Predictive Value of CD8 Expression and FoxP3 Methylation in Nasopharyngeal Carcinoma Patients Treated with Chemoradiotherapy in a Non-endemic Area.

Undifferentiated Nasopharyngeal Carcinoma (UNPC) is associated with Epstein-Barr Virus (EBV) and characterized by an abundant immune infiltrate potentially influencing the prognosis. Thus, we retrospectively assessed the significance of immunosuppression in the UNPC microenvironment as prognostic biomarker of treatment failure in a non-endemic area, and monitored the variation of systemic EBV-specific immunity before and after chemoradiotherapy (CRT). DNA and RNA were extracted from diagnostic biopsies obtained by tumor and adjacent mucosa from 63 consecutive EBV+ UNPC patients who underwent radical CRT. Among these patients 11 relapsed within 2 years. The expression of the EBV-derived UNPC-specific BARF1 gene and several immune-related genes was monitored through quantitative RT-PCR and methylation-specific PCR analyses. Peripheral T cell responses against EBV and BARF1 were measured in 14 patients (7 relapses) through IFN-γ ELISPOT assay. We found significantly higher expression levels of BARF1, CD8, IFN-γ, IDO, PD-L1, and PD-1 in UNPC samples compared to healthy tissues. CD8 expression was significantly reduced in both tumor and healthy tissues in UNPC patients who relapsed within two years. We observed a hypomethylated FOXP3 intron 1 exclusively in relapsed UNPC patients. Finally, we noticed a significant decrease in EBV- and BARF1-specific T-cells after CRT only in relapsing patients. Our data suggest that a high level of immunosuppression (low CD8, hypomethylated FoxP3) in UNPC microenvironment may predict treatment failure and may allow an early identification of patients who could benefit from the addition of immune modulating strategies to improve first line CRT.

Exploiting a new strategy to induce immunogenic cell death to improve dendritic cell-based vaccines for lymphoma immunotherapy.

Although promising, the clinical benefit provided by dendritic cell (DC)-based vaccines is still limited and the choice of the optimal antigen formulation is still an unresolved issue. We have developed a new DC-based vaccination protocol for aggressive and/or refractory lymphomas which combines the unique features of interferon-conditioned DC (IFN-DC) with highly immunogenic tumor cell lysates (TCL) obtained from lymphoma cells undergoing immunogenic cell death. We show that treatment of mantle cell lymphoma (MCL) and diffuse large B-cell lymphoma (DLBCL) cell lines with 9-cis-retinoic acid and IFNα (RA/IFNα) induces early membrane exposure of Calreticulin, HSP70 and 90 together with CD47 down-regulation and enhanced HMGB1 secretion. Consistently, RA/IFNα-treated apoptotic cells and -TCLs were more efficiently phagocytosed by DCs compared to controls. Notably, cytotoxic T cells (CTLs) generated with autologous DCs pulsed with RA/IFNα-TCLs more efficiently recognized and specifically lysed MCL or DLBCL cells or targets loaded with several HLA-A*0201 cyclin D1 or HLA-B*0801 survivin epitopes. These cultures also showed an expansion of Th1 and Th17 cells and an increased Th17/Treg ratio. Moreover, DCs loaded with RA/IFNα-TCLs showed enhanced functional maturation and activation. NOD/SCID mice reconstituted with human peripheral blood lymphocytes and vaccinated with autologous RA/IFNα-TCL loaded-IFN-DCs showed lymphoma-specific T-cell responses and a significant decrease in tumor growth with respect to mice treated with IFN-DC unpulsed or loaded with untreated TCLs. This study demonstrates the feasibility and efficacy of the use of RA/IFNα to generate a highly immunogenic TCL as a suitable tumor antigen formulation for the development of effective anticancer DC-based vaccines.

Cross talk between EBV and telomerase: the role of TERT and NOTCH2 in the switch of latent/lytic cycle of the virus.

Epstein-Barr virus (EBV)-associated malignancies, as well as lymphoblastoid cell lines (LCLs), obtained in vitro by EBV infection of B cells, express latent viral proteins and maintain their ability to grow indefinitely through inappropriate activation of telomere-specific reverse transcriptase (TERT), the catalytic component of telomerase. Our previous studies demonstrated that high levels of TERT expression in LCLs prevent the activation of EBV lytic cycle, which is instead triggered by TERT silencing. As lytic infection promotes the death of EBV-positive tumor cells, understanding the mechanism(s) by which TERT affects the latent/lytic status of EBV may be important for setting new therapeutic strategies. BATF, a transcription factor activated by NOTCH2, the major NOTCH family member in B cells, negatively affects the expression of BZLF1, the master regulator of viral lytic cycle. We therefore analyzed the interplay between TERT, NOTCH and BATF in LCLs and found that high levels of endogenous TERT are associated with high NOTCH2 and BATF expression levels. In addition, ectopic expression of TERT in LCLs with low levels of endogenous telomerase was associated with upregulation of NOTCH2 and BATF at both mRNA and protein levels. By contrast, infection of LCLs with retroviral vectors expressing functional NOTCH2 did not alter TERT transcript levels. Luciferase reporter assays, demonstrated that TERT significantly activated NOTCH2 promoter in a dose-dependent manner. We also found that NF-κB pathway is involved in TERT-induced NOTCH2 activation. Lastly, pharmacologic inhibition of NOTCH signaling triggers the EBV lytic cycle, leading to the death of EBV-infected cells. Overall, these results indicate that TERT contributes to preserve EBV latency in B cells mainly through the NOTCH2/BAFT pathway, and suggest that NOTCH2 inhibition may represent an appealing therapeutic strategy against EBV-associated malignancies.

Exposure to animals and increased risk of marginal zone B-cell lymphomas of the ocular adnexae.

BACKGROUND: Ocular adnexal marginal zone B-cell lymphoma (OAMZL) has been associated with Chlamydophila psittaci, an infection that may be transmitted by carrier animals. However, it is still unclear whether exposure to animals affects the risk of OAMZL in comparison with other lymphoma histotypes. We therefore investigated the role of professional and/or domestic exposures to animals in the occurrence of OAMZL, as compared with other types of lymphoma. METHODS: A hospital-based case-control study was carried out on 43 consecutive OAMZL patients (cases) and 87 consecutive patients with nodal non-Hodgkin's lymphomas (NHLs; controls). Multiple logistic regression (MLR) odds ratios (ORs), and 95% confidence intervals (CIs) were used to estimate the association between exposures to animals and OAMZL risk. RESULTS: A higher proportion of cases reported a lifetime exposure to household animals (79.1% vs 64.4% among controls), with a non-statistical significant MLR-OR of 2.18 (95% CI: 0.85-5.62). The OAMZL cases more frequently reported a history of occupation in breeding and/or slaughtering than controls (34.9% vs 6.9%), with an overall increased risk of 7.69 (95%CI: 2.65-22.34). CONCLUSION: These results indicate that, compared with nodal NHLs, the risk of OAMZL is markedly increased by contact with animals, particularly by occupational exposures.

Immunoglobulin gene repertoire in ocular adnexal lymphomas: hints on the nature of the antigenic stimulation.

Evidence from certain geographical areas links lymphomas of the ocular adnexa marginal zone B-cell lymphomas (OAMZL) with Chlamydophila psittaci (Cp) infection, suggesting that lymphoma development is dependent upon chronic stimulation by persistent infections. Notwithstanding that, the actual immunopathogenetical mechanisms have not yet been elucidated. As in other B-cell lymphomas, insight into this issue, especially with regard to potential selecting ligands, could be provided by analysis of the immunoglobulin (IG) receptors of the malignant clones. To this end, we studied the molecular features of IGs in 44 patients with OAMZL (40% Cp-positive), identifying features suggestive of a pathogenic mechanism of autoreactivity. Herein, we show that lymphoma cells express a distinctive IG repertoire, with electropositive antigen (Ag)-binding sites, reminiscent of autoantibodies (auto-Abs) recognizing DNA. Additionally, five (11%) cases of OAMZL expressed IGs homologous with autoreactive Abs or IGs of patients with chronic lymphocytic leukemia, a disease known for the expression of autoreactive IGs by neoplastic cells. In contrast, no similarity with known anti-Chlamydophila Abs was found. Taken together, these results strongly indicate that OAMZL may originate from B cells selected for their capability to bind Ags and, in particular, auto-Ags. In OAMZL associated with Cp infection, the pathogen likely acts indirectly on the malignant B cells, promoting the development of an inflammatory milieu, where auto-Ags could be exposed and presented, driving proliferation and expansion of self-reactive B cells.

HLA DR-DQ combination associated with the increased risk of developing human HCV positive non-Hodgkin's lymphoma is related to the type II mixed cryoglobulinemia.

This investigation was focused on the contribution of individual human leukocyte antigen (HLA)-DR and -DQ alleles to the human hepatitis C virus (HCV)(+) non-Hodgkin's lymphoma (NHL), with and without mixed cryoglobulinemia (MC), to study whether individual HLA class II alleles are expressed preferentially or equally in human HCV-specific NHL. For this purpose, peripheral blood mononuclear cells were obtained from two groups of patients with HCV(+) NHL and with or without MC (70 and 71 cases, respectively), and from 4575 blood donors. Eighty-three subjects with HCV infection only, and 118 patients with MC, only without lymphoma, were added as additional control groups. Individual HLA-DR and -DQ alleles were determined using high-resolution sequence-based typing and then data were collected by considering the HLA-DRB1 and DQB1 supertypes on the basis of common structural and functional features, proposed by in silico Bioinformatic studies. From the data, it is evidenced that the DR5-DQ3 HLA combination was strongly associated with the HCV (+) MC (+) NHL group of patients compared with bone marrow donor population (P

Ocular adnexal MALT lymphoma: an intriguing model for antigen-driven lymphomagenesis and microbial-targeted therapy.

Non-Hodgkin's lymphomas constitute one half of malignancies arising in the orbit and the ocular adnexae. Mucosa-associated lymphoid tissue (MALT)-type lymphoma is the most common histological category in this anatomic region. The incidence of ocular adnexal lymphoma of mucosa-associated lymphoid tissue-type (OAML) is increasing and recent studies offered new relevant insights in molecular, pathogenetic and therapeutic issues on these neoplasms. A pathogenetic model of antigen-driven lymphoproliferation similar to that reported for Helicobacter pylori-related gastric MALT lymphomas has been hypothesized for OAML. This notion is supported by the association between OAML and Chlamydophila psittaci infection, an association that is of likely pathogenetic relevance and may influence both the biological behavior and the therapeutic management of these neoplasms. However, this association displays evident geographical variability indicating that other etiopathogenic agents could be involved. These recent acquisitions coupled with the occurrence of chromosomal translocations and other genetic alterations, as well as additional risk factors like autoimmune disorders have contributed to render OAML an exciting challenge for a broad group of physicians and scientists. OAML is an indolent and rarely lethal malignancy that, in selected patients, can be managed with observation alone. Lymphomatous lesions are frequently responsible for symptoms affecting patient's quality of life, requiring, therefore, immediate treatment. Several therapeutic strategies are available, often associated with relevant side-effects. However, the therapeutic choice in OAML is not supported by consolidated evidence due to the lack of prospective trials. In this review, we analyze the most relevant biological, molecular, pathological and clinical features of OAML and propose some therapeutic guidelines for patients affected by this malignancy.

Genetic insights into the disease mechanisms of type II mixed cryoglobulinemia induced by hepatitis C virus.

The ability of the immune system to distinguish between self and non-self is critical to the functioning of the immune response. A breakdown in these mechanisms can lead to the onset of autoimmune disease. Clinical and molecular data suggest that shared immunogenetic mechanisms lead to the autoimmune process. The most studied part of the autoimmune process is the human leukocyte antigen (HLA) region. Recently, progress has been made in narrowing down HLA cluster classifications based on structural and functional features of HLA alleles. Using this approach we have investigated 175 patients with hepatitis C virus (HCV)-induced type II cryoglobulinemia (MC), and compared them to a control group of 14,923 bone marrow donors. Additionally, we investigated the frequency of HLA homozygosity in the same groups of subjects. Our results provide evidence of a role for DR5 and DQ3 HLA class II clusters and a higher frequency of HLA homozygous leading to the clinical outcome of type II mixed cryoglobulinemic autoimmune disease. The DR5 cluster is characterized by a Glu in beta 9 and its polymorphism is connected with preferred anchors at beta 9 of the binding peptide, while the DQ3 cluster is characterized by Glu B86 and Leu B87, which allows the binding of large hydrophobic amino acids at p1 of the binding peptide. The mechanisms by which variations in HLA lead to autoimmunity remain unknown, although they are likely to be mediated by continuous presentation of HCV epitopes to T cells and a genetic background that limits the effective clearance of HCV. The results presented in this paper have increased our knowledge of the mechanism of autoimmune disease and B-cell lymphoproliferation during HCV infection. The work was performed in accordance with the principles of the 1983 Declaration of Helsinki. There is no conflict of interest.

Proteins specifically hyperexpressed in a coeliac disease patient with aberrant T cells.

An aberrant T cell population is the basis for diagnosis of refractory coeliac disease and determines the risk of enteropathy-associated T cell lymphoma. This disease is serious with a poor survival. Pathogenetic mechanisms sustaining aberrant T cell proliferation remain unknown. Recently, alemtuzumab has been proposed as a promising new approach to treat these patients. Only few single cases have been tested at present; nevertheless, in all the cases a clinical improvement was observed. However, whether intraepithelial lymphocytes have been targeted effectively by alemtuzumab is still debated. This study reports, using two-dimensional difference gel electrophoresis (2D DIGE), hyperexpressed proteins associated specifically with aberrant T cells found in a patient with coeliac disease by comparison of the protein expression of this sample with that of patients with coeliac disease and polyclonal T cells or with control subjects. The data demonstrated a significantly higher expression of IgM, apolipoprotein C-III and Charcot-Leyden crystal proteins in a duodenal biopsy specimen of the patient with clonal T cells compared with that of other patients. These preliminary results allow hypothesizing different clinical effects of alemtuzumab in patients with coeliac disease and aberrant T cell proliferation, because as well as the probable effect on T cells, alemtuzumab could exert its effect by acting on inflammatory associated CD52(+) IgM(+) B cells and eosinophil cells, known to produce IgM and Charcot-Leyden crystal proteins, that we demonstrated to be altered in this patient. The results also emphasize the possible association of apolipoprotein with aberrant T cell proliferation.

Clinical implications of hepatitis C virus infection in MALT-type lymphoma of the ocular adnexa.

BACKGROUND: A pathogenic link between hepatitis C virus (HCV) and MALT-type lymphomas has been suggested. However, studies assessing the role of HCV infection separately in different forms of MALT lymphomas are not available. PATIENTS AND METHODS: The prevalence and clinical implications of HCV seropositivity were analyzed in 55 patients with ocular adnexa lymphoma (OAL) of MALT-type. RESULTS: HCV seropositivity was detected in seven (13%) patients. At presentation, HCV infection was significantly associated with concomitant extra-orbital disease, lymph node dissemination and involvement of additional extranodal organs. HCV seropositivity was associated also with a higher relapse rate and worse progression-free survival. In fact, 16 patients experienced relapse after first-line treatment: five (71%) were HCV-seropositive and 11 (23%) were HCV-seronegative, with a median TTP of 31 and 50+ months (P = 0.01), and a 5-year progression-free survival of 43 +/- 18% and 77 +/- 7% (P = 0.005), respectively. HCV-seropositive patients experienced frequent relapses despite further lines of therapy; relapses were systemic in all cases but one; multiple subcutaneous nodules were common at relapse. CONCLUSIONS: HCV seropositivity is present in 13% of OAL of MALT-type. Concomitant HCV infection is associated with more disseminated disease and aggressive behavior in OAL, with a consequent potential negative impact in patients managed with radiotherapy alone.

Hepatitis C virus-related hepatocellular carcinoma and B-cell lymphoma patients show a different profile of major histocompatibility complex class II alleles.

Comparison of human leukocyte antigen (HLA) frequencies in patients with hepatitis C virus (HCV)-associated hepatocellular carcinoma (HCC) and in patients with HCV-associated non-Hodgkin's lymphoma (NHL) has not been addressed previously. To this aim, we investigated the distribution of HLA class II alleles in two selected groups of HCV-infected patients. Group 1 included 50 patients with HCV-associated NHL; group 2 included 29 patients with HCV-associated HCC. A control group included 144 hospitalized patients without NHL or HCC and who were negative for HCV, hepatitis B virus, and human immunodeficiency virus antibodies. Polymerase chain reaction sequence DRB1 and DQB1 specific-primer methods were used. DRB1*1101/DQB1*0301 haplotype, which mainly favors the spontaneous clearance of HCV infection, was lower in HCC subjects than in controls, whereas HLA-DRB1*1104/DQB1*0301, was higher in NHL patients. These findings suggest different pathogenic pathways in HCC and in NHL development. In patients with HCV-associated HCC, a major protective role of DQB1*0301 allele, rather than DRB1*11, was found, probably because of a better HLA class II-associated virus clearance. By contrast, the same allele as HLA-DRB1*04 showed an increase in HCV-associated NHL. These data suggest that NHL and HCC development may be associated to a different response with respect to chronic HLA class II-restricted antigen presentation (perhaps a switch toward CD4+Th2 response in NHL?) or, alternatively, that these alleles could be in linkage disequilibrium to unrelated gene(s), or are in synergy with other immunomodulatory genes that may confer increased risk for NHL.

The Italian multi-centre project on evaluation of MRI and other imaging modalities in early detection of breast cancer in subjects at high genetic risk.

This report presents the preliminary results of the first phase (21 months) of a multi-centre, non-randomised, prospective study, aimed at evaluating the effectiveness of contrast-enhanced magnetic resonance imaging (MRI), X-ray mammography (XM) and ultrasound (US) in early diagnosis of breast cancer (BC) in subjects at high genetic risk. This Italian national trial (coordinated by the Istituto Superiore di Sanità, Rome) so far recruited 105 women (mean age 46.0 years; median age 51.0; age range 25-77 years), who were either proven BRCA1 or BRCA2 mutation carriers or had a 1 in 2 probability of being carriers (40/105 with a previous personal history of BC). Eight cases of breast carcinomas were detected in the trial (mean age 55.3 years, median age 52.5; age range 35-70 years; five with previous personal history of BC). All trial-detected BC cases (8/8) were identified by MRI, while XM and US correctly classified only one. MRI had one false positive case, XM and US none. Seven "MRI-only" detected cancers (4 invasive, 3 in situ) occurred in both pre- (n = 2) and post-menopausal (n = 5) women. With respect to the current XM screening programmes addressed to women in the age range 50-69 years, the global incidence of BC in the trial (7.6%) was over ten-fold higher. The cost per "MRI-only" detected cancer in this particular category of subjects at high genetic risk was substantially lower than that of an XM-detected cancer in the general women population. These preliminary results confirmed that MRI is a very useful tool to screen subjects at high genetic risk for breast carcinoma, not only in pre-, but also in post-menopausal age, with a low probability of false positive cases.

Microsatellite instability and high content of activated cytotoxic lymphocytes identify colon cancer patients with a favorable prognosis.

Colorectal cancers with high-frequency microsatellite instability show peculiar clinicopathological features and a favorable clinical outcome. We investigated whether the improved prognosis for these cancers is related to the content of activated cytotoxic intraepithelial T lymphocytes. Microsatellite instability and the amount of activated cytotoxic lymphocytes were analyzed according to clinicopathological features, survival, and disease recurrence in 109 right-sided colon carcinomas from 245 consecutive patients with stage II/III colon cancer that underwent radical surgery. High-frequency microsatellite instability was found in 43% of stage II/III proximal colon cancers and was associated with significantly higher numbers of activated cytotoxic lymphocytes. High-frequency microsatellite instability, as well as the content of intratumoral-activated cytotoxic T lymphocytes correlated with improved overall and disease-free survival, particularly in patients with stage III tumors. Multivariate analysis revealed that patients with both features had a risk of death and relapse markedly lower than that associated with microsatellite status or intratumoral cytotoxic lymphocytes separately. The presence of local cytotoxic immune responses is probably the major determinant of the good clinical course of patients with microsatellite unstable colon cancer. Furthermore, high-frequency microsatellite instability coupled with a high content of intratumoral cytotoxic lymphocytes may identify a subset of colon cancer patients with a favorable clinical outcome, particularly in stage III disease.

Pathogenetic and histogenetic features of HIV-associated Hodgkin's disease.

Compared with the cases in the general population, Hodgkin's disease (HD) arising in the HIV setting shows distinctive features in terms of epidemiology, aetiopathogenesis, histopathology and clinical behaviour. Although HD does not represent an AIDS-defining condition, recent evidence consistently indicates that HIV-infected individuals have a significantly increased risk of developing HD. HIV-related HD is characterised by the preponderance of aggressive histological subtypes, advanced stage at presentation, and highly malignant clinical course. Moreover, unlike HD in the general population, the large majority of HIV-related HD cases are pathogenetically linked to Epstein-Barr virus (EBV), with rates of EBV positivity ranging from 80 to 100%. Hodgkin and Reed-Sternberg cells of these cases invariably show a strong expression of the EBV-encoded latent membrane protein-1 (LMP-1), which functions as a constitutively activated tumour necrosis factor (TNF) receptor-like molecule. Usurpation of physiologically relevant pathways by LMP-1 may lead to the simultaneous or sequential activation of signalling pathways involved in the promotion of cell activation, growth, and survival, contributing thus to most of the features of HIV-related HD.