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Missing in Metastasis (MIM), or Metastasis Suppressor 1 (MTSS1), is a highly conserved protein, which links the plasma membrane to the actin cytoskeleton. MIM has been implicated in various cancers, however, its modes of action remain largely enigmatic. Here, we performed an extensive in silico characterisation of MIM to gain better understanding of its function. We detected previously unappreciated functional motifs including adaptor protein (AP) complex interaction site and a C-helix, pointing to a role in endocytosis and regulation of actin dynamics, respectively. We also identified new functional regions, characterised with phosphorylation sites or distinct hydrophilic properties. Strong negative selection during evolution, yielding high conservation of MIM, has been combined with positive selection at key sites. Interestingly, our analysis of intra-molecular co-evolution revealed potential regulatory hotspots that coincided with reduced potentially pathogenic polymorphisms. We explored databases for the mutations and expression levels of MIM in cancer. Experimentally, we focused on chronic lymphocytic leukaemia (CLL), where MIM showed high overall expression, however, downregulation on poor prognosis samples. Finally, we propose strong conservation of MTSS1 also on the transcriptional level and predict novel transcriptional regulators. Our data highlight important targets for future studies on the role of MIM in different tissues and cancers.

Chronic lymphocytic leukemia (CLL) patients with differential somatic hypermutation status of the immunoglobulin heavy variable genes, namely mutated or unmutated, display fundamental clinico-biological differences. Considering this, we assessed prognosis separately within mutated (M-CLL) and unmutated (U-CLL) CLL in 3015 patients, hypothesizing that the relative significance of relevant indicators may differ between these two categories. Within Binet A M-CLL patients, besides TP53 abnormalities, trisomy 12 and stereotyped subset #2 membership were equivalently associated with the shortest time-to-first-treatment and a treatment probability at five and ten years after diagnosis of 40% and 55%, respectively; the remaining cases exhibited 5-year and 10-year treatment probability of 12% and 25%, respectively. Within Binet A U-CLL patients, besides TP53 abnormalities, del(11q) and/or SF3B1 mutations were associated with the shortest time-to-first-treatment (5- and 10-year treatment probability: 78% and 98%, respectively); in the remaining cases, males had a significantly worse prognosis than females. In conclusion, the relative weight of indicators that can accurately risk stratify early-stage CLL patients differs depending on the somatic hypermutation status of the immunoglobulin heavy variable genes of each patient. This finding highlights the fact that compartmentalized approaches based on immunogenetic features are necessary to refine and tailor prognostication in CLL., (Copyright © 2019 Ferrata Storti Foundation.)

Monoclonal B-cell lymphocytosis (MBL) is defined by the presence of a monoclonal B-cell population in the peripheral blood (PB) at a concentration of <5 × 10 9 /l and no signs or symptoms of a lymphoproliferative disorder. In around 75% of cases, the immunophenotype of the clonal B-cell expansions is superimposable to that of chronic lymphocytic leukemia (CLL), thus defined "CLL-like". Other cases may coexpress CD19, CD5, bright CD20, and lack CD23 ("atypical CLL"), while others are CD5-negative ("non-CLL"). Beside the immunophenotypic profile, a key distinction is based on the B-cell count, stratifying the MBL category in low (<0.5 × 10 9 /l) or high-count (≥0.5 × 10 9 /l). Low-count (LC) MBL is recognized in general population studies and it is not associated with lymphocytosis. High-count (HC) MBL is identified during the clinical work-up for lymphocytosis and carries a risk of progression to CLL requiring therapy of 1-2% per year in most series, warranting clinical monitoring over time. At the time of MBL diagnosis, the key point is the careful evaluation and exclusion of differential diagnoses. After the initial workup, the follow-up at a referral center by a hematologist would be desirable as, in addition to the obvious risk of progression to clinically relevant CLL, the appropriate management of MBL individuals should take into account the risk of developing infections, other cancers and autoimmune disorders. For those cases who indeed bear a risk, though limited, of clinical consequences, such as the majority of HC-MBL cases, current evidences suggest that they may benefit from a tailored and specialized approach., (Copyright © 2018 European Federation of Internal Medicine. Published by Elsevier B.V. All rights reserved.)

PI3Kδ (idelalisib) and BTK (ibrutinib) inhibitors have demonstrated significant clinical activity in chronic lymphocytic leukemia (CLL) interfering with the cross-talk between CLL cells and the lymph node microenviroment, yet their mechanism of action remains to be fully elucidated. Here, we developed an ex vivo model with the aim of reproducing the effects of the microenvironment that would help shed light on the in vivo mechanism of action of idelalisib and ibrutinib and predict their clinical efficacy in individual patients. First we explored the effects of various cell-extrinsic elements on CLL apoptosis and proliferation and found that the combination of CpG+IL2+HS5 stromal cell line + human serum +CLL plasma and erythrocyte fractions represented the best co-culture conditions to test the effects of the novel inhibitors. Then, using this assay, we investigated the impact of idelalisib and ibrutinib on both survival and proliferation in 30 CLL patients. While both drugs had a limited direct pro-apoptotic activity, a potent inhibition of proliferation was achieved at clinically achievable concentrations. Notably, up to 10% of CLL cells still proliferated even at the highest concentrations, likely mirroring the known difficulty to achieve complete responses in vivo . Altogether, this novel assay represents an appropriate ex vivo drug testing system to potentially predict the clinical response to novel inhibitors in particular by quantifying the antiproliferative effect., Competing Interests: CONFLICTS OF INTEREST KS receives unrestricted research support from Gilead Sciences, Janssen Pharmaceuticals and Novartis SA. PG receives unrestricted research support from Gilead Sciences, GSK/Novartis and Celgene. Vivia has received unrestricted research support from Gilead.

In chronic lymphocytic leukemia (CLL), the non-hematopoietic stromal microenvironment plays a critical role in promoting tumor cell recruitment, activation, survival, and expansion. However, the nature of the stromal cells and molecular pathways involved remain largely unknown. Here, we demonstrate that leukemic B lymphocytes induce the activation of retinoid acid synthesis and signaling in the microenvironment. Inhibition of RA-signaling in stromal cells causes deregulation of genes associated with adhesion, tissue organization and chemokine secretion including the B-cell chemokine CXCL13. Notably, reducing retinoic acid precursors from the diet or inhibiting RA-signaling through retinoid-antagonist therapy prolong survival by preventing dissemination of leukemia cells into lymphoid tissues. Furthermore, mouse and human leukemia cells could be distinguished from normal B-cells by their increased expression of Rarγ2 and RXRα, respectively. These findings establish a role for retinoids in murine CLL pathogenesis, and provide new therapeutic strategies to target the microenvironment and to control disease progression.

Despite the recent discovery of recurrent driver mutations in chronic lymphocytic leukemia, the genetic factors involved in disease onset remain largely unknown. To address this issue, we performed whole-genome sequencing in 11 individuals with monoclonal B- cell lymphocytosis, both of the low-count and high-count subtypes, and 5 patients with ultra-stable chronic lymphocytic leukemia (>10 years without progression from initial diagnosis). All three entities were indistinguishable at the genomic level exhibiting low genomic complexity and similar types of somatic mutations. Exonic mutations were not frequently identified in putative chronic lymphocytic leukemia driver genes in all settings, including low-count monoclonal B-cell lymphocytosis. To corroborate these findings, we also performed deep sequencing in 11 known frequently mutated genes in an extended cohort of 28 monoclonal B-cell lymphocytosis/chronic lymphocytic leukemia cases. Interestingly, shared mutations were detected between clonal B cells and paired polymorphonuclear cells, strengthening the notion that at least a fraction of somatic mutations may occur before disease onset, likely at the hematopoietic stem cell level. Finally, we identified previously unreported non-coding variants targeting pathways relevant to B-cell and chronic lymphocytic leukemia development, likely associated with the acquisition of the characteristic neoplastic phenotype typical of both monoclonal B-cell lymphocytosis and chronic lymphocytic leukemia., (Copyright © 2018 Ferrata Storti Foundation.)

Chronic lymphocytic leukemia cells strongly depend on external stimuli for their survival. Both antigen receptor and co-stimulatory receptors, including Toll-like receptors, can modulate viability and proliferation of leukemic cells. Toll-like receptor ligands, and particularly the TLR9 ligand CpG, mediate heterogeneous responses in patients' samples reflecting the clinical course of the subjects. However, the molecular framework of the key signaling events underlying such heterogeneity is undefined. We focused our studies on a subset of chronic lymphocytic leukemia cases characterized by expression of CD38 and unmutated immunoglobulin genes, who respond to CpG with enhanced metabolic cell activity. We report that, while CpG induces NFKBIZ mRNA in all the samples analyzed, it induces the IκBζ protein in a selected group of cases, through an unanticipated post-transcriptional mechanism. Interestingly, IκBζ plays a causal role in sustaining CpG-induced cell viability and chemoresistance, and CpG stimulation can unleash immunoglobulin secretion by IκBζ-positive malignant cells. These results identify and characterize IκBζ as a marker and effector molecule of distinct key pathways in chronic lymphocytic leukemia., (Copyright© Ferrata Storti Foundation.)

Toll interleukin-1 receptor 8 (also known as TIR8, SIGIRR, or IL1R8) is a transmembrane receptor that inhibits inflammation. Accordingly, genetic inactivation of this protein exacerbates chronic inflammation and inflammation-associated tumors in mice. In particular, lack of TIR8 triggers leukemia progression in a mouse model of chronic lymphocytic leukemia (CLL), supporting its role as a novel tumor restrainer. The aim of this study was to measure the amount of TIR8 mRNA and protein in CLL cells, and to analyze its regulation of expression. Circulating leukemic cells expressed lower levels of TIR8 compared to normal B-lymphocytes. Treatment of CLL cells with Azacytidine restored higher levels of TIR8 suggesting that DNA methylation may be involved in modulating TIR8 expression, with implications for novel therapeutic strategies.

Chronic lymphocytic leukemia (CLL) is characterized by the expansion of malignant CD5 + B lymphocytes in blood, bone marrow, and lymphoid organs. CD1d-restricted invariant natural killer T (iNKT) cells are innate-like T lymphocytes strongly implicated in tumor surveillance. We investigated the impact of iNKT cells in the natural history of the disease in the Eμ-Tcl1 (Tcl1) CLL mouse model and 68 CLL patients. We found that Tcl1-CLL cells express CD1d and that iNKT cells critically delay disease onset but become functionally impaired upon disease progression. In patients, disease progression correlates with high CD1d expression on CLL cells and impaired iNKT cells. Conversely, disease stability correlates with negative or low CD1d expression on CLL cells and normal iNKT cells, suggesting indirect leukemia control. iNKT cells indeed hinder CLL survival in vitro by restraining CD1d-expressing nurse-like cells, a relevant proleukemia macrophage population. Multivariable analysis identified iNKT cell frequency as an independent predictor of disease progression. Together, these results support the contribution of iNKT cells to CLL immune surveillance and highlight iNKT cell frequency as a prognostic marker for disease progression., (© 2017 by The American Society of Hematology.)

Cell-autonomous B-cell receptor (BcR)-mediated signalling is a hallmark feature of the neoplastic B lymphocytes in chronic lymphocytic leukaemia (CLL). Here we elucidate the structural basis of autonomous activation of CLL B cells, showing that BcR immunoglobulins initiate intracellular signalling through homotypic interactions between epitopes that are specific for each subgroup of patients with homogeneous clinicobiological profiles. The molecular details of the BcR-BcR interactions apparently dictate the clinical course of disease, with stronger affinities and longer half-lives in indolent cases, and weaker, short-lived contacts mediating the aggressive ones. The diversity of homotypic BcR contacts leading to cell-autonomous signalling reconciles the existence of a shared pathogenic mechanism with the biological and clinical heterogeneity of CLL and offers opportunities for innovative treatment strategies.

Background: Patients with relapsed diffuse large B-cell lymphoma (DLBCL) not eligible for autologous stem cell transplantation (ASCT) or having relapse after ASCT have a low likelihood of cure. Single-drug maintenance after salvage therapy might be an attractive strategy to prolong survival in these patients. Lenalidomide is a suitable candidate for long-lasting maintenance as it is an oral drug, active against DLBCL that can be taken for years with an acceptable toxicity profile. We designed a study to investigate safety and efficacy of lenalidomide maintenance in patients with chemosensitive relapse of DLBCL not eligible for ASCT or having relapse after ASCT., Methods: In this open-label, single group, multicentre phase 2 trial, we recruited HIV-negative adults with de novo or transformed DLBCL and relapsed disease responsive to conventional rituximab-containing salvage therapy from 12 oncology-haematology centres in Italy. All patients were given oral lenalidomide 25 mg per day for 21 of 28 days until lymphoma progression or unacceptable toxicity (severely compromises organ function, quality of life, or both). Primary endpoint was 1-year progression-free survival. The estimated sample size was 47 patients; maintenance was deemed efficacious if at least 19 patients were progression-free survivors at 1 year. All enrolled patients were included in primary analyses, with the exception of patients who post-hoc objectively did not meet the eligibility criteria (modified intention-to-treat). This study is registered with clinicaltrials.gov registry, number NCT00799513., Findings: Between March 24, 2009, and Dec 22, 2015, we recruited 48 patients. 46 of 48 enrolled patients were assessable (two patients had unconfirmed diagnoses). 36 (78%) of 46 patients had de novo DLBCL and ten (22%) of 46 patients had transformed DLBCL. At a median follow-up of 25 months (IQR 12-56), 556 lenalidomide courses had been delivered, with an average mean of 12 courses (range 3-41) per patient; 19 patients were still in treatment at a median follow-up of 25 months. Lenalidomide was well tolerated; with the exception of neutropenia, grade 3-4 toxicities were uncommon. We recorded ten severe adverse events in nine patients due to febrile neutropenia (n=4), diarrhoea (n=2), melena, stroke, vomiting, and intestinal infarction; all but one patient recovered, and six of these patients continued with lenalidomide treatment. The exception was the only death due to toxicity (intestinal infarction). At 1 year from trial registration, 28 patients were progression free, which was much higher than the predetermined efficacy threshold. During the whole observation period, 21 events occurred: progressive lymphoma in 19 patients, death due to toxicity in one, death while off therapy in one, 1-year progression-free survival was 70% (95% CI 57-83)., Interpretation: With the limitations of a non-randomised design, this trial supports the use of lenalidomide maintenance in patients with chemo-sensitive relapse of DLBCL who are not eligible for ASCT or who had relapse after ASCT. These results warrant further investigation of immunomodulatory drugs as maintenance in high-risk patients with DLBCL., Funding: Celgene Corp., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Chronic lymphocytic leukaemia (CLL) is the most common leukaemia among the adults in the Western World. CLL (and the corresponding nodal entity small lymphocytic lymphoma, SLL) is classified as a lymphoproliferative disorder characterised by the relentless accumulation of mature B-lymphocytes showing a peculiar immunophenotype in the peripheral blood, bone marrow, lymph nodes and spleen. CLL clinical course is very heterogeneous: the majority of patients follow an indolent clinical course with no or delayed treatment need and with a prolonged survival, while others experience aggressive disease requiring early treatment followed by frequent relapses. In the last decade, the improved understanding of CLL pathogenesis shed light on premalignant conditions (i.e., monoclonal B-cell lymphocytosis, MBL), defined new prognostic and predictive markers, improving patient stratification, but also broadened the therapeutic armamentarium with novel agents, targeting fundamental signaling pathways., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

We report on markedly different frequencies of genetic lesions within subsets of chronic lymphocytic leukemia patients carrying mutated or unmutated stereotyped B-cell receptor immunoglobulins in the largest cohort (n=565) studied for this purpose. By combining data on recurrent gene mutations (BIRC3, MYD88, NOTCH1, SF3B1 and TP53) and cytogenetic aberrations, we reveal a subset-biased acquisition of gene mutations. More specifically, the frequency of NOTCH1 mutations was found to be enriched in subsets expressing unmutated immunoglobulin genes, i.e. #1, #6, #8 and #59 (22-34%), often in association with trisomy 12, and was significantly different (P<0.001) to the frequency observed in subset #2 (4%, aggressive disease, variable somatic hypermutation status) and subset #4 (1%, indolent disease, mutated immunoglobulin genes). Interestingly, subsets harboring a high frequency of NOTCH1 mutations were found to carry few (if any) SF3B1 mutations. This starkly contrasts with subsets #2 and #3 where, despite their immunogenetic differences, SF3B1 mutations occurred in 45% and 46% of cases, respectively. In addition, mutations within TP53, whilst enriched in subset #1 (16%), were rare in subsets #2 and #8 (both 2%), despite all being clinically aggressive. All subsets were negative for MYD88 mutations, whereas BIRC3 mutations were infrequent. Collectively, this striking bias and skewed distribution of mutations and cytogenetic aberrations within specific chronic lymphocytic leukemia subsets implies that the mechanisms underlying clinical aggressiveness are not uniform, but rather support the existence of distinct genetic pathways of clonal evolution governed by a particular stereotyped B-cell receptor selecting a certain molecular lesion(s)., (Copyright© Ferrata Storti Foundation.)

Chronic lymphocytic leukemia (CLL) is characterized by the accumulation of leukemic B cells in peripheral blood, bone marrow (BM) and lymphoid tissues, and by their recirculation between these compartments. We observed that circulating chromogranin A (CgA) and its N-terminal fragment (called vasostatin-1, CgA1-76), two neuroendocrine secretory polypeptides that enhance the endothelial barrier function, are present in variable amounts in the blood of CLL patients. Studies in animal models showed that daily administration of full-length human CgA1-439 (0.3 μg, i.v., or 1.5 μg/mouse, i.p.) can reduce the BM/blood ratio of leukemic cells in Eμ-TCL1 mice, a transgenic model, and decrease BM, lung and kidney infiltration in Rag2-/-γc-/- mice engrafted with human MEC1 CLL cells, a xenograft model. This treatment also reduced the loss of body weight and improved animal motility. In vitro, CgA enhanced the endothelial barrier integrity and the trans-endothelial migration of MEC1 cells, with a bimodal dose-response curve. Vasostatin-1, but not a larger fragment consisting of N-terminal and central regions of CgA (CgA1-373), inhibited CLL progression in the xenograft model, suggesting that the C-terminal region is crucial for CgA activity and that the N-terminal domain contains a site that is activated by proteolytic cleavage. These findings suggest that circulating full-length CgA and its fragments may contribute to regulate leukemic cell trafficking and reduce tissue infiltration in CLL., Competing Interests: No competing financial interest.

Hypoxia-inducible transcription factors (HIFs) regulate a wide array of adaptive responses to hypoxia and are often activated in solid tumors and hematologic malignancies due to intratumoral hypoxia and emerging new layers of regulation. We found that in chronic lymphocytic leukemia (CLL), HIF-1α is a novel regulator of the interaction of CLL cells with protective leukemia microenvironments and, in turn, is regulated by this interaction in a positive feedback loop that promotes leukemia survival and propagation. Through unbiased microarray analysis, we found that in CLL cells, HIF-1α regulates the expression of important chemokine receptors and cell adhesion molecules that control the interaction of leukemic cells with bone marrow and spleen microenvironments. Inactivation of HIF-1α impairs chemotaxis and cell adhesion to stroma, reduces bone marrow and spleen colonization in xenograft and allograft CLL mouse models, and prolongs survival in mice. Of interest, we found that in CLL cells, HIF-1α is transcriptionally regulated after coculture with stromal cells. Furthermore, HIF-1α messenger RNA levels vary significantly within CLL patients and correlate with the expression of HIF-1α target genes, including CXCR4, thus further emphasizing the relevance of HIF-1α expression to CLL pathogenesis., (© 2016 by The American Society of Hematology.)

Monoclonal B-cell lymphocytosis (MBL) is defined as a laboratory abnormality where small (<5 x 10(9)/L) clonal B-cell populations are detected in the peripheral blood of otherwise healthy subjects. According to the immunophenotype, MBL is labeled as chronic lymphocytic leukemia (CLL)-like (75% of cases), atypical CLL, and CD5-negative. Concentration of clonal B cells differentiates low- (LC) and high-count (HC)-MBL (< or ≥ 0.5 x 10(9)/L, respectively). Thanks to technical improvements, we are able to identify CLL-like clonal B-cell populations at increased frequency with age, but we are still far from understanding its relationship with clinically overt CLL. LC-MBL, requiring high-throughput screening technique to be identified in population studies, seems to be a bird of a different feather and several hints suggest that LC-MBL is related to aging and/or chronic antigenic stimulation. Immunogenetic, cytogenetic and genetic data support the notion that HC-MBL, usually identified in the clinical setting, is a premalignant condition and, based on biological parameters, it is frequently difficult to differentiate it from early stage CLL. The rapid improvement and widespread availability of cutting-edge technology, in particular next-generation sequencing (NGS), raises hope that we are getting closer to unveiling the fundamental nature of MBL and CLL and how they are related to each other., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Recent studies on splenic marginal zone lymphoma identified distinct mutations in genes belonging to the B-cell receptor and Toll-like receptor signaling pathways, thus pointing to their potential implication in the biology of the disease. However, limited data is available regarding the exact role of TLRs. We aimed at characterizing the expression pattern of TLRs in splenic marginal zone lymphoma cells and their functional impact on the activation, proliferation and viability of malignant cells in vitro. Cells expressed significant levels of TLR1, TLR6, TLR7, TLR8, TLR9 and TLR10 mRNA; TLR2 and TLR4 showed a low, variable pattern of expression among patients whereas TLR3 and TLR5 mRNAs were undetectable; mRNA specific for TLR signaling molecules and adapters was also expressed. At the protein level, TLR1, TLR6, TLR7, TLR9 and TLR10 were detected. Stimulation of TLR1/2, TLR2/6 and TLR9 with their respective ligands triggered the activation of IRAK kinases, MAPK and NF-κB signaling pathways, and the induction of CD86 and CD25 activation molecules, although in a heterogeneous manner among different patient samples. TLR-induced activation and cell viability were also inhibited by a specific IRAK1/4 inhibitor, thus strongly supporting the specific role of TLR signaling in these processes. Furthermore, TLR2/6 and TLR9 stimulation also significantly increased cell proliferation. In conclusion, we demonstrate that splenic marginal zone lymphoma cells are equipped with functional TLR and signaling molecules and that the stimulation of TLR1/2, TLR2/6 and TLR9 may play a role in regulating disease pathobiology, likely promoting the expansion of the neoplastic clone., (Copyright© Ferrata Storti Foundation.)

The most effective strategy to prevent central nervous system (CNS) dissemination in diffuse large B-cell lymphoma (DLBCL) remains an important, unmet clinical need. Herein, we report a retrospective analysis of risk-tailored CNS prophylaxis in 200 human immunodeficiency virus-negative adults with DLBCL treated with rituximab-CHOP (cyclophosphamide, doxorubicin, vincristine, prednisone) or similar. High risk of CNS relapse was defined by involvement of specific extranodal organs, or simultaneous presence of advanced stage and high serum lactate dehydrogenase level; CNS prophylaxis with high-dose methotrexate ± intrathecal chemotherapy (IT) was routinely used in high-risk patients diagnosed after 2007. CNS relapse risk was low in 93 patients and high in 107; 40 high-risk patients received prophylaxis, which consisted of IT alone in 7. At a median follow-up of 60 months, one low-risk and nine high-risk patients (1% vs. 8%; P = 0·01) experienced CNS relapse. In the high-risk group, CNS relapses occurred in 8/67 (12%) patients who did not receive prophylaxis and in 1/40 (2·5%) patients who did; the latter occurred in a patient managed with IT alone. CNS relapse rate was 12% (9/74) for patients treated with "inadequate" prophylaxis (none or IT only) and 0% (0/33) for patients managed with intravenous prophylaxis (P = 0·03). In conclusion, high-dose methotrexate-based prophylaxis significantly reduces CNS failures in high-risk patients stratified by involvement of specific extranodal sites and International Prognostic Index., (© 2014 John Wiley & Sons Ltd.)

Introduction: The clinical features, management, and prognosis of stage I-II diffuse large B-cell lymphoma of the bone (PB-DLBCL) included in an international database of 499 lymphoma patients with skeletal involvement were reviewed., Methods: HIV-negative patients (n = 161) with diffuse large B-cell lymphoma of the bone (PB-DLBCL) after complete staging workup were considered. The primary objective of this study was to identify the most effective treatment modality; the secondary objectives were to define the contribution of irradiation fields and doses and the pattern of relapse., Results: Median age was 55 years (range, 18-99 years), with a male/female ratio of 1:2; 141 (87%) patients had stage I, 14 (9%) had B symptoms, 37 (23%) had bulky lesion, 54 (33%) showed elevated lactate dehydrogenase serum levels, and 25 (15%) had fracture. Thirteen (8%) patients received chemotherapy alone, 23 (14%) received radiotherapy alone, and 125 (78%) received both treatments. The response to the first-line treatment was complete in 131 of 152 assessed patients (complete response rate, 86%; 95% confidence interval [CI], 81%-91%) and partial in 7, with an overall response rate of 91% (95% CI, 87%-95%). At a median follow-up of 54 months (range, 3-218), 107 (67%) patients remained relapse-free, with a 5-year progression-free survival of 68% (SE: 4). Four (2.5%) patients had meningeal relapse; 119 patients were alive (113 disease-free), with a 5-year overall survival of 75% (SE: 4). Patients managed with primary chemotherapy, whether followed by radiotherapy or not, had a significantly better outcome than patients treated with primary radiotherapy, whether followed by chemotherapy or not. The addition of consolidative radiotherapy after primary chemotherapy was not associated with improved outcome; doses >36 Gy and the irradiation of the whole affected bone were not associated with better outcome., Conclusion: Patients with PB-DLBCL exhibit a favorable prognosis when treated with primary anthracycline-based chemotherapy whether followed by radiotherapy or not. In patients treated with chemoradiotherapy, the use of larger radiation fields and doses is not associated with better outcome. Central nervous system dissemination is a rare event in PB-DLBCL patients.

'Multifocal bone lymphoma' or 'polyostotic lymphoma' is a neoplasm with exclusive multifocal involvement of the skeleton, without affecting lymph nodes or other soft tissues. Knowledge on this uncommon condition is limited because the related literature is sparse and fragmentary. We reviewed cases of multifocal bone diffuse large B-cell lymphoma (MB-DLBCL) registered in a clinico-pathological database of the International Extranodal Lymphoma Study Group that includes 499 cases of bone lymphoma. Clinical features, management and prognosis of 37 MB-DLBCL patients and 63 'controls' (stage-IV DLBCL and skeletal involvement) were analysed. Presentation and treatment of MB-DLBCL and controls were identical. At a median follow-up of 52 months (10-189), MB-DLBCL patients exhibited a significantly better response rate (92% vs. 65%; P = 0·002), progression-free survival (5-year: 56 ± 9% vs. 34 ± 6%; P = 0·003) and overall survival (5-year: 74 ± 8% vs. 36 ± 7%; P = 0·002). Among MB-DLBCL patients, the use of post-chemo radiotherapy was associated with better overall survival (5-year: 83 ± 12% vs. 55 ± 16%; P = 0·003). Two MB-DLBCL patients (5·4%) with spine and skull involvement experienced central nervous system (CNS) relapse. Thus, MB-DLBCL patients exhibit a significantly better prognosis compared to patients with advanced-stage DLBCL, and should be treated with conventional anthracycline-based chemotherapy, keeping intensified treatment for relapsing cases, considering involved-field radiotherapy, and CNS prophylaxis in high-risk patients., (© 2014 John Wiley & Sons Ltd.)

The finding of monoclonal B-cell populations not fulfilling criteria for a lymphoid malignancy has given great impulse to study mechanisms involved in lymphomagenesis and factors responsible for the transition from B-cell precursor states to overt lymphoproliferative disorders. Monoclonal B cell expansions were initially recognized in peripheral blood of otherwise healthy subjects (thus defined monoclonal B-cell lymphocytosis, MBL) and in most cases share the immunophenotypic profile of chronic lymphocytic leukemia (CLL). The clinical relevance of this phenomenon is different according to B-cell count: high-count MBL is considered a preneoplastic condition and progresses to CLL requiring treatment at a rate of 1-2% per year, while low-count MBL, though persisting over time, has not shown a clinical correlation with frank leukemia so far. MBL other than CLL-like represent a minority of cases and are ill-defined entities for which clinical and biological information is still scanty. In situ follicular lymphoma (FL) and mantle cell lymphoma (MCL) are characterized by the localization of atypical lymphoid cells, carrying t(14;18)(q32;q21) or t(11;14)(q13;q32), only in the germinal centers and mantle zones respectively, where their normal counterparts are localized. The localization of these cells indicates that germinal centers or mantle zones provide appropriate microenvironments for cells carrying these oncogenic alterations to survive or proliferate. The progression of these lesions to overt lymphomas occurs rarely and may require the accumulation of additional genetic events. Individuals with these lymphoid proliferations should be managed with caution., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Purpose: Immunoglobulin G-switched chronic lymphocytic leukemia (G-CLL) is a rare variant of CLL, whose origin and ontogenetic relationship to the common IgM/IgD (MD-CLL) variant remains undefined. Here, we sought for clues about the ontogeny of G-CLL versus MD-CLL by profiling the relevant IG gene repertoires., Experimental Design: Using purpose-built bioinformatics methods, we performed detailed immunogenetic profiling of a multinational CLL cohort comprising 1,256 cases, of which 1,087 and 169 expressed IG mu/delta and gamma heavy chains, respectively., Results: G-CLL has a highly skewed IG gene repertoire that is distinct from MD-CLL, especially in terms of (i) overuse of the IGHV4-34 and IGHV4-39 genes and (ii) differential somatic hypermutation (SHM) load. Repertoire differences were also found when comparing subgroups with similar SHM status and were mainly attributed to the exclusive representation in G-CLL of two major subsets with quasi-identical (stereotyped) B-cell receptors. These subsets, namely #4 (IGHV4-34/IGKV2-30) and #8 (IGHV4-39/IGKV1(D)-39), were found to display sharply contrasting SHM and clinical behavior., Conclusions: G-CLL exhibits an overall distinct immunogenetic signature from MD-CLL, prompting speculations about distinct ontogenetic derivation and/or immune triggering. The reasons underlying the differential regulation of SHM among G-CLL cases remain to be elucidated., (©2013 AACR.)

The BCL2 family members play a central role in regulating programmed cell death (apoptosis) and arbitrating the cellular fate through an accurate balance between pro-apoptotic (BAX, BAK, and BH3-only proteins) and pro-survival (BCL2 and its closest homologues, BCLXL, BCLW and MCL-1) factors. Deregulation of BCL2 family proteins contributes to programmed cell death evasion, that is a hallmark of human cancers and it is often related to (chemo)therapy resistance. High BCL2 levels have been detected in most human lymphoid malignancies, not limited to follicular lymphoma (where the role of BCL2 overexpression is driven by the t[14;18] translocation) but also B-cell chronic lymphocytic leukemia (CLL) and multiple myeloma. For all these reasons, the opportunity to induce apoptosis by targeting BCL2 proteins is considered a potentially promising therapeutic approach in hematological malignancies. BCL2 family inhibition strategies currently explored in phase 1, 2 and 3 clinical trials are essentially two: (1) the use of antisense-based strategies to knockdown BCL2 or BCLXL expression (e.g. oblimersen) or (2) the use of synthetic BH3 mimetics i.e. small molecules binding to anti-apoptotic inhibitors thereby allowing the pro-apoptotic activity of BH3-only molecules (e.g. obatoclax, AT-101, ABT-737 and its derivatives ABT-263 and ABT-199). Several of these drugs demonstrated relevant clinical activity as single-agent or in combination therapy, with the most significant drawbacks in clinical use being represented by challenging pharmacokinetic profile (e.g. iv administration, high-levels of plasma proteins binding) and on-target side effects (e.g. gastrointestinal toxicity and thrombocytopenia). Further clinical development of the current compounds (e.g. ABT-199), showing high efficacy but devoid of the most threatening drug-related toxicities, is eagerly awaited. Hopefully, in the next future, BCL2 inhibitors (alone or in combination with immuno- and/or chemo-therapeutic agents) will represent target-specific drugs expanding our therapeutic armamentarium in the fight against hematologic malignancies., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Chronic lymphocytic leukemia (CLL) -like monoclonal B-cell lymphocytosis (MBL) shares common immunophenotype and cytogenetic abnormalities with CLL, from which it is discriminated by a cutoff value of 5 × 10(9)/L circulating clonal B cells. However, the clonal size in MBL is extremely variable and allows discrimination of two distinct entities (high-count [HC] and low-count [LC]-MBL) based on a cutoff value of 0.5 × 10(9)/L clonal B cells. HC-MBL is associated with lymphocytosis and progresses to CLL requiring treatment at a rate of 1.1% per year, whereas LC-MBL is found in the general population only through high-sensitivity techniques and carries limited, if any, risk of progression. We performed an immunogenetic profiling of 333 cases with CLL-like MBL supplemented by detailed comparisons with CLL, focusing especially on CLL Rai stage 0 (CLL-0). LC- and HC-MBL had similar somatic hypermutation status, yet different IGHV gene repertoires and frequencies of B-cell receptor (BcR) stereotypy. In particular, stereotyped BcRs were infrequent in LC-MBL and were often not CLL specific. In contrast, HC-MBL exhibited clear immunogenetic similarities to CLL-0. These findings indicate that LC-MBL may not represent a true preleukemic condition, thus differing from HC-MBL/CLL-0 in which the identification of factors endowing malignant potential is strongly warranted.

B-cell receptor (BCR) triggering and responsiveness have a crucial role in the survival and expansion of chronic lymphocytic leukemia (CLL) clones. Analysis of in vitro response of CLL cells to BCR triggering allowed the definition of 2 main subsets of patients and lack of signaling capacity was associated with constitutive activation of extracellular-regulated kinases 1/2 (ERK1/2) and nuclear factor of activated T cells c1 (NF-ATc1), consistent with the idea that at least one group of CLL patients derives from the abnormal expansion of anergic B cells. In the present work, we further investigated the anergic subset of CLL (defined as the one with constitutive ERK1/2 phosphorylation) and found that it is characterized by low levels of surface immunoglobulin M and impairment of calcium mobilization after BCR engagement in vitro. Chronic BCR triggering promoted CLL cell survival selectively in phosphorylated ERK1/2 samples and the use of mitogen-activated protein kinase and NF-AT signaling inhibitors specifically induced apoptosis in this group of patients. Apoptosis induction was preceded by an initial phase of anergy reversal consisting in the loss of ERK phosphorylation and NF-AT nuclear translocation and by the restoration of BCR responsiveness, reinforcing the idea that the anergic program favors the survival of leukemic lymphocytes.

HS1 (hematopoietic cell-specific Lyn substrate-1) is a cytoskeletal interactor in the B-cell receptor (BCR) signaling pathway whose phosphorylation correlates with prognosis in Chronic Lymphocytic Leukemia (CLL). The differentially phosphorylated sites and the kinases that regulate HS1 activity in CLL remain poorly understood. We demonstrate that HS1 activity is differentially regulated by LYN kinase that, in a subset of patients, phosphorylates HS1 on Tyrosine (Y)397, resulting in its activation. This correlates with increased cytoskeletal functionality in terms of migration, adhesion and F-actin polymerization. In these patients, LYN is also activated on Y396 residue and its inhibition with the tyrosine kinase inhibitor Dasatinib abrogates HS1-Y397 phosphorylation. This results in the reduction of HS1 activation along with that of cytoskeletal effector VAV1 and the downstream kinase ERK also in the presence of BCR and CXC chemokine receptor CXCR4 stimulation. Interestingly, targeting the LYN/HS1 axis in vitro leads to the concomitant reduction of cytoskeletal activity, BCR signaling and cell survival in the subset of patients with activated LYN/HS1. In a transplantable mouse model based on the EμTCL1 transgenic mouse, LYN/HS1 signaling inhibition interferes with CLL progression and lymphoid organ infiltration. Thus LYN/HS1 axis marks distinct signaling profiles and cytoskeletal-related features that may represent valuable targets for cytoskeleton-targeted therapeutic intervention in CLL.

Purpose: The emerging role of Toll-like receptors (TLR) in the pathogenesis of chronic lymphocytic leukemia (CLL) led us to ask whether TLR stimulation may protect CLL cells from drug-induced apoptosis., Experimental Design: We cultured in vitro malignant B cells freshly isolated from 44 patients with CLLs in the presence or the absence of different concentrations of fludarabine before or after 24-hour TLR stimulation with specific ligands and evaluated cell viability, apoptosis, and molecular pathways involved., Results: Heterogeneity was observed among samples. In leukemic cells from patients bearing adverse prognostic factors, TLR stimulation caused a significant increase of protection to fludarabine treatment, whereas this did not occur in the cells from patients with good prognosis. To identify novel molecular mechanisms accounting for the dichotomy of response between the two groups of patients, we conducted an apoptosis gene expression profile on leukemic cells either unstimulated or stimulated with TLR9 ligand. Strikingly, TLR9 stimulation specifically upregulated the expression of lymphotoxin-α in cells where an increased protection to fludarabine treatment was observed. Also, the expression of miR-155-3p was significantly increased after stimulation of distinct TLR in cells where fludarabine treatment was less effective., Conclusions: These results suggest that at least in a proportion of patients, in vitro sensitivity to fludarabine may be modulated by the stimulation of TLR, likely mimicking microenvironmental signals occurring in vivo., (©2012 AACR.)

Primary splenic small B-cell lymphomas mostly comprise the distinct entity of splenic marginal-zone lymphoma (SMZL) and the provisional category of splenic lymphoma/leukemia unclassifiable, mainly represented by the hairy cell leukemia variant and splenic diffuse red pulp small B-cell lymphoma (SDRL). Until recently, histopathologic examination of splenectomy specimens was considered mandatory for the diagnosis of SMZL. However, nowadays, mainly because of advances in chemoimmunotherapy, splenectomy is performed much less frequently. We evaluated the diagnostic efficacy of bone marrow biopsy (BMB) histopathology in the diagnostic approach toward SMZL and SDRL and tested whether it may serve as a substitute for spleen histopathology in the differential diagnosis between these 2 entities. To this end, we conducted a paired assessment of BMB and spleen diagnostic samples from 46 cases with a diagnosis of SMZL (n=32) or SDRL (n=14) based on spleen histopathology. We demonstrate that detailed immunohistopathologic BMB evaluation offers adequate evidence for the confirmation of these entities and their differential diagnosis from other small B-cell lymphoma histotypes. Notably, the immunophenotypical profile of SMZL and SDRL was identical in both BMB and spleen specimens for 21 evaluated markers. Paired assessment of BMB and spleen specimens did not identify discriminating patterns of BMB infiltration, cytology, and/or immunohistology between SMZL and SDRL. Accordingly, bone marrow histopathology contributes significantly in confirming the diagnosis of SMZL and SDRL. However, presently it is not possible to distinguish SMZL from SDRL on the basis of BMB evaluation alone; hence, histopathologic examination of the spleen remains the "gold standard" approach.

CLL-like monoclonal B-cell lymphocytosis (MBL) shares a unique immunophenotype with chronic lymphocytic leukemia (CLL), and represents the vast majority of clonal B-cell expansions found in the peripheral blood of otherwise healthy subjects. Along with the improvement of laboratory techniques and the widespread availability of multiparameter flow cytometry, the finding of tiny aberrant B-cell populations became more frequent, prompting the need for clinical and biological definition of the nature of this condition and its relationship with leukemia development. MBL seems to be a melting-pot containing several entities, identical in terms of phenotype but with extremely different risks of leukemia development (from low to none) that seem to correlate with the number of B lymphocytes. CLL-like MBL observed in the clinical setting ("Clinical MBL"), usually being characterized by lymphocytosis, demonstrated a sizeable, even if low (1.1-1.4% per year), risk of leukemic progression, but represents a minority of all MBL cases. The vast majority of CLL-like MBL are detected in general population screenings and do not likely have a risk of CLL that is substantially higher than that of unaffected individuals. Interestingly, MBL frequency increases with age, being virtually undetectable under 40 years of age but being present in 50-75% of the people older than 90 years. It has been proposed that MBL could be interpreted as an epiphenomenon of a chronic and persistent antigenic stimulation. The (rare) possibility to evolve into a frank leukemia might then depend on biological and molecular factors insofar unknown that may modify the modality of cell reaction as well as the potential to acquire further genetic abnormalities. Therefore, the real challenge of the next years in the MBL research field is not to increase the sensitivity of detection, neither to implement screening protocols to be applied to the general population, rather to unravel the biologic features that, at individual level, will identify those (few) cases that are at risk of developing a progressive disease., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

We investigated functional relationships between microRNA 221/222 (miR-221/222) cluster and p27, a key regulator of cell cycle, in chronic lymphocytic leukemia (CLL). The enforced expression of miR-221/222 in the CLL cell line MEC1 induced a significant down-regulation of p27 protein and conferred a proliferative advantage to the transduced cells that exhibited faster progression into the S phase of the cell cycle. Accordingly, expression of miR-221/miR-222 and p27 was found to be inversely related in leukemic cells obtained from peripheral blood (PB) of 38 patients with CLL. Interestingly, when miR-221/222 and p27 protein were evaluated in different anatomic compartments (lymph nodes or bone marrow) of the same patients, increased expression of the 2 miRNAs became apparent compared with PB. This finding was paralleled by a low expression of p27. In addition, when CLL cells were induced in vitro to enter cell cycle (eg, with cytosine phosphate guanine oligodeoxynucleotide), a significant increase of miR-221/222 expression and a marked down-regulation of p27 protein were evident. These data indicate that the miR-221/222 cluster modulates the expression of p27 protein in CLL cells and lead to suggest that miR-221/222 and p27 may represent a regulatory loop that helps maintaining CLL cells in a resting condition.

Easily reproducible animal models that allow for study of the biology of chronic lymphocytic leukemia (CLL) and to test new therapeutic agents have been very difficult to establish. We have developed a novel transplantable xenograft murine model of CLL by engrafting the CLL cell line MEC1 into Rag2(-/-)gamma(c)(-/-) mice. These mice lack B, T, and natural killer (NK) cells, and, in contrast to nude mice that retain NK cells, appear to be optimal recipient for MEC1 cells, which were successfully transplanted through either subcutaneous or intravenous routes. The result is a novel in vivo model that has systemic involvement, develops very rapidly, allows the measurement of tumor burden, and has 100% engraftment efficiency. This model closely resembles aggressive human CLL and could be very useful for evaluating both the biologic basis of CLL growth and dissemination as well as the efficacy of new therapeutic agents.

Background: Monoclonal B cell lymphocytosis (MBL) is a preclinical condition characterized by an expansion of clonal B cells in the absence of B lymphocytosis (BALC < 5 × 10(9)/L) in the peripheral blood, without clinical signs, suggestive of a lymphoproliferative disorder. B cell clonal expansions are also associated with hepatitis C virus (HCV) infection and they can evolve into lymphoproliferative disorders such as mixed cryoglobulinemia and non-Hodgkin lymphomas (NHL). The relationship between MBL and HCV infection has not been established yet., Methods: By five-colour flow cytometry, we analyzed 123 HCV positive subjects with diagnosis of chronic hepatitis (94) or cirrhosis (29); 16 of those with cirrhosis had a diagnosis of hepatocellular carcinoma., Results: MBL were identified in 35/123 (28.5%), at significantly higher frequency than in the general population. Sixteen/thirty-five were atypical-chronic lymphocytic leukemia (CLL) MBL (CD5(+), CD20(bright)), 13/35 were CLL-like MBL (CD5(bright), CD20(dim)), and 6/35 were CD5(-) MBL. Twenty-four/ninety-four (25.5%) patients affected by chronic hepatitis had MBL, whereas 11/29 (37.9%) patients with cirrhosis showed a B cell clone. A biased usage of IGHVgenes similar to HCV-associated NHL was evident., Conclusions: All three types of MBL can be identified in HCV-infected individuals at a higher frequency than in the general population, and their presence appears to correlate with a more advanced disease stage. The phenotypic heterogeneity is reminiscent of the diversity of NHL arising in the context of HCV infection. The persistence of HCV may be responsible for the dysregulation of the immune system and in particular of the B cell compartment., (© 2010 International Clinical Cytometry Society.)