the T315I-mutated cells through unknown mechanisms. These observations suggest that OMA could allow a safe TKI rechallenge in patients with resistant chronic-phase CML.

Background Information: miRNAs (microRNAs) are a class of non-coding RNAs that inhibit gene expression by binding to recognition elements, mainly in the 3' UTR (untranslated region) of mRNA. A single miRNA can target several hundred mRNAs, leading to a complex metabolic network. miR-16 (miRNA-16), located on chromosome 13q14, is involved in cell proliferation and apoptosis regulation; it may interfere with either oncogenic or tumour suppressor pathways, and is implicated in leukaemogenesis. These data prompted us to search for and validate novel targets of miR-16., Results: In the present study, by using a combined bioinformatics and molecular approach, we identified two novel putative targets of miR-16, caprin-1 (cytoplasmic activation/proliferation-associated protein-1) and HMGA1 (high-mobility group A1), and we also studied cyclin E which had been previously recognized as an miR-16 target by bioinformatics database. Using luciferase activity assays, we demonstrated that miR-16 interacts with the 3' UTR of the three target mRNAs. We showed that miR-16, in MCF-7 and HeLa cell lines, down-regulates the expression of caprin-1, HMGA1a, HMGA1b and cyclin E at the protein level, and of cyclin E, HMGA1a and HMGA1b at the mRNA levels., Conclusions: Taken together, our data demonstrated that miR-16 can negatively regulate two new targets, HMGA1 and caprin-1, which are involved in cell proliferation. In addition, we also showed that the inhibition of cyclin E expression was due, at least in part, to a decrease in its mRNA stability.

miR-16, a miRNA involved in cell proliferation and apoptosis regulation, may interfere with either oncogenic or tumor-suppressor pathways and is implicated in leukemogenesis. We then explored its expression in 93 childhood acute lymphoblastic leukemia (ALL) cases. A high miR-16 expression was associated with hyperleukocytosis and poor cytogenetic groups. In the whole group and in B-cell ALLs, disease-free survival (DFS) was significantly shorter for miR-16 above quartile 75. In T-cell ALLs, for both DFS and overall survival, a significant trend was found with a survival shortening from the lowest to the highest miR-16 levels. miR-16 expression neither significantly correlated with normal and malignant lymphocyte proliferation nor varied according to lymphocyte differentiation. The prognostic value of miR-16 in childhood ALL highlighted the complexity of miR-16 functions.

We report the emergence of a chronic myeloid leukaemia (CML) during the course of a JAK2V617F-positive chronic idiopathic myelofibrosis (CIMF) in the absence of any myelosuppressive treatment. Although a response to imatinib was observed, the underlying myelofibrosis persisted after treatment and hydroxyurea was finally added to control the persistent thrombocytosis. Such rare patients with co-existing BCR-ABL translocation and JAK2V617F mutation must be identified in view of the possibility of targeted therapies. Moreover, the detection of BCR-ABL translocation appears to be crucial especially in the case of treated CIMF with an atypical course to identify CML before acute transformation.

The presence of circulating villous lymphocytes (VLs) in lymphoma patients usually points to splenic marginal zone B-cell lymphoma (SMZL), even if the VLs can be found occasionally in other small B-cell lymphomas. However, those cells are variably described, and detailed cytologic characterization is often lacking. We identified lymphoma cases with numerous basophilic VLs among the large group of splenic lymphoma with VLs, and for further delineation, 37 cases with this particular cytology were analyzed. Patients, predominantly older men, presented with moderate lymphocytosis and splenomegaly without pancytopenia. The monoclonal B cells expressed IgM + D, IgM + G, IgM or IgG, as well as CD76 and CD11c, frequently CD103, and rarely CD123. Spleen sections were peculiar, with atrophic white pulp and a monomorphic diffuse lymphoma infiltration in a congested red pulp. Bone marrow infiltration was interstitial and intrasinusoidal without extensive fibrosis. Cytogenetic analysis showed a frequent absence of clonal aberrations (68%). Most cases (79%) were IgH mutated, with an overrepresentation of V(H)3 and V(H)4 gene families. These results, as well as the clinical evolution, show that those lymphoma cases represent a homogeneous group distinct from SMZL and reminiscent of hairy cell leukemia variant, perhaps corresponding to a separate lymphoma entity.

High-resolution multicolor banding (mBAND) analysis was applied to precisely fine-map the genomic extent of 7q deletions in a series of 26 marginal zone lymphoma patients displaying the abnormality on conventional karyotypes. Using this approach, the breakpoints and the extent of deletions revealed by conventional banding techniques had to be re-defined in 70% of cases. Although no common minimal region of deletion was delineated, mBAND demonstrated the involvement of the 7q32 region in more than 90% of cases. In addition, unsuspected translocations and intrachromosomal changes could be identified in four cases. Taken together, these data demonstrate that mBAND represents an alternative cytogenetic tool in the comprehensive analysis of chromosome aberrations in hematologic malignancies, allowing rapid screening and precise delineation of structural rearrangements of a defined chromosome. This also confirms the localization in the vicinity of band 7q32 of putative candidate gene(s) involved in the pathogenic development of the disease.

Dasatinib is efficient in vitro against most of CML cells harboring ABL kinase domain mutations and induces high rates of response in imatinib-resistant CML patients. Here, we monitored the mutated BCR-ABL transcripts during the follow-up of 12 CML patients treated with dasatinib. We identified four groups of patients based on their sensitivity to dasatinib. Clinical responses were correlated to the in vitro sensitivity of BCR-ABL mutants to dasatinib, however, some discrepancies were observed in a subfraction of CML patients, suggesting subtle differences between in vitro observations and clinical entities and/or the onset of other mechanisms responsible for dasatinib resistance.

In most cases of lymphomas with blood dissemination, the careful cytological analysis of peripheral blood smears provides a rapid orientation to diagnosis, even if the final subtyping is achieved by histology and eventually other techniques. Here, we evaluated if the analysis of blood smears may suggest the blood dissemination of angioimmunoblastic T-cell lymphoma (AITL) and if CD10 expression on neoplastic T cells, as recently reported on AITL, may contribute to the diagnosis. In all, 11 lymph nodes and six peripheral blood samples from 12 patients with AITL were studied using four-colour flow cytometry associated to histological, cytological and molecular data. According to previous results, a fraction of T cells expressed CD10 in 10/11 lymph nodes. Interestingly, all blood smears showed atypical lymphoid cells and a fraction of T cells expressed CD10 with a mean percentage of 18.75% (range 5.00-47.00%), regardless of lymphocytosis level and of rate of CD10 T cells in corresponding lymph node. In contrast, in all control samples (100), none CD10-positive T cell was identified. This is to our knowledge the first description of circulating CD10 neoplastic T cells in AITL. Therefore, they ought to be explored in further studies when aggressive lymphoma, in particular with lymphopenia and circulating atypical cells, is suspected.

The purpose of this study was to document the frequency and distribution of karyotypic changes present at diagnosis in 103 non-MALT marginal zone cell lymphoma (MZL) patients. This cytogenetic analysis of a large cohort extends previous observations and allows the identification of new cytogenetic features. Abnormalities identified in more than 15% of patients included +3/+3q (37%), 7q deletions (31%), +18/+18q (28%), 6q deletions (19%), +12/+12q (15%) and 8p deletions (15%). Trisomy 3/3q, 7q deletions, +18 and +12 were seen in different combinations in more than 30% of patients in comparison to 2% in lymphocytic lymphomas/chronic lymphocytic leukemias, 1% in mantle cell lymphomas and 7% in follicular lymphomas. The marked propensity of these abnormalities to be recurrently associated with the same tumoral clone of individual karyotypes allowed the delineation of a cytogenetic profile that may help to distinguish non-MALT MZL among other mature B-cell neoplasms. If +3/3q, +12/+12q, and 6q, 7q and 8p deletions were significantly associated with clinical prognostic factors previously reported to influence survival and time to progression, patients displaying these abnormalities did not experience a significantly shorter time to progression.

The immunological profile of lymphoproliferative disorders is usually conserved whatever the involved site, thus allowing a reliable diagnosis from peripheral blood analysis, especially in small lymphocytic lymphoma/chronic lymphocytic lymphoma (SLL/CLL). Here we present a case wherein the cytology and immunophenotype of blood specimen and bone marrow argue in favor of SLL/CLL with a typical Matutes score (5/5), whereas the cyto-histology and immunophenotype of spleen specimen led to the diagnosis of splenic marginal zone B-cell lymphoma (SMZL). Moreover genomic analysis showed that the splenic cells displayed a SMZL signature. Whereas these data suggested the presence of 2 B-cell clones, the study of the mutational status of IgVH gene in blood and spleen demonstrated the presence of a single clone, which likely developed simultaneously along two distinct ways of differentiation according to the anatomic site suggesting here the predominant role of a micro-environmental factor in cell differentiation. Although rare, this kind of event must be kept in mind as a cause of discrepancies between diagnoses from different sites.

Quantitative monitoring of imatinib mesylate (IM)-resistant, mutated BCR-ABL(+) cells during the follow-up of CML could be useful for optimizing therapeutic management. We retrospectively analyzed T315I mutated BCR-ABL clones throughout the CML history of two patients by nested-PCR-RFLP. At the time of progression, the T315I mutation represented 100% of the BCR-ABL transcripts. During follow-up, we showed that (i) despite a molecular response to IM, a high proportion of T315I transcripts were present (>85%) and predictive of relapse, (ii) interruption of IM and switching to other therapies resulted in a significant reduction in mutant transcript level while total BCR-ABL(+) transcripts remained stable.

More than 35 different partner genes with the mixed lineage leukemia (MLL) gene have been cloned from leukemia cells with translocations involving chromosome 11 band q23. In this study, we report on a novel fusion partner of the MLL gene, AF4p12, which we have identified as the human homologue to the furry gene of Drosophila. AF4p12, highly conserved in evolution, encodes a large protein of 3,105 amino acids. The expression of AF4p12 has been preferentially detected in colon, placenta, and brain tissues and in tumor cells of lymphoid origin. We show that the t(4;11)(p12;q23) translocation results in the creation of a chimeric RNA encoding a putative fusion protein containing 1,362 amino acids from the NH2-terminal part of MLL and 712 amino acids from the COOH-terminal part of AF4p12. FLT3 and HOXA9 genes are overexpressed in this leukemia. We found that the COOH-terminal part of AF4p12 fused to MLL contains a leucine zipper motif and exhibits transcriptional activation properties when fused to Gal4 DNA-binding domains in transient transfection assays. The AF4p12 fragment fused to MLL may contribute to the oncogenic activation of MLL, possibly through specific recruitment of the transcriptional machinery.

Waldenstrom's macroglobulinemia (WM) is considered in the World Health Organization classification as a clinical syndrome associated with monoclonal immunoglobulin (Ig) M secretion, mainly observed in patients with lymphoplasmacytic lymphoma (LPL) and occasionally with other small B-cell lymphomas. Some authors consider it a rare distinct lymphoproliferative disorder with primary bone marrow infiltration and IgM monoclonal gammopathy. As LPL shares important morphologic and immunophenotypic overlaps with marginal zone B-cell lymphomas (MZLs) in cases showing plasmacytic maturation, it remains unclear if they constitute unique or distinct entities. Both diseases are composed of lymphocytes, lymphoplasmacytoid cells, and tumoral plasma cells with a surface (s) IgM-positive sIgD+/ cytoplasmic IgMpositive CD19+ CD20+ CD27+/ CD5 CD10 CD23 phenotype, without a specific marker. Extranodal mucosa-associated lymphoid tissue (MALT) lymphoma, nodal MZL (NMZL), and splenic MZL (SMZL) are distinct entities displaying common morphologic, immunophenotypic, and genetic characteristics. MALT lymphoma is clearly distinct from LPL, although bone marrow infiltration and IgM paraprotein are not rare. Splenic MZL and NMZL are incompletely characterized, but a plasmacytoid/plasmacytic differentiation, autoimmune manifestations, and monoclonal component are frequent in both diseases. Bone marrow involvement is constant in SMZL and present in 60% of NMZLs. Molecular IgVH gene analysis has confirmed this heterogeneity, particularly within SMZL, with mutated and unmutated cases. Further studies are needed to clarify the pathogenesis of these MZLs and their relationship with LPL.

The CCR4-associated protein CAF1 has been demonstrated to play several roles in the control of transcription and of mRNA decay. To gain further insight into its physiological function, we generated CAF1-deficient mice. They are viable, healthy, and normal in appearance; however, mCAF1(-/-) male mice are sterile. The crossing of mCAF1(+/-) mice gave a Mendelian ratio of mCAF1(+/+), mCAF1(+/-), and mCAF1(-/-) pups, indicating that haploid mCAF1-deficient germ cells differentiate normally. The onset of the defect occurs during the first wave of spermatogenesis at 19 to 20 days after birth, during progression of pachytene spermatocytes to haploid spermatids and spermatozoa. Early disruption of spermatogenesis was evidenced by Sertoli cell vacuolization and tubular disorganization. The most mature germ cells were the most severely depleted, but progressively all germ cells were affected, giving Sertoli cell-only tubes, large interstitial spaces, and small testes. This phenotype could be linked to a defect(s) in germ cells and/or to inadequate Sertoli cell function, leading to seminiferous tubule disorganization and finally to a total disappearance of germ cells. The mCAF1-deficient mouse provides a new model of failed spermatogenesis in the adult that may be relevant to some cases of human male sterility.

As autophagic inclusions accumulate in senescent fibroblasts, we wondered whether an increase in cellular fragility during in vitro lymphocyte aging may be related to an autolysosome accumulation. We established that, during long-term cultures, repeatedly stimulated T-lymphocytes acquired characteristics of replicative senescence and became progressively intolerant to activation. Cell death following stimulations: (i) corresponded to apoptosis, associated with necrosis at the end of the culture; (ii) was not, for its main part, mediated through CD95/CD178 or TNFRII/TNF alpha interactions; and (iii) occurred in spite of bcl-2 increased expression. After 14 weeks of culture, the percentage of lymphocytes containing at least one autophagic inclusion (p<0.0001) and the lipofuscin autofluorescence in lymphocytes (p<0.0001) were significantly increased. The expression of several genes regulating autophagy did not significantly vary with the age of the culture. Forty-eight hours after each stimulation, the percentage of induced cell death rose while, in the remaining living cells, the percentage of lymphocytes with autophagic vacuoles (p<0.05), with beta-galactosidase activity and the lipofuscin autofluorescence (p<0.001) significantly decreased, suggesting the preferential death of cells with autophagy. Our data support the view that the accumulation of autolysosomes in senescent lymphocytes might aggravate cellular fragility, leading to apoptosis and necrosis mainly induced by lymphocyte activation.