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Patients with chronic lymphocytic leukemia (CLL) progressing on ibrutinib constitute an unmet clinical need. Though BTK and PLCG2 mutations are associated with ibrutinib resistance, their frequency and relevance to progression are not fully understood. In this multicenter retrospective observational study, we analyzed 98 patients with CLL treated with ibrutinib (49 relapsing after an initial response and 49 still responding after ≥1 year of continuous treatment) using a next-generation sequencing (NGS) panel (1% sensitivity) comprising 13 CLL-relevant genes including BTK and PLCG2. BTK hotspot mutations were validated by droplet digital PCR (ddPCR) (0.1% sensitivity). By integrating NGS and ddPCR results, 32/49 relapsing cases (65%) carried at least 1 hotspot BTK mutation and/or PLCG2 mutation(s); in 6/32, BTK mutations were only detected by ddPCR [variant allele frequency (VAF) 0.1-1.2%]. BTK/PLCG2 mutations were also identified in 6/49 responding patients (12%; 5/6 VAF

Chronic lymphocytic leukemia (CLL) is characterized by the existence of subsets of patients with (quasi)identical, stereotyped B-cell receptor (BcR) immunoglobulins. Patients in certain major stereotyped subsets often display remarkably consistent clinicobiological profiles, suggesting that the study of BcR immunoglobulin stereotypy in CLL has important implications for understanding disease pathophysiology and refining clinical decision-making. Nevertheless, several issues remain open, especially pertaining to the actual frequency of BcR immunoglobulin stereotypy and major subsets, as well as the existence of higher-order connections between individual subsets. To address these issues, we investigated clonotypic IGHV-IGHD-IGHJ gene rearrangements in a series of 29 856 patients with CLL, by far the largest series worldwide. We report that the stereotyped fraction of CLL peaks at 41% of the entire cohort and that all 19 previously identified major subsets retained their relative size and ranking, while 10 new ones emerged; overall, major stereotyped subsets had a cumulative frequency of 13.5%. Higher-level relationships were evident between subsets, particularly for major stereotyped subsets with unmutated IGHV genes (U-CLL), for which close relations with other subsets, termed “satellites,” were identified. Satellite subsets accounted for 3% of the entire cohort. These results confirm our previous notion that major subsets can be robustly identified and are consistent in relative size, hence representing distinct disease variants amenable to compartmentalized research with the potential of overcoming the pronounced heterogeneity of CLL. Furthermore, the existence of satellite subsets reveals a novel aspect of repertoire restriction with implications for refined molecular classification of CLL. Key Points: • In a series of 29 856 CLL patients, the incidence of BcR stereotypy peaked at 41%. • Higher-order relations exist between stereotyped subsets, particularly for those from U-CLL, for which satellite subsets were identified.

The expression of BCL6 in B-cell lymphoma can be deregulated by chromosomal translocations, somatic mutations in the promoter regulatory regions, or reduced proteasome-mediated degradation. FBXO11 was recently identified as a ubiquitin ligase that is involved in the degradation of BCL6, and it is frequently inactivated in lymphoma or other tumors. Here, we show that FBXO11 mutations are found in 23% of patients with Burkitt lymphoma (BL). FBXO11 mutations impaired BCL6 degradation, and the deletion of FBXO11 protein completely stabilized BCL6 levels in human BL cell lines. Conditional deletion of 1 or 2 copies of the FBXO11 gene in mice cooperated with oncogenic MYC and accelerated B-cell lymphoma onset, providing experimental evidence that FBXO11 is a haploinsufficient oncosuppressor in B-cell lymphoma. In wild-type and FBXO11-deficient BL mouse and human cell lines, targeting BCL6 via specific degraders or inhibitors partially impaired lymphoma growth in vitro and in vivo. Inhibition of MYC by the Omomyc mini-protein blocked cell proliferation and increased apoptosis, effects further increased by combined BCL6 targeting. Thus, by validating the functional role of FBXO11 mutations in BL, we further highlight the key role of BCL6 in BL biology and provide evidence that innovative therapeutic approaches, such as BCL6 degraders and direct MYC inhibition, could be exploited as a targeted therapy for BL.

In chronic lymphocytic leukemia (CLL), the hypoxia-inducible factor 1 (HIF-1) regulates the response of tumor cells to hypoxia and their protective interactions with the leukemic microenvironment. In this study, we demonstrate that CLL cells from TP53-disrupted (TP53 dis) patients have constitutively higher expression levels of the α-subunit of HIF-1 (HIF-1α) and increased HIF-1 transcriptional activity compared to the wild-type counterpart. In the TP53 dis subset, HIF-1α upregulation is due to reduced expression of the HIF-1α ubiquitin ligase von Hippel-Lindau protein (pVHL). Hypoxia and stromal cells further enhance HIF-1α accumulation, independently of TP53 status. Hypoxia acts through the downmodulation of pVHL and the activation of the PI3K/AKT and RAS/ERK1-2 pathways, whereas stromal cells induce an increased activity of the RAS/ERK1-2, RHOA/RHOA kinase and PI3K/AKT pathways, without affecting pVHL expression. Interestingly, we observed that higher levels of HIF-1A mRNA correlate with a lower susceptibility of leukemic cells to spontaneous apoptosis, and associate with the fludarabine resistance that mainly characterizes TP53 dis tumor cells. The HIF-1α inhibitor BAY87-2243 exerts cytotoxic effects toward leukemic cells, regardless of the TP53 status, and has anti-tumor activity in Em-TCL1 mice. BAY87-2243 also overcomes the constitutive fludarabine resistance of TP53 dis leukemic cells and elicits a strongly synergistic cytotoxic effect in combination with ibrutinib, thus providing preclinical evidence to stimulate further investigation into use as a potential new drug in CLL.

Context Two variants of Richter Syndrome (RS) are recognized, namely the Diffuse Large B-Cell Lymphoma (DLBCL) and the Hodgkin's Lymphoma (HL) variant. Clonal relationship, defined as an identity of the immunoglobulin heavy chain variable (IGHV) region sequence between CLL and RS clones, characterizes patients with a poor prognosis. Objective To describe and molecularly profile the peculiar case of a 63-year-old patient with a CD5+ Monoclonal B-Cell Lymphocytosis (MBL), who sequentially developed a DLBCL-RS followed by a HL-RS. Patients & Methods DLBCL-RS and HL-RS were diagnosed by histological and immunohistochemical evaluation of lymph node sections. Standard IGHV clonality was assessed by Sanger sequencing (SS) analysis. Allele-Specific Oligonucleotide-droplet digital PCR (ASO-ddPCR) with tumor-specific primers was performed to quantitatively investigate the presence of MBL and RS clones during disease evolution. The mutational profiles of MBL and DLBCL were assessed by next-generation sequencing (NGS). Results Three years after the MBL diagnosis, DLBCL-RS developed. Based on standard IGHV clonality analysis, the lymphoma was considered as a de novo DLBCL and treated accordingly, achieving a persistent complete response. Three years later, the patient developed a clonally unrelated HL-RS, refractory to bendamustine, but successfully treated with brentuximab vedotin and radiotherapy. Results from a more sensitive ASO-ddPCR quantitative analysis showed that the RS-DLBCL clone was already present - although undetectable by standard SS - in the peripheral blood at MBL diagnosis, thus deriving from the transformation of a pre-existing CD5+ clone. Both the MBL and DLBCL-RS clones were carrying low-risk genetic features [i.e., del(13q) and hypermutated IGHV], and NGS analysis resulted in negative for all the evaluated mutations, including TP53 and NOTCH1. IGHV clonality analysis, performed by ASO-ddPCR on the lymph node of HL-RS, confirmed its unrelatedness with both MBL and DLBCL-RS. Conclusions In this case, characterized by a high degree of complexity due to the presence of three sequentially occurring neoplastic clones, the application of modern and sensitive molecular technologies uncovered that DLBCL-RS, initially considered as a de novo lymphoma, was instead the result of the transformation of a pre-existing ancillary clone, already present at the time of first MBL diagnosis.

Dear Editor, Myelodysplastic syndrome with isolated del (5q) and JAK2 mutation is a distinct entity within the World Health Organization (WHO) category myelodysplastic syndromes (MDS) [1]. JAK2 is a gain of function mutation, typically associated with myeloproliferative neoplasms (MPN) [2]. However, approximately 5 % of patients with isolated del (5q) also harbours the JAK2 mutation [3, 4]. No correlation was found between isolated del (5q) JAK2 mutated cases and clinical presentation, morphological features, disease transformation or patient outcome [4, 5], although a few cases presented with higher platelet and WBC count [3]. Lenalidomide was reported to be an effective treatment for the disease [6]. We have recently studied a patient with del (5q) and JAK2 mutation transformed to acute myeloid leukaemia (AML) with complex karyotype and increased JAK2 mutation load. A 66-year-old female presented with moderate anaemia and high platelet count (haemoglobin 100 g/l, WBC 7.2 × 10/l, platelets 800 × 10/l). Neutrophils were 4.95×10/l, eosinophils 0.21×10/l, monocytes 0.14×10/l and lymphocytes 1.9×10/l. No blast cell was present in the peripheral blood. There was no splenoor hepatomegaly. On marrow aspirate, only eight metaphases showing a normal karyotype (46,XX) could be analysed, due to the lack of growth of an adequate number of metaphases and the poor cellularity of the sample. FISH analysis was limited to the study of 5q and demonstrated 5q31 deletion in 12 % of the nuclei. Thus, additional aberrations could not be identified. BCR/ABL rearrangements (p210, p190) were absent, while JAK2 mutation displayed an allele burden of 4.28 %. Bone marrow (BM) biopsy showed a 70 % cellularity, slight dyserythropoiesis, moderate myeloid hyperplasia and marked proliferation of the megakaryocytic lineage with some dysplasia. A few megakaryocytes were large with hyperlobulated nuclei; others were medium-sized, with round and/or hypolobulated nuclei. There were 3 % CD34-positive blasts and moderate reticulin fibrosis (WHOMF-2). (Fig. 1a– c, e). A strong nuclear p53 immunostaining was detected in 2 % haematopoietic cells (Fig. 1d). We proposed a diagnosis of MDS with del (5q) and JAK2 mutation. The patient did not receive any therapy. No organomegaly nor leukoerythroblastosis was reported during the course of the disease. Twenty-seven months later, she was admitted to the hospital because of persistent fever, severe anaemia and neutropenia (Hb level 7.5 g/l, WBC 2.3×10/l, neutrophils 0.75×10/l, platelets 137×10/l). There were 5 % blast cells in the peripheral blood. Chromosome banding analysis showed the following: 46, XX [1] / 45,XX, −7, del(5)(q13q33), del(13)(q14q34) [7] / 45, idem, del(2)(p12) [2]. FISH analysis demonstrated −7, del(5)(q31), del(13)(q14) (80 %) without BCR/ABL, AML1/ETO, PML/RARA, inv(16), MLL(11q23) translocation and +8. Molecular analysis did not identify FLT3 (ITD, D835), NPM1 (exon 12) or MLL-PTD mutations. Real-time quantitative polymerase chain reaction (RQ-PCR) detected 4164 WT1 copies/10 ABL copies and 55.6 % JAK2 mutation load. p53 sequencing analysis of exons 2–11 showed * Achille Pich achille.pich@unito.it

Hepatosplenic T cell lymphoma (HSTCL) is a type of hematologic neoplasia with a poor prognosis and a high frequency of refractoriness to conventional chemotherapy. The results obtained by high dose chemotherapy followed by autologous stem cells transplantation seem to be a more effective option but still unsatisfactory. Also the role of allogeneic stem cell transplantation is still unclear, although the few cases reported on the literature would seem to show good results in overall survival rates. In this paper, we reported the patient׳s medical history affected by a αβ variant of hepatosplenic T cell successfully rescued with a haploidentical transplant., Highlights • Hepatosplenic T-cell lymphoma is associated with a poor prognosis. • Allogeneic stem cell transplantation has a role in treatment of refractory lymphoma. • The haploidentical SCT may offer a strategy in patients without a matched donor.

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive disease with an average survival of 4-6 months following diagnosis. Surgical resection is the only treatment with curative intent, but resectable PDAC patients are in the minority. Also, unlike other neoplasms, PDAC is resistant to conventional and targeted chemotherapy. Innovative treatments, such as immunotherapy, can be very important and the study of the immune response is fundamental. We previously demonstrated that PDAC patients show tumor-infiltrating T cells specific to α-enolase (ENO1), a glycolytic enzyme over-expressed by pancreatic tumor cells, which plays an important role in promoting cell migration and cancer metastasis. In the present study, we evaluate the functional anticancer proprieties of ENO1-specific T cells isolated from the peripheral blood of PDAC patients. Furthermore, comparing the T cell receptor repertoire of ENO1-specific peripheral and infiltrating tumor T cells from the same patient suggests that ENO1-specific T cells, despite having a different functional profile, can recirculate from the tumor to the periphery. Finally, of clinical relevance, the presence of peripheral ENO1-specific T cells has a prognostic value and significantly correlates with a longer survival.

Composite lymphoma (CL) is a rare occurrence of 2 or more morphologically and immunophenotypically distinct lymphoma clones in a single anatomic site. A retrospective analysis of 1,722 solid tissue samples clinically suggestive of lymphoma was carried out in our institute during a 12-year period to evaluate the efficacy of flow cytometry (FC) in identifying CL. We report 17 CL cases. A strong correlation between morphologic findings and FC was observed in 13 cases (76%). In the 4 cases diagnosed as non-Hodgkin lymphoma plus Hodgkin lymphoma, although FC did not detect Reed-Sternberg cells, it accurately identified the neoplastic B- or T-cell component. In 3 cases, FC indicated the need to evaluate an additional neoplastic component that was not morphologically evident. Our data demonstrate that FC immunophenotyping of tissues may enhance the performance of the diagnostic morphologic evaluation of CL. To the best of our knowledge, this is the first report in the literature of a wide series of CL studied also by FC.

Homing of chronic lymphocytic leukemia (CLL) cells to sites favoring growth, a critical step in disease progression, is principally coordinated by the CXCL12/CXCR4 axis. A cohort of 62 CLL patients was divided into migrating and nonmigrating subsets according to chemotaxis toward CXCL12. Migrating patients phosphorylated extracellular signal-regulated kinase 1/2 (ERK1/2) proteins more than nonmigrating patients (P0.0002). CD38 expression was the parameter most strongly associated with heightened CXCL12 signaling (P0.0001), confirmed by independent statistical approaches. Consistent with this observation, CD38(-) CLL cells in samples with bimodal CD38 expression responded less to CXCL12 than the intact clone (P=0.003). Furthermore, lentivirus-induced de novo expression of CD38 was paralleled by increased responses to CXCL12, as compared with cells infected with a control virus. CD38 ligation with agonistic monoclonal antibodies (mAbs) enhanced CXCL12 signaling, whereas blocking anti-CD38 mAbs inhibited chemokine effects in vitro. This is attributed to physical proximity on the membrane between CD38 and CXCR4 (the CXCL12 receptor), as shown by (i) coimmunoprecipitation and (ii) confocal microscopy experiments. Blocking anti-CD38 mAbs significantly compromised homing of CLL cells from blood to lymphoid organs in a mouse model. These results indicate that CD38 synergizes with the CXCR4 pathway and support the working hypothesis that migration is a central step in disease progression.

Objective Overall diversity of the T-cell receptor (TCR) repertoire can be regarded as a recapitulatory signature of a host's immunocompetence status. We aimed to establish a time- and cost-saving multiplex polymerase chain reaction (PCR) method for determining the TCR repertoire of conventional αβ T cells in small T-cell samples. Materials and Methods The method estimates the length distribution of the complementarity-determining regions 3 (CDR3) of β variable (BV) gene segments (TCRBV repertoire) by multiplex PCR, followed by fluorescent run-off reactions to visualize BV-BC and/or BV-BJ rearrangements. Run-off products are separated on a capillary sequencer and subsequently analyzed with GeneScan or Genotyper programs. Detection-limit studies with normal T cells, KMS27 cells, and regulatory T cells were carried out to evaluate sensitivity and reproducibility. Results Head-to-head comparison of the method with conventional immunoscope assay has shown that it is a time- and cost-saving approach to characterize TCRBV and TCRBJ repertoires, including the presence of oligoclonal T cells in samples containing as few as 1 × 10 5 T cells. Conclusion We have developed a multiplex PCR method that allows comprehensive assessment of the TCRBV repertoire at the BV-BC and BV-BJ levels, and saves a considerable amount of time, reagents, and cell input.

Our interest in chronic lymphocytic leukemia (CLL) derives primarily from the exploitation of human diseases as strategic models for defining the in vivo biological roles of CD38. Using this model, we showed that CD38 triggers robust proliferation/survival signals modulated through the interactions with the CD31 ligand expressed by nurselike cells and by the stromal/endothelial components. By analyzing a cohort of 56 patients with clinically and molecularly characterized CLL, we show that (1) patients with CD38+/ZAP-70+ are characterized by enhanced migration toward Stromal derived factor-1α (SDF-1α)/CXCL12; (2) CD38 ligation leads to tyrosine phosphorylation of ZAP-70, showing that these markers are functionally linked; (3) ZAP-70 represents a limiting factor for the CD38 pathway in the CLL context, as shown by studying CD38-mediated signal transduction in 26 molecularly characterized patients; and (4) the CLL subgroup of patients defined on the basis of migratory potential is marked by a specific genetic signature, with a significant number of differentially expressed genes being involved in cell-cell interactions and movement. Altogether, the results of this work provide biological evidence for why the combined analysis of CD38 and ZAP-70 expression as determined in several clinical trials results in more dependable identification of patients with CLL who have aggressive disease.

A real-time reverse transcriptase-polymerase chain reaction (RT-PCR) method for detection of cytokeratin 20-positive cells in blood characterized by two novel features was developed and tested on 99 patients with colorectal cancer, 110 with breast cancer, and 150 healthy subjects. To optimize the specificity and sensitivity of the method, two novel features were used. First, a primer overlapping two adjacent exons was generated to inhibit nonspecific amplification both in healthy donors and cancer patients; second, a non-end-point first-round amplification was used to increase sensitivity. The number of first-round cycles was chosen to reach the highest level of sensitivity while conserving quantitative characteristics. PCR efficiency increased from 88.9% in single-round RT-PCR to 99.0% in nested real-time RT-PCR. To establish sensitivity and specificity of the method, HT29 cells were serially diluted with normal blood. Detection limit improved from 100 HT29 cells (single-round RT-PCR) to 1 to 10 cells (nested real-time RT-PCR) per 3 ml of whole blood. None of the healthy subjects was positive, whereas 22 and 29% of all colorectal and breast cancer patients, respectively, had cytokeratin 20 cell equivalents in blood. The association between cytokeratin 20 cell equivalents and metastasis was statistically significant for breast (P = 0.026) but not colorectal cancer patients (P = 0.361). Negativity of all 150 healthy controls examined confers diagnostic potential to the method.

Summary Background A dominant T-cell clone can be detected by polymerase chain reaction (PCR) in 40–90% of cutaneous samples from patients with cutaneous T-cell lymphoma (CTCL). Materials and methods From 1996 to 2003 we analysed 547 cutaneous biopsies performed to exclude CTCL (mycosis fungoides, MF/Sezary syndrome, SS). The final diagnosis was benign inflammatory disease (BID) in 353 samples (64·5%) and CTCL in 194 (35·5%). T-cell receptor (TCR)-γ gene rearrangement was studied by using a multiplex PCR/heteroduplex (HD) analysis. The PCR results were correlated with the clinical picture, the histological pattern and the presence of T-cell lineage antigen loss, using univariate and multivariate logistic regression analyses. Objective To determine the sensitivity and specificity of the multiplex PCR/HD analysis and to identify which are the clinical, histopathological or immunophenotypical features significantly associated with a positive T-cell clonality. Results A clonality was demonstrated in 83·5% of CTCL and in 2·3% of BID (P

Most follicular lymphomas (FLs) have a phenotype consistent with the origin from CD5-, CD10+, bcl-6+ follicular center cells and can progress to diffuse large B-cell lymphoma (DLBCL). CD5 is expressed in about 10% of DLBCLs, showing prognostic value, whereas expression is rare in FL. We present 6 cases with coexisting features of CD5+ FL and CD5+ DLBCL, supporting a follicular origin for some CD5+ DLBCLs. The follicular areas showed a meshwork of CD21+ follicular dendritic cells that were lacking in the DLBCL areas. All cases showed a clonal CD19+, CD20+, CD5+, and CD10+ population in both follicular and diffuse areas. Molecularly, 4 of 6 cases demonstrated immunoglobulin heavy chain rearrangements and 1 case, a bcl-2/immunoglobulin heavy chain gene rearrangement. Somatic hypermutations were high in all 4 cases, in keeping with their germinal center origin. Four of five patients died of disease within 42 months, consistent with the proposed prognostic value of CD5 expression in DLBCL. Our data describe an aggressive variant of CD5+ FL suggesting the follicular origin of some CD5+ DLBCLs.

In non-small cell lung cancer (NSCLC), receptor tyrosine kinases (RTKs) stand out among causal dominant oncogenes, and the ablation of RTK signaling has emerged as a novel tailored therapeutic strategy. Nonetheless, long-term RTK inhibition leads invariably to acquired resistance, tumor recurrence and metastatic dissemination. In ALK+ cell lines, inhibition of ALK signaling was associated with coactivation of several RTKs, whose pharmacological suppression reverted the partial resistance to ALK blockade. Remarkably, ERBB2 signaling synergized with ALK and contributed to the neoplastic phenotype. Moreover, the engagement of wild-type epidermal growth factor receptor or MET receptors could sustain cell viability through early growth response 1 (EGR1) and/or Erk1/2; Akt activation and EGR1 overexpression prevented cell death induced by combined ALK/RTK inhibition. Membrane expression of ERBB2 in a subset of primary naive ALK+ NSCLC could be relevant in the clinical arena. Our data demonstrate that the neoplastic phenotype of ALK-driven NSCLC relays ‘ab initio' on the concomitant activation of multiple RTK signals via autocrine/paracrine regulatory loops. These findings suggest that molecular and functional signatures are required in de novo lung cancer patients for the design of efficacious and multi-targeted ‘patient-specific' therapies.

Mounting evidence indicates that grouping of chronic lymphocytic leukemia (CLL) into distinct subsets with stereotyped BCRs is functionally and prognostically relevant. However, several issues need revisiting, including the criteria for identification of BCR stereotypy and its actual frequency as well as the identification of “CLL-biased” features in BCR Ig stereotypes. To this end, we examined 7596 Ig VH (IGHV-IGHD-IGHJ) sequences from 7424 CLL patients, 3 times the size of the largest published series, with an updated version of our purpose-built clustering algorithm. We document that CLL may be subdivided into 2 distinct categories: one with stereotyped and the other with nonstereotyped BCRs, at an approximate ratio of 1:2, and provide evidence suggesting a different ontogeny for these 2 categories. We also show that subset-defining sequence patterns in CLL differ from those underlying BCR stereotypy in other B-cell malignancies. Notably, 19 major subsets contained from 20 to 213 sequences each, collectively accounting for 943 sequences or one-eighth of the cohort. Hence, this compartmentalized examination of VH sequences may pave the way toward a molecular classification of CLL with implications for targeted therapeutic interventions, applicable to a significant number of patients assigned to the same subset.

Contains fulltext : 107785.pdf (Publisher’s version ) (Open Access) PCR-based immunoglobulin (Ig)/T-cell receptor (TCR) clonality testing in suspected lymphoproliferations has largely been standardized and has consequently become technically feasible in a routine diagnostic setting. Standardization of the pre-analytical and post-analytical phases is now essential to prevent misinterpretation and incorrect conclusions derived from clonality data. As clonality testing is not a quantitative assay, but rather concerns recognition of molecular patterns, guidelines for reliable interpretation and reporting are mandatory. Here, the EuroClonality (BIOMED-2) consortium summarizes important pre- and post-analytical aspects of clonality testing, provides guidelines for interpretation of clonality testing results, and presents a uniform way to report the results of the Ig/TCR assays. Starting from an immunobiological concept, two levels to report Ig/TCR profiles are discerned: the technical description of individual (multiplex) PCR reactions and the overall molecular conclusion for B and T cells. Collectively, the EuroClonality (BIOMED-2) guidelines and consensus reporting system should help to improve the general performance level of clonality assessment and interpretation, which will directly impact on routine clinical management (standardized best-practice) in patients with suspected lymphoproliferations.

Gain-of-function (GOF) mutations of Janus kinase 2 (JAK2) are frequently seen in myeloproliferative disorders (MPDs). Meanwhile, JAK3 activating substitutions have been found in a few megakaryocytic cell lines and in primary myeloid leukemia (AMKL). Here, we sought to discover novel leukemogenetic mutations in de novo acute myeloid leukemia of non-Down syndrome (N-DS) by DNA sequencing. A total of 191 normal Caucasian individuals were studied to define single nucleotide polymorphisms (SNPs) within the JH2 and JH6 domains. Although known activating substitutions were observed in rare cases of acute myeloid leukemia (AML) (V722I [2/134] or P132T [1/119]), all samples were wild-type (WT) for the oncogenic A572V (119/119). Interestingly, a novel homozygous mutation (P132A) was discovered in a patient with acute megakaryoblastic leukemia and in vivo studies demonstrated that its ectopic expression was oncogenic in a mouse xenotransplant model. This study defines a novel JAK3 mutation among patients with N-DS AML and demonstrates that normal individuals can also display germline JAK3 substitutions, previously proven to have oncogenic properties, in vitro and in vivo. The discovery of these substitutions in normal donors encourages future studies to define new risk factors among patients with MPDs.

In the present study, we investigated the association of PDGFRA and KIT mutations as well as PDGFRA immunohistochemical expression with clinicopathologic features and prognosis in a series of gastrointestinal stromal tumors (GISTs). Tumor DNA from 40 GISTs was sequenced for the presence of mutations in KIT exons 9, 11, 13 and 17, and in PDGFRA exons 12 and 18. Tissue sections were stained with polyclonal anti-PDGFRA antibody. KIT mutations occurred in 26 cases. There were 13 deletions, 6 substitutions, 3 deletion-substitutions, 3 duplications and 1 insertion. Tumors with KIT deletions/insertion were large with a high mitotic index (MI), and were associated with a high rate of symptoms at diagnosis, invasion into adjacent organs, distant metastasis, relapse and a short disease-free survival (DFS). PDGFRA mutations occurred in 6 gastric GISTs. There were 4 deletions and 2 substitutions. Tumors with PDGFRA mutations were small, with a low MI and Ki67 score, and were associated with a very low rate of symptoms at diagnosis, invasion into adjacent organs and distant metastasis. PDGFRA immunopositivity was found in 23 cases: a peculiar 'dotlike' staining was found in 5 out of 6 PDGFRA mutated cases. Patients with positive PDGFRA immunostaining had a longer DFS than those with negative staining. Our data confirm that the type of KIT mutation is associated with various clinicopathologic features of GISTs, and indicate that PDGFRA mutations are associated with rather indolent tumors. PDGFRA immunopositivity reflects PDGFRA mutational status and is associated with a favorable outcome.

GeneScan (GS) analysis is a highly sensitive method for the early detection of cutaneous T-cell lymphoma (CTCL) and allows the identification of clonal heterogeneity, defined as the coexistence of two or more different T-cell clones in multiple samples from the same patient. We analyzed by GS the incidence and the significance of long-lived oligoclonal expansions in multiple skin and blood samples from 24 Sézary syndrome (SS) patients, and tried to correlate them with the clinical outcome. A skin clonal heterogeneity with additional reproducible TCRgamma-gene rearrangements (TCRgamma-GRs) was detected at diagnosis in 19/24 patients, 13 of whom had a constant prevalence of pathological TCRgamma-GRs in both skin and blood (dominant clonal pattern). During follow-up, an increase in oligoclones that were present at diagnosis or the appearance of new oligoclones was observed in 10 patients; all of them achieved a clinical response to treatment with extracorporeal photochemotherapy (ECP). The TCRgamma pattern (homogeneity or heterogeneity) in the skin at diagnosis showed a relevant prognostic value, and patients with an oligoclonal pattern had a significantly longer survival than those with a homogeneous pattern. In conclusion, multiple-sample approach GS analysis allows the identification of clonal heterogeneity and could also help in identifying SS patients with a potential higher response to therapy.

We examined the in vitro effects of imatinib (Novartis Pharma AG, Basel, Switzerland) as a possible inhibitor of PDGFR pathway on cells derived from a recurrence of a pleural malignant solitary fibrous tumor (SFT). Primary cell culture was characterised by immunofluorescence. SFT-derived cells were treated with imatinib at different time points. Western blotting for PDGFR-beta, phospho-PDGFR-beta or smooth muscle actin (SMA) was performed before and after 96 h of treatment with imatinib. SFT-derived cells treated with imatinib for 96 h showed a dose dependent decrease of Ki67 expression. Results were confirmed by growth curve. Western blotting showed that PDGFR-beta was highly expressed and phosphorylated in SFT-derived cells and imatinib treatment reduced PDGFR-beta phosphorylation and SMA expression. With the limit of experimental findings, our results support a possible future application of imatinib as a candidate molecule in the target therapy of malignant SFTs over-expressing wild-type PDGFR.

CD38 rules proliferation signals in chronic lymphocytic leukemia (CLL) cells, suggesting that the molecule is not merely a prognostic marker but also a key element in the pathogenetic network underlying the disease. CD38 has a genetic polymorphism, characterized by a C>G variation in the regulatory region of intron 1. The working hypothesis is that the presence of different alleles in CLL patients marks (or accounts for) some of the clinical heterogeneity. CD38 allele distribution in 248 Italian patients overlapped with that of the controls (n = 232), suggesting that susceptibility to CLL is not influenced by CD38 genotype. Stratification of patients according to markers of unfavorable prognosis constantly resulted in a significantly higher frequency of the rare G allele. Furthermore, analysis of clinical parameters showed that G allele is independently associated with nodal/splenic involvement. The highest G allele frequency was observed in the 16 patients of the cohort that developed Richter syndrome (RS). Five-year cumulative incidence of transformation was significantly higher in G allele carriers than in CC homozygotes. Multivariate analysis on a total of 30 RS patients confirmed that the probability of transformation is strongly associated with G allele, likely representing an independent risk factor for RS development.

BACKGROUND: Bone forming metastases are a common and disabling consequence of prostate cancer (CaP). The potential role of osteoclast activity in CaP bone metastases is not completely explained. In this study, we investigated ex vivo whether the osteolytic activity is present and how it is ruled in CaP patients with bone forming metastases. METHODOLOGY: Forty-six patients affected by newly diagnosed CaP and healthy controls were enrolled. At diagnosis, 37 patients had a primary tumour only, while 9 had primary tumour and concomitant bone forming metastases. In all patients there was no evidence of metastasis to other non-bone sites. For all patients and controls we collected blood and urinary samples. We evaluated patients' bone homeostasis; we made peripheral blood mononuclear cell (PBMC) cultures to detect in vitro osteoclastogenesis; we dosed serum expression of molecules involved in cancer induced osteoclatogenesis, such as RANKL, OPG, TNF-alpha, DKK-1 and IL-7. By Real-Time PCR, we quantified DKK-1 and IL-7 gene expression on micro-dissected tumour and healthy tissue sections. PRINCIPAL FINDINGS: CaP bone metastatic patients showed bone metabolism disruption with increased bone resorption and formation compared to non-bone metastatic patients and healthy controls. The CaP PBMC cultures showed an enhanced osteoclastogenesis in bone metastatic patients, due to an increase of RANKL/OPG ratio. We detected increased DKK-1 serum levels and tissue gene expression in patients compared to controls. IL-7 resulted high in patients' sera, but its tissue gene expression was comparable in patients and controls. CONCLUSIONS: We demonstrated ex vivo that osteoclastogenesis is an active mechanism in tumour nesting of bone forming metastatic cancer and that serum DKK-1 levels are increased in CaP patients, suggesting to deeply investigate its role as tumour marker.

Lung cancer is a widespread disease and its incidence is growing. Since therapies have increased the life expectancy of lung cancer patients, the development of bone osteolytic metastases is becoming a common cause of morbidity. Osteolysis is caused by an increased osteoclast activity and may be reduced by inhibiting their formation and activity. We studied 60 male patients affected by NSCLC, divided in early and advanced stage disease. Patients' blood and urinary samples were collected at tumor diagnosis and at follow-up. PBMCs were cultured to investigate the spontaneous osteoclastogenesis. IL-7 was dosed in serum and its quantitative gene expression was evaluated on tumor and healthy tissues by RQ-PCR. Both at diagnosis and follow-up, osteolytic bone patients showed high spontaneous osteoclastogenesis level compared to non-bone metastatic and healthy controls. The presence of spontaneous osteoclastogenesis correlated with urinary crosslinks increase. Serum IL-7 levels were higher in bone metastatic patients than in patients without bone lesions and healthy controls. The serum IL-7 increase correlated with the osteoclastogenesis and, at least in part, depended on an increased IL-7 production by tumor cells. At follow-up, patients with increased osteoclastogenesis and serum IL-7 levels, were subjected to standard clinical analysis, which showed early secondary bone lesions. The in vitro assay for spontaneous osteoclastogenesis and serum IL-7 dosage could be useful for diagnostic purposes and it might be able to monitor cancer patients with a high risk to develop osteolytic metastases at follow-up, especially after a curative treatment.

In non–small cell lung cancer (NSCLC), epidermal growth factor receptor (EGFR) mutational analysis is an excellent predictor of responsiveness to treatment with tyrosine kinase inhibitors, such as gefitinib. In up to 80% of NSCLCs, cytologic samples or endoscopic biopsies are the only specimens available for molecular analysis, but PCR amplification of DNA from small fixed and paraffin-embedded samples may create artifactual mutations. Fluorescence in situ hybridization (FISH) of EGFR and HER2 has been proposed as an alternative method of analysis. This project aimed to determine the optimal scoring method for FISH or chromogenic in situ hybridization (CISH) assays when analyzing small NSCLC samples to predict response. FISH or CISH analysis of EGFR and HER2 genes was done on 42 small samples derived from NSCLC patients treated with gefitinib. EGFR mutational analysis was done after quantity and quality controls of DNA. In seven of seven cases, a balanced increase in EGFR gene and chromosome 7 number was found to correlate with the presence of specific EGFR mutations. In addition, seven of seven cases with balanced EGFR/HER2 polysomy and two of three cases with balanced EGFR/HER2 trisomy responded to gefitinib (75% of responders). Instead, the EGFR mutations predicted only 7 of 12 (58%) of gefitinib-responsive patients. When only endoscopic biopsies or cytologic specimens are available, we propose using FISH/CISH for EGFR and HER2 as the test of choice for selecting patients for treatment with gefitinib and to consider as negative predictive factor the absence of EGFR/HER2 gene gain. [Mol Cancer Ther 2007;6(4):1223–9]

Background Persistent infections with high-risk types of human papillomavirus (HR-HPV) favour integration of viral DNA into the host cells and are associated with cervical carcinoma. HPV16 is the prevalent HR-type worldwide associated to cervical cancer. Integration of viral DNA promotes a selective cell growth advantage, resulting a risk factor for cancer development. Objectives To test physical status of HPV16 infection in pre-neoplastic cervical lesions using a quantitative real time-PCR (QRT-PCR) based method. To investigate reliability of this method in identification of HPV16 integrated sequences, by detection of integrated papillomavirus sequences (DIPS-PCR) assay and sequencing. Study design One hundred and seventy HR-HPV positive archival cervical specimens were tested for presence of HPV16 DNA. In HPV16 positive samples, viral load and physical status were evaluated. Results HPV16 DNA was detected in 74/170 (43%) HR-HPV positive specimens. In 52/74 a QRT-PCR was performed, and 3 integrated, 13 mixed and 36 episomal forms were detected. Presence of integrated forms was confirmed by DIPS-PCR and sequencing. Conclusions Presence of HPV integrated forms was detected and confirmed in pre-neoplastic cervical lesions. The QRT-PCR method we used is sensitive and specific for identification of HPV integration in cervical samples, and may be suitable for large scale investigations with prognostic and clinical implications in management of cervical cancer.

The selection of proper tissues from formalin-fixed and paraffin-embedded tumors before diagnostic molecular testing is responsibility of the pathologist and represents a crucial step to produce reliable test results. The international guidelines suggest two cut-offs, one for the percentage and one for the number of tumor cells, in order to enrich the tumor content before DNA extraction. The aim of the present work was two-fold: to evaluate to what extent a low percentage or absolute number of tumor cells can be qualified for somatic mutation testing; and to determine how assay sensitivities can guide pathologists towards a better definition of morphology-based adequacy cut-offs. We tested 1797 tumor specimens from melanomas, colorectal and lung adenocarcinomas. Respectively, their BRAF, K-RAS and EGFR genes were analyzed at specific exons by mutation-enriched PCR, pyrosequencing, direct sequencing and real-time PCR methods. We demonstrate that poorly cellular specimens do not modify the frequency distribution of either mutated or wild-type DNA samples nor that of specific mutations. This observation suggests that currently recommended cut-offs for adequacy of specimens to be processed for molecular assays seem to be too much stringent in a laboratory context that performs highly sensitive routine analytical methods. In conclusion, new cut-offs are needed based on test sensitivities and documented tumor heterogeneity.

Background Interleukin-7 (IL-7) is a potent regulator of lymphocyte development, which has also significant effects on bone; in fact it is a potent osteoclastogenic factor. Some human solid tumors produce high IL-7 levels, suggesting a potential IL-7 role on tumor development and progression. Methodology We studied 50 male patients affected by solid tumors, and their blood samples were collected at tumor diagnosis. PBMCs were isolated and cultured with/without IL-7 to study its influence on osteoclastogenesis. Serum and cell culture supernatant IL-7 levels were measured by ELISA. The quantitative analysis of IL-7 expression on T and B cells was performed by Real-Time PCR. Principal findings Serum IL-7 levels were highest in osteolytic cancer patients, followed by cancer patients without bone lesions, and then healthy controls. We showed the IL-7 production in PBMC cultures and particularly in monocyte and B cell co-cultures. A quantitative analysis of IL-7 expression in T and B cells confirmed that B cells had a high IL-7 expression. In all cell culture conditions, IL-7 significantly increased osteoclastogenesis and an anti-IL-7 antibody inhibited it. We demonstrated that IL-7 supports OC formation by inducing the TNF-alpha production and low RANKL levels, which synergize in promoting osteoclastogenesis. Conclusions We demonstrated the presence of high serum IL-7 levels in patients with bone metastasis, suggesting the use of serum IL-7 level as a clinical marker of disease progression and of bone involvement. Moreover, we showed the capability of IL-7 to stimulate spontaneous osteoclastogenesis of bone metastatic patients and to induce osteoclastogenesis in cancer patients without bone involvement. These findings add further details to the disclosure of the mechanisms controlling bone metastasis in solid tumors.

This work addresses the question whether CD38, a negative prognostic marker in B-cell chronic lymphocytic leukemia (B-CLL), plays a role in neoplastic B-cell growth and survival. We show that CD38+ B-CLL cells bind to murine fibroblasts transfected with the CD31 ligand. The interaction triggers an extensive remodeling of the B-CLL membrane, with relocalization of BCR/CD19 to the CD38/CD31 contact areas, and it also increases cell survival and proliferation. A second event is the up-modulation of the survival receptor CD100, restricted to proliferating cells, and a concomitant decrease of CD72 (low-affinity CD100 ligand and negative regulator of immune responses). The most efficient signals are delivered through sequential interactions between CD38/CD31 and CD100/plexin-B1 (high-affinity CD100 ligand), as inferred by coculture experiments using specific transfectants and blocking monoclonal antibodies (mAbs). The finding that nurselike cells from B-CLL patients express CD31 and plexin-B1, which deliver growth and survival signals to CD38+/CD100+ B-CLL cells, further confirms the model proposed. These findings show that a set of normal receptors and ligands ruling physiologic signaling pathways in B lymphocytes becomes detrimental when expressed in the context of B-CLL cells, ultimately leading to the generation of a tumor reservoir.

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Lung cancer is the most common cancer in the world, and is lethal in 90% of the cases. In non-small cell lung cancer (NSCLC), deregulated receptor tyrosine kinases (RTKs) stand out among the causal dominant oncogenes. Consequently, the genomic and/or pharmacological ablation of RTK signaling has emerged as a novel tailored therapeutic strategy. Nevertheless, this approach has serious constrains, probably due to the frequent co-activation of multiple RTKs in a considerable subsets of solid tumors. It has become also evident that during TK inhibitor-based regimens, RTK switching and/or the selection of resistant clones can frequently occur representing a serious and problematic drawback. In a subset of NSCLC, the Anaplastic Lymphoma Kinase (ALK) gene has been described to be translocated and fused to EML4. This determines the ALK constitutive dimerization and autophosphorylation leading to cellular transformation. Here, we investigated the ALK oncogenic addiction of human NSCLC and studied the putative co-operative role of other kinases. We first demonstrated that the ectopic expression of EML4-ALK in ALK positive NSCLC cell lines (H2228 and H3122) resulted in the activation of multiple signaling pathways, in manner similar to that described for other known ALK fusions. Although EML4-ALK can induce transformation in lung in vitro and in vivo, ALK inhibition via shRNA or small molecule inhibitors (CEPs, Cephalon) induced only the apoptosis of H3122 cells, whereas in H2228 it caused cell growth arrest. Moreover, the treatment with ALK inhibitors led to tumor regression, but not tumor eradication, in vivo. Based on Phosphoproteomic analyses we demonstrated that the phosphorylation status of several tyrosine kinases (such as, EGFR, Met, FGFR, Jak1 or IGFR) was affected by the ALK inhibition only in H2228 cell line, but not in H3122. Notably, the combined treatment with anti-ALK ([CEP14083][1], CEP2550, [CEP28122][2]) and -EGFR inhibitors resulted in an increased cell death of H2228 cells to values similar to those observed for ALK treated H3122 cells. Finally, gene expression analyses showed that known EGFR substrates were specifically down regulated upon the combined treatment in ALK positive H2228 cells. Our findings suggest that ALK signaling is required for the maintenance of the neoplastic phenotype of some ALK positive NSCLC cells and that its abrogation could represent a novel strategy for the treatment of a well-defined subset of human lung cancer (complete ALK addiction). More importantly, we showed that the tumor survival and maintenance of ALK positive neoplastic cells might relay on the concomitant activation of multiple RTKs. These findings further support the notion that molecular and functional signatures are required for designing molecular-based “patient specific” therapeutic protocols in lung cancer patients. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-309. [1]: /lookup/external-ref?link_type=GENPEPT&access_num=CEP14083&atom=%2Fcanres%2F70%2F8_Supplement%2FLB-309.atom [2]: /lookup/external-ref?link_type=GENPEPT&access_num=CEP28122&atom=%2Fcanres%2F70%2F8_Supplement%2FLB-309.atom

Abstract 2328 Poster Board II-305 Chronic lymphocytic leukemia (CLL) is characterized by the expansion of a monoclonal population of mature B lymphocytes expressing surface CD5, CD23 and low levels of immunoglobulin (Ig). Originating in the bone marrow (BM), neoplastic cells accumulate in blood (the circulating pool) and in lymphoid organs (the residential pool). Data obtained ex vivo and in patients suggest that the proliferative core of the disease dwells within the residential pool, while little or no proliferation is seen among circulating CLL cells. Threrefore, homing of chronic lymphocytic leukemia (CLL) cells to sites favoring growth is a critical step in disease progression. Human CD38 is a pleiotropic glycoprotein belonging to a complex family of genes coding for enzymes of the cell surface and involved in the catabolism of extracellular nucleotides. Along with the absence of mutations in the immunoglobulin variable region heavy chain gene (IgVH) and expression of the cytoplasmic kinase ZAP-70, CD38 expression above a critical threshold represents a reliable negative prognostic marker for CLL patients. The working hypothesis of our group is that CD38 is not merely a marker in CLL, but a cell surface receptor and adhesion molecule directly involved in the delivery of growth signals. Prolonged interactions between CD38 and its non-substrate ligand CD31 under static conditions increase survival of the CLL clone and sustain its proliferation in vitro. Here we show that CD38, is directly involved in chemokine-mediated homing of CLL cells. CD38 signals facilitate short- (ERK1/2 phosphorylation) and long-term (chemotaxis) effects induced by the CXCL12 chemokine. This was first shown by testing functional responses to CXCL12 in a large cohort of clinically and molecularly characterized patients. The superior chemotactic performance of CD38+ CLL cells was confirmed by comparing the CD38+ and CD38- leukemic components within the same patient presenting with a bimodal clone. Assays using i) a specific inhibitor of CD38 enzymatic activities, ii) a panel of agonistic and blocking monoclonal antibodies (mAbs) and iii) microarray technology indicate that increased homing depends on the receptor functions of CD38. Indeed, interactions between CD38 and its non-substrate ligand CD31 define a genetic signature characterized by modulation of pathways involved in proliferation and migration of CLL cells. Conversely, blocking CD38 with domain-specific mAbs significantly weakens CXCL12 responses. This is attributed to physical proximity on the cell membrane between CD38 and CXCR4 (the CXCL12 receptor). The existence of a CD38/CXCR4 complex was demonstrated by i) co-immunoprecipitation, ii) confocal microscopy and iii) direct measurement of CXCL12 binding to its receptor. The relevance of CD38 in homing is substantiated by results from an in vivo mouse model, where CLL cells are allowed to home to lymphoid organs for a short period of time. The compromised homing ability of CLL cells to spleen and BM in the presence of blocking anti-CD38 mAbs confirms in vivo the in vitro evidence. Furthermore, the lack of effects induced by an isotype-matched mAb with the same anti-CD38 specificity convincingly rules out the possibility that the effects observed are secondary to sequestration of mAb-coated cells. Future studies will address the issue of whether CD38 may become a therapeutic target for selected CLL patients. Disclosures: No relevant conflicts of interest to declare.

Human CD38, an ectoenzyme involved in the catabolism of NAD, can also bind the non-substrate ligand CD31, resulting in activation and proliferation of distinct lymphocyte populations. CD38 is clinically used as a negative prognostic marker for chronic lymphcytic leukemia (CLL) patients. We adopted CLL as a model to study the relevance of CD38 in vivo. CD38 signals trigger proliferation and increase survival of CLL cells. Further, the simultaneous expression of CD38 and ZAP-70 defines a genetically distinct subset of CLL cells with heightened responses to CXCL12, a critical chemokine in CLL homing from blood to lymphoid organs. Here we show that CD38 is directly involved in CXCL12-mediated homing. CD38 signals facilitate short- (ERK1/2 phosphorylation) and long-term (chemotaxis) effects induced by CXCL12. Assays using i) a specific inhibitor of CD38 enzymatic activities, ii) agonistic mAbs, and iii) microarray technology indicate that increased homing depends on the receptor functions of CD38. Conversely, blocking anti-CD38 mAbs significantly weaken CXCL12 responses. This is due to physical association on the membrane between CD38 and CXCR4 (the CXCL12 receptor), as shown by i) co-immunoprecipitation, ii) confocal microscopy and iii) measurement of CXCL12 binding to its receptor. These results indicate that CD38 modulates CXCL12 signals, representing a novel mechanism in CLL chemotaxis and homing.

Chronic lymphocytic leukemia (CLL), the most common leukemia in Europe and North America, is characterized by a highly heterogeneous clinical behavior. Absence of mutations in the immunoglobulin variable (IgV) genes, expression of the surface receptor CD38 and the cytoplasmatic kinase ZAP-70 represent independent molecular markers of unfavorable prognosis. Recent data indicates that the proliferative core of the disease resides within peripheral lymphoid organs. There, the neoplastic cells have access to the antigen as well as to microenvironmental signals necessary to the maintenance and expansion of the clone. Our group has investigated the role of CD38 in CLL not only as a disease marker but also as a pathogenetic agent. We showed that CD38 ligation by agonistic monoclonal antibodies (mAbs) or the CD31 ligand is followed by proliferation and blast transformation of a subset of CLL cells. CD38/CD31 interactions occur predominantly in the lymph nodes and spleen where CD31 is abundantly expressed by stromal and nurse like cells (NLC). The signaling pathway initiated upon CD38 engagement relies upon tyrosine phosphorylation of ZAP-70 as a necessary step. CLL cells expressing CD38 and ZAP-70 also show the highest migration propensity towards CXCL12, a critical chemokine produced by NLC to attract CLL cells to lymph nodes. Collectively, these results point to a crucial role for the CD38/ZAP-70 pathway in directing CLL cells from the blood to peripheral lymphoid organs and suggest that targeting CD38 might impair CLL cells homing to sites favorable for neoplastic growth. In order to obtain a genome-wide signature of the transcriptional events taking place upon CD38/CD31 contacts CLL cells from 10 patients were co-cultured with CD31 transfected (L-CD31+) cells and L-mock controls for 5 days. Microarray analyses revealed 1,645 sequences selectively modulated as a consequence of CD38/CD31 interactions with 84 differentially regulated pathways: 12 of these pathways regulated cell adhesion/movement phenomena and 14 lymphocyte signalling. Attention was next focused on the impact of blocking anti-CD38 mAbs on CLL homing in vivo. To investigate the homing of CLL cells from blood to spleen and BM, PBMC were injected in the tail vein of female NOD/SCID mice and left to home for 24 hours. Next, mice were sacrificed and human cells tracked in spleen and BM by staining with specific reagents. Homing was significantly reduced when human cells were pre-incubated with anti-CD38 mAbs immediately prior to in vivo delivery. The in vivo data provided a rationale for a more in depth in vitro investigation of the effects of anti-CD38 mAbs on the CXCL12/CXCR4 signaling pathway. Results indicate that CXCL12-mediated chemotaxis can be blocked by anti-CD38 mAbs. Pre-treatment of CLL cells with anti-CD38 mAbs impairs CXCR4 signaling, as indicated by lack of Ca2+ mobilization, ERK1/2 activation and receptor internalization. These effects are specific for CD38+ samples, as no interference in migration was recorded when anti-CD38 mAbs were incubated with CD38− cells. The molecular explanation may be found at least in part in a physical proximity between CD38 and CXCR4 on the cell membrane, as shown by confocal microscopy and coimmunoprecipitation studies. In line with this working hypothesis, CXCL12 binding to CXCR4 is effectively inhibited following pre-incubation with anti-CD38 mAbs. In conclusion, these results offer biological evidence of a functional cross-talk between CD38 and CXCR4 signaling pathways. They also provide a preliminary rationale for the use of anti-CD38 ligands in therapy of a subset of CLL patients, representing a novel approach interfering with deleterious growth circuits and increasing the susceptibility of leukemic cells to conventional chemotherapy.

The absence of mutations in the IgVH genes along with the expression of the cell surface receptor CD38 and the cytoplasmic kinase ZAP-70 are independent molecular markers of unfavourable outcome in CLL patients. Our group has been interested in unravelling the role of CD38 in CLL not only as disease marker but also as pathogenetic agent. We have shown that CD38 can positively affect proliferation and survival of CLL cells through binding CD31, the ligand expressed by stromal, endothelial and nurse-like cells (NLC). Starting points of our current investigation are that NLC produce high levels of the attractant stromal cell-derived factor-1 (SDF-1α/CXCL12), CD38 is an essential mediator of migration and homing in murine neutrophils, and ZAP-70 is a key element in CXCR4 (SDF-1α receptor) pathway in T cells. The consequent working hypothesis is that CD38 and ZAP-70 intertwine to influence CLL cell migration. The analysis of a cohort of 56 clinically and molecularly characterized CLL patients demonstrated that CD38+/ZAP-70+ samples migrate significantly better towards CXCL12 than all the other CLL subgroups, including the CD38+/ZAP-70− one, suggesting a functional association between CD38 and ZAP-70. CD38 engagement by agonistic monoclonal antibodies (mAbs) leads to phosphorylation of the activatory tyrosines present in ZAP-70 in 26 CLL cell cases and in an ad hoc modified B line expressing a ZAP-70-myc construct. More importantly, ZAP-70 is required for CD38 signalling, as the CD38 pathway is impaired in CD38+/ZAP-70− CLL cells. Microarray analyses performed on a subgroup of 20 CLL patients indicate the existence of a genetic signature marking CLL samples endowed with a high migratory potential. These studies resulted in the identification of a panel of 24 differently expressed genes, many of which are known to be involved in adhesion and migration. Taken together, the above data point to a direct role for the CD38/ZAP-70 pathway in CLL migration towards CXCL12. Indeed, CXCL12-mediated chemo taxis of CLL cells and of the NALM-6 pre-B cell line can be efficiently blocked by anti-CD38 mAbs in a dose-dependent fashion. Out of the 9 different anti-CD38 mAbs tested, only the ones binding epitopes close to the C terminus of the molecule interfere with migration, while the ones binding closer to the plasma membrane do not. Pre-treatment of CLL and NALM-6 cells results in the almost complete block of the CXCR4 signalling pathway. This was shown by monitoring Ca2+ mobilization, actin polymerization and ligand-induced internalization. Further, upon exposure to CXCL12, CD38 and CXCR4 become physically associated, as shown using confocal microscopy and co-immunoprecipitation approaches. CD38 however, does not internalize upon CXCL12 biding to CXCR4. Collectively, these data suggest that the blocking effects of the anti-CD38 mAbs are due to steric hindrance. In conclusion, we believe that these results offer biological evidence explaining why the combination of CD38 and ZAP-70 results in a more efficient identification of CLL patients with aggressive disease. Further, it may provide a rationale for using anti-CD38 ligands or inhibitors as an adjunct tool for the therapy of aggressive CLL.

Introduction: the addition of Rituximab to standard chemotherapy CHOP improves the outcome of advanced stage B-DLCL, however pts at high risk continue to have a poor outcome. The combination of Rituximab with dose-dense and intensified chemotherapy is still poorly studied so far. Patients and methods: from January 2001 to June 2004, 92 previously untreated pts Results: Median age was 47 years (19–60); 6% were at LI risk (enrolled because of BM involvement), 53% at IH and 41% at H risk according to IPI; 27% had BM involvement and 80% LDH level >normal. So far, 72 pts who completed the treatment are evaluable for response and toxicity. Fifty-seven pts (79%) achieved a CR;one a PR (1.4%); ten pts (14%) progressed before (7 pts) or shortly after (3 pts) ASCT. Four pts (5.6%) died of infectious toxicities (2 bacterial sepsis and 2 pneumonia) which occurred during neutropenic period post MAD (3 pts) or after ASCT (1 pt). Sixteen pts did not completed the planned treatment because of disease progression before ASCT (7 pts) or inadequate PBSC yield (5 pts) or toxicity (4 pts). With a median follow-up of 2 yrs, only 2 pts relapsed. Two-yrs FFS and OS were: 72% and 77%. Fourteen pts developed infections (WHO>2): 2 interstitial pneumonia, 5 neutropenic Gram- and 2 Gram+ sepsis, 1 disseminated herpes zoster, 1 Ps. Aeruginosa and 1 Staph. aureus pneumonia, 1 bacterial pancreatitis and 1 perianal abscess. A molecular marker has been detected at diagnosis in 27 patients. So far, 12 PBSC harvest has been analyzed and 11/12 were PCR negative. Conclusions: this interim analysis suggests that the concurrent use of Rituximab with dose-dense and high dose chemotherapy followed by myeloablative therapy with ASCT support is effective to achieve a high CR and a high 2-yr FFS rate in a group of B-DLCL patients at poor prognosis. Furthermore, the addition of Rituximab during mobilitation procedure with R-MAD may allow to collect lymphoma-free PBSC harvest.

Ieext generation sequencing studies in Homo sapiens have identified novel immunoglobulin heavy variable (IGHV) genes and alleles necessitating changes in the international ImMunoGeneTics information system (IMGT) GENE-DB and reference directories of IMGT/V-QUEST. In chronic lymphocytic leukaemia (CLL), the somatic hypermutation (SHM) status of the clonotypic rearranged IGHV gene is strongly associated with patient outcome. Correct determination of this parameter strictly depends on the comparison of the nucleotide sequence of the clonotypic rearranged IGHV gene with that of the closest germline counterpart. Consequently, changes in the reference directories could, in principle, affect the correct interpretation of the IGHV mutational status in CLL. To this end, we analyzed 8066 productive IG heavy chain (IGH) rearrangement sequences from our consortium both before and after the latest update of the IMGT/V-QUEST reference directory. Differences were identified in 405 cases (5 % of the cohort). In 291/405 sequences (71.9 %), changes concerned only the IGHV gene or allele name, whereas a change in the percent germline identity (%GI) was noted in 114/405 (28.1 %) sequences; in 50/114 (43.8 %) sequences, changes in the %GI led to a change in the mutational set. In conclusion, recent changes in the IMGT reference directories affected the interpretation of SHM in a sizeable number of IGH rearrangement sequences from CLL patients. This indicates that both physicians and researchers should consider a re-evaluation of IG sequence data, especially for those IGH rearrangement sequences that, up to date, have a GI close to 98 %, where caution is warranted.