Simona Zupo

Micro-RNAs (miR genes) are a large family of highly conserved noncoding genes thought to be involved in temporal and tissue-specific gene regulation. MiRs are transcribed as short hairpin precursors ( approximately 70 nt) and are processed into active 21- to 22-nt RNAs by Dicer, a ribonuclease that recognizes target mRNAs via base-pairing interactions. Here we show that miR15 and miR16 are located at chromosome 13q14, a region deleted in more than half of B cell chronic lymphocytic leukemias (B-CLL). Detailed deletion and expression analysis shows that miR15 and miR16 are located within a 30-kb region of loss in CLL, and that both genes are deleted or down-regulated in the majority ( approximately 68%) of CLL cases.

Chronic lymphocytic leukemia (CLL) is the most common human leukemia and is characterized by predominantly nondividing malignant B cells overexpressing the antiapoptotic B cell lymphoma 2 (Bcl2) protein. miR-15a and miR-16-1 are deleted or down-regulated in the majority of CLLs. Here, we demonstrate that miR-15a and miR-16-1 expression is inversely correlated to Bcl2 expression in CLL and that both microRNAs negatively regulate Bcl2 at a posttranscriptional level. BCL2 repression by these microRNAs induces apoptopsis in a leukemic cell line model. Therefore, miR-15 and miR-16 are natural antisense Bcl2 interactors that could be used for therapy of Bcl2-overexpressing tumors.

Little is known about the expression levels or function of micro-RNAs (miRNAs) in normal and neoplastic cells, although it is becoming clear that miRNAs play important roles in the regulation of gene expression during development [Ambros, V. (2003) Cell 113, 673-676; McManus, M. T. (2003) Semin. Cancer Biol. 13, 253-258]. We now report the genomewide expression profiling of miRNAs in human B cell chronic lymphocytic leukemia (CLL) by using a microarray containing hundreds of human precursor and mature miRNA oligonucleotide probes. This approach allowed us to identify significant differences in miRNome expression between CLL samples and normal CD5+ B cells; data were confirmed by Northern blot analyses and real-time RT-PCR. At least two distinct clusters of CLL samples can be identified that were associated with the presence or absence of Zap-70 expression, a predictor of early disease progression. Two miRNA signatures were associated with the presence or absence of mutations in the expressed Ig variableregion genes or with deletions at 13q14, respectively. These data suggest that miRNA expression patterns have relevance to the biological and clinical behavior of this leukemia.

MicroRNAs (miRNAs) are a class of small noncoding RNA genes recently found to be abnormally expressed in several types of cancer. Here, we describe a recently developed methodology for miRNA gene expression profiling based on the development of a microchip containing oligonucleotides corresponding to 245 miRNAs from human and mouse genomes. We used these microarrays to obtain highly reproducible results that revealed tissue-specific miRNA expression signatures, data that were confirmed by assessment of expression by Northern blots, real-time RT-PCR, and literature search. The microchip oligolibrary can be expanded to include an increasing number of miRNAs discovered in various species and is useful for the analysis of normal and disease states.

Noncoding RNA (ncRNA) transcripts are thought to be involved in human tumorigenesis. We report that a large fraction of genomic ultraconserved regions (UCRs) encode a particular set of ncRNAs whose expression is altered in human cancers. Genome-wide profiling revealed that UCRs have distinct signatures in human leukemias and carcinomas. UCRs are frequently located at fragile sites and genomic regions involved in cancers. We identified certain UCRs whose expression may be regulated by microRNAs abnormally expressed in human chronic lymphocytic leukemia, and we proved that the inhibition of an overexpressed UCR induces apoptosis in colon cancer cells. Our findings argue that ncRNAs and interaction between noncoding genes are involved in tumorigenesis to a greater extent than previously thought.

Due to its relatively slow clinical progression, B cell chronic lymphocytic leukemia (B-CLL) is classically described as a disease of accumulation rather than proliferation. However, evidence for various forms of clonal evolution suggests that B-CLL clones may be more dynamic than previously assumed. We used a nonradioactive, stable isotopic labeling method to measure B-CLL cell kinetics in vivo. Nineteen patients drank an aliquot of deuterated water (2H2O) daily for 84 days, and 2H incorporation into the deoxyribose moiety of DNA of newly divided B-CLL cells was measured by gas chromatography/mass spectrometry, during and after the labeling period. Birth rates were calculated from the kinetic profiles. Death rates were defined as the difference between calculated birth and growth rates. These analyses demonstrated that the leukemic cells of each patient had definable and often substantial birth rates, varying from 0.1% to greater than 1.0% of the entire clone per day. Those patients with birth rates greater than 0.35% per day were much more likely to exhibit active or to develop progressive disease than those with lower birth rates Thus, B-CLL is not a static disease that results simply from accumulation of long-lived lymphocytes. Rather, it is a dynamic process composed also of cells that proliferate and die, often at appreciable levels. The extent to which this turnover occurs has not been previously appreciated. A correlation between birth rates and disease activity and progression appears to exist, which may help identify patients at risk for worsening disease in advance of clinical deterioration.

Two decades after the discovery of the first animal microRNA (miRNA), the number of miRNAs in animal genomes remains a vexing question. Here, we report findings from analyzing 1,323 short RNA sequencing samples (RNA-seq) from 13 different human tissue types. Using stringent thresholding criteria, we identified 3,707 statistically significant novel mature miRNAs at a false discovery rate of ≤ 0.05 arising from 3,494 novel precursors; 91.5% of these novel miRNAs were identified independently in 10 or more of the processed samples. Analysis of these novel miRNAs revealed tissue-specific dependencies and a commensurate low Jaccard similarity index in intertissue comparisons. Of these novel miRNAs, 1,657 (45%) were identified in 43 datasets that were generated by cross-linking followed by Argonaute immunoprecipitation and sequencing (Ago CLIP-seq) and represented 3 of the 13 tissues, indicating that these miRNAs are active in the RNA interference pathway. Moreover, experimental investigation through stem-loop PCR of a random collection of newly discovered miRNAs in 12 cell lines representing 5 tissues confirmed their presence and tissue dependence. Among the newly identified miRNAs are many novel miRNA clusters, new members of known miRNA clusters, previously unreported products from uncharacterized arms of miRNA precursors, and previously unrecognized paralogues of functionally important miRNA families (e.g., miR-15/107). Examination of the sequence conservation across vertebrate and invertebrate organisms showed 56.7% of the newly discovered miRNAs to be human-specific whereas the majority (94.4%) are primate lineage-specific. Our findings suggest that the repertoire of human miRNAs is far more extensive than currently represented by public repositories and that there is a significant number of lineage- and/or tissue-specific miRNAs that are uncharacterized.

Peripheral T cell lymphoma, unspecified (PTCL/U), the most common form of PTCL, displays heterogeneous morphology and phenotype, poor response to treatment, and poor prognosis. We demonstrate that PTCL/U shows a gene expression profile clearly distinct from that of normal T cells. Comparison with the profiles of purified T cell subpopulations (CD4+, CD8+, resting [HLA-DR-], and activated [HLA-DR+]) reveals that PTCLs/U are most closely related to activated peripheral T lymphocytes, either CD4+ or CD8+. Interestingly, the global gene expression profile cannot be surrogated by routine CD4/CD8 immunohistochemistry. When compared with normal T cells, PTCLs/U display deregulation of functional programs often involved in tumorigenesis (e.g., apoptosis, proliferation, cell adhesion, and matrix remodeling). Products of deregulated genes can be detected in PTCLs/U by immunohistochemistry with an ectopic, paraphysiologic, or stromal location. PTCLs/U aberrantly express, among others, PDGFRalpha, a tyrosine-kinase receptor, whose deregulation is often related to a malignant phenotype. Notably, both phosphorylation of PDGFRalpha and sensitivity of cultured PTCL cells to imatinib (as well as to an inhibitor of histone deacetylase) were found. These results, which might be extended to other more rare PTCL categories, provide insight into tumor pathogenesis and clinical management of PTCL/U.

Angioimmunoblastic lymphoma (AILT) is the second most common subtype of peripheral T-cell lymphoma (PTCL) and is characterized by dismal prognosis. Thus far, only a few studies have dealt with its molecular pathogenesis. We performed gene expression profile (GEP) analysis of six AILT, six anaplastic large cell lymphomas (ALCL), 28 PTCL-unspecified (PTCL/U), and 20 samples of normal T lymphocytes (including CD4(+), CD8(+), and activated and resting subpopulations), aiming to (a) assess the relationship of AILT with other PTCLs, (b) establish the relationship between AILT and normal T-cell subsets, and (c) recognize the cellular programs deregulated in AILT possibly looking for novel potential therapeutic targets. First, we found that AILT and other PTCLs have rather similar GEP, possibly sharing common oncogenic pathways. Second, we found that AILTs are closer to activated CD4(+), rather than to resting or CD8(+) lymphocytes. Furthermore, we found that the molecular signature of follicular T helper cells was significantly overexpressed in AILT, reinforcing the idea that AILT may arise from such cellular counterpart. Finally, we identified several genes deregulated in AILT, including PDGFRA, REL, and VEGF. The expression of several molecules was then studied by immunohistochemistry on tissue microarrays containing 45 independent AILT cases. Notably, we found that the vascular endothelial growth factor (VEGF) was expressed not only by reactive cells, but also by neoplastic cells, and that nuclear factor-kappaB (NF-kappaB) activation is uncommon in AILT, as suggested by frequent exclusively cytoplasmic c-REL localization. Our study provides new relevant information on AILT biology and new candidates for possible therapeutic targets such as PDGFRA (platelet-derived growth factor alpha) and VEGF.

The importance of post-transcriptional mechanisms for the regulation of the homoeostasis of the immune system and the response to challenge by microorganisms is becoming increasingly appreciated. We investigated the contribution of microRNAs (miRNAs) to macrophage activation induced by lipopolysaccharide (LPS). We first observed that Dicer knockout in bone marrow-derived macrophages (BMDMs) increases the LPS-induced expression of some inflammation mediators. miRNA microarray analysis in BMDMs revealed that LPS significantly induces the expression of a single miRNA, miR-155, and this induction depends on enhanced miR-155 maturation from its precursors. The single-strand RNA-binding protein KH-type splicing regulatory protein (KSRP) binds to the terminal loop of miR-155 precursors and promotes their maturation. Both inhibition of miR-155 and KSRP knockdown enhance the LPS-induced expression of select inflammation mediators, and the effect of KSRP knockdown is reverted by mature miR-155. Our studies unveil the existence of an LPS-dependent post-transcriptional regulation of miR-155 biogenesis. Once induced, miR-155 finely tunes the expression of select inflammation mediators in response to LPS.

The expression of CD38 by B cells chronic lymphocytic leukemia (B-CLL) was studied in 20 untreated patients. The cells expressed abundant CD38 (relative fluorescence intensity range, 6 to 15) in 6 cases (group I patients), whereas CD38 expression was low to absent (relative fluorescence intensity range, 0 to 3) in the remaining cases (group II patients). Exposure of the cells from group I patients to goat antihuman mu chain antibodies (Ga mu-ab) resulted in the elevation of intracellular free Ca2+ concentration([Ca2+]i) followed by apoptosis. In contrast, exposure of group II cells to Ga mu-ab was not followed by increased levels of [Ca2+]i, programmed cell death or cell proliferation. No differences in the expression of surface IgM were noted in the two groups of B-CLL cells. Normal peripheral blood B cells, which expressed low to absent CD38, were capable of mobilizing [Ca2+]i and of proliferating after exposure to Ga mu-ab. The collected data suggest that, although group I B-CLL cells were able to transduce the signals delivered by IgM crosslinking, this pathway was severely impaired in group II B-CLL cells. However, unlike that observed in normal circulating B cells, stimulation of group I cells with Ga mu-ab resulted in apoptosis rather than proliferation. CD38 did not appear to be directly involved in [Ca2+]i mobilization induced by Ga mu-ab in group I B-CLL cells because their exposure to anti-CD38 monoclonal antibodies failed to cause [Ca2+]i mobilization or to block the [Ca2+]i response induced by Ga mu-ab. These data indicate that CD38 expression identified a particular subset of B-CLL cells with defined functional properties, including the propensity to undergo apoptosis.

Abstract The present study demonstrates that an agonistic anti‐CD38 monoclonal antibody (mAb) (IB4) is capable of preventing apoptosis of human tonsillar germinal center (GC) B cells as measured by either morphological methods on Giemsa‐stained cytospin preparations or flow cytometry on propidium iodidestained cells. Two other anti‐CD38 mAb (Leu‐17 and OKT10) consistently failed to prevent apoptosis in the same cells, even when tested over a wide range of concentrations. Furthermore, exposure of GC B cells to IB4 mAb up‐regulates the bcl‐2 proto‐oncogene product in a manner similar to that observed with CD40 ligand (CD40L). The ability of IB4 mAb to prevent apoptosis of GC B cells was inferior to that of both anti‐CD40 mAb and CD40L. No synergistic or additive effects were observed when IB4 mAb was used together with CD40L. Unlike anti‐CD40 mAb or CD40L, IB4 mAb neither induced a proliferation of GC B cells nor increased their proliferative response to anti‐CD40, CD40L or recombinant interleukin‐4, used alone or in combination. The present results are consistent with the recent findings on either the feature of the CD38 molecules to deliver activation signals and on the mechanisms of selection of B cells that operates in the GC.

Uveal melanoma is the most frequent primary tumour of the eye. It is molecularly clearly distinct from cutaneous melanoma and shows a different pattern of driver mutations. The influence of sunlight ultraviolet (UV) exposure on the aetiology of uveal melanoma is a matter of debate. The recent identification of driver mutations in the promoter of the telomerase reverse transcriptase (TERT) gene with UV-induced cytidine-to-thymidine transitions in cutaneous melanoma prompted us to investigate whether these mutations also occur in uveal melanoma. We analysed 50 cases of uveal melanoma obtained from enucleation surgery for mutations in the genes GNAQ, GNA11, BAP1, SF3B1, EIFAX1 and TERT, measured gene expression using microarrays and analysed gene copy numbers by SNP arrays. We detected a TERT mutation in only one case of a 57-year-old white male patient with clinical and histopathological features typical for uveal melanoma. The tumour showed mutations in GNA11 and EIF1AX that are typical for uveal melanoma and absent from cutaneous melanoma. No mutations were detected in GNAQ, BAP1 and SF3B1 that are frequently mutated in uveal melanoma. Both copies of chromosome 3 were retained. Several tumours among which the one carrying the TERT promoter mutation showed elevated TERT expression. Consistent with previous reports, GNAQ is inversely associated with chromosome 3 monosomy and metastasis. BAP1 mutations are significantly associated with chromosome 3 monosomy but not with relapse. These data indicate that TERT mutations are rare in uveal melanoma. No conclusion can be drawn on their potential influence on tumour progression.

Abstract To extent our knowledge on the cytokines possibly involved in the pathophysiology of B-cell chronic lymphocytic leukemia (B-CLL), the mRNA expression of a panel of 10 cytokines was investigated on purified B-CLL cells using a reverse-transcriptase polymerase chain reaction method. Whereas negative RT-PCR signals were recorded for interleukin-1 alpha (IL-1 alpha), IL-2, IL-3, IL-4, IL-5, IL-7, tumor necrosis factor beta (TNF beta), and granulocyte-macrophage colony-stimulating factor, we detected the expression of IL-1 beta, IL-6 and TNF alpha. Furthermore, the constitutive expression of IL-8 mRNA was observed in all 17 B-CLL samples analyzed. mRNA expression was associated with the capacity of the leukemic cells to release IL-8 both constitutively (4.6 +/- 8.1 SD ng/mL) and, to a further extent, after stimulation (14.5 +/- 19.4 ng/mL). The circulating levels of IL-8 were also evaluated in 12 untreated B-CLL sera samples and the overall mean level was significantly higher (P <.01) than in normal sera. In addition, supernatants of purified B-CLL cells cultured in the presence of 12-O- tetradecanoylphorbol-13-acetate showed chemotactic activity towards neutrophils; this activity was neutralized in the presence of an anti- IL-8 antiserum. The mRNA for IL-8 was absent in five B-cell preparations from hairy cell leukemia cases and in four B-cell lines. Normal tonsil CD5+ B cells showed a low expression of IL-8 mRNA only in two of the nine preparations tested and the overall quantity of IL-8 released by these cells after 3 days' incubation was significantly lower compared with that released by B-CLL cells (0.4 +/- 0.3 and 1.6 +/- 0.9 ng/mL under basal and stimulated conditions, respectively). These findings point to an involvement of a member of the proinflammatory chemokine supergene family in human CD5+ B lymphocytes. The different IL-8 behavior observed between B-CLL cells and their normal counterpart is likely to reflect an activation state of the leukemic population.

Interleukin-21 (IL-21) is a member of the IL-2 cytokine family, which mediates proliferation or growth arrest and apoptosis of normal B cells, depending on their activation state. Here we demonstrate that surface IL-21 receptor (R) is expressed at variable levels by chronic lymphocytic leukemia (CLL) B cells freshly isolated from 33 different patients. IL-21R expression was up-regulated following cell stimulation via surface CD40. Therefore, IL-21 effects were more evident in CD40-activated CLL B cells. IL-21 induced an early signaling cascade in CLL B cells, which included JAK-1 and JAK-3 autophosphorylation and tyrosine phosphorylation of STAT-1, STAT-3, and STAT-5. IL-21 signaling failed to stimulate CLL B-cell proliferation, but induced their apoptosis. In addition, IL-21 counteracted the proliferative and antiapoptotic signals delivered by IL-15 to CLL B cells. IL-21-mediated apoptosis involved activation of caspase-8 and caspase-3, cleavage of Bid to its active form t-Bid, and cleavage of PARP and of p27Kip-1. Recent data indicate that CLL B cells require interaction with the microenvironment for their survival and expansion. The present findings thus provide a set of new mechanisms involved in the balance between cell-survival and apoptotic signals in CLL B cells.

Abstract Influenza vaccination is generally recommended for non-Hodgkin’s lymphoma (NHL) patients, but no data are available about the activity of this vaccine after treatment with rituximab-containing regimens. We evaluated the humoral response to the trivalent seasonal influenza vaccine in a group of NHL patients in complete remission for ≥6 mo (median, 29 mo) after treatment with rituximab-containing regimens (n = 31) compared with age-matched healthy subjects (n = 34). B cell populations and incidence of influenza-like illness were also evaluated. For each viral strain, the response was significantly lower in patients compared with controls and was particularly poor in patients treated with fludarabine-based regimens. In the patient group, the response to vaccination did not fulfill the immunogenic criteria based on the European Committee for Medicinal Products for Human Use requirements. Among the patients, CD27+ memory B cells were significantly reduced, and their reduction correlated with serum IgM levels and vaccine response. Episodes of influenza-like illness were recorded only in patients. These results showed that NHL patients treated with rituximab-containing regimens have persisting perturbations of B cell compartments and Ig synthesis and may be at particular risk for infection, even in long-standing complete remission.

The VH4 genes expressed by both resting and in vivo-activated subepithelial (SE) B cells from human tonsils were studied. Resting SE B cells were subdivided according to the presence (IgDlow) or absence (IgM-only) of surface IgD. CD27 was abundant on activated SE B cells and low on resting IgM-only B cells. Resting IgDlow SE B cells could be subdivided into CD27low and CD27high cell fractions. Resting IgDlow SE B cells displayed VH4 genes with a substantial number of mutations (13/29 of the molecular clones were mutated), whereas 25/26 of the clones from resting IgM-only SE B cells were unmutated. Moreover, mutated VH4 genes were detected mainly within the CD27high cell fraction of the IgDlow SE B cells. Several identical unmutated VH4DJH sequences (11/32) were found in different molecular clones from resting IgM-only SE B cells, suggesting local cellular expansion. Both unmutated (14/25) and mutated (11/25) sequences were found in mu transcripts of activated SE B cells. Extensive mutation was observed in the gamma transcripts of activated SE B cells. Therefore, SE B cells are heterogeneous, being comprised of B cells with mutated Ig VH4 genes, that are Ag-experienced B cells, and a subset of B cells with unmutated VH4 genes that are either virgin cells or cells driven by Ags that did not induce or select for V gene mutations.

Helsinki.Genomic DNA was isolated from whole blood, and exon 10 was amplified in standard polymerase chain reaction (PCR) conditions.The primer sequences used for PCR were the same as those used by Krijanovski et al. 1 DNA sequences were determined on an ABI 3130 ϫ 1 Genetic Analyzer (Applied Biosystems, Foster City, CA).A single cytidine nucleotide insertion was found at nucleotide position 1217 (Figure 1B). 6There were 4 consecutive cytidine nucleotides before the insertion of an extra cytidine.This insertion resulted in a frameshift in codon 406, and a premature stop signal (TGA) was generated at codon 415.We next examined the proband's family members and found that her parents and brother were heterozygous for this mutation (Figure 1B).These results indicate that a cytidine nucleotide insertion found in the proband is responsible for the isolated HK deficiency inherited in this family.In conclusion, we identified a novel frameshift mutation in exon 10 of the kininogen gene in a Japanese family with isolated HK deficiency.

Objectives: To determine (i) whether primary (idiopathic) follicular mucinosis (PFM) and lymphoma‐associated follicular mucinosis (LAFM) are distinct or related entities and whether there are reliable criteria that allow the two forms to be distinguished, (ii) the histochemical properties and consequently the type of mucin that accumulates in the follicle in PFM and LAFM, and (iii) whether there is any difference between the staining properties of mucin in patients with PFM and LAFM. Methods: Thirty‐one patients were divided into two groups. Group 1 comprised 20 patients with no associated mycosis fungoides or Sézary syndrome (PFM) and group 2 was made up of the other 11 patients who had clinicopathological evidence of cutaneous T‐cell lymphoma (LAFM). The biopsy specimens of the patients were studied with histopathological, histochemical and immunohistochemical methods. Molecular biology studies were also performed. Results: Patients with PFM were more frequently younger (mean age 39 years), women (F:M=3:1), and presented with a solitary lesion involving the head/neck area more often than patients with LAFM who were older (mean age 54 years), men (M:F=2:1), and presented with multiple lesions on areas of the body other than the head/neck area. As for histopathological findings, large cystic spaces filled with mucin and a slight perivascular and periadnexal polyclonal infiltrate of mostly non‐atypical lymphocytes without epidermotropism and with an equivalent CD4+/CD8+ cell rate were more suggestive of PFM. On the contrary, patients with LAFM were more probably to present with a dense band‐like infiltrate with some atypical lymphocytes and sign of epidermotropism, a prominent CD4+ immunophenotype and a monoclonal rearrangement of the infiltrate. Mucin proved to be a dermal‐type mucin, composed of both hyaluronic acid and sulfated glycosaminoglycans. No differences were found in the composition of the follicular mucin in the PFM compared with LAFM. Conclusions: Although no single, indisputable feature can reliably differentiate PFM from LAFM and a considerable overlapping among the two groups exists, the use of multiple clinical, histological and immunopathological criteria associated with gene rearrangement analysis can be useful in evaluation of those patients.

Splenectomy in addition to immunotherapy with rituximab can provide quick and sometimes durable disease control in patients with splenic marginal zone lymphoma (SMZL). However, systemic chemotherapy is ultimately required in many cases. The BRISMA (Bendamustine-rituximab as first-line treatment of splenic marginal zone lymphoma)/IELSG (International Extranodal Lymphoma Study Group)36 trial is an open-label, single arm phase II study designed by the IELSG in cooperation with the Fondazione Italiana Linfomi and the lymphoma Study Association according to Simon's two-stage method. The primary endpoint was complete response rate. Fifty-six patients with SMZL diagnosis confirmed on central revision were treated with bendamustine (90 mg/m2 days 1, 2) and rituximab (375 mg/m2 day 1) every 28 days for six cycles (B-R). The overall response and CR rates were 91% and 73%, respectively. Duration of response, progression-free survival and overall survival at 3 years were 93% (95% confidence interval [CI] 81-98), 90% (95% CI 77-96) and 96% (95% CI 84-98), respectively. Toxicity was mostly haematological. Neutropenia grade ≥3 was recorded in 43% of patients; infections and febrile neutropenia in 5·4% and 3·6%. Overall, 14 patients (25%) experienced serious adverse events. Five patients (9%) went off-study because of toxicity and one patient died from infection. In conclusion, B-R resulted in a very effective first-line regimen for SMZL. Based on the results achieved in the BRISMA trial, B-R should be considered when a chemotherapy combination with rituximab is deemed necessary for symptomatic SMZL patients.

Previously, we demonstrated that B-chronic lymphocytic leukemia (B-CLL) cells could be divided into 2 groups depending on the expression of CD38 by the malignant cells. The 2 groups differed in their signal-transducing capacities initiated by cross-linking of surface IgM; only in CD38-positive cells was an efficient signal delivered, invariably resulting in cell apoptosis. In this study, we investigated the effect of surface IgD cross-linking in 10 patients with CD38-positive B-CLL. Exposure of the malignant cells to goat antihuman δ-chain antibodies (Gaδ-ab) caused [Ca++]i mobilization and tyrosine kinase phosphorylation in a manner not different from that observed after goat antihuman μ-chain antibody (Gaμ-ab) treatment in vitro. However, Gaδ-ab-treated cells failed to undergo apoptosis and instead displayed prolonged survival in culture and differentiated into plasma cells when rIL2 was concomitantly present. Cross-linking of surface IgD failed to induce proliferation of the malignant cells in vitro. Moreover, treatment with Gaδ-ab did not prevent apoptosis of B-CLL cells induced by Gaμ-ab. Collectively, these experiments demonstrated that IgM and IgD expressed by the same cell may deliver opposite signals under particular circumstances and provide some clues for the understanding of the pathophysiology of B-CLL.

Abstract This study describes the purification of a subset of tonsillar B cells which share phenotypic, morphologic and cytochemical features with subepithelial (SE) B cells. These cells, which represented the 5–10% of the total tonsillar B cells, were found in the Percoll gradient fraction of highest density, together with resting follicular mantle (FM) B cells. The latter B cells, however, expressed surface CD5 and could be removed by an immune rosetting procedure. The remaining small CD5 − B cells had a surface phenotype (IgM + , IgD + , CD23 − , CD38 ± , CD10 − , CD44 + ) that was different from that of FM (IgM + , IgD + , CD23 + , CD39 + , CD38 − , CD10 − , CD44 ± ) and of germinal center (GC) (CD23 − , CD39 − , CD38 + , CD10 + , CD44 ± , IgG + ) B cells isolated from the same cell suspensions. Furthermore, the absence of surface activation markers (CD71 and CD69) and of surface IgG allowed us to distinguish small CD5 − B cells from activated and memory cells migrating within Percoll fractions of lower density. In situ immunohistochemical studies revealed that B cells with an identical phenotype as that of small CD5 − B cells could be detected predominantly in the SE region (lamina propria) of the tonsil, and also within the epithelium lining the cryptae. This area was also comprised of a relatively minor proportion of activated B cells, not found in the small CD5 − B cell fraction owing to the separation procedure used. Consistent with the notion that the SE area could be a site of B cell activation was also the presence of activated macrophages and of plasma cells. Thirty to forty percent of small CD5 − B cells isolated in suspension were positive for the endogeneous alkaline phosphatase (ALP) activity. In contrast, only a few FM B cells were ALP + , while GC cells were consistently ALP − . In situ studies also demonstrated a prevalent expression of ALP activity by the B cells in the SE area. At the ultrastructural level, small CD5 − B cells were clearly different from both FM and GC B cells. They displayed a cytoplasm more extended than that of FM B cells with abundant endosomes and plasma membrane projections, and a speckled pattern of nuclear heterochromatin distribution. When fixed tissue sections were examined, cells with identical ultrastructural features could be demonstrated in the tonsillar lamina propria. Collectively, the above data demonstrate an identity of features between the small CD5 − B cells isolated in suspension and SE B cells analyzed in situ . Since tonsillar SE B cells are generally thought to represent the homolog of the extrafollicular B cells (including those of the splenic marginal zone), these studies may provide new opportunities for functional studies on this so far incompletely characterized B cell subset.

In the last two decades, many efforts have been made to better understand the biology of B-lymphoproliferative disorders through the knowledge of physiology and function of the postulated normal counterpart. The follicular mantle B-cells express a typical CD23+ IgM+ IgD+ phenotype and surround the germinal center area in secondary lymphoid organs. CD5+ B-cells with FM phenotype can be isolated from different sources and all share similar morphologic, phenotypic and functional features (small cells, scanty nucleus/cytoplasm ratio, unmutated VH genes, response to polyclonal activators but not to T independent antigens, production of “natural” antibodies). While the CD5+ B-cells predominate in fetal life, their number decreases with age. However, the CD5+ B-cells have been demonstrated to increase again in elderly both in man and mouse. This finding may explain the incidence of B-CLL and of MCL that are believed to represent the malignant transformation of the normal CD5+ B-cells, among elderly and middle aged individuals, respectively. Cell facts A major function of CD5+ B-cells is to produce low-affinity polyreactive antibodies; CD5+ cells comprise 15–25% of the B-cell population in adult secondary lymphoid organs; They mostly display a follicular mantle phenotype (CD23+ IgM+ IgD+); They are indicated as a self-replenishing B-cell subpopulation; CD5+ B-cells show an increased propensity to malignant transformation.

Summary IGHV mutational status and ZAP‐70 or CD38 expression correlate with clinical course in B‐cell chronic lymphocytic leukaemia (CLL). The three markers may be discordant in the single case and there is no consensus on their combined use in clinical practise. This multicenter study investigated this issue. Two‐hundred and sixty‐two Binet stage A patients were studied for the three markers. Sixty patients were profiled with HG‐U133A gene expression chips. Disease progression was determined by time from diagnosis to treatment (TTT). The probability of being treatment‐free at 3 years was significantly shorter in patients with unmutated IGHV genes ( IGHV unmut 66% vs. 93%, chi square of log‐rank = 30, P < 0·0001), ZAP‐70 positive (ZAP‐70pos 73% vs. 96%, chi square of log‐rank = 8·2, P = 0·004) or CD38‐positive cells (CD38pos 68% vs. 91%, chi square of log‐rank = 21, P < 0·0001). Cox multivariate regression analysis showed that the three markers had an independent predictive value for TTT of similar power. A prognostic system based on presence of none (low‐risk), one (intermediate‐risk) or two or three (high‐risk) markers was generated. Based on such criteria, 56%, 23% and 21% of cases were clustered in low (HR = 1), intermediate [HR = 2·8, 95% confidence interval (CI) 2·4–5·8] and high‐risk group (HR = 8·0, 95% CI 3·9–16·2). Specific transcriptional patterns were significantly associated with risk groups.

Several chemokines/chemokine receptors such as CCR7, CXCR4 and CXCR5 attract chronic lymphocytic leukemia (CLL) cells to specific microenvironments. Here we have investigated whether the CX3CR1/CX3CL1 axis is involved in the interaction of CLL with their microenvironment. CLL cells from 52 patients expressed surface CX3CR1 and CX3CL1 and released constitutively soluble CX3CL1. One third of these were attracted in vitro by soluble CX3CL1. CX3CL1-induced phosphorylation of PI3K, Erk1/2, p38, Akt and Src was involved in induction of CLL chemotaxis. Leukemic B cells upregulated CXCR4 upon incubation with CX3CL1 and this was paralleled by increased chemotaxis to CXCL12. Akt phosphorylation was involved in CX3CL1-induced upregulation of CXCR4 on CLL. In proliferation centers from CLL lymph node and bone marrow, CX3CL1 was expressed by CLL cells whereas CX3CR1 was detected in CLL and stromal cells. Nurselike cells (NLCs) generated from CLL patient blood co-expressed surface CX3CR1 and CX3CL1, but did not secrete soluble CX3CL1. Only half of NLC cell fractions were attracted in vitro by CX3CL1. In conclusion, the CX3CR1/CX3CL1 system may contribute to interactions between CLL cells and tumor microenvironment by increasing CXCL12-mediated attraction of leukemic cells to NLC and promoting directly adhesion of CLL cells to NLC.

Hepatocyte growth factor, produced by stromal and follicular dendritic cells, and present at high concentrations in the sera of patients with chronic lymphocytic leukemia, prolongs the survival of leukemic B cells by interacting with their receptor, c-MET. It is, however, unknown whether hepatocyte growth factor influences microenvironmental cells, such as nurse-like cells, which deliver survival signals to the leukemic clone. We evaluated the expression of c-MET on nurse-like cells and monocytes from patients with chronic lymphocytic leukemia and searched for phenotypic/functional features supposed to be influenced by the hepatocyte growth factor/c-MET interaction. c-MET is expressed at high levels on nurse-like cells and at significantly higher levels than normal on monocytes from patients. Moreover, the hepatocyte growth factor/c-MET interaction activates STAT3(TYR705) phosphorylation in nurse-like cells. Indoleamine 2,3-dioxygenase, an enzyme modulating T-cell proliferation and induced on normal monocytes after hepatocyte growth factor treatment, was detected together with interleukin-10 on nurse-like cells, and on freshly-prepared patients' monocytes. Immunohistochemical/immunostaining analyses demonstrated the presence of c-MET(+) and indoleamine 2,3-dioxygenase(+) cells in lymph node biopsies, co-expressed with CD68 and vimentin. Furthermore nurse-like cells and chronic lymphocytic monocytes significantly inhibited T-cell proliferation, prevented by anti-transforming growth factor beta and interleukin-10 antibodies and indoleamine 2,3-dioxygenase inhibitors, and supported CD4(+)CD25(high+)/FOXP3(+) T regulatory cell expansion. We suggest that nurse-like cells display features of immunosuppressive type 2 macrophages: higher hepatocyte growth factor levels, produced by leukemic or other microenvironmental surrounding cells, may cooperate to induce M2 polarization. Hepatocyte growth factor may thus have a dual pathophysiological role: directly through enhancement of survival of the leukemic clone and indirectly by favoring T-cell immunosuppression.

Interleukin (IL)-23 and IL-27 are pro-inflammatory cytokines that share functional and structural similarities and may exert anti-tumor activities against solid and hematological malignancies. Here, we asked whether IL-23 and IL-27, alone or in combination, may act directly against human follicular lymphoma (FL) and diffuse large B-cell lymphoma (DLBCL) cells. In this study, we demonstrated for the first time that human primary FL and DLBCL cells expressed complete and functional IL-23 and IL-27 receptors (R) and that IL-23 and IL-27 exerted anti-tumor activities in vitro and in vivo through different and complementary mechanisms. In vivo studies using severe combined immunodeficiency /non-obese diabetic mice-injected subcutaneously with human SU-DHL-4 cell line revealed that IL-23 inhibited directly tumor-cell proliferation, whereas IL-27 impaired the angiogenic program of lymphoma cells resulting in strong reduction of cell growth. In addition, combined treatment of IL-23 and IL-27 amplified the anti-tumor effects in vivo as compared with administration of each cytokine alone. These anti-tumor mechanisms were confirmed by in vitro experiments performed with primary lymphoma cells and cell lines. Our results strongly encourage the development of future clinical trials to evaluate the toxicity and efficacy of the IL-23 and IL-27 in lymphoma patients.

Chronic lymphocytic leukemia (CLL) clones are characterized by loss of a critical region in 13q14.3, (del(13)(q14)) involving the microRNA (miRNA) cluster miR-15a and miR-16-1. We have investigated the effects of replacement of miR-15a and miR-16-1. CLL cells transfected with these miRNA mimics exhibited a decrease in cell viability in vitro and impaired capacity for engraftment and growth in NOD/Shi-scid,γcnull (NSG) mice. No synergistic effects were observed when the two miRNA mimics were combined. The phenomena were not restricted to CLL with the del(13)(q14) lesion. Similar effects induced by miRNA mimics were seen in cells with additional chromosomal abnormalities with the exception of certain CLL clones harboring TP53 alterations. Administration of miRNA mimics to NSG mice previously engrafted with CLL clones resulted in substantial tumor regression. CLL cell transfection with miR-15a and miR-16-1-specific inhibitors resulted in increased cell viability in vitro and in an enhanced capacity of the engrafted cells to grow in NSG mice generating larger splenic nodules. These data demonstrate that the strong control by miR-15a and miR-16-1 on CLL clonal expansion is exerted also at the level of full-blown leukemia and provide indications for a miRNA-based therapeutic strategy.

Abstract Deficiency of glycosylphosphatidylinositol (GPI)–anchored molecules on blood cells accounts for most features of paroxysmal nocturnal hemoglobinuria (PNH) but not for the expansion of PNH (GPI−) clone(s). A plausible model is that PNH clones expand by escaping negative selection exerted by autoreactive T cells against normal (GPI+) hematopoiesis. By a systematic analysis of T-cell receptor beta (TCR-β) clonotypes of the CD8+ CD57+ T-cell population, frequently deranged in PNH, we show recurrent clonotypes in PNH patients but not in healthy controls: 11 of 16 patients shared at least 1 of 5 clonotypes, and a set of closely related clonotypes was present in 9 patients. The presence of T-cell clones bearing a set of highly homologous TCR-β molecules in most patients with hemolytic PNH is consistent with an immune process driven by the same (or similar) antigen(s)—probably a nonpeptide antigen, because patients sharing clonotypes do not all share identical HLA alleles. These data confirm that CD8+ CD57+ T cells play a role in PNH pathogenesis and provide strong new support to the hypothesis that the expansion of the GPI− blood cell population in PNH is due to selective damage to normal hematopoiesis mediated by an autoimmune attack against a nonpeptide antigen(s) that could be the GPI anchor itself.

Interleukin (IL)-31A binds to an heterodimer composed of IL-31 receptor A (IL-31RA) and Oncostatin M Receptor (OSMR). The IL-31/IL-31R complex is involved in the pathogenesis of various skin diseases, including cutaneous T-cell lymphoma. No information is available on the relations between the IL-31/IL-31R complex and B-cell lymphoma. Here we have addressed this issue in follicular lymphoma (FL), a prototypic germinal center(GC)-derived B-cell malignancy. IL-31 enhanced primary FL cell proliferation through IL-31R-driven signal transducer and activator of transcription factor 1/3 (STAT1/3), extracellular signal-regulated kinase 1/2 (ERK1/2) and Akt phosphorylation. In contrast, GC B cells did not signal to IL-31 in spite of IL-31R expression. GC B cells expressed predominantly the inhibitory short IL-31RA isoform, whereas FL cells expressed predominantly the long signaling isoform. Moreover, GC B cells lacked expression of other IL-31RA isoforms potentially involved in the signaling pathway. IL-31 protein expression was significantly higher in surface membrane than in cytosol of both FL and GC B cells. IL-31 was detected in plasma membrane microvesicles from both cell types but not released in soluble form in culture supernatants. IL-31 and IL-31RA expression was higher in lymph nodes from FL patients with grade IIIa compared with grade I/II, suggesting a paracrine and/or autocrine role of IL-31/IL-31RA complex in tumor progression through microvesicle shedding.

This study describes the features of the marginal zone (MZ) B cells of human tonsils and spleens and compares them with those of the follicular mantle (FM) B cells from the same tissues. The two B cell subpopulations displayed marked differences in phenotype, in response capacity to T cell-independent antigens and polyclonal B cell activators, and in presentation of antigens to T cells. FM B cells expressed surface CD5, and hence should be considered as B1 cells by current nomenclature. Fractionation of MZ B cells according to the presence or absence of surface IgD revealed the presence of two subsets. These subsets were characterized by different properties, including the presence of Ig V(H) gene mutations and the response capacity to TI-2 antigens, this latter property being associated with IgD-positive cells. Comparison of the data with those reported for mice revealed that human MZ B cells had strong analogies with both the murine MZ and B1 cells. In contrast, human B1 cells (that is, CD5-positive FM cells) were considerably different, an observation that should prompt further studies. Indeed, B cells with characteristics analogous to those of murine B1 cells were detected in small but definite proportions in the peripheral blood and tonsils. If the current distinction into B1 and B2 cells has to be maintained also for humans, it is likely that only these CD5-positive cells rather than the FM B cells should be called B1 cells.

The demonstration of EGFR T790M gene mutation in plasma is crucial to assess the eligibility of Non Small Cell Lung Cancer (NSCLC) patients, who have acquired resistance to first or second generation Tyrosine Kinase Inhibitors (TKIs), to receive a subsequent treatment with osimertinib. Since circulating tumor DNA (ctDNA) is present in very low amounts in plasma, high sensitive and specific methods are required for molecular analysis. Improving sensitivity of T790M mutation detection in plasma ctDNA enables a larger number of NSCLC patients to receive the appropriate therapy without any further invasive procedure. A tag-based next generation sequencing (NGS) platform capable of tagging rare circulating tumor DNA alleles was employed in this study for the identification of T790M mutation in 42 post-TKI NSCLC patients. Compared to Real Time PCR, tag-based NGS improved the T790M detection rate (42.85% versus 21.4%, respectively), especially in those cases with a low median mutation abundance (i.e. 0.24, range 0.07–0.78). Moreover, the tag-based NGS identified EGFR activating mutations more efficiently than Real Time PCR (85.7% versus 61.9% detection rate, respectively), particularly of the L858R variant type (0.06–0.75 mutation abundance range). Patients in whom the T790M mutation was detected in plasma, achieved an objective response to osimertinib (9/14, 64.28%). Tag-based NGS represents an accurate and sensitive tool in a clinical setting for non-invasive assessment and monitoring of T790M variant in NSCLC patients.

Roughly 40% of germinal mutations in melanoma families (MF) affect p16(INK4a) and p14(ARF). We investigated the association between INK4/ARF alterations and the occurrence of pancreatic cancer in MF and in sporadic pancreatic cancer (SPC) patients.Forty-nine MF, 66 SPC cases and 54 controls were enrolled. The INK4/ARF locus was screened.As compared with the general population, the risk of pancreatic cancer (PC) was increased 9.4-fold [95% confidence interval (CI) 2.7-33.4] and 2.2-fold (95% CI 0.8-5.7) in G101W-positive and -negative MF, respectively, while mean ages at onset were 61 and 77 years, respectively. A 1.7 (95% CI 1.06-2.79) increased risk of cancer at any site was observed among first-degree relatives of SPC cases as compared with controls. The G101W founder mutation was detected in 4% of SPC cases but the rate increased to 13% when tumor clustering in either branch of families was taken into account. One G101W-positive PC patient with a melanoma in a first-degree relative harbored a germline deletion of the second allele, including exon 1B.The presence of a deletion including exon 1B in two PC patients points to the involvement of p14(ARF) in the development of PC and may suggest that the increased risk of PC in MF is caused by impairment of both loci.

Metastatic colorectal cancer (mCRC) is frequently characterized by the presence of mutations of the KRAS oncogene, which are generally associated with a poor response to treatment with anti-epidermal growth factor receptor (anti-EGFR) monoclonal antibodies. With the methods currently used, a case is classified as KRAS-mutated when approximately 20% of the cells bear an activating KRAS mutation. These considerations raise the question of whether cells with a mutated KRAS can be found in mCRC cases classified as KRAS wild-type when more sensitive methods are used. In addition, the issue arises of whether these mCRC cases with low proportion of KRAS-mutated cells could account at least in part for the therapeutic failure of anti-EGFR therapies that occur in 40-60% of cases classified as KRAS wild type. In this study, we compared the classical assays with a very sensitive test, a locked nucleic acid (LNA) polymerase chain reaction (PCR), capable of detecting KRAS-mutated alleles at extremely low frequency (detection sensitivity limit 0.25% mutated DNA/wild-type DNA). By analyzing a cohort of 213 mCRC patients for KRAS mutations, we found a 20.6% discordance between the sequencing/TheraScreen methods and the LNA-PCR. Indeed, 44 mCRC patients initially considered KRAS wild type were reclassified as KRAS mutated by using the LNA-PCR test. These patients were more numerous among individuals displaying a clinical failure to anti-EGFR therapies. Failure to respond to these biological treatments occurred even in the absence of mutations in other EGFR pathway components such as BRAF.

Reactive oxygen species (ROS) are metabolism by-products that may act as signaling molecules to sustain tumor growth. Antioxidants have been used to impair cancer cell survival. Our goal was to determine the mechanisms involved in the response to antioxidants of a human cell culture (PT4) containing glioblastoma (GBM) tumorigenic initiating cells (TICs). ROS production in the absence or presence of N-acetyl-L-cysteine (NAC), tiron, and trolox was evaluated by flow cytometry (FCM). The effects of these antioxidants on cell survival and apoptosis were evaluated by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay (MTT) and FCM. The biological processes modulated by these drugs were determined by oligonucleotide microarray gene expression profiling. Our results showed that NAC, tiron and trolox impaired PT4 cell survival, had minor effects on ROS levels and caused wide deregulation of cell cycle genes. Furthermore, tiron and trolox caused inhibition of cell survival in two additional cell cultures containing TICs, FO-1 and MM1, established from a melanoma and a mesothelioma patient, respectively. NAC, instead, impaired survival of the MM1 cells but not of the FO-1 cells. However, when used in combination, NAC enhanced the inhibitory effect of PLX4032 (BRAF V600E inhibitor) and Gefitinib (EGFR inhibitor), on FO-1 and PT4 cell survival. Collectively, NAC, tiron and trolox modulated gene expression and impaired the growth of cultures containing TICs primarily by inhibiting cell cycle progression.

Richter syndrome is the name given to the transformation of the most frequent type of leukemia, chronic lymphocytic leukemia, into an aggressive lymphoma. Patients with Richter syndrome have limited response to therapies and dismal survival. The underlying mechanisms of transformation are insufficiently understood and there is a major lack of knowledge regarding the roles of microRNA that have already proven to be causative for most cases of chronic lymphocytic leukemia. Here, by using four types of genomic platforms and independent sets of patients from three institutions, we identified microRNA involved in the transformation of chronic lymphocytic leukemia to Richter syndrome. The expression signature is composed of miR-21, miR-150, miR-146b and miR-181b, with confirmed targets significantly enriched in pathways involved in cancer, immunity and inflammation. In addition, we demonstrated that genomic alterations may account for microRNA deregulation in a subset of cases of Richter syndrome. Furthermore, network analysis showed that Richter transformation leads to a complete rearrangement, resulting in a highly connected microRNA network. Functionally, ectopic overexpression of miR-21 increased proliferation of malignant B cells in multiple assays, while miR-150 and miR-26a were downregulated in a chronic lymphocytic leukemia xenogeneic mouse transplantation model. Together, our results suggest that Richter transformation is associated with significant expression and genomic loci alterations of microRNA involved in both malignancy and immunity.

Abstract In this study the mode of expression of CD5 by human tonsillar CD5 − B cells after stimulation with different agents was investigated. Resting B cells were separated into CD5 + and CD5 − cells and the two cell fractions exposed to phorbol 12‐myristate 13‐acetate (PMA). CD5 − B cells expressed CD5 and maximum CD5 expression was achieved after approximately 60 h of culture. Based upon the proportions of cells that express CD5 as well as those of the cells surviving in culture, it was calculated that 15–25 % of the total CD5 − B cells were induced to express CD5. Unlike CD5 − B cells, CD5 + B cells proliferated vigorously in response to PMA as assessed by [ 3 H] thymidine incorporation and cell cycle analysis in vitro . However, the expression of CD5 by CD5 − B cells was not related to the selective expansion of some CD5 + B cells left over as contaminant cells since this occurred in the absence of cell proliferation. Upon exposure to PMA, CD5 − B cells remained in the G0‐G1 phases of the cell cycle and did not express the Ki67 antigen or incorporate [ 3 H] thymidine. Furthermore, mitomycin C treatment of the CD5 − B cells did not prevent CD5 expression. Phenotypic studies disclosed that CD5 + B cells but not CD5 − B cells expressed CD39. This finding offered the opportunity to carry out an additional control experiment. Separation of the two populations according to the expression of CD39 confirmed the finding obtained by fractionating the cells into CD5 + and CD5 − B cells. The cells induced to express CD5 also expressed CD38 that was not detected on resting CD5 − B cells. In this respect, the CD5 − B cells that converted into CD5 + cells (inducible CD5 + B cells) resembled the cells from the CD5 + B cell fractions that up‐regulated CD5 and also expressed CD38 upon exposure to PMA alone. Another example of coordinate expression of these two antigens was the finding that exposure to PMA in the presence of recombinant interleukin‐4 (rIL‐4) resulted in inhibition of the expression of CD5 and CD38. Although virtually all of the tonsillar CD5 − B cells expressed the CD69 activation marker, no cells other than those co‐expressing CD5 and CD38 were induced to express CD5 by PMA alone. Resting CD5 − B cells failed to express CD5 and/or CD38 when cultured with PMA in the presence of EL4 T cells and IL‐4‐free T cell supernatants. Although this combination of stimuli induced a vigorous cell proliferation, the failure to express CD5 and CD38 was not related to cell cycling, since mitomicyn C‐treated CD5 − B cells also failed to express CD5 or CD38 when exposed to PMA in the presence of EL4 cells with or without T cell supernatants. Thus, exposure to T cells alone was sufficient to down‐regulate CD5 and CD38 expression. Collectively, the above findings indicate that mature CD5 − B cells can follow distinct pathways of differentiation depending upon the nature of the stimuli encountered, and that CD5 expression may identify a special B cell subset or a particular stage of B cell differentiation.

The effects of short‐chain fatty acids (SCFA) produced by anaerobic bacteria, namely propionic, butyric and iso‐butyric on T‐cell proliferation was investigated. A dose‐dependent inhibition of both phytohemagglutinin‐induced blastogenesis and mixed lymphocyte culture was observed in the millimolar range of SCFA concentrations. The tested SCFA displayed different levels of suppression. The degree of activity was in the following order: butyrate > propionate > iso‐butyrate. T‐cell inhibition was partially reversed, at least for propionic and iso‐butyric acids, by increasing the concentration of macrophages in the assay system. Furthermore, butyric acid displayed an interesting biphasic stimulation of monocyte interleukin‐1 beta production, a cytokine with a powerful bone‐resorbing activity. Since millimolar concentrations of SCFA are present in gingival fluid from periodontal pockets, the observed results support the role of these byproducts of anaerobic metabolism in the pathogenesis of periodontal diseases.

Abstract Tonsillar resting B cells were separated into CD5 + and CD5 − cell subsets and stimulated with the thymus‐independent mitogens, Staphylococcus aureus Cowan strain I (SAC) or insolubilized anti‐μ monoclonal antibodies (aμAb). CD5 + cells incorporated [ 3 H]thymidine more efficiently than unfractionated cells when stimulated with SAC and their response was augmented by the addition of interleukin (IL)2 to the cultures. CD5 + cells also proliferated in response to aμAb provided that IL 2 was present. SAC‐, but not aμAb‐stimulated CD5 + cells produced IgM and IgG molecules when IL 2 was added to the cultures and also secreted autoantibodies with rheumatoid factor activity and sometimes also with anti‐single‐stranded, but not double‐stranded, DNA activity. The efficient response of CD5 + cells was not explained by the fact that they contained cells already activated in vivo. Thus, they did not express the CD23, CD69, CD71 and CD39 activation markers, failed to incorporated [ 3 H]thymidine and to secrete Ig spontaneously or in response to IL 2 and were found to be in a quiescent state by cell cycle flow cytometric analysis. In contrast to CD5 + cells, CD5 − cells displayed very little or no [ 3 H]thymidine incorporation in response to SAC or to aμAb and their poor responsiveness was not altered by changing either the doses of the stimulants, the timing of the cultures, by co‐culturing the cells together with CD5 + cells, or by adding IL 2 or IL4. Immunofluorescence studies showed that freshly prepared CD5 − cells did not have surface activation markers but that they expressed them following SAC stimulation. Thus, unlike that observed for CD5 + cells, SAC seems to be capable of activating CD5 − cells but does not appear to be a sufficient stimulus for driving the cells into the subsequent phases of the cell cycle. The above findings, that demonstrate marked differences in the response to CD5 + and CD5 − cells to thymus‐independent stimuli, may bear relevance for the understanding of the normal clonal expansion of CD5 + cells as well as for the pathogenesis of autoimmune diseases.

Total and specific IgE were assessed in serum, bronchial lavage (BL) and broncho‐alveolar lavage (BAL) of allergic asthmatics and healthy controls. Serum total IgE were found to be correlated with total IgE in BAL but not in BL. Total IgE/K + ratio in serum and BL was higher in asthmatics than in controls, while the total IgE/albumin ratio was significantly higher in asthmatics than in controls in serum but not in BL and BAL. The mean of specific IgE in serum and BL was significantly higher in the group of patients with positive specific bronchial provocation test (sBPT) than in the group with negative sBPT. Similar results were observed between specific IgE serum level and BL and prick tests (PT). which show that BL does not always reflect the total IgE level of serum; in asthmatics, albumin can not be used to determine the degree of dilution in the recovered fluids; as in the serum, there is agreement between specific IgE in BL and PT or sBPT results.

This study investigates the main functional features of subepithelial (SE) B cells and compares them with those of purified germinal center (GC) and follicular mantle (FM) B cells isolated from the same tonsils. Unlike FM B cells, SE B cells failed to produce polyspecific antibodies in vitro; unlike GC B cells, SE B cells expressed high levels of Bcl-2 and failed to undergo spontaneous apoptosis in vitro. The most striking function of SE B cells was their ability to produce IgM antibodies to T cell-independent type-2 (TI-2) (but not to TI-1) antigens (Ag). These antibodies could not be detected when both FM and GC B cells were stimulated with TI-2 Ag in vitro. Moreover, B cells isolated from peripheral blood were unable to mount a response to TI-2 Ag. The latter finding is consistent with the observation that B cells with the phenotypic features of SE B cells were virtually absent in the peripheral blood and emphasizes the notion that SE B cells belong to a subset of non-recirculating B cells. SE B cells were by far superior to FM B cells in mixed lymphocyte reaction (MLR) stimulation of allogeneic T cells in vitro, although they were not as efficient as dendritic cells (DC). In order to stimulate T cells efficiently, SE B cells had to be exposed to anti-mu antibody, a treatment which induced expression of activation markers such as CD80, CD86, CD69 and CD39, usually absent in resting SE B cells. CD80 and CD86 molecules expressed by SE B cells participated in the chain of events required to promote the proliferation of allogeneic T cells as demonstrated by inhibition tests with the appropriate mAb. The expression of CD80 and CD86 by anti-mu-treated SE B cells was not, however, the sole explanation for their good antigen presenting capacities since the exposure of FM B cells to anti-mu antibody also induced expression of these surface structures. Nevertheless, these cells failed to become good MLR stimulators. Collectively, the above data contribute further to the characterization of a distinct subset of tonsillar B cells which resemble, both phenotypically and functionally, the B cells of the splenic marginal zone.

Objective Interleukin (IL)-21, a member of the IL-2 family, has antitumor activity and is now being tested in non-Hodgkin's lymphoma in combination with anti-CD20 antibodies. IL-21 may either induce apoptosis or promote growth in different lymphoid malignancies. We therefore investigated the IL-21/IL-21R system in follicular lymphoma (FL) cells. Materials and Methods IL-21R expression was studied by reverse transcription polymerase chain reaction, immunofluorescence, and Western blot analyses. Apoptosis was measured by Annexin-V−propidium iodide staining. Signaling via IL-21R was studied using antibodies specific for phosphorylated Janus-activating kinase and signal transducers and activators of transcription proteins by Western Blot. Results IL-21R was found on primary FL cells in 15 of 15 cases at diagnosis and IL-21 increased apoptosis in 10 of 10 FL samples. However, cells from areas of diffuse growth in FL and from two diffuse lymphomas evolved from previous FL, showed low IL-21R expression. The latter were also resistant to IL-21−mediated apoptosis. Among lymphoma cell lines bearing the t(14;18) translocation, only 1 of 7 showed increased apoptosis in response to IL-21 stimulation. This cell line was IL-21R−positive, whereas five of six nonresponsive cell lines showed very low IL-21R expression. Intriguingly, one of the IL-21-resistant cell lines (DOHH2) expressed high levels of IL-21R. Treatment with IL-21 or IL-4 upregulated suppressor of cytokine signaling 3 gene expression in the IL-21−responsive cell line, but not in DOHH2 cells, which showed defective Janus-activating kinase/signal transducers and activators of transcription signaling in response to IL-21, in relationship to the lack of Janus-activating kinase 3 gene expression. Conclusion These data indicate that low IL-21R expression or defective signal transduction downstream IL-21R may cause refractoriness to IL-21−mediated effects in some FL cells. Interleukin (IL)-21, a member of the IL-2 family, has antitumor activity and is now being tested in non-Hodgkin's lymphoma in combination with anti-CD20 antibodies. IL-21 may either induce apoptosis or promote growth in different lymphoid malignancies. We therefore investigated the IL-21/IL-21R system in follicular lymphoma (FL) cells. IL-21R expression was studied by reverse transcription polymerase chain reaction, immunofluorescence, and Western blot analyses. Apoptosis was measured by Annexin-V−propidium iodide staining. Signaling via IL-21R was studied using antibodies specific for phosphorylated Janus-activating kinase and signal transducers and activators of transcription proteins by Western Blot. IL-21R was found on primary FL cells in 15 of 15 cases at diagnosis and IL-21 increased apoptosis in 10 of 10 FL samples. However, cells from areas of diffuse growth in FL and from two diffuse lymphomas evolved from previous FL, showed low IL-21R expression. The latter were also resistant to IL-21−mediated apoptosis. Among lymphoma cell lines bearing the t(14;18) translocation, only 1 of 7 showed increased apoptosis in response to IL-21 stimulation. This cell line was IL-21R−positive, whereas five of six nonresponsive cell lines showed very low IL-21R expression. Intriguingly, one of the IL-21-resistant cell lines (DOHH2) expressed high levels of IL-21R. Treatment with IL-21 or IL-4 upregulated suppressor of cytokine signaling 3 gene expression in the IL-21−responsive cell line, but not in DOHH2 cells, which showed defective Janus-activating kinase/signal transducers and activators of transcription signaling in response to IL-21, in relationship to the lack of Janus-activating kinase 3 gene expression. These data indicate that low IL-21R expression or defective signal transduction downstream IL-21R may cause refractoriness to IL-21−mediated effects in some FL cells.

Several studies have indicated that stimulation via surface immunoglobulin (sIg) may be an important promoting factor for clonal expansion in chronic lymphocytic leukaemia (CLL) (Chiorazzi & Ferrarini, 2003; Stevenson & Caligaris-Cappio, 2004; Chiorazzi et al, 2005). CLL clones expressing unfavourable prognostic markers, such as ZAP-70 or CD38, have viable sIg-dependent signal transducing pathways, while cells from cases with favourable cellular markers are often anergic to sIg cross-linking. In addition, cases with a more aggressive disease are characterized by the expression of sIg encoded by unmutated IGHV/KV/LV genes. These molecules often have polyspecific activity and bind to a battery of antigens, including self-antigens. sIg from indolent cases are encoded by somatically mutated IGHV/VL genes and do not have polyspecific activity, suggesting that continuous stimulation by self-antigens in vivo may drive the clonal expansion in aggressive CLL cases (Chiorazzi & Ferrarini, 2003; Stevenson & Caligaris-Cappio, 2004; Chiorazzi et al, 2005). Finally, neoplastic cells from a substantial number of patients with aggressive clinical courses share ‘stereotyped receptors’, encoded for by identical IGHV DJ and IGHV JL genes and very similar Complimentary-Determining Region 3, suggesting exposure to selective pressure by special antigens at certain points of lymphomagenesis (Messmer et al, 2004; Widhopf et al, 2004; Murray et al, 2008). Most CLL clones express both sIgM and IgD and the two isotypes share the same antigen-specific combining site. The available evidence supports the notion that sIgM actively participates in the process of clonal expansion, but information regarding the role of sIgD is scanty (Zupo et al, 2000; Lanham et al, 2003). The present study investigated the effect of stimulating CLL cells in vitro by cross-linking their sIgD in a cohort of patients collected through a cooperative multicenter study (Gruppo Italiano Studio Linfomi). The data were correlated with clinical features and the findings suggested the involvement of sIgD in clonal expansion. CLL cells, purified from 106 patients, were exposed to anti delta antibody (aδAb) in vitro and apoptosis was measured after 48 h by propidium iodide staining and flow-cytometry (Zupo et al, 2000). Patients were subdivided into three groups, depending upon the type of response observed. In one group (33 cases, I group), inhibition of spontaneous apoptosis was seen, when the apoptosis values following sIgD cross-linking were at least 20% lower than those observed in the absence of cell exposure to aδAb. In a second group (eight cases, D group), exposure to aδAb caused 20% greater cell death than that spontaneously observed in absence of the antibody. In a third group (65 cases, N group), exposure to aδAb failed to modify spontaneous apoptosis below or above 20% of the spontaneous values. Patient clinical courses were correlated with their classification into one of the three groups defined above. Patients in group D or I were grouped together and classified as ‘responders’. Disease progression was measured as the time elapsed from diagnosis to first treatment (TFS). After a median follow up of 4 years, 36 patients were treated. Cox univariate analysis showed that a cell response to aδAb (group D + I) conferred a hazard ratio (HR) for disease progression greater than the absence of response (Fig 1A). All patients were studied for ZAP-70 and CD38 expression, and IGHV mutational status (Fig 1B). At Cox multivariate analysis, response to aδAb remained an independent variable also in the presence of the three other markers (P = 0·009). Response to sIgD cross-linking by CLL cells purified from 106 patients. (A) Cells were purified and cultured with aδAb [goat antihuman δ-chain antibodies (Sigma, St Louis, MO, USA), at the concentration of 10 μg/ml] for 48 h, as described (Zupo et al, 2000). Apoptosis was determined by propidium iodide staining of permeabilized cells at the end of culture period. Cases were divided into three groups depending upon whether there was no change in apoptosis (N) or whether apoptosis was inhibited (I) or enhanced (D) by more than 20% compared to controls not exposed to aδAb. The data of I and D groups were pooled when TFS was considered. By Cox univariate analysis, the risk of treatment start was significantly higher for I + D group. Cox-derived estimated survival curves according to the response to IgD cross linking is also represented. (B) The risk of therapy requirement for patients whose cells expressed for CD38 and ZAP-70 and utilizing unmutated IGHV genes. Overall, these observations suggest that stimulation via sIgD, perhaps by self-antigens, may have pathogenic relevance by facilitating clonal expansion in vivo. Stimulation via sIgD may prevent cell apoptosis in the majority of cases, although this hypothesis may be somewhat speculative, because the outcome of cell stimulation may be related to the number of apoptotic/antiapoptotic signals delivered in vivo. From the standpoint of our experimental design, the outcome of cell stimulation (apoptosis inhibition or induction) following sIgD cross-linking represents a readout system for cell susceptibility to the stimulus. Next, we investigated possible correlations between protein tyrosine phosphorylation following sIgD cross ligation and in vitro response. Preliminary data showed that the maximum tyrosine phosphorylation was observed after 1 min of cell exposure to aδΑb (P = 0·003) (Fig 2A). Tyrosine phosphorylation at 1 min was significantly different in 10 N and 14 I + D cases (Fig 2B). As stimulation may be influenced by sIgD density, we measured sIgD density as mean fluorescence intensity (MFI) by flow-cytometry in N (65 cases) and I + D (38 cases) patients (Fig 2C). The mean MFI value ± SEM was 27 ± 6·7 (median 9) in the whole patient cohort (106). When cases with an MFI ≥ 9 were analysed separately from those with an MFI < 9, it was clear that there was more N cases with lower density surface IgD (42/56 [75%] vs. I + D 14/56, [25%]). Among cases with higher density sIgD, the N cases were 23/47 (49%) and I + D cases were 24/47 (51%). This difference was statistically significant (P = 0·014). (A, B) Protein tyrosine phosphorylation following exposure to aδAb. (A) Cells from 24 cases were exposed to aδAb for various times and protein tyrosine phosphorylation determined as described in (Cutrona et al, 2008). The total amount of phosphorylation was calculated by summing up the phosphorylation of the single bands (1D Image Analysis Software version 3.5; Kodak, Rochester, NY USA) (Cutrona et al, 2008). The Friedman test, which compares the distributions of more related variables, demonstrated significant differences in protein tyrosine phosphorylation after 1 and 10 min or 5 and 10 min of stimulation. (B) Protein tyrosine phosphorylation bands following exposure to aδAb for 1 min was measured in CLL cells from 14 patients from I + D and 10 patients from N group. (C) Correlation between stimulation via surface IgD capacity and cell surface IgD density measured as mean fluorescence intensity (MFI), in N (65 cases) and I + D (38 cases) patient groups. (D) Patients with high and low surface IgD were studied for their TFS. The values recorded were significantly different. Finally, patients with high cellular IgD expression (MFI ≥ 9) had a shorter TFS (Fig 2D) and a 2·6-fold higher HR, determined by Cox univariate analysis, than the group with IgD MFI < 9. sIgD density maintained an independent predictive value when analysed together with ZAP-70, CD38 and IGHV status (P = 0·045). Studies on the same cohort revealed a correlation between an apoptotic response of the CLL cells in vitro to sIgM cross-linking and an unfavourable clinical course. Again, sIgM density correlated with both cellular response and clinical course. Although the issue goes beyond the scope of this letter, it is of note that response to IgM cross-linking is not independent of other prognostic factors (CD38, ZAP-70 and IGHV status) at multivariate analysis. In addition, comparative analysis of the cellular responses to stimulation via sIgM and sIgD revealed heterogeneous cases capable of responding to both, one or none of the stimuli. sIgD density and response to sIgD cross-linking may represent an additional predictor of disease progression, although these parameters may not be of great practical value for everyday clinical practice. Nevertheless, both studies of sIgD density and particularly of the response to stimulation via sIgD appear to provide useful information on the mechanisms that potentially lead to disease progression. Supported from Associazione Italiana Ricerca sul Cancro (AIRC) (to FM, AN and MF), FIRB (Grant no RBIP06LCA9, to MF) MIUR, CIPE (2006-8, CBA project, to SZ), ISS (to SZ), progetto Regione Liguria (to SZ). Progetti Progetti Strategici – Ricerca Finalizzata Ministero Italiano della Salute ‘RFPS_2006_3_33_99_60’ (to GC) and ‘RFPS_2006_340196’ (to FM and MF); progetto ordinario ricerca finalizzata Ministero Italiano della salute-2007 (to GC), progetto Compagnia San Paolo (to GC), the Fondazione Internazionale Ricerche Medicina Sperimentale (FIRMS) provided financial and administrative assistance, Fondazione ‘Amelia Scorza’ onlus, Cosenza, Italy; GISL (Gruppo Italiano Studio Linfomi); and Associazione Italiana contro le Leucemie -Milano. SM is supported by fellowship from the Fondazione Italiana Ricerca sul Cancro (FIRC). We thank Laura Veroni and Brigida Gulino for their valuable secretarial assistance.

Several factors support CLL cell survival in the microenvironment. Under different experimental conditions, IL21 can either induce apoptosis or promote CLL cell survival. To investigate mechanisms involved in the effects of IL21, we studied the ability of IL21 to modulate gene and miRNA expressions in CD40-activated CLL cells. IL21 was a major regulator of chemokine production in CLL cells and it modulated the expression of genes involved in cell movement, metabolism, survival and apoptosis. In particular, IL21 down-regulated the expression of the chemokine genes CCL4, CCL3, CCL3L1, CCL17, and CCL2, while it up-regulated the Th1-related CXCL9 and CXCL10. In addition, IL21 down-regulated the expression of genes encoding signaling molecules, such as CD40, DDR1 and PIK3CD. IL21 modulated a similar set of genes in CLL and normal B-cells (e.g. chemokine genes), whereas other genes, including MYC, TNF, E2F1, EGR2 and GAS-6, were regulated only in CLL cells. An integrated analysis of the miRNome and gene expression indicated that several miRNAs were under IL21 control and these could, in turn, influence the expression of potential target genes. We focused on hsa-miR-663b predicted to down-regulate several relevant genes. Transfection of hsa-miR-663b or its specific antagonist showed that this miRNA regulated CCL17, DDR1, PIK3CD and CD40 gene expression. Our data indicated that IL21 modulates the expression of genes mediating the crosstalk between CLL cells and their microenvironment and miRNAs may take part in this process.

The present study describes the case of a 45-year-old man diagnosed with metastatic lung adenocarcinoma, which harbored a deletion within exon 19 of the epidermal growth factor receptor (EGFR) gene. The patient was subsequently treated with gefitinib (250 mg/day orally from May 2013 to March 2014), but developed acquired resistance to the drug following 11 months of treatment. Tumor burden molecular analysis was performed on a tumor rebiopsy and plasma sample, and histological analysis was also performed on the tumor rebiopsy. A small cell transformation retaining the original EGFR mutation was detected in the tumor rebiopsy, while the T790M mutation together with the activating ex19del mutation were identified only in the plasma sample. The patient was treated with cytotoxic chemotherapy (off-label schedule with epirubicin 80 mg/mq and paclitaxel 160 mg/mq every 21 days for 6 cycles) and radiation (50.4 Gy administered in 28 fractions of 1.8 Gy once daily for 5.5 weeks) specific for small cell lung cancer, and may also have benefitted from treatment with a third generation T790M-specific EGFR-TKI. To better describe the mechanisms of resistance to TKI inhibitors and to optimize therapeutic regimens, the simultaneous analysis of tumor biopsies and circulating tumor DNA should be considered.

Oncogene-targeted therapies based on mutated BRAF-and/or MEK-specific inhibitors have been developed for melanoma treatment.Although these drugs induce tumor regression in a high percentage of patients, clinical responses are frequently limited in time and tumors often recur.Recent studies suggested that the combination of BRAF/MEK inhibition with immunotherapy could represent a promising strategy for the cure of melanoma.NK cells are suitable effectors for tumor immunotherapy.Here we show that PLX4032 (a mutant BRAF V600 inhibitor) had no effect on the functional properties of NK cells cultured in the presence of IL-2 or IL-15.In contrast, PD0325901 (a MEK inhibitor) induced the down-regulation of the main activating NK receptors and inhibited NK cell function.Importantly, PD0325901 did not affect the anti-tumor activity of NK cells that had been exposed to a combination of IL-15 and IL-18.In addition, both PLX4032 and PD0325901 did not exert any inhibitory effect on in vitro IL-2 or IL-15 pre-activated NK cells.Our data may provide a rationale for future clinical protocols that combine IL-15/IL-18 cytokine administration with MEK inhibitors.In addition, they suggest that oncogene-targeting drugs are compatible with NK-based adoptive therapy.

Repair of the oxidized purine 8-oxo-7,8-dihydroguanine (8-oxoGua) is inefficient in cells belonging to the B complementation group of Cockayne syndrome (CS-B), a developmental and neurological disorder characterized by defective transcription-coupled repair. We show here that cells belonging to the A complementation group (CS-A) are also defective in repair of 8-oxoGua and we demonstrate that expression of the Escherichia coli formamidopyrimidine DNA glycosylase (FPG) completely corrects the repair deficiency in both CS-A and CS-B cells. Phenotypically, CS-A cells are normally sensitive to toxicity and micronuclei induced by the oxidizing agent potassium bromate. CS-B cells display sensitivity to elevated concentrations of potassium bromate but this is not compensated by FPG expression, suggesting toxicity of lesions that are not FPG substrates. The data indicate that 8-oxoGua is not a major toxic and clastogenic lesion in CS cells.

To the editor: In their work Yri et al show that non-Hodgkin lymphoma (NHL) patients undergoing or having received rituximab (anti-CD20 mAb)–containing regimens within the previous 6 months are unable to generate an antibody response to the AS-03–adjuvanted A/H1N1-2009 pandemic influenza

A risk score based on three biological features (CD38, ZAP‐70, and IGHV mutational status) was previously developed to predict progression‐free survival (PFS) in untreated Binet A CLL patients. Here we perform a score validation analysis in a prospective and independent cohort of patients. Biological markers (CD38, ZAP‐70, and IGHV mutational status) and gene expression profiles (GEP) of leukemic cells from CLL patients included in a prospective multicenter observational study (O‐CLL1‐GISL protocol, clinicaltrial.gov ID:NCT00917549) were used to assess the value and reproducibility of this score. To date, 468 Binet A patients were classified as low‐ (0 positive marker), intermediate‐ (1 positive marker), or high‐risk (2 or 3 positive markers) using the progression risk score. The 3‐year PFS probability was 91.7%, 82.9%, and 57.4% for low‐, intermediate‐, and high‐risk ( P &lt; 0.0001) cases, respectively. These values were similar to those found in the original cohort. At Cox multivariate analysis, Rai stage, absolute lymphocyte count, progression risk score, and β‐2 microglobulin maintained an independent prognostic impact on PFS. This score remained a predictor of progression when analysis was limited to 371 Rai 0 cases ( P &lt; 0.0001). Finally, the cells from the different CLL risk groups showed differences in their gene expression patterns. These results confirm the ability of this progression risk score to predict PFS among Binet A patients. The utility of the score was also extended by demonstrating that it retains prognostic value when applied exclusively to Rai 0 patients. Specific transcriptional patterns were significantly associated with risk groups. Am. J. Hematol. 89:743–750, 2014. © 2014 Wiley Periodicals, Inc.

An autocrine/paracrine loop involving IL-23 and the IL-23R complex drives CLL cell expansion and represents a potential therapeutic target.

In order to investigate the role of microRNAs in the pathogenesis of different B-cell lymhoma subtypes, we have applied an array-based assay to a series of 76 mixed non-Hodgkin B-cell lymphomas, including Burkitt's lymphoma (BL), diffuse large B-cell lymphoma, primary mediastinal B-cell lymphoma, mantle cell lymphoma (MCL) and follicular lymphoma. Lymphomas clustered according to histological subtypes, driven by two miRNA clusters (the miR-29 family and the miR-17-92 cluster). Since the two miRNA clusters are known to be MYC-regulated, we investigated whether this would be supported in MYC-driven experimental models, and found that this signature separated BL cell lines and a MYC-translocated MCL cell lines from normal germinal center B-cells and other B-cell populations. Similar results were also reproduced in tissue samples comparing BL and reactive lymph node samples. The same series was then quantitatively analyzed for MYC expression by immunohistochemistry and MYC protein levels were compared with corresponding miRNA signatures. A specific metric was developed to summarize the levels of MYC-related microRNAs and the corresponding protein levels. We found that MYC-related signatures are directly related to MYC protein expression across the whole spectrum of B-cells and B-cell lymphoma, suggesting that the MYC-responsive machinery shows predominantly quantitative, rather than qualitative, modifications in B-cell lymphoma. Novel MYC-related miRNAs were also discovered by this approach. Finally, network analysis found that in BL MYC-related differentially expressed miRNAs could control, either positively or negatively, a limited number of hub proteins, including BCL2, CDK6, MYB, ZEB1, CTNNB1, BAX and XBP1.

We report a case of an elderly woman presenting with a huge cervical mass invading the tracheal lumen. Diagnosed as invasive poorly differentiated thyroid cancer, after an endotracheal biopsy, stenting and radiotherapy, it was judged eligible for total thyroidectomy, but surgery was delayed due to pulmonary thromboembolism. The patient was therefore treated with lenvatinib with a neoadjuvant intent until hemodynamic stability was obtained. Thyroidectomy and radioiodine therapy were then performed and the postdose scan revealed an area of modest uptake in the anterior part of the neck. The patient is now in a good clinical status and she continues her follow-up program without any adjuvant therapy.

Abstract This study investigated the response of different CD5 − B cell subsets to CD40 monoclonal antibody (mAb) in various combinations with interleukin (IL)‐4 or rabbit anti‐human μ chain antibody (a‐μ‐Ab). The different CD5 B cell subsets were isolated from tonsillar B cell suspensions depleted of CD5 + B cells and subsequently fractionated on Percoll density gradients. While resting CD5 + B cells proliferated and produced IgM molecules in response to a‐μ‐Ab, IL‐4 and CD40 mAb as well as to Staphylococcus aureus Cowan strain I (SAC) and IL‐2, resting CD5 − B cells, which were co‐purified in the same 60% Percoll fractions, consistently failed to respond. These cells were, however, activated by the stimuli employed, as demonstrated by their capacity to express the surface activation markers CD69, CD25 and CD71. Resting CD5 + B cells had the typical phenotype of mantle zone B cells (IgM + IgD + CD39+ CD38 − CD10 − CDw75 dim ), whereas resting CD5 − B cells were CD38 − CD39 − CD 10 − CDw75 intermediate and expressed surface IgM but relatively little surface IgD and could not be classified as mantle zone or germinal center cells. The finding that purified germinal center cells (CD38 + CD10 + CD39 − CDw75 bright , IgG + ) responded to CD40 mAb and IL‐4 and also to SAC plus IL‐2 further underlined the differences to resting CD5 − B cells. However, some of the data collected suggest possible relationships between CD5 − B cells and germinal center cells. The CD5 − B cells isolated from the 50 % Percoll fraction proliferated in response to a‐μ‐Ab, CD40 mAb and IL‐4 as well as to SAC and IL‐2. These cells had the same mantle zone B cell phenotype as the CD5 + B cells, but their capacity to respond to the stimuli in vitro was unrelated to a possible contamination with CD5 + B cells, as documented by the appropriate controls. Furthermore, upon exposure to SAC or phorbol esters, the large majority of CD5 − B cells from the 50 % Percoll fraction did not express surface CD5 and there was very little if any accumulation of CD5 mRNA. Finally, most of the cycling cells in the stimulated CD5 − B cells did not express CD5. The CD5 − B cells from the 50 % Percoll fraction were comprised of a consistent proportion of cells that expressed surface activation markers. The removal of these cells abrogated the capacity of the suspensions to respond to the stimuli in vitro , possibly suggesting that these cells additional activation signals in vivo which were essential to acquire the capacity to respond and that could not be reproduced in vitro . The present study underlines the phenotypic and functional heterogeneity of CD5 − B cells and contributes to the identification of two subsets of these cells which differ in phenotype, tissue distribution and in vitro responses to different stimuli.

Hippolyte inermis Leach 1814 is a benthic shrimp characterized by a peculiar mechanism of sex reversal influenced by diatom foods. In fact, the appearance of primary females in spring is due to an apoptotic early disruption of the androgenic gland and of the male gonad, triggered by still unknown compounds present in diatoms of the genus Cocconeis. The influence of diatoms on the reproductive ecology and life cycle of planktonic crustaceans has been demonstrated previously: some planktonic diatoms produce aldehydes inducing apoptosis in the embryos and in the larvae of marine copepods, reducing their viability. Both benthic and planktonic diatoms therefore produce compounds having an apoptotic effect on some tissues of target crustaceans, although the ecological significance of the two processes is different: deleterious for copepod populations, regulative for shrimps associated with Posidonia oceanica. In the present article we experimentally administered specific planktonic diatoms, their fractions and compounds known to induce apoptosis in planktonic copepods, to H. inermis postlarvae, to check whether the apoptotic effect is due to an identical family of diatom compounds, and to establish whether the processes observed in the plankton and in the benthos, respectively, are analogous or homologous, from an ecological point of view. Our results indicated that diatom compounds acting in the two systems are different, since both planktonic diatoms and their aldehydes had negligible effects on the sex ratios of cultured shrimps.

The cytology of 130 indeterminate nodules (Thy 3) was retrospectively reviewed according to the British Thyroid Association 2014 classification. Nodules were divided into Thy 3a (atypical features) and Thy 3f (follicular lesion) categories. Histology was available as a reference for 97 nodules. Pre-surgical evaluations comprised biochemical tests, color-Doppler ultrasonography (US), semi-quantitative elastography-US (USE), contrast-enhanced US (CEUS), and mutation analysis from cytological slides. Thyroid malignancy was the final diagnosis for 19% of surgically-treated nodules. No statistically significant difference in the risk of malignancy was found between Thy 3a (26%) and Thy 3f (14%) nodules. Histology of the Thy 3a and Thy 3f nodules showed a higher incidence of Hurtle cell adenomas in Thy 3f (29%) than in Thy 3a (3%) nodules (P=0.01). The only pre-surgical difference concerned the BRAF V600E mutation, which was positive in some Thy 3a but not in any Thy 3f nodules (P=0.04). Receiver-operating characteristic (ROC) analysis was used to obtain cut-off values from US (score), USE (ELX 2/1 strain index), and CEUS (time-to-peak index and peak index) data. The cut-off values were similar for Thy 3a and Thy 3f nodules. Data showed that malignancy can be suspected if the US score is >2, ELX 1/2 strain index >1, time-to-peak index >1, and peak index <1. In a sub-group of 24 revised nodules (12 Thy 3a and 12 Thy 3f) with histology as a reference, the diagnostic power of cumulative pre-surgical analysis by means of US, USE, and CEUS showed high positive and negative predictive values (83% and 100%, respectively) for the presence of malignancy in Thy 3a and Thy 3f nodules. In conclusion, in our series of revised Thy 3 nodules, malignancy was low and displayed no significant differences between Thy 3a and Thy 3f categories. The use of cut-offs based on histology as a reference could reduce surgery. Our data support the conviction that, in mutation-negative Thy 3a and Thy 3f nodules, observation should be the first choice when not all instrumental results are suspect.

In the late B cell differentiation stages, miRNAs expression modifications promoting or inhibiting key pathways are only partially defined.We isolated 29 CD19 + human B cell samples at different stages of differentiation: B cells from peripheral blood; naïve, germinal center (GC) and subepithelial (SE) B cells from tonsils.SE cells were further split in activated and resting B cell.The miRNA expression profile of these B cells was assessed by microarray analysis and selected miRNAs were validated by quantitative RT-PCR and in situ hybridization on normal tonsils.The comparison of all samples showed changes in 107 miRNAs in total.Among 48 miRNAs differentially expressed in naïve, GC and SE cells, we identified 8 miRNAs: mir-323, mir-138, mir-9*, mir-211, mir-149, mir-373, mir-135a and mir-184, strictly specific to follicular cells that had never been implicated before in late stages of B cell development.Moreover, we unveiled 34 miRNAs able to discriminate between CD5 -activated B cells and resting B cells.The miRNAs profile of CD5 -resting B cells showed a higher similarity to naïve CD5 + than CD5 -activated B cells.Finally, network analysis on shortest paths connecting gene targets suggested ZEB1 and TP53 as key miRNA targets during the follicular differentiation pathway.These data confirm and extend our knowledge on the miRNAs-related regulatory pathways involved in the late B cell maturation.

First line drug treatment of follicular lymphoma (FL) patients is followed by a highly variable disease-free time before relapse in about one third of patients. No molecular marker is able to predict efficiently the risk of relapse. We investigated the expression profile of microRNAs (miRNAs) by microarrays and of the tumor microenvironment by immunohistochemistry in 26 FLs and 12 reactive lymph nodes (rLN) as reference. Twenty-nine miRNAs were differentially expressed in FLs compared to rLNs and some of them discriminated grade 1 from 3a FLs. Both FLs and rLNs displayed molecular heterogeneity. FLs grouped into two clusters mostly driven by the tumor T-cell content. Among 21 drug-treated FL patients with an average follow-up of 13.5 years, eight cases relapsed. Twenty-six miRNAs discriminated between relapsed and non-relapsed FLs. Ten miRNAs also correlated with Foxp3+ cells number. Notably, Foxp3+ cells were significantly less in relapsed patients and lower Foxp3+ cell number associated with shorter time-to-relapse. Foxp3+ cells did not co-expressed follicular helper T-cell markers and were therefore classified as regulatory T cells rather than follicular regulatory T-cells. These findings introduce new knowledge about the relationship between miRNA alterations and infiltrating immune cells and show that Foxp3+ cells might be predictive of disease relapse.

We have previously characterized mouse H4 (mH4), a surface glycoprotein recognized by the C398.4A monoclonal antibody. We now show that C398.4A also binds its human putative homolog (hpH4). Both hpH4 and mH4 (1) are selectively expressed by activated T cells and mature thymocytes, (2) are disulfide-linked dimers of two chains (29/37 kDa in humans, 25/29 kDa in mice), whose N-deglycosylation produces a single band at 20 – 21 kDa, and (3) display a low association with CD4 and the TCR. The expression pattern of hpH4 and its biochemical features showed that it is different from other known activation molecules, and this was confirmed when analysis of the tryptic digest of the hpH4 29-kDa band by peptide mass searching using matrix-assisted laser desorption ionization mass spectrometry did not reveal any significant homology with other molecules. In normal lymphoid tissue, hpH4 is expressed by T cells located at the periphery of lymph node germinal centers and paracortical areas. In T cell neoplasia, expression of hpH4 clusters with a subset of peripheral T cell lymphomas with a large-cell component, and with cases of angioimmunoblastic T cell lymphomas. Overall, these data provide evidence for a novel T cell activation molecule that could help in the phenotypic categorization of T cell malignancies.

Abstract In this study, we investigated the c‐myc expression by tonsillar germinal center (GC) B cells using reverse transcriptase‐polymerase chain reaction, flow cytometry, Western blot and in situ immunohistochemical methods. The results obtained demonstrate elevated levels of c‐myc mRNA and of Myc protein in GC B cells compared to those of the other resting or activated tonsillar B cells. Separation of GC B cells into centroblasts and centrocytes revealed that, while differing in their cell cycle status, surface marker expression and morphology, the two cell types had the same propensity to apoptosis and elevated Myc protein expression, thus reinforcing the notion of a close correlation between these two events. Based upon these observations and other considerations it is proposed that elevation of Myc proteins confers to GC B cells a particular propensity to apoptosis, while the subsequent decision between progression into the cell cycle or programmed cell death is dictated by other signals that are delivered in the GC and perhaps operate at the level of other proto‐oncogenes.

We read with great interest the paper by Bernal et al[1][1] on the ability of anti–human IgM antibodies to prevent spontaneous apoptosis of B-cell chronic lymphocytic leukemia (B-CLL) cells in vitro. These findings suggest that receptor engagement by certain antigens, perhaps autoantigens that are

Total body computed tomography (TB‐CT) scan is not mandatory in the diagnostic/staging algorithm of chronic lymphocytic leukemia (CLL). The aim of this study was to determine the value and prognostic significance of TB‐CT scan in early stage CLL patients. Baseline TB‐CT scan was performed in 240 Binet stage A CLL patients (179 Rai low‐ and 61 Rai intermediate‐risk) included in a prospective multicenter observational study ( clinicaltrial.gov ID:NCT00917549). The cohort included 69 clinical monoclonal B lymphocytosis (cMBLs). Patients were restaged considering only radiological data. Following TB‐CT scans, 20% of cases reclassified as radiologic Binet (r‐Binet) stage B. r‐Binet B patients showed a higher incidence of unfavorable cytogenetic abnormalities ( P = 0.027), as well as a shorter PFS ( P = 0.001). At multivariate analysis, r‐Binet stage [HR = 2.48; P = 0.004] and IGHV mutational status [HR = 3.01; P = 0.002] retained an independent predictive value for PFS. Among 179 Rai low‐risk cases, 100 were redefined as r‐Rai intermediate‐risk based upon TB‐CT scan data, showing a higher rate of cases with higher ZAP‐70 ( P = 0.033) and CD38 expression ( P = 0.029) and β2‐microglobulin levels ( P &lt; 0.0001), as well as a shorter PFS than those with r‐Rai low‐risk ( P = 0.008). r‐Rai stage [HR = 2.78; P = 0.046] and IGHV mutational status [HR = 4.25; P = 0.009] retained a significant predictive value for PFS at multivariate analysis. Forty‐two percent of cMBL patients were reclassified as r‐small lymphocytic lymphomas (r‐SLLs) by TB‐CT scan. TB‐CT scan appears to provide relevant information in early stage CLL related to the potential and the timing of patients to progress towards the more advanced disease stages. Am. J. Hematol. 88:539–544, 2013. © 2013 Wiley Periodicals, Inc.

We analyzed individual VH gene rearrangements in 55 consecutive B-chronic lymphocytic leukemia (B-CLL) patients collected from a northeastern region of Italy, stressing the possible differences related to geographic characteristics of the cohorts studied. Considering the percentage of somatic mutations present in the VH gene sequences and using the 98% cut-off value, 38 of the 55 B-CLL (69%) patients displayed somatic hypermutations and 17 (31%) had a germline configuration. Our results confirm and extend the observations of a bias in the use of certain VH, DH, and JH genes among B-CLL cells. The most frequently used VH genes were VH1-69 (12.7%) with VH3-23 (12.7%) and VH4-34 (10.9%). Collectively these genes accounted for 36.3% of the cases. In the mutated cases, the range of mutations varied from 2% to 15%, with a median of 6.5%. VH1-69 (7 cases, all unmutated) carried few mutations as opposed to VH3-23 (7 cases, 5 of which mutated), VH4-34 (6 cases, all mutated), and VH3-30 (5 cases, all mutated), which show a high load of mutations. D3 family genes were found frequently (38.1%) followed by D2 (27.2%) and D6 (18.1%). The individual D segment most frequently used was D3-3, which was present in 16.3% of cases. There was predominance of the JH4 gene (49%) followed by JH6 (40%). Analysis of the distribution of replacement and silent mutations in the mutated sequences using the method of Lossos showed in 39.4% of cases evidence of antigen selection in the framework region and/or complementary determining regions. In comparison with a recent study on B-CLL patients from the Mediterranean area, the VH4-34 gene was significantly overused in the mutated group at a percentage double that of the Italian cohort reported in this study (10.9% vs. 5%), but at a frequency similar to the entire Mediterranean region (10.7%). We also found an over-representation of VH1-69 usage in the germline group, at a frequency (12.7%) higher than previously described by the same authors (Italian 8%, Mediterranean 10.7%). On the contrary, VH3-07 and VH3-49 were not much used in our study (5.4% and 1.8%, respectively) compared with the Italian group (8% and 5.1%). In our study, VH3-23 gene segment was frequently expressed, at frequency as high as that of VH1-69, a finding in keeping with reported B-CLL Italian data, but higher than the entire series of the Mediterranean area (12.7% vs. 9.2%); VH3-21 gene, frequently expressed in northern European CLL but rarely in the Mediterranean area, was completely absent. This biased usage of VH family genes may reflect a geographic leukemic repertoire, perhaps owing to a peculiar genetic background, depending on variations in germline composition of the IgVH locus or to the effect of a potential environmental element less frequently encountered in different regions.

Background . Molecular diagnostics has offered new techniques for searching for mutations in thyroid indeterminate lesions. The study’s aim was to evaluate the BRAF mutations’ incidence in an Italian regional population. Subjects and Methods . 70 Caucasian patients born in Liguria with indeterminate or suspicious cytological diagnoses. Results. A BRAF gene mutation was successfully analyzed in 56/70 patients. The mutation was BRAF V600E in 12/56 cases (21%) and BRAF K601E in 2/56 (4%). Of the BRAF mutated samples on cytological diagnosis (14/56 cases), 2/14 cases (14%) were benign on final histology and 12/14 (86%) were malignant. All BRAF-mutated cases on cytology that were found to be benign on histological examination carried the K601E mutation. Of the nonmutated BRAF cases (42/56, 75%) which were later found to be malignant on definitive histology, 5 cases were follicular carcinomas (36%), 3 cases were incidentally found to be papillary microcarcinomas (22%), 2 were cases papillary carcinomas (14%), 1 was case follicular variant of papillary carcinoma (7%), 1 was case medullary carcinoma (7%), 1 case was Hurtle cell tumor (7%), and 1 case was combined cell carcinoma and papillary oncocytic carcinoma (7%). Conclusions . The presence of the BRAF V600E mutation may suggest a more aggressive surgical approach. BRAF K601E mutation did not correlate with malignancy indexes.

EN4 MoAb was originally described as a MoAb that reacts specifically with human endothelial cells, and the reagent was not assigned to any of the presently known CD. Here, we provide evidence indicating that EN4 reacts with the CD31 antigen. Thus, EN4 stains strongly murine fibroblasts transfected with the human CD31 gene. Furthermore, SDS-PAGE analysis of immunoprecipitates of cell lysates from surface-iodinated Jurkart T cells demonstrated that EN4 and reference CD31 MoAb recognized the same antigen, of 130 kD mol. wt. Finally, both EN4 and CD31 gave the same pattern of reactivity when tested on tonsillar or peripheral blood lymphoid cells by FACS analysis or by immunohistochemistry on sections of a variety of human tissues. EN4, however, proved consistently more efficient than the reference anti-CD31 MoAb as judged by both the intensity of fluorescence or of tissue staining. This property has thus allowed a better characterization of the tissue and cellular distribution of CD31.

Repair of some oxidized purines such as 8-oxo-7,8-dihydroguanine (8-oxoG) is inefficient in human cells in comparison to repair of other major endogenous lesions (e.g. uracil, abasic sites or oxidized pyrimidines). This is due to the poor catalytic properties of hOGG1, the major DNA glycosylase involved in 8-oxoG removal. The formamidopyrimidine DNA glycosylase (FPG) protein from E. coli is endowed with a potent 8-oxoG glycolytic activity coupled with a beta,delta-AP lyase. In this study, we have expressed FPG fused to the enhanced green fluorescent protein (EGFP) in human bladder cells to accelerate the repair of oxidative DNA damage. Cells expressing the fusion protein EGFP-FPG repaired 8-oxoG and AP sites at accelerated rates, in particular via the single-nucleotide insertion base excision repair (BER) pathway and were resistant to mutagenicity of the oxidizing carcinogen potassium bromate. FPG may stably protect human cells from some harmful effects of oxidative DNA damage.

The majority of splenic marginal zone lymphoma (SMZL) patients experience an indolent clinical course; however, some cases transform to a high-grade lymphoma. Cytogenetic analyses have shown that chromosome 7 is the most frequently altered chromosome and, in some cases, 7q deletion has been found as a single aberration, suggesting its association with the development of SMZL. We studied one patient showing clinical features of SMZL with an aggressive course. Immunophenotypic, conventional and molecular cytogenetic techniques were applied to support the diagnosis. The immunophenotype of peripheral blood mononuclear cells showed the presence of 90% B-lymphocytes. Cytogenetic analysis indicated the presence of a stem-line lacking normal chromosomes 7, but showing a der(7) and a ring, and a side-line with additional aberrations: t(2;22), add(8). Fluorescence in situ hybridization analysis revealed a loss of the 7q32 region. Nonclonal rearrangements involving chromosome 7 were also detected. Chromosome 7 rearrangements were studied to investigate their evolution during the development of the pathology. We have shown that in this patient both chromosomes 7 underwent different rearrangements leading to a loss of the 7q32 region and that the ring chromosome originated from chromosome 7 and was associated with a t(7;7) (p22;q31). We conclude that not only the 7q deletion but also the proneness of chromosome 7 to rearrange might have played a role in the progression of this SMZL.

Click to increase image sizeClick to decrease image sizeKeywordsFludarabineCyclophosphamideMitoxantroneFollicular non-Hodgkin's lymphoma

Abstract Objective We performed an external and multicentric validation of the predictive value of abdominal computed tomography ( aCT ) on time to first treatment ( TTFT ) in early stage chronic lymphocytic leukemia ( CLL ) patients. Methods aCT was performed at diagnosis in 181 Rai 0 patients enrolled in the O‐ CLL 1‐ GISL trial (clinicaltrial.gov ID : NCT 00917549). Results Fifty‐five patients showed an abnormal aCT . Patients with an abnormal aCT showed a significantly shorter TTFT than those with normal aCT ( P &lt; 0.0001). At multivariate analysis, aCT ( P = 0.011), β ‐2 microglobulin ( P = 0.019), and CD 38 expression ( P = 0.047) correlated with TTFT . Following IWCLL 2008 criteria, 112 (61.9%) cases remained at Rai 0, while 69 (38.1%) satisfied the criteria of clinical monoclonal B‐cell lymphocytosis ( cMBL ). Reclassified Rai 0 patients with an abnormal aCT showed a significantly shorter TTFT than those with a normal aCT ( P &lt; 0.0001). At multivariate analysis, only aCT ( P = 0.011) correlated with TTFT . Eleven cMBL cases (15.9%) showed an abnormal aCT and were reclassified as small lymphocytic lymphomas ( SLL ); nonetheless, TTFT was similar for cMBL s and SLLs. Conclusion Our results confirm the ability of the abnormal aCT to predict progression in early stage cases.

Heterogeneous expression of the collagen receptor DDR1 in chronic lymphocytic leukaemia and correlation with progression

Due to its relatively slow clinical progression, B cell chronic lymphocytic leukemia (B-CLL) is classically described as a disease of accumulation rather than proliferation.However, evidence for various forms of clonal evolution suggests that B-CLL clones may be more dynamic than previously assumed.We used a nonradioactive, stable isotopic labeling method to measure B-CLL cell kinetics in vivo.Nineteen patients drank an aliquot of deuterated water ( 2 H 2 O) daily for 84 days, and 2 H incorporation into the deoxyribose moiety of DNA of newly divided B-CLL cells was measured by gas chromatography/mass spectrometry, during and after the labeling period.Birth rates were calculated from the kinetic profiles.Death rates were defined as the difference between calculated birth and growth rates.These analyses demonstrated that the leukemic cells of each patient had definable and often substantial birth rates, varying from 0.1% to greater than 1.0% of the entire clone per day.Those patients with birth rates greater than 0.35% per day were much more likely to exhibit active or to develop progressive disease than those with lower birth rates Thus, B-CLL is not a static disease that results simply from accumulation of long-lived lymphocytes.Rather, it is a dynamic process composed also of cells that proliferate and die, often at appreciable levels.The extent to which this turnover occurs has not been previously appreciated.A correlation between birth rates and disease activity and progression appears to exist, which may help identify patients at risk for worsening disease in advance of clinical deterioration. Nonstandard abbreviations used:

note that Fig. 1C incorrectly shows the direct correlation between the Bc12 levels and levels of miR-15a and miR-16-1 instead of the indirect correlation, as presented in the article.

Mycosis Fungoides (MF) is a rare malignant T-cell lymphoma, involving mainly the skin. In 50%–75% of cases, it can involve organs other than skin, with a 11%–14% Central Nervous System involvement (CNS). A 82-year-old woman presented to our Department with a 15-years history of MF with skin lesions. Neurological examination showed dysarthria and a left facio-brachial-crural hemiparesis. A CT scan showed a right fronto-rolandic lesion. A MRI, including DWI, confirmed the presence of the “neoplastic” lesion with slight hemorrhagic component and leptomeningeal contrast enhancement. Molecular TCR rearrangement test by PCR analysis was performed on skin biopsy, showed the presence of a single peak which fits with a monoclonal TCRG gene rearrangement (size 67). Molecular TCR test was also performed on the cerebrospinal fluid (CSF), which confirmed the presence of lymphocyte clone T g/ more expressed with the same size of that observed in the skin biopsy A total body CT scan did not show any lymphnodal or extranodal disease. The patient died after ten days. MF usually occurs in the context of advanced and often histologically transformed cutaneous disease. Isolated CNS involvement is remarkably rare. This case highlights the need for regular neurologic follow-up after the diagnosis of MF, in particular when features that suggest risk of disease progression are present. Furthermore, the analysis of the skin biopsy and above all of CSF by PCR technique, based on our experience, should always be executed in MF patients with signs or symptoms suggesting CNS involvement.

After removal of an ovarian mass in a 43-year-old woman, a struma ovarii was diagnosed.Within this teratoma, a papillary thyroid cancer was found.The tumor was negative for BrAF, NrAs, KrAs, PIK3cA and c-KIT mutations on molecular analysis.Thyroid function and morphology were normal.Thyroidectomy, l-T4 TsH-suppressive therapy and rhTsH-induced radioiodine ablation were performed.so far, the follow-up has been favorable.This is the first case of thyroid cancer in a struma ovarii in which mutations of PIK3cA exons 9 and 20, and c-KIT exons 9, 11 and 13 have been evaluated and the third in which ablation has been performed under rhTsH.The prognosis of patients with thyroid cancer in a struma ovarii is generally poor.In our patient, as in those who undergo ablative radioactive iodine therapy, this was not the case.

note that Fig. 1C incorrectly shows the direct correlation between the Bc12 levels and levels of miR-15a and miR-16-1 instead of the indirect correlation, as presented in the article.

<b>Background:</b> The course of chronic lymphocytic leukaemia/small lymphocytic lymphoma (CLL/SLL) partly depends on the mutational status of the variable region of immunoglobulin heavy chain genes (IgV_H), which defines two subgroups of tumours: mutated and unmutated. The expression of zeta-associated protein 70 (ZAP70) is significantly associated with the more aggressive unmutated forms. <b>Aims:</b> To assess the feasibility of the ZAP70 immunohistochemical test on bone-marrow biopsy (BMB) specimens and to compare the results with those of western blotting (WB) and IgV_H mutational status assessed on neoplastic cells from peripheral blood. <b>Methods:</b> 26 patients with CLL/SLL detected on BMB and with known IgV_H mutational status were selected. ZAP70 was determined by immunohistochemistry (IHC) comparing three antibodies from different sources (Upstate, Cell Signaling, Santa Cruz, California, USA) and two different methods (APAAP and EnVision⁺). In 23 cases, ZAP70 WB results were also available. <b>Results:</b> ZAP70 determination on BMB specimens turned out to be easily feasible with routine procedures with reagents from Upstate and Cell Signaling. The results were concordant with those obtained with WB and mutational status analysis in &gt;80% of the cases with both reagents. Three of four discordant cases were mutated/ZAP70 positive, with two staining weakly for ZAP70 on both WB and IHC. <b>Conclusions:</b> The study confirms the role of ZAP70 as a possible surrogate of mutational status and emphasises its application in routine diagnostics; it discloses a small subset of discordant cases (mutated/ZAP70 weakly positive) that clinically cluster with the more favourable forms.

Abstract We are reporting the outcome of 39 patients with idiopathic myelofibrosis (IMF) who received an allogeneic hemopoietic stem cell transplant (HSCT) at a single Institution. Patients. The conditioning was a thiotepa - cyclophosphamide based reduced intensity regimen, and graft versus host disease (GvHD) prophylaxis was cyclosporin and methotrexate. 30 patients received marrow, 9 peripheral blood. The donor was an HLA identical sibling (ISD) (n=22) or an alternative related or unrelatd donor (UD) (n=17). The median age was 51 (32–67), the median interval diagnosis HSCT was 1015 days (225–7178) and the Lille risk score at the time of transplant was intermediate in 23 and high in 16 patients. Splenectomy was performed in 25 patients as a preparation for transplant. Results. The overall actuarial survival is 50% with a median follow up of 823 days (221– 4356). In univariate analysis favourable factors for survival were a Karnowsky score of 100% (93% vs 37%), an interval diagnosis transplant of less than 1 year (85% vs 55%), an HLA identical sibling donor (68% vs 47%) and splenectomy pre-transplant (60% vs 50%). When combined together, 17 and 22 patients has 0–1 or 2–3 favourable factors pre-transplant: their 4 year actuarial survival is respectively 0% vs 83% (p=.0001). Patients with 0–1 favourable factors, had higher transplant related mortality (41% vs 0%; p=0.009) and higher relapse related death (35% vs 4%, p=0.01). Conclusions. We conclude that allogeneic transplantation is a curative option for patients with idiopathic myelofibrosis. We have identified factors which predict a favourable outcome and may be used to select patients for this procedure. Figure Figure

Differentiated thyroid cancer arising from thyroid follicular epithelial cells is the most frequent endocrine malignancy, and skin metastases are very rare. We describe a case of a 70-year-old women with a history of an indeterminate thyroid nodule on cytology. A painless, erythematous skin nodule of about 7 mm diameter was removed from the scalp and diagnosed as a metastasis from thyroid cancer. After total thyroidectomy, a histological diagnosis of follicular thyroid cancer was made. Two cycles of radioactive iodine were performed. Both the follicular thyroid carcinoma (FTC) and the metastasis were investigated for the presence of BRAF/RAS and TERT promoter mutations. The results showed that the cutaneous metastasis was BRAF wild-type and TERT promoter-mutated (position g.1,295,228 C>T); in contrast, the primary thyroid lesion was negative for both molecular markers.

Using immunofluorescence, RT-PCR, and Western blotting, we have demonstrated the ability of human B cells to express CD4. In each of the 10 lymphoblastoid cell lines (LCL) tested there was variable, but definite, proportion of CD4-positive B cells. Expression of CD4 was related to the cell cycle; CD4 was expressed in the G1 phase and continued at later phases of the cell cycle. CD4 was in part internalized and degraded by the LCL B cells. Surface CD4 was associated tolckand its crosslinking resulted in tyrosine phosphorylation. Additional experiments conducted on freshly prepared tonsillar B cells demonstrated that CD4 was expressed by large activated B cells, but not by small resting B cells. However, not all the activated tonsillar B cells had surface CD4 since germinal center cells were CD4-negative. Crosslinking of CD4 on LCL or on tonsillar activated B cells resulted in apoptosisin vitro,a finding that indicates the capacity of CD4 to deliver functional signals to B cells and to play a regulatory function in their physiology. Exposure of CD4 expressing B cells to gp120 under conditions that resulted in CD4 crosslinking also caused apoptosis suggesting some implications for the pathophysiology of AIDS.

Human tonsillar subepithelial B cells, which are a marginal zone-equivalent B cell subset, respond readily to T-independent type 2 antigens, but not to polyclonal B cell activators in vitro. In this study, subepithelial (SE) B cells were induced to proliferate and mature into plasma cells when co-cultured with activated T cells. The response of SE B cells was not observed when co-cultures were carried out in transwell chambers or in the presence of blocking anti-LFA-1 antibodies, demonstrating the need for a close T-B cell interaction. The presence of soluble CD40 also prevented the B cell response in vitro suggesting a pivotal role of CD40-CD40 ligand interactions. The data are discussed in terms of the T cell dependence of marginal zone (MZ) B cell response and the possible existence of various MZ B cell subsets.

Supplementary Tables 1-5 from Gene Expression Analysis of Angioimmunoblastic Lymphoma Indicates Derivation from T Follicular Helper Cells and Vascular Endothelial Growth Factor Deregulation

&lt;div&gt;Abstract&lt;p&gt;Angioimmunoblastic lymphoma (AILT) is the second most common subtype of peripheral T-cell lymphoma (PTCL) and is characterized by dismal prognosis. Thus far, only a few studies have dealt with its molecular pathogenesis. We performed gene expression profile (GEP) analysis of six AILT, six anaplastic large cell lymphomas (ALCL), 28 PTCL-unspecified (PTCL/U), and 20 samples of normal T lymphocytes (including CD4&lt;sup&gt;+&lt;/sup&gt;, CD8&lt;sup&gt;+&lt;/sup&gt;, and activated and resting subpopulations), aiming to (&lt;i&gt;a&lt;/i&gt;) assess the relationship of AILT with other PTCLs, (&lt;i&gt;b&lt;/i&gt;) establish the relationship between AILT and normal T-cell subsets, and (&lt;i&gt;c&lt;/i&gt;) recognize the cellular programs deregulated in AILT possibly looking for novel potential therapeutic targets. First, we found that AILT and other PTCLs have rather similar GEP, possibly sharing common oncogenic pathways. Second, we found that AILTs are closer to activated CD4&lt;sup&gt;+&lt;/sup&gt;, rather than to resting or CD8&lt;sup&gt;+&lt;/sup&gt; lymphocytes. Furthermore, we found that the molecular signature of follicular T helper cells was significantly overexpressed in AILT, reinforcing the idea that AILT may arise from such cellular counterpart. Finally, we identified several genes deregulated in AILT, including &lt;i&gt;PDGFRA, REL&lt;/i&gt;, and &lt;i&gt;VEGF&lt;/i&gt;. The expression of several molecules was then studied by immunohistochemistry on tissue microarrays containing 45 independent AILT cases. Notably, we found that the vascular endothelial growth factor (VEGF) was expressed not only by reactive cells, but also by neoplastic cells, and that nuclear factor-κB (NF-κB) activation is uncommon in AILT, as suggested by frequent exclusively cytoplasmic c-REL localization. Our study provides new relevant information on AILT biology and new candidates for possible therapeutic targets such as PDGFRA (platelet-derived growth factor α) and VEGF. [Cancer Res 2007;67(22):10703–10]&lt;/p&gt;&lt;/div&gt;

Supplementary File 1 from Gene Expression Analysis of Angioimmunoblastic Lymphoma Indicates Derivation from T Follicular Helper Cells and Vascular Endothelial Growth Factor Deregulation

Supplementary File 1 from Gene Expression Analysis of Angioimmunoblastic Lymphoma Indicates Derivation from T Follicular Helper Cells and Vascular Endothelial Growth Factor Deregulation

&lt;div&gt;Abstract&lt;p&gt;Angioimmunoblastic lymphoma (AILT) is the second most common subtype of peripheral T-cell lymphoma (PTCL) and is characterized by dismal prognosis. Thus far, only a few studies have dealt with its molecular pathogenesis. We performed gene expression profile (GEP) analysis of six AILT, six anaplastic large cell lymphomas (ALCL), 28 PTCL-unspecified (PTCL/U), and 20 samples of normal T lymphocytes (including CD4&lt;sup&gt;+&lt;/sup&gt;, CD8&lt;sup&gt;+&lt;/sup&gt;, and activated and resting subpopulations), aiming to (&lt;i&gt;a&lt;/i&gt;) assess the relationship of AILT with other PTCLs, (&lt;i&gt;b&lt;/i&gt;) establish the relationship between AILT and normal T-cell subsets, and (&lt;i&gt;c&lt;/i&gt;) recognize the cellular programs deregulated in AILT possibly looking for novel potential therapeutic targets. First, we found that AILT and other PTCLs have rather similar GEP, possibly sharing common oncogenic pathways. Second, we found that AILTs are closer to activated CD4&lt;sup&gt;+&lt;/sup&gt;, rather than to resting or CD8&lt;sup&gt;+&lt;/sup&gt; lymphocytes. Furthermore, we found that the molecular signature of follicular T helper cells was significantly overexpressed in AILT, reinforcing the idea that AILT may arise from such cellular counterpart. Finally, we identified several genes deregulated in AILT, including &lt;i&gt;PDGFRA, REL&lt;/i&gt;, and &lt;i&gt;VEGF&lt;/i&gt;. The expression of several molecules was then studied by immunohistochemistry on tissue microarrays containing 45 independent AILT cases. Notably, we found that the vascular endothelial growth factor (VEGF) was expressed not only by reactive cells, but also by neoplastic cells, and that nuclear factor-κB (NF-κB) activation is uncommon in AILT, as suggested by frequent exclusively cytoplasmic c-REL localization. Our study provides new relevant information on AILT biology and new candidates for possible therapeutic targets such as PDGFRA (platelet-derived growth factor α) and VEGF. [Cancer Res 2007;67(22):10703–10]&lt;/p&gt;&lt;/div&gt;

Supplementary Tables 1-5 from Gene Expression Analysis of Angioimmunoblastic Lymphoma Indicates Derivation from T Follicular Helper Cells and Vascular Endothelial Growth Factor Deregulation

Annals of the New York Academy of SciencesVolume 815, Issue 1 p. 171-181 Phenotypic and Functional Characterization of Human Tonsillar Subepithelial (SE) B Cellsa MARIELLA DONO, MARIELLA DONO Servizio di Immunologia Clinica Istituto Nazionale per la Ricerca sul Cancro IST 16132 Genova, ItalySearch for more papers by this authorSIMONA ZUPO, SIMONA ZUPO Servizio di Immunologia Clinica Istituto Nazionale per la Ricerca sul Cancro IST 16132 Genova, ItalySearch for more papers by this authorVITO L. BURGIO, VITO L. BURGIO Istituto di Ia Clinica Medica Generale e Terapia Medica Università“La Sapienza”00100 Roma, ItalySearch for more papers by this authorADRIANO AUGLIERA, ADRIANO AUGLIERA Servizio di Immunologia Clinica Istituto Nazionale per la Ricerca sul Cancro IST 16132 Genova, ItalySearch for more papers by this authorCARLO TACCHETTI, CARLO TACCHETTI Istituto di Anatomia Umana Normale Università di Genova 16132 Genova, ItalySearch for more papers by this authorANNA FAVRE, ANNA FAVRE Istituto di Anatomia Umana Normale Università di Genova 16132 Genova, ItalySearch for more papers by this authorCARLO E. GROSSI, CARLO E. GROSSI Istituto di Anatomia Umana Normale Università di Genova 16132 Genova, Italy Servizio di Patologia Clinica Istituto Nazionale per la Ricerca sul Cancro IST 16132 Genova, ItalySearch for more papers by this authorNICHOLAS CHIORAZZI, NICHOLAS CHIORAZZI Department of Medicine North Shore University Hospital New York University School of Medicine Manhasset, New York 11030Search for more papers by this authorMANLIO FERRARINI, MANLIO FERRARINI Servizio di Immunologia Clinica Istituto Nazionale per la Ricerca sul Cancro IST 16132 Genova, ItalySearch for more papers by this author MARIELLA DONO, MARIELLA DONO Servizio di Immunologia Clinica Istituto Nazionale per la Ricerca sul Cancro IST 16132 Genova, ItalySearch for more papers by this authorSIMONA ZUPO, SIMONA ZUPO Servizio di Immunologia Clinica Istituto Nazionale per la Ricerca sul Cancro IST 16132 Genova, ItalySearch for more papers by this authorVITO L. BURGIO, VITO L. BURGIO Istituto di Ia Clinica Medica Generale e Terapia Medica Università“La Sapienza”00100 Roma, ItalySearch for more papers by this authorADRIANO AUGLIERA, ADRIANO AUGLIERA Servizio di Immunologia Clinica Istituto Nazionale per la Ricerca sul Cancro IST 16132 Genova, ItalySearch for more papers by this authorCARLO TACCHETTI, CARLO TACCHETTI Istituto di Anatomia Umana Normale Università di Genova 16132 Genova, ItalySearch for more papers by this authorANNA FAVRE, ANNA FAVRE Istituto di Anatomia Umana Normale Università di Genova 16132 Genova, ItalySearch for more papers by this authorCARLO E. GROSSI, CARLO E. GROSSI Istituto di Anatomia Umana Normale Università di Genova 16132 Genova, Italy Servizio di Patologia Clinica Istituto Nazionale per la Ricerca sul Cancro IST 16132 Genova, ItalySearch for more papers by this authorNICHOLAS CHIORAZZI, NICHOLAS CHIORAZZI Department of Medicine North Shore University Hospital New York University School of Medicine Manhasset, New York 11030Search for more papers by this authorMANLIO FERRARINI, MANLIO FERRARINI Servizio di Immunologia Clinica Istituto Nazionale per la Ricerca sul Cancro IST 16132 Genova, ItalySearch for more papers by this author First published: 17 December 2006 https://doi.org/10.1111/j.1749-6632.1997.tb52058.xCitations: 5 a This work was supported in part by grants from AIRC, ISS Progetto AIDS, CNR (P. F. Ingegneria Genetica and ACRO), Fondazione Andrea Cesalpino, and NIH (No. AI 10811). Read the full textAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Citing Literature Volume815, Issue1B‐Lymphocytes and AutoimmunityApril 1997Pages 171-181 RelatedInformation

Common variable immunodeficiency (CVID) disorders are the most common form of clinically significant primary immunodeficiencies found in adults. There is now clear evidence that CVID includes a group of clinically and genetically heterogenous conditions. In addition to recurrent infections, some patients are highly prone to granulomatous lesions. Rarely, CVID may be characterized by an increased number of circulating CD8+ T cells with tissue infiltration. We report a unique case of CVID associated with a sarcoidosis-like disease and polyclonal CD8+ T cell expansion with multiple tissue infiltration occurring in a subject with the chromosome translocation t(1;16). No sequence variant in TACI, APRIL, BAFF, ICOS or BTK genes was discovered. Cytometric analysis showed that the chemokine receptors expressed on peripheral and tissue CD8+ T cells are responsible for the tissue homing of the cells. Moreover, CD8+ T cells produced high amounts of IFN-γ, but not IL-4 and IL-17, with regular expression of the transcription factor Vav1. Genes coding for IL-32 and FRAP1, both involved in the regulation of memory T cell differentiation, are located in the translocation breakpoint. This suggests that a chromosomal abnormality plays a role in the clinical features of this phenotypic variant of CVID.

503 Background: LS is largely underdiagnosed although Universal Screening (US) in colorectal cancer (CRC) patients through MisMatch Repair deficiency (MMR-d) testing is widely endorsed. Low adherence to guidelines among oncologists may be partly due to a lack of consensus on whether all MMR-d patients should be referred to GC/Genetic Testing (GT). As BRAF mutation rules out LS, we estimated the increased yield of LS diagnosis from GC /GT which could be obtained by selecting candidates for GC through BRAF testing. Methods: From 2011 to 2016, 1447 consecutive stage I-IV CRC surgical patients at a single institution, underwent immunohistochemistry (IHC) for LS using anti MLH1, MSH2, MSH6 and PMS2 antibodies. Oncologists were invited to refer all MMR-d patients to GC/GT. BRAFV600E testing was carried out only in case of MLH1 protein loss at IHC. Results: MMR-d was found in 194 patients (13%), with 171 showing loss of MLH1 expression (88%). Oncologists referred 27 (16%) to GC. Among the 21 who underwent GC, BRAF testing and GT, 9 were BRAF wild type (wt) (43%) and none had LS. Among the 23 MMR-d patients with loss of expression of MSH2, MSH6 or PMS2 (≠MLH1), oncologists referred 9 to GC (39%): 7 underwent GC / GT and 3 carried LS (43%) at GT. Median age was 76 years (range 30-97) in the MMR-d group, 78 (range 41-97) in the MLH1 group and 63 (range 30-86) in the ≠MLH1 group. Overall, LS was diagnosed in 3 of the 28 MMR-d patients (11%) who underwent GC /GT, possibly an underestimate due to the advanced median age of our MLH1 loss patients. Had we only offered GC to the 9 BRAF wt patients among the 21 with MLH1 loss, we could have avoided 12 (57%) of the GC sessions conducted, increasing the yield of LS diagnosis from 3/28 (11%) to 3/16 (19%) (75% increase). Conclusions: When US for LS is adopted, a GC referral rate reduction of 57% among MLH1 loss patients, and an overall increase in the yield of GC of about 75% can be obtained by testing for BRAF mutation before oncologist referral to GC rather than after. As multistep selection of patients by oncologists may be unfeasible, CRC pathology reports with combined MMR-d and BRAF testing (for MLH1 loss at IHC) and an ‘LS suspicion alert’ could improve oncologists’ awareness of LS and compliance with guidelines.

Universal Screening (US) for Lynch Syndrome (LS) in colorectal cancer (CRC) patients through DNA Mismatch Repair deficiency (dMMR) testing is widely endorsed. However, LS is still largely underdiagnosed mainly because of the low adherence to guidelines among oncologists. Genetic counseling (GC) and testing (GT) are always recommended for CRC patients with MSH2, MSH6 or PMS2 expression loss. However, approximately 80% of all dMMR cases have MLH1 expression loss which is not caused by a germline mutation, but rather by epigenetic methylation of the MLH1 promoter. As BRAF mutation and hypermethylation of the MLH1 promoter (reflex testing) rule out LS in these patients, we estimated the increased rate of LS diagnosis from GC/GT which is obtained by selecting candidates for GC through reflex testing. From April 2011 to February 2020, all consecutive stage I-IV CRC surgical patients at our hospital, underwent immunohistochemistry (IHC) for dMMR using anti MLH1, MSH2, MSH6, and PMS2 antibodies. Oncologists were asked to refer all dMMR patients to GC/GT. Starting in 2019, reflex testing was implemented for all patients with MLH1 protein loss at IHC, therefore oncologists were asked to refer only the patients with wild-type BRAF who had no hypermethylation of the MLH1 promoter. BRAFV600E was tested using both IHC and real-time PCR Easy BRAF kit or Sanger sequencing. Overall, 2532 CRCs were analyzed and dMMR was found in 340 (13%), with 302 showing loss of MLH1 expression (89%). Oncologists referred 47 of the patients (14%) to GC. Among the 38 dMMR patients with loss of expression of MSH2, MSH6 or PMS2 (≠MLH1), oncologists referred seven to GC (18%), three of whom were diagnosed with LS (43%). Among 302 dMMR patients with loss of MLH1 expression, oncologists referred 40 to GC (13%), 8 of whom were LS (20%). Between February 2019 and February 2020, a total of 37 dMMR patients were identified. Their median age was 77 (range 44-100). Only one had loss of MSH6 and underwent GC and GT is ongoing. Reflex testing was therefore carried out in 36 with loss of MLH1 (97%). Among these, 30 showed BRAFV600E mutation (83%). Those with wild-type BRAF were tested for hypermethylation of the MLH1 promoter: two showed hypermethylation and were not referred to GC; two were not hypermethylated and were referred to GC/GT, one of whom was diagnosed with LS; the other two are ongoing. Therefore, by implementing reflex testing, we greatly improved the appropriateness of our referrals to GC, avoiding unnecessary sessions (32/36, 89%) and increasing the yield of LS diagnosis from 1/34 (3%) to 1/2 (50%). Reflex testing can drastically improve the appropriateness of GC referrals, decreasing the number of unnecessary GC sessions and significantly increasing the diagnostic yield of GC/GT. As stepwise patient selection by oncologists may be impractical in routine clinical practice, patients with MLH1 loss at IHC should undergo reflex testing and an ‘LS suspicion alert’ on the pathology report could improve oncologists’ awareness of LS and their compliance with guidelines.

In this study we have investigated the ability of human B lymphocytes to produce granulocyte-macrophage colony stimulating factor (GM-CSF) and, in preliminary experiments, granulocyte CSF (G-CSF). The sources of human B cells were surgically removed tonsils from normal individuals and peripheral blood from patients with B cell chronic lymphocytic leukemia (B-CLL). Tonsil B lymphocytes were purified by E rosetting and complement-mediated cytotoxicity with selected monoclonal antibodies and subsequently fractionated by a Percoll density gradient into in vivo activated and resting cells. The latter cell fractions were subsequently cultured with or without stimuli. GM-CSF was detected by a bioassay, G-CSF by an enzyme-linked immunoassay. In vivo and in vitro activated B cells produced GM-CSF, whereas in vivo activated, but not in vitro activated, B lymphocytes produced G-CSF. These results were confirmed by Northern blot experiments with cDNA probes specific for GM-CSF and G-CSF genes. Many B cell suspensions from B-CLL patients produced GM-CSF or G-CSF only following Staphylococcus Aureus Cowan I (SAC) stimulation; in some cases, a spontaneous production or no production at all of the two cytokines was detected. The possible implications of these results for B cell physiology and for the pathogenesis of immunologically mediated diseases will be discussed.

Although lymphoma involvement of the gallbladder, especially by MALT and large-cell types, is rare, this possibility should be considered in patients with symptoms of acute cholecystitis. A cholecystectomy was performed in a 79-year-old male patient with a clinical diagnosis of chronic cholecystitis. Histologically, the specimen showed an incidental finding of a small lymphocytic lymphoma (CLL) by morphologic and immunophenotyping studies, subsequently confirmed with flow cytometric analysis of blood. During follow-up, multiple lymph node enlargement was detected. An axillary node, excised and submitted to our department, was positive for lymphoma involvement. The bone marrow was negative.

e18026 Background: Gefitinib was approved in Italy for treatment of NSCLC pts carrying mutant EGFR in May 2010. Methods: The EGFR FASTnet program was designed to facilitate the exchange of biological material, clinico-pathological data and diagnostic reports between medical oncologists, primary pathologists and referral laboratories through a dedicated website (www.egfrfastnet.it), a call center and a courier. EGFR mutational analysis was carried by Sanger sequencing, Real Time PCR, Pyrosequencing, Fragment Analysis and High resolution melting (HRM). The Italian Association of Medical Oncology (AIOM) and the Italian Society of Surgical Pathology and Cytopathology (SIAPEC-IAP) have full access to the anonymous database of EGFR FASTnet. Results: The EGFR FASTnet network started in July 2010 and as of January 2011, 272 oncologists, 75 primary pathologists and 12 referral laboratories joined the program. In this period 658 pts were enrolled. The cohort of pts was significantly enriched for adenocarcinoma histology (73.4%), female sex (37.5%) and smoking history (never smoker 25.7%, former smoker>15 yrs 21.4%, light smoker 6.1%). Mutational analysis was feasible in 593 pts (90.1%), on the primary tumor (444, 74.8%) or metastases (149, 25.2%). At the registration, 54,4% of the pts had not received yet treatment for advanced disease. Mutational analysis was carried by Sanger sequencing in 67% of the pts. EGFR mutations were found in 84 cases (14.2%), mainly in exons 19 (69%) and 21 (29.8%). A higher mutation rate was found in never smokers (28.6%), former smokers >15 yrs (15.2%) and light smokers (16.7%), as well as in adenocarcinoma/BAC (15.4%) and females (23.2%). Conclusions: The EGFR FASTnet network allows assessment of EGFR mutations in a population of Italian NSCLC pts in centers with or without molecular biology facilities. The pts to be tested for EGFR mutations are spontaneously selected by medical oncologists according to known predictive factors. The results of the mutational analysis from clinical practice are consistent with data from literature. This program is supported by AstraZeneca Italy.

Annals of the New York Academy of SciencesVolume 815, Issue 1 p. 436-438 C-Myc Proto-oncogene Expression by Germinal Center B Cells Isolated from Human Tonsilsa GIOVANNA CUTRONA, GIOVANNA CUTRONA Servizio di Immunologia Clinica Istituto Nazionale per la Ricerca sul Cancro IST 16132 Genova, ItalySearch for more papers by this authorMARIELLA DONO, MARIELLA DONO Servizio di Immunologia Clinica Istituto Nazionale per la Ricerca sul Cancro IST 16132 Genova, ItalySearch for more papers by this authorSANDRA PASTORINO, SANDRA PASTORINO Servizio di Immunologia Clinica Istituto Nazionale per la Ricerca sul Cancro IST 16132 Genova, ItalySearch for more papers by this authorMASSIMO ULIVI, MASSIMO ULIVI Servizio di Immunologia Clinica Istituto Nazionale per la Ricerca sul Cancro IST 16132 Genova, ItalySearch for more papers by this authorVITO LELIO BURGIO, VITO LELIO BURGIO Istituto di Ia Clinica Medica Generale e Terapia Medica Università“La Sapienza'’00100 Roma, ItalySearch for more papers by this authorSIMONA ZUPO, SIMONA ZUPO Servizio di Immunologia Clinica Istituto Nazionale per la Ricerca sul Cancro IST 16132 Genova, ItalySearch for more papers by this authorSILVIO RONCELLA, SILVIO RONCELLA Servizio di Immunologia Clinica Istituto Nazionale per la Ricerca sul Cancro IST 16132 Genova, ItalySearch for more papers by this authorMANLIO FERRARINI, MANLIO FERRARINI Servizio di Immunologia Clinica Istituto Nazionale per la Ricerca sul Cancro IST 16132 Genova, ItalySearch for more papers by this author GIOVANNA CUTRONA, GIOVANNA CUTRONA Servizio di Immunologia Clinica Istituto Nazionale per la Ricerca sul Cancro IST 16132 Genova, ItalySearch for more papers by this authorMARIELLA DONO, MARIELLA DONO Servizio di Immunologia Clinica Istituto Nazionale per la Ricerca sul Cancro IST 16132 Genova, ItalySearch for more papers by this authorSANDRA PASTORINO, SANDRA PASTORINO Servizio di Immunologia Clinica Istituto Nazionale per la Ricerca sul Cancro IST 16132 Genova, ItalySearch for more papers by this authorMASSIMO ULIVI, MASSIMO ULIVI Servizio di Immunologia Clinica Istituto Nazionale per la Ricerca sul Cancro IST 16132 Genova, ItalySearch for more papers by this authorVITO LELIO BURGIO, VITO LELIO BURGIO Istituto di Ia Clinica Medica Generale e Terapia Medica Università“La Sapienza'’00100 Roma, ItalySearch for more papers by this authorSIMONA ZUPO, SIMONA ZUPO Servizio di Immunologia Clinica Istituto Nazionale per la Ricerca sul Cancro IST 16132 Genova, ItalySearch for more papers by this authorSILVIO RONCELLA, SILVIO RONCELLA Servizio di Immunologia Clinica Istituto Nazionale per la Ricerca sul Cancro IST 16132 Genova, ItalySearch for more papers by this authorMANLIO FERRARINI, MANLIO FERRARINI Servizio di Immunologia Clinica Istituto Nazionale per la Ricerca sul Cancro IST 16132 Genova, ItalySearch for more papers by this author First published: 17 December 2006 https://doi.org/10.1111/j.1749-6632.1997.tb52096.xCitations: 2 a This work was supported in part by grants from AIRC, ISS Progetto AIDS, CNR (P. F. Ingegneria Genetica and ACRO), Fondazione Andrea Cesalpino, and NIH (No. AI 10811). Read the full textAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL REFERENCES 1 Liu, Y. J., G. D. Jonson & I. C. M. MacLennan. 1992. Immunol. Today 13: 17– 21. 2 Berek, C. 1992. Immunol. Rev. 126: 5– 19. 3 Küppers, R., M. Zhao, M-L. Hansmann & K. Rajewski. 1993. EMBO J. 12: 4955– 4967. 4 Osmond, D. G. 1993. The turnover of B-cell population. Immunol. Today 14: 34– 37. 5 Cutrona, G., M. Ulivi, F. Fais, S. Roncella & M. Ferrarini. 1995. J. Exp. Med. 181: 699– 711. 6 Dono, M., S. Zupo, R. Masante, G. Taborelli, N. Chiorazzi & M. Ferrarini. 1993. Eur. J. Immunol. 23: 873– 881. 7 Zupo, S., E. Rugari, M. Dono, G. Taborelli, F. Malavasi & M. Ferrarini. 1994. Eur. J. Immunol. 24: 1218– 1222. 8 Valdez, H. M., C. Guret, O. De Bouteiller, I. Fugier, J. Banchereau & Y. J. Liu. 1996. J. Exp. Med. 183: 971– 977. Citing Literature Volume815, Issue1B‐Lymphocytes and AutoimmunityApril 1997Pages 436-438 ReferencesRelatedInformation

To extent our knowledge on the cytokines possibly involved in the pathophysiology of B-cell chronic lymphocytic leukemia (B-CLL), the mRNA expression of a panel of 10 cytokines was investigated on purified B-CLL cells using a reverse-transcriptase polymerase chain reaction method. Whereas negative RT-PCR signals were recorded for interleukin-1 alpha (IL-1 alpha), IL-2, IL-3, IL-4, IL-5, IL-7, tumor necrosis factor beta (TNF beta), and granulocyte-macrophage colony-stimulating factor, we detected the expression of IL-1 beta, IL-6 and TNF alpha. Furthermore, the constitutive expression of IL-8 mRNA was observed in all 17 B-CLL samples analyzed. mRNA expression was associated with the capacity of the leukemic cells to release IL-8 both constitutively (4.6 +/- 8.1 SD ng/mL) and, to a further extent, after stimulation (14.5 +/- 19.4 ng/mL). The circulating levels of IL-8 were also evaluated in 12 untreated B-CLL sera samples and the overall mean level was significantly higher (P &lt;.01) than in normal sera. In addition, supernatants of purified B-CLL cells cultured in the presence of 12-O- tetradecanoylphorbol-13-acetate showed chemotactic activity towards neutrophils; this activity was neutralized in the presence of an anti- IL-8 antiserum. The mRNA for IL-8 was absent in five B-cell preparations from hairy cell leukemia cases and in four B-cell lines. Normal tonsil CD5+ B cells showed a low expression of IL-8 mRNA only in two of the nine preparations tested and the overall quantity of IL-8 released by these cells after 3 days' incubation was significantly lower compared with that released by B-CLL cells (0.4 +/- 0.3 and 1.6 +/- 0.9 ng/mL under basal and stimulated conditions, respectively). These findings point to an involvement of a member of the proinflammatory chemokine supergene family in human CD5+ B lymphocytes. The different IL-8 behavior observed between B-CLL cells and their normal counterpart is likely to reflect an activation state of the leukemic population.

Abstract Background: Over the last two decades several phenotypic, molecular, and chromosomal markers have been identified that are significantly associated with the prognosis of CLL patients. Therefore, clinicians managing CLL patients would benefit from a simplified prognostic index. Methods: We analyzed prospectively collected data from 337 Binet A CLL patients enrolled in Italian the O-CLL1-GISL protocol with the aim of developing scores capable of predicting treatment free survival (TFS). Factors independently associated with TFS were included in the prognostic indexes. To account for differences in the magnitude of the association between the individual independent factors and TFS, we assigned a weighted risk score to each factor based on ranges of their corresponding hazard ratios (HRs) (i.e., 1 point for HR 1.1-1.9; 2 points for HR 2.0-2.9, etc.). The total risk score was then calculated by the sum of the ratings of each factor on individual basis. Risk groups were identified combining risk categories with a non-statistically different TFS. Results: We developed two scores based on weighted multivariable models: the first included clinical and laboratory parameters [clinical score (c-score)], while the second was based on biological markers [biological score (b-score)] (Table 1). The c-score allowed to predict the TFS of patients through the combination of Rai stage, b2-microglobulin and absolute lymphocyte count (ALC), while the b-score predicted TFS by IGHV mutational status and CD38 expression. The c-score showed a C-statistic of 0.72, while the b-score was 0.67, although cases stratified according to the b-score showed a more specific mRNA/microRNA profile. When the two scores were forced in a multivariate analysis, both showed an independent predictive value on TFS with a similar HR, demonstrating their complementarity. Thus, we attempted to integrate the two scores performing a further multivariate analysis in which all parameters, significantly associated with TFS at univariate analysis, were tested (Table 1). ALC, Rai stage, b2-microglobulin together with IGHV mutational status, resulted independently associated with TFS. We constructed a weighted score [comprehensive score (co-score)], including all the above 4 variables, which allowed the identification of 3 different risk groups with significantly different TFS (Figure 1). The C-statistic of the g-score was 0.75, showing a better concordance than the other two scores. Moreover, its validity was externally validated in a series of 297 newly diagnosed Binet A CLL patients from the Mayo Clinic, USA. Conclusions: Using this multistep process and external validation, we developed a score with high discriminatory power and predictive significance on the individual patient level. Table 1. Univariate and multivariate Cox proportional Hazards Models Variable Univariate analysis Multivariate analysis Clinical model Biological model Comprehensive model HR (95% CI) P HR (95% CI) P score HR (95% CI) P score HR (95% CI) P score Age (years) &lt;60/&gt;60 1.12 (0.73-1.74) 0.59 - - - - - - - - - Sex Male/Female 0.93 (0.6-1.44) 0.93 - - - - - - - - - Rai stage 0/I-II 2.30 (1.47-3.50) &lt;0.0001 2.13 (1.24-3.03) 0.004 0/2 - - - 1.76 (1.11-2.78) 0.015 0/1 ALC (109/L) &lt;10/&gt;10 3.43 (1.99-5.92) &lt;0.0001 2.91 (1.85-5.20) &lt;0.0001 0/2 - - - 2.70 (1.54-4.72) 0.001 0/2 b-2 microglobulin normal/elevated 3.04 (1.96-4.70) &lt;0.0001 2.78 (1.79-4.30) &lt;0.0001 0/2 - - - 2.65 (1.66-4.21) &lt;0.0001 0/2 LDH normal/elevated 1.25 (0.57-2.71) 0.57 - - - - - - - - - CD38 negative/positive 3.22 (2.06-5.02) &lt;0.0001 - - - 2.11 (1.15-3.16) 0.02 0/2 1.40 (0.80-2.42) 0.24 - ZAP-70 negative/positive 2.34 (1.51-3.61) &lt;0.0001 - - - 1.21 (0.70-2.16) 0.485 - 1.0 (0.98-1.01) 0.72 - IGHV mutated/unmutated 3.57 (2.32-5.50) &lt;0.0001 - - - 2.10 (1.12-3.90) 0.019 0/2 2.39 (1.27-4.50) 0.007 0/2 FISH risk low+int/high 2.93 (1.46-5.90) 0.002 - - - 1.65 (0.76-3.34) 0.216 - 1.80 (0.84-3.88) 0.13 - Abbreviations: ALC: absolute lymphocyte count; CI: confidential interval; HR: hazard ratio. Figure 1. TFS according to comprehensive progression risk score. Figure 1. TFS according to comprehensive progression risk score. Disclosures Shanafelt: Cephalon: Research Funding; Glaxo-Smith-Kline: Research Funding; Genentech: Research Funding; Hospira: Research Funding; Celgene: Research Funding; Jannsen: Research Funding; Polyphenon E International: Research Funding; Pharmacyclics: Research Funding. Kay:Genentech: Research Funding; Celgene Corporation: Membership on an entity's Board of Directors or advisory committees, Research Funding; Gilead: Membership on an entity's Board of Directors or advisory committees, Research Funding; Hospira: Research Funding; Tolero Pharma: Research Funding; Pharmacyclics: Research Funding.