Valli De Re

Pegylated interferon-α, ribavirin, and rituximab combined therapy of hepatitis C virus–related mixed cryoglobulinemia: a long-term study

This study illustrates the use and efficacy of a combination of pegylated interferon-alpha (Peg-IFN-alpha) and ribavirin (RBV), with or without rituximab (RTX), in hepatitis C virus (HCV)-related mixed cryoglobulinemia (MC). Twenty-two patients with HCV-related MC received Peg-IFN-alpha (2a: 180 mug or 2b: 1.5 mug/kg) weekly plus RBV (1000 or 1200 mg) daily for 48 weeks, and RTX (375 mg/m(2)) once a week for 1 month followed by two 5-monthly infusions (termed PIRR). Fifteen additional patients received Peg-IFN-alpha/RBV with the same modalities as the PIRR schedule. Complete response was achieved in 54.5% (12/22) and in 33.3% (5/15) of patients who received PIRR and Peg-IFN-alpha/RBV, respectively (P < .05). Clearance of HCV RNA and conversion of B-cell populations from oligoclonal to polyclonal in liver, bone marrow, and peripheral blood was maintained for up to 3 years in 10 of 12 (83.3%) and in 2 of 5 (40%) patients receiving PIRR and Peg-IFN-alpha/RBV, respectively (P < .01). Cryoproteins in 22.7% (5/22) of patients with PIRR and in 33.3% (5/15) with Peg-IFN-alpha/RBV persisted despite sustained HCV RNA clearance. No response occurred in remaining 5 patients of both groups. PIRR therapy is well tolerated and more effective than Peg-IFN-alpha/RBV combination in HCV-related MC. Its effect may last for more than 3 years.

Oligoclonal non-neoplastic B cell expansion is the key feature of type II mixed cryoglobulinemia: clinical and molecular findings do not support a bone marrow pathologic diagnosis of indolent B cell lymphoma.

OBJECTIVE Type II mixed cryoglobulinemia (type II MC) is often characterized by features of indolent B cell lymphoma (IBCL) found on pathologic examination of bone marrow, whereas the clinical evidence does not indicate a neoplastic disorder. To better address the issue of indolent malignant versus nonmalignant bone marrow lymphoproliferation underlying type II MC, molecular analyses of B cell clonality were performed in the present study, in conjunction with clinical and pathologic characterization. METHODS Polymerase chain reaction DNA amplification of immunoglobulin heavy chain genes was performed in bone marrow biopsy specimens obtained from 15 selected patients with type II MC, all infected with hepatitis C virus. Five of them had also developed overt B cell lymphoma (OBCL) during followup. Bone marrow features were consistent with IBCL in 9 of the 15 patients (group 1) and with reactive lymphoplasmacytosis in 6 of the 15 (group 2). RESULTS An oligoclonal B cell expansion was detected in 6 of 9 baseline bone marrow lesions from group 1 patients (biclonal or monoclonal expansion in the remaining 3 cases), and in 6 of 6 from group 2 patients. OBCL was always monoclonal. Selected lesions were analyzed by clonospecific hybridization and by cloning and sequence analysis in patients who had developed OBCL at followup. In 4 of 5 cases, OBCL did not originate from the dominant B cell clones that were overexpanded in the putative neoplastic baseline bone marrow lesions. OBCL clones showed significant homology with rheumatoid factor database sequences. CONCLUSION Based on the present results, as well as on evidence from previous studies of liver lesions, oligoclonal non-neoplastic B cell proliferation in the course of chronic infection-related inflammation appears to be the key feature of type II MC. Of note, molecular evidence from target tissues supports the clinical findings both at the time of type II MC diagnosis and in cases of OBCL complication. Bone marrow pathologic findings resembling those of IBCL should thus be considered in the light of clinical and molecular evidence.

Pre‐malignant and malignant lymphoproliferations in an HCV‐infected type II mixed cryoglobulinemic patient are sequential phases of an antigen‐driven pathological process

Type II mixed cryoglobulinemia (MC) is a systemic vasculitis characterized by the presence in the serum of a monoclonal cryoprecipitable IgM with rheumatoid factor (RF) activity. Hepatitis C virus (HCV) has been recognized as its major etiologic factor. Because MC frequently evolves into overt B‐cell non‐Hodgkins lymphoma (NHL), chronic HCV infection is hypothesized to lead to both benign and malignant lymphoproliferative disease. In this study, we investigated mutations in the VH and VK genes of the B‐cell clone originating the overt B‐cell lymphoma in a subject with MC. Mutational patterns were analyzed longitudinally in two bone marrow biopsies obtained at the stage of MC, as well as in multiple involved tissues (bone marrow, liver, and peripheral blood cells) at the stage of overt NHL. Hybridization of variable‐diversity‐joining (VDJ) PCR products with a probe specific for the neoplastic clone indicated that the lymphoma originated from one of the clones over‐stimulated during MC. This clone producing an IgM highly homologous to a protein with RF specificity may explain the MC syndrome in the patient. Moreover, the presence of an IgH ongoing mutation process and the expression of an Ig antigen receptor significantly homologous to an anti‐HCV protein support the hypothesis that the MC syndrome and the subsequent evolution to NHL are antigen‐driven lymphoproliferative processes possibly sustained by HCV. Furthermore, the marked reduction in intra‐clonal diversity in the last bone marrow biopsy obtained at the stage of overt NHL points out a minor dependence of the cells on the antigen‐driven mechanism, although an intrinsic propensity of the neoplastic cell to undergo replacement mutations cannot be excluded. Int. J. Cancer 87:211–216, 2000.

Salivary gland B cell lymphoproliferative disorders in Sjögren's syndrome present a restricted use of antigen receptor gene segments similar to those used by hepatitis C virus-associated non-Hodgkins's lymphomas

Sjögrens syndrome (SS) represents a pathological model of the evolution from polyclonal B lymphocyte activation to oligoclonal / monoclonal B cell expansion, which may culminate in the development of a malignant lymphoproliferative disease. The different phases of this process are usually marked by the appearance of antigen‐driven activated B cell clones, which are commonly IgM‐positiveand with rheumatoid factor (RF) activity. However, the agent(s) able to trigger B cell proliferation is still unknown. A similar pathogenetic mechanism exist in mixed cryoglobulinemia, another autoimmune disease that often evolves to non‐Hodgkins lymphoma (NHL) and in which hepatitis C virus (HCV) infection has been demonstrated to play an etiopathogenetic role. In the present study, we cloned and sequenced the antigen receptor (IgR) variable region genes of SS‐associated monoclonal non‐neoplastic lymphoproliferations and compared them with those of our previous reported HCV‐associatedNHL, to derive clues on the antigen(s) that sustains SS. The results obtained showed remarkable homologies between the antigen combinatory regions of the IgR expressed by both diseases. These homologies concern: a) the specific combinations of heavy and light variable region genes; b) the limited length of complementarity‐determining regions (CDR3); c) the homology with antibodies with RF activity; d)the amino acid sequences of CDR3 in which common somatic mutations are present that possibly determine the antigen‐binding specificity. In conclusion, although there are significant differences between SS and HCV‐associated lymphoproliferative diseases, they share many molecular characteristics, which suggest an immunological cross‐reactivity or molecular mimicry among the agents that underlie these disorders.

Intrahepatic B cell clonal expansions and extrahepatic manifestations of chronic HCV infection

B cell repertoire in three biological compartments (liver, bone marrow and peripheral blood) of 30 unselected patients chronically infected with HCV has been characterized. Restriction of humoral immune response defined by enrichment of B cell clonal expansions occurred in the liver of 15 patients (50%), in the bone marrow and peripheral blood of 2 (6.7%) and 8 (26.7%) patients, respectively. An in situ hybridization technique was developed for the detection of dominant B cell clones in patients with monoclonal expansions. It was shown that morphologically distinct B cell expansion contributes to the formation of intraportal follicle‐like structures. Sequence analyses of CDRH3 gene segments revealed a wide range of variations. Clones derived from the same founder were demonstrated simultaneously in the three compartments explored. The occurrence of B cell clonal expansions profoundly influenced the clinical expression of HCV infection, since it was associated with extrahepatic manifestations. In sharp contrast, no extrahepatic signs or disease occurred in patients without evidence of intrahepatic B cell clonalities. These findings emphasize the profound B cell function derangement in at least half of HCV‐infected patients. Thus, the restriction of V gene usage has a direct impact on the clinical spectrum of HCV infection.

A clinicopathologic study of lymphoid neoplasias associated with human immunodeficiency virus infection in Italy

The clinicopathologic features of 45 human immunodeficiency virus (HIV)‐infected patients (mainly intravenous drug users [IVDU]) with lymphoid neoplasias seen from September 1984 through July 1990 at an Italian cancer center are reviewed. Thirty‐five had systemic non‐Hodgkins lymphoma (NHL), and ten had Hodgkins disease (HD). Histologically, 27 NHL cases were intermediate grade (five cases) or high grade (22 cases, 14 of the small noncleaved cell type), according to the Working Formulation. Eight NHL cases, including four anaplastic large cell (ALC) BerH2 (CD30)‐positive lymphomas, were in the miscellaneous group. Immunohistologic and/or gene rearrangement analysis showed the B‐cell origin of 20 of the 24 NHL cases studied. At presentation, 71% of NHL patients had advanced stages (Stage III or IV), and 85% had extranodal disease (predominantly gastrointestinal tract and marrow). Of the 23 patients evaluable for treatment, only seven had a complete clinical response after lymphoma therapy; the median survival of 34 evaluable patients was 22 months after the diagnosis of NHL. Fifteen patients died; most deaths were attributable to progressive lymphoma and opportunistic infections. As with NHL, advanced disease, extranodal involvement, aggressive histologic findings, and poor response to therapy were also observed in patients with HD. This study shows that lymphoid neoplasias occurring in Italian IVDU with HIV infection and those previously reported in North American homosexual men with HIV infection share similar clinicopathologic features. However, some features such as the absence of history of Kaposis sarcoma at diagnosis, the lack of detection of primary brain and rectal NHL, and the occurrence of B‐cell ALC BerH2 (CD30)‐positive NHL were observed uniquely in this series of patients.

Antibody Production and In Vitro Behavior of CD27-Defined B-Cell Subsets: Persistent Hepatitis C Virus Infection Changes the Rules

ABSTRACT There is growing interest in the tendency of B cells to change their functional program in response to overwhelming antigen loading, perhaps by regulating specific parameters, such as efficiency of activation, proliferation rate, differentiation to antibody-secreting cells (ASC), and rate of cell death in culture. We show that individuals persistently infected with hepatitis C virus (HCV) carry high levels of circulating immunoglobulin G (IgG) and IgG-secreting cells (IgG-ASC). Thus, generalized polyclonal activation of B-cell functions may be supposed. While IgGs include virus-related and unrelated antibodies, IgG-ASC do not include HCV-specific plasma cells. Despite signs of widespread activation, B cells do not accumulate and memory B cells seem to be reduced in the blood of HCV-infected individuals. This apparent discrepancy may reflect the unconventional activation kinetics and functional responsiveness of the CD27+ B-cell subset in vitro. Following stimulation with T-cell-derived signals in the absence of B-cell receptor (BCR) engagement, CD27+ B cells do not expand but rapidly differentiate to secrete Ig and then undergo apoptosis. We propose that their enhanced sensitivity to BCR-independent noncognate T-cell help maintains a constant level of nonspecific serum antibodies and ASC and serves as a backup mechanism of feedback inhibition to prevent exaggerated B-cell responses that could be the cause of significant immunopathology.

Pegylated-interferon plus ribavirin for HCV-positive indolent non-Hodgkin lymphomas.

Hepatitis C virus (HCV) causes not only chronic liver disease, but is also implicated in several lymphoproliferative disorders (Pozzato et al, 1994). The identification of CD81 as the main HCV receptor could explain this lymphotropism because this receptor is expressed in B lymphocytes (Pileri et al, 1998). The CD81 engagement determines cell activation with overexpression of CD69, CD71, CD86, and the chemokinereceptor CXCR3 (Rosa et al, 2005). In addition, an increased prevalence of t(14,18) translocation has been reported in HCV-patients, even in the absence of lymphoproliferative disorders (Zignego et al, 2002). This interaction of HCV with the immune system might explain the role of HCV in several extra-hepatic diseases, such as non-Hodgkin lymphoma (NHL). As HCV-positive lymphoproliferative disorders have been successfully treated with antiviral therapy (Gisbert et al, 2005), we compared the traditional treatment of interferon plus ribavirina (IFN + RIBA) with the newest antiviral therapy i.e. pegylated interferon alpha plus ribavirina (PEG-IFN +RIBA) in a small group of indolent NHL. Eighteen patients affected by HCV-related B-NHL were consecutively included in the study from February 1998 to January 2004. Criteria for patient’s selection were: (i) lowgrade B-NHL at first diagnosis or relapse, (ii) indolent course, (iii) HCV infection (HCV-RNA positivity in serum). Patients that carried the hepatitis B surface antigen (HBsAg) or were positive for anti-human immunodeficiency virus antibodies were excluded. All patients gave their informed consent before entry in the study. B cell monoclonality was analysed in the peripheral blood mononuclear cells and bone marrow samples of four cases by the previously described isotype-specific IGH VDJ Gene Scanning technique: briefly, a forward primer labelled with 6-carboxyfluorescein (FAM) fluorochrome in the second VDJ polymerase chain reaction (PCR) round was used. The VDJ PCR product and the internal size standard GeneScan 400 HD (Applied Biosystem, Forster City, CA, USA) were added to deionized formamide, and then subjected to capillary electrophoresis. The detection of IGHV gene segments was performed using VDJ FR1 PCR protocol, in which a second multiplexing PCR, IGHV3, IGHV4, IGHV5, IGHV6 primers and the IGHJ region were added. IGHV1-IGHV4, and IGHV3-IGHV6 primers were each labelled with a different fluorescent dye: FAM, HEX, NED or ROX. PCR products were added to the internal GeneScan 500 LIZ size standard. This mixture was subjected to capillary electrophoresis. When a monoclone was found, the PCR product was sequenced by standard methods. Eight patients (Group A) received 3 MU of IFN alpha-2b three times a week for 48 weeks. Ten patients (Group B) received PEG-IFN alpha-2b 1Æ5 lg/kg once a week for 48 weeks. Both groups received ribavirin 1000 mg or 1200 mg daily according to body weight (lower or greater than 75 kg) for 48 weeks (Manns et al, 2001). The patients were randomized on the basis of a computer-generated random list. The patient diagnoses were: one follicular lymphoma, one splenic lymphoma with villous lymphocytes, and 16 lymphoplasmacytoid lymphomas. The follicular lymphoma and the splenic lymphoma had node involvement (axillary and mediastinal/retroperitoneal, respectively). HCV genotyping showed genotype 1b in 11 cases (61%) while genotype 2a/2c was detected in the others. Eleven cases had chronic hepatitis at liver biopsy. Thirteen patients had dosable levels of cryoglobulins (from 2% to 35%) and high rheumatoid factor levels (from 30 to 21 700 U/l). Therapy outcome (Table I) was as follows:

Histopathologic, immunophenotypic, and genotypic analysis of Ki‐1 anaplastic large cell lymphomas that express histiocyte‐associated antigens

CD30/Ki‐1 antigen expression in 243 cases of malignant lymphomas was examined using Ber‐H2 monoclonal antibody. Among them 20 cases were categorized as Ki‐1 anaplastic large cell lymphoma. in two of these cases histiocyte‐associated markers were also expressed. in these cases histopathologic and extensive in situ immunophenotypic analyses were used with genotypic studies in the determination of cell lineage. A sinusoid histologic pattern of involvement with partial lymph node infiltration by pleomorphic neoplastic cells was noticed in the nodes from both patients. Solid areas of node replacement resembling metastatic carcinoma were seen in Patient 1. Immunohistologically, tumor cells of both cases were positive for CD30, CD25, CD71, LN3 (HLA‐DR), EMA, CD45, CD74, vimentin, alpha‐1‐antichymotrypsin, and CD68. Patient 1 was also CD45RO+, CD43+, whereas Patient 2 was positive for alpha‐1‐antitrypsin and CD4 tumor cells. Genotypic studies revealed that TCRβ and TCRγ chain genes were clonally rearranged in Patient 1, whereas no rearrangements were detected in Patient 2. This study supports the view that some Ki‐1 anaplastic large cell lymphomas may express multiple histiocyte‐associated antigens and confirms that this group of neoplasms have immunophenotypic heterogeneity. the results of genotypic analyses used with immunophenotyping does not exclude that the tumor cells in these cases may be of true histiocytic origin despite the Ki‐1‐positive phenotype.

Successful vaccination induces multifunctional memory T-cell precursors associated with early control of hepatitis C virus.

BACKGROUND & AIMS T cells are an important component for development of a vaccine against hepatitis C virus (HCV), but little is known about the features of successful vaccine-induced T cells. METHODS We compared the phenotype, function, and kinetics of vaccine-induced and infection-induced T cells in chimpanzees with HCV infection using multicolor flow cytometry and real-time polymerase chain reaction. RESULTS In chimpanzees successfully vaccinated with recombinant adenovirus and DNA against HCV NS3-5, HCV-specific T cells appeared earlier, maintained better functionality, and persisted at higher frequencies for a longer time after HCV challenge, than those of mock-vaccinated chimpanzees. Vaccine-induced T cells displayed higher levels of CD127, a marker of memory precursors, and lower levels of programmed death-1 (PD-1) than infection-induced T cells. Vaccine-induced, but not infection-induced, T cells were multifunctional; their ability to secrete interferon gamma and tumor necrosis factor α correlated with early expression of CD127 but not PD-1. Based on a comparison of vaccine-induced and infection-induced T cells from the same chimpanzee, the CD127(+) memory precursor phenotype was induced by the vaccine itself rather than by low viremia. In contrast, induction of PD-1 correlated with viremia, and levels of intrahepatic PD-1, PD-L1, and 2,5-OAS-1 messenger RNAs correlated with peak titers of HCV. CONCLUSIONS Compared with infection, vaccination-induced HCV-specific CD127(+) T cells with high functionality that persisted at higher levels for a longer time. Control of viremia prevented up-regulation of PD-1 on T cells and induction of PD-1, PD-L1, and 2,5-OAS-1 in the liver. Early development of a memory T-cell phenotype and, via control of viremia, attenuation of the inhibitory PD1-PD-L1 pathway might be necessary components of successful vaccine-induced protection against HCV.