Lymphoid Tumorigenesis Research Articles

Serum Level Kinetic of Key Cytokines in Newly Diagnosed Lymphoma Patients during First Line Treatment: Correlation with PET/CT Scan Results and Treatment Outcome

Background: Cytokines are soluble proteins that play a key role in mediating and regulating inflammation and immune responses. Further, they are involved in lymphoid development and differentiation, as well as in lymphoid tumorigenesis. Aim of the present study was to verify if the serum-cytokine levels could be an early marker of treatment response. With this purpose, we analyzed the serum concentration kinetic of key cytokines in diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL) and Hodgkin lymphoma (HL) pts comparing it with PET/CT scan results and treatment outcome. Patients and Methods: After Ethic Committee approval, we start to enroll lymphoma pts treated at Modena Cancer Center. Until now, 29 patients entered the study, including 12 pts with DLBCL, 11 with FL, and 6 with HL. DLBCL lymphoma were treated with R-CHOP and FL patients with R-CVP or R-Benda or R-CHOP, while HL patients with ABVD + ABVD or BEACOP following indications of intermediate PET. A panel of eight serum cytokines/chemokines (IL6, IL2, IL8, IL10, IFNγ, TNFα, CXCL10/IPI10, CCL17/TARC) was analyzed using the Simple Plex system (Protein Simple, San Jose, CA, USA) according to the manufacturer's recommendations. Serum samples were collected at the day of staging (PET-0), at the day of intermediate PET (I-PET) and at the day of end treatment PET (EOT-PET). Samples were analyzed in duplicate. Continuous variables were compared by Mann-Whitney test and categorical variables by Chi2 test. Matched data were analyzed by paired Wilcoxon or Friedman test. Statistical significance was defined as p < 0.05. Results: The median age of all 29 pts was 60 yr (range 21-86), and 65 % were male. Patients with HL were younger (50 yr) than pts with FL (61 yr) and DLBCL (68 yr). In all 29 pts, median concentration of IL10, IL6, TNFα at EOT-PET decreased in comparison with the levels at PET-0, while IFNγ and IPI10 increased. IL8 increased in DLBCL and in HL, while decreased in FL. IL2 levels did not show relevant changes. No statistically significant differences were found between the concentrations of these cytokines at PET-0 and EOT-PET. The mean serum TARC level at PET-0 were 854 pg/mL (range 130-5829), 1209 pg/mL (range 134-6903) and 8327 pg/mL (range 6488-10885) in DLBC, FL and HL pts, respectively. At EOT-PET the mean concentration of TARC were 562 pg/mL (range 130-854) (p=0.49), 989 pg/mL (range 134-6903) (p=0.74), 591 pg/mL (range 353-932) (p=0.03 ) in DLBCL, FL and HL pts, respectively. In HL patients, TARC values were significantly lower at I-PET with a mean of 525 pg/mL (range 210-1062) compared to PET-0 (p=0.016), with a p value along the 3 timepoint =0.009. EOT-PET and I-PET, evaluated by Deauville score shows that almost all patients achieved CR at the end of treatment: 11 out of 12 DLBCL pts have a Deauville score between 1 and 3, as well as all FL and HL pts. Only 1 DLBCL patient had a Deauville score of 4 at the end of treatment, achieving CR after further radiotherapy. Conclusion: Our study, despite the limitations due to the small number of pts enrolled to date, shows that the concentrations of cytokines/chemokines, with the exception of TARC, do not undergo significant changes, before and after first-line therapy. The TARC values, on the other hand, correlate well with the results of PET/CT and therefore also with the response to therapy in all subtypes of lymphoma. This correlation is statistically significant in pts with HL. TARC assessment could therefore become a useful, inexpensive and easy to determine predictive marker of response to therapy and may be important for determining treatment strategy in lymphoma patients. Further, the TARC levels assessed during the follow-up could be early markers of relapse. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

B-cell lymphomas

Among the hematologic tumors, lymphoid malignancies, especially B-cell lymphomas (BLs), are categorized according to the WHO classification into a large number of entities, which are aggregates of clinically and biologically diverse diseases. It is thought that various molecular genetic abnormalities are involved in lymphoid tumorigenesis. Recent advances in genetic analysis technology have resulted in the discovery of many novel and recurrent genetic abnormalities. In particular, those associated with diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), and mantle cell lymphoma have been identified utilizing molecular pathology techniques, which is important aspect for the future development of novel therapeutic drugs and indications for clinical trials. In this section, the possibility of patient-stratification based on genetic abnormalities of DLBCL and FL, which are particularly prevalent among mature BLs, are described. I also introduce the genetic features and clinical importance of BL and MCL identified in a small number of patients in Japan.

Aberrant Expression of Golgin-84 in Non-Hodgkin Lymphomas and Plasma Cell Myeloma

Abstract 4633 Background:Golgins are proteins of the Golgi complex. Several Golgins have been implicated in apoptosis. Expression of Golgin-84, a Golgin protein, is altered in apoptotic WEHI-231, a B-cell lymphoma line, suggesting that Golgin-84 may play a role in lymphoid tumorigenesis. Here, we aimed to determine the expression levels of Golgin-84 in human primary non-Hodgkin lymphomas and plasma cell myeloma. Design:Golgin-84 expression was investigated in non-Hodgkin lymphoma cell lines by using Western blot analysis and polyclonal antibodies. Using immunohistochemical stains, Western blotting analysis and Q-PCR, Golgin-84 expression was assessed in 5 reactive lymph nodes, 149 cases of primary non-Hodgkin lymphoma and 28 cases of primary plasma cell myeloma. Results:Immunohistochemical stains, Western blotting analysis and Q-PCR on 5 reactive lymph nodes demonstrated that Golgin-84 was expressed at low levels in lymphoid cells of germinal centers, mantle cells, marginal zones, and interfollicular areas. Golgin-84 was variably expressed in non-Hodgkin lymphoma cell lines tested, with the highest levels in cells from high-grade tumors (e.g. anaplastic large cell lymphoma; ALCL, Diffuse large B-cell lymphoma (DLBCL), ALCL and peripheral T-cell lymphoma unspecified (PTCL)) and the lowest levels in mantle cell lymphoma (MCL) cells. DLBCL, ALCL and PTCL frequently showed high expression of Golgin-84. Most lymphoplasmacytic lymphomas (LPL) and plasma cell myeloma (PCM) expressed high levels of Golgin-84. Expression levels of Golgin-84 were lower in MCL and low-grade B-cell non-Hodgkin lymphomas, including chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL), follicular lymphoma (FL), and marginal zone lymphoma (MZL). Conclusions:Golgin-84 expression levels are low in lymphoid cells of normal lymph nodes. Most (>90%) cases of LPL and PCM, and at least half of cases of DLBCL, ALCL and PTCL express high levels of Golgin-84. These findings suggest that Golgin-84 may be involved in tumorigenesis or lymphoma progression, particularly in neoplasms with plasmacytic differentiation. Disclosures:No relevant conflicts of interest to declare.

Pattern of Mir-155 and PU.1 Expression in CLL/SLL and Aggressive Lymphomas

Abstract 4600MicroRNA miR-155 represents a candidate pathogenic factor in chronic lymphocytic leukemia (CLL) and lymphomas (Eis PS, et al. 2005, Fulci V, et al. 2007, Calin GA, et al. 2005, Kluiver J, et al. 2005, Metzler M, et al. 2004, van den Berg A, et al. 2003., Vargova K. et al 2011). In diffuse large B-cell lymphoma (DLBCL) expression of miR-155 was associated with NFkB pathway (Rai et al., 2008). In addition, increased miR-155 levels were associated with more aggressive (Activated B-cell like) DLBCL (Eis et al. 2005). The targets of up-regulated miR-155 apparently include a key hematopoietic transcription factor PU.1. Down-regulation of PU.1 represents an important and critical step for both myeloid as well as lymphoid tumorigenesis. We herein found the increased levels of miR-155 coupled with downregulation of its target PU.1 are frequent events in B cells of a vast majority of CLL patients (N=169). To advance understanding of miR-155 and PU.1 in lymphoid malignancies we studied lymph node biopsies derived from patients with lymphomas. We show that increased miR-155 levels are also found in DLBCL (N=31, P < 0.0001), Hodgkin lymphoma (HL, N=22, P < 0.0001), follicular lymphoma (FL, N=23, P < 0.001), small lymphocytic lymphoma (SLL, N=12, P < 0.01), and marginal zone lymphoma (MZL, N=11, P < 0.01). The miR-155-overexpressing tumors display downregulation of PU.1. In contrast, peripheral T cell lymphoma (T-NHL, N=6) and mantle cell lymphoma (MCL, N=7) expressed miR-155 and PU.1 comparably as control lymph nodes. Our data indicate that expression pattern of miR-155 and its target PU.1 represents a hallmark of some but not all lymphoid malignancies. Interestingly, the miR-155/PU1 levels vary substantially within each diagnosis, therefore, our aim is to further analyze the occurrence of this relationship in different prognostic groups and analyze the clinical outcome as well. (Grants: NT10310-3/2009 & NT11218-6/2010, MPO FR-TI2/509, GAUK251135 82210 & 251070 45410, SVV-2011-262507) Disclosures:No relevant conflicts of interest to declare.

HMGA1 drives stem cell, inflammatory pathway, and cell cycle progression genes during lymphoid tumorigenesis.

BackgroundAlthough the high mobility group A1 (HMGA1) gene is widely overexpressed in diverse cancers and portends a poor prognosis in some tumors, the molecular mechanisms that mediate its role in transformation have remained elusive. HMGA1 functions as a potent oncogene in cultured cells and induces aggressive lymphoid tumors in transgenic mice. Because HMGA1 chromatin remodeling proteins regulate transcription, HMGA1 is thought to drive malignant transformation by modulating expression of specific genes. Genome-wide studies to define HMGA1 transcriptional networks during tumorigenesis, however, are lacking. To define the HMGA1 transcriptome, we analyzed gene expression profiles in lymphoid cells from HMGA1a transgenic mice at different stages in tumorigenesis.ResultsRNA from lymphoid samples at 2 months (before tumors develop) and 12 months (after tumors are well-established) was screened for differential expression of > 20,000 unique genes by microarray analysis (Affymetrix) using a parametric and nonparametric approach. Differential expression was confirmed by quantitative RT-PCR in a subset of genes. Differentially expressed genes were analyzed for cellular pathways and functions using Ingenuity Pathway Analysis. Early in tumorigenesis, HMGA1 induced inflammatory pathways with NFkappaB identified as a major node. In established tumors, HMGA1 induced pathways involved in cell cycle progression, cell-mediated immune response, and cancer. At both stages in tumorigenesis, HMGA1 induced pathways involved in cellular development, hematopoiesis, and hematologic development. Gene set enrichment analysis showed that stem cell and immature T cell genes are enriched in the established tumors. To determine if these results are relevant to human tumors, we knocked-down HMGA1 in human T-cell leukemia cells and identified a subset of genes dysregulated in both the transgenic and human lymphoid tumors.ConclusionsWe found that HMGA1 induces inflammatory pathways early in lymphoid tumorigenesis and pathways involved in stem cells, cell cycle progression, and cancer in established tumors. HMGA1 also dyregulates genes and pathways involved in stem cells, cellular development and hematopoiesis at both early and late stages of tumorigenesis. These results provide insight into HMGA1 function during tumor development and point to cellular pathways that could serve as therapeutic targets in lymphoid and other human cancers with aberrant HMGA1 expression.

Coupling of V(D)J Recombination to the Cell Cycle Suppresses Genomic Instability and Lymphoid Tumorigenesis

V(D)J gene segment recombination is linked to the cell cycle by the periodic phosphorylation and destruction of the RAG-2 protein at the G1-to-S cell cycle transition. To examine the function of this coupling, we constructed mice in which the phosphorylation site at threonine 490 of RAG-2 was mutated to alanine. The RAG-2(T490A) mutation uncoupled DNA cleavage from cell cycle and promoted aberrant recombination. Similar aberrant recombination products were observed in mice deficient in the Skp2 ubiquitin ligase subunit, which is required for periodic destruction of RAG-2. On a p53-deficient background, the RAG-2(T490A) mutation induced lymphoid malignancies characterized by clonal chromosomal translocations involving antigen receptor genes. Taken together, these observations provide a direct link between the periodic destruction of RAG-2 and lymphoid tumorigenesis. We infer that cell cycle control of the V(D)J recombinase limits the potential genomic damage that could otherwise result from RAG-mediated DNA cleavage.

Cooperation between deficiencies of IRF-4 and IRF-8 promotes both myeloid and lymphoid tumorigenesis

AbstractInterferon regulatory factor 4 (IRF-4) plays important functions in B- and T-cell development and immune response regulation and was originally identified as the product of a proto-oncogene involved in chromosomal translocations in multiple myeloma. Although IRF-4 is expressed in myeloid cells, its function in that lineage is not known. The closely related family member IRF-8 is a critical regulator of myelopoiesis, which when deleted in mice results in a syndrome highly similar to human chronic myelogenous leukemia. In early lymphoid development, we have shown previously that IRF-4 and IRF-8 can function redundantly. We therefore investigated the effects of a combined loss of IRF-4 and IRF-8 on hematologic tumorigenesis. We found that mice deficient in both IRF-4 and IRF-8 develop from a very early age a more aggressive chronic myelogenous leukemia-like disease than mice deficient in IRF-8 alone, correlating with a greater expansion of granulocyte-monocyte progenitors. Although these results demonstrate, for the first time, that IRF-4 can function as tumor suppressor in myeloid cells, interestingly, all mice deficient in both IRF-4 and IRF-8 eventually develop and die of a B-lymphoblastic leukemia/lymphoma. Combined losses of IRF-4 and IRF-8 therefore can cooperate in the development of both myeloid and lymphoid tumors.

Inactivation of the INK4A/ARF (CDKN2) locus Cooperates with HMGA1 in T-Cell Leukemogenesis.

Abstract 3969Poster Board III-905Because T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive lymphoid leukemia with a high rate of relapse in both children and adults, research is urgently needed to discover molecular pathways that could be targeted in therapy. Recent studies have identified NOTCH1 activating mutations, genomic classifiers, and other molecular pathways that are disrupted in T-ALL, although these findings have not yet translated into better therapy or improved long-term survival. Thus, we are investigating molecular pathways that play a causative role in T-ALL and could be modulated by therapy. We previously showed that HMGA1a is overexpressed in human T-ALL. Further, HMGA1a transgenic mice develop aggressive T-ALL with complete penetrance, indicating that HMGA1a induces leukemogenic transformation in vivo. Disruption of the CDKN2A (INK4A/ARF) tumor suppressor locus has been reported in up to 90% of T-ALL, suggesting that this contributes to leukemic transformation in T-cells. To determine if loss of function of the INK4A/ARF tumor suppressor locus could cooperate with HMGA1a in T-ALL, we crossed our HMGA1a transgenics with mice that are null for the INK4A/ARF locus. We found that T-ALL was markedly accelerated in the HMGA1a transgenics that are also null at this tumor suppressor locus. These mice died significantly earlier than the HMGA1 transgenics that are wildtype at the INK4A/ARF locus (4.8 months+/- 1.0 month versus 10.5 +/- 2.5 months, p < 0.0001 by the student's t-test). The HMGA1a transgenic, INK4A/ARF null mice recapitulate salient clinical and pathologic features of human T-ALL. All mice appeared ill and exhibit marked organomegaly with extensive leukemic infiltration. Immunophenotyping by fluorescence activated cell sorting showed T-ALL in all cases with identical T-cell markers (CD3, CD8, and αβ-TCR) to those previously reported for the HMGA1a transgenics at 8-10 months. Interestingly, the HMGA1a-INK4A/ARF+/- mice also developed early lymphoid malignancy in 6/17 cases. The accelerated lymphoid malignancy in these mice suggests that there is loss of heterozygosity at the INK4A/ARF locus. Alternatively, haploinsufficiency of INK4A and/or ARF may be sufficient to promote lymphoid tumorigenesis in the setting of HMGA1a overexpression. To determine if HMGA1 overexpression and loss of INK4A/ARF expression occur in human T-ALL, we analyzed gene expression profile analysis in leukemic blasts from children and young adults with T-ALL. We found high levels of HMGA1 expression and low levels of INK4A/ARF expression in most patients. Taken together, our results indicate that overexpression of HMGA1 and loss of function at the INK4A/ARF (CDKN2A) tumor suppressor locus cooperate in T-cell leukemogenesis and these pathways could represent rational therapeutic targets. Further, our mice provide a novel preclinical model for T-ALL. Disclosures:No relevant conflicts of interest to declare.

Developmental Phases of Inflammation-Induced Massive Lymphoid Hyperplasia and Extensive Changes in Epithelium in an Experimental Model of Allergy

Direct evidence that inflammation is linked to carcinogenesis has yet to be established. Very few data are available on the developmental phases of inflammation-induced immune dysfunction that may lead to tumorigenesis. In a series of studies conducted in the 1980s and 1990s, an experimental model of acute and chronic inflammation was established in guinea pig conjunctiva by topical application of fluoresceinyl ovalbumin (FLOA) for up to 30 months. In this updated report, some of the findings are reanalyzed and expanded to identify that at lease 3 developmental phases were involved during the entire course of inflammatory responses including (1) an acute response (phase A) involving IgE-mast cell sensitization and degranulation; (2) an intermediate phase (phase B), a desensitization phenomenon and loss of mast cell function and neovascularization; (3) a chronic response (phase C) and induction of massive lymphoid hyperplasia, follicular formation with germinal centers, increased swollen goblet cells, extensive epithelial thickening and thinning, and angiogenesis. The results suggest evidence of a direct association between inflammation and the development of tumor-like lesions in lymphoid tissues and extensive changes in adjacent epithelia. Confirmation that inflammation induces irreversible changes in lymphoid and epithelial tissues leading to lymphoid tumorigenesis and/or carcinogenesis requires further studies. Understanding the developmental phases of immune dysfunction may provide unique opportunities for diagnosis and treatment of inflammatory diseases, autoimmune disorders, and cancers including lymphomas associated with Sjogren syndrome, squamous cell carcinoma of the conjunctiva, and other lymphomas or epithelial cancers. It is suggested that inflammatory mediators are ideal targets (biomarkers) for diagnosis, chemoprevention, and therapy for several cancers.

Hyper IgM syndromes.

The immune defense against extracellular pathogens is largely dependent on antibody production. Class switch recombination and somatic hypermutation shape the secondary antibody repertoire in peripheral lymphoid tissue. In the past few years, a series of primary immune deficiencies characterized by defects in these processes and collectively referred to as hyper-IgM syndromes, have been described. Careful investigation of these rare "experiments of nature" has enabled to identify novel genes and molecular events that drive terminal B-cell differentiation. Abnormalities in these genes are likely involved also in lymphoid tumorigenesis and autoimmunity.

Cancer risk and the ATM gene: a continuing debate.

Deficiencies in the ability of cells to sense and repair damage in individuals with rare genetic instability syndromes increase the risk of developing cancer. Ataxia-telangiectasia (A-T), such a condition, is associated with a high incidence of leukemia and lymphoma that develop in childhood. Although A-T is an autosomal recessive disorder, some penetrance appears in individuals with one mutated ATM gene (A-T carriers), namely, an increased risk of developing breast cancer. The gene mutated in A-T, designated ATM, is homologous to several DNA damage recognition and cell cycle checkpoint control genes from other organisms. Recent studies suggest that ATM is activated primarily in response to double-strand breaks, the major cytotoxic lesion caused by ionizing radiation, and can directly bind to and phosphorylate c-Abl, p53, and replication protein A (RPA). Analysis of ATM mutations in patients with A-T or with sporadic non-A-T cancers has suggested the existence of two classes of ATM mutation: null mutations leading to A-T and dominant negative missense mutations predisposing to cancer in the heterozygous state. Studies with A-T mouse models have helped determine the basis of lymphoid tumorigenesis in A-T and have shown that ATM plays a critical role in maintaining genetic stability by ensuring high-fidelity execution of chromosomal events. Thus, ATM appears to act as a caretaker of the genome.

ATM in Lymphoid Development and Tumorigenesis

This chapter discusses various applications of the ataxia-telangiectasia (A-T) mouse model, the Atm -/- mice, to elucidate the basis of lymphoid defects and tumorigenesis in A-T. Immune defects in A-T patients are characterized by thymic hypoplasia or absence of thymus, reduced circulating T cells, and a selective deficiency of immunoglobulin isotypes, including IgA, IgE, IgG2, and IgG. Genetic evidence suggests that ataxia-telangiectasia mutation (ATM) plays a crucial role in the regulation of V(D)J recombination and abnormality that could contribute to the genetic instability and tumorigenesis in ATM -/- lymphocytes. Therefore, the elucidation of the exact mechanism of such potential functions of ATM is crucial for the understanding of the A-T lymphoid tumorigenesis. The V(D)J recombination process involves the introduction of DNA double-stranded breaks by RAG-1/2 at the recombination signal sequences flanking the V(D)J gene segments. Subsequently, the recombination signaling sequences are precisely joined to form signal joints and the coding ends are usually imprecisely joined to form coding joints, sometimes with the addition of N regions. ATM might play two possible roles during V(D)J recombination. Based on its structural similarity to DNA-PKcs that is essential in the joining processes of the DNA double-stranded break repair, ATM might perform a similar function in the joining processes of the V(D)J gene segments.

An E mu-v-abl transgene elicits plasmacytomas in concert with an activated myc gene.

To clarify how the v-abl oncogene of Abelson murine leukemia virus contributes to lymphoid tumorigenesis, we introduced the gene linked to an immunoglobulin heavy chain enhancer (E mu) into the mouse germline. Although lymphoid development was not detectably affected in young E mu-v-abl mice, three transgenic lines shared a high predisposition to develop clonal plasmacytomas that secreted IgA or IgG. The unexpected absence of pre-B lymphomas suggests that Abelson virus generates such tumors by infecting an early lymphoid progenitor cell that has not yet activated the heavy chain enhancer. Most plasmacytomas bore a rearranged c-myc gene, apparently as a result of spontaneous translocation to the Igh locus. Moreover, progeny of a cross with analogous E mu-myc mice rapidly developed oligoclonal plasmacytomas. Thus, the collusion of v-abl with c-myc is stage specific, efficiently transforming plasma cells but not pre-B cells or B cells.

Molecular Genetics of Lymphoid Tumorigenesis

The body of this work illustrates the utility of the combined cytogenetic and molecular approach to lymphoid tumorigenesis. A number of tumor-specific translocations have proven amenable to dissection by molecular techniques. We have a firm grasp of the general principles that underlie lymphoid neoplasia; in particular, the activation of cellular oncogenes by translocation into genes of the immunoglobulin superfamily is a widespread phenomenon. However, numerous lymphopoietic malignancies are only poorly understood. These remain a challenge for the continued application of these methodologies.