Serum antibodies to huntingtin interacting protein-1: a new blood test for prostate cancer

Huntingtin-interacting protein 1 (HIP1) is frequently overexpressed in prostate cancer. HIP1 is a clathrin-binding protein involved in growth factor receptor trafficking that transforms fibroblasts by prolonging the half-life of growth factor receptors. In addition to human cancers, HIP1 is also overexpressed in prostate tumors from the transgenic adenocarcinoma of the mouse prostate (TRAMP) mouse model. Here we provide evidence that HIP1 plays an important role in mouse tumor development, as tumor formation in the TRAMP mice was impaired in the Hip1null/null background. In addition, we report that autoantibodies to HIP1 developed in the sera of TRAMP mice with prostate cancer as well as in the sera from human prostate cancer patients. This led to the development of an anti-HIP1 serum test in humans that had a similar sensitivity and specificity to the anti-alpha-methylacyl CoA racemase (AMACR) and prostate-specific antigen tests for prostate cancer and when combined with the anti-AMACR test yielded a specificity of 97%. These data suggest that HIP1 plays a functional role in tumorigenesis and that a positive HIP1 autoantibody test may be an important serum marker of prostate cancer.     

Huntingtin interacting protein 1 is a novel brain tumor marker that associates with epidermal growth factor receptor

Huntingtin interacting protein 1 (HIP1) is a multidomain oncoprotein whose expression correlates with increased epidermal growth factor receptor (EGFR) levels in certain tumors. For example, HIP1-transformed fibroblasts and HIP1-positive breast cancers have elevated EGFR protein levels. The combined association of HIP1 with huntingtin, the protein that is mutated in Huntington's disease, and the known overexpression of EGFR in glial brain tumors prompted us to explore HIP1 expression in a group of patients with different types of brain cancer. We report here that HIP1 is overexpressed with high frequency in brain cancers and that this overexpression correlates with EGFR and platelet-derived growth factor beta receptor expression. Furthermore, serum samples from patients with brain cancer contained anti-HIP1 antibodies more frequently than age-matched brain cancer-free controls. Finally, we report that HIP1 physically associates with EGFR and that this association is independent of the lipid, clathrin, and actin interacting domains of HIP1. These findings suggest that HIP1 may up-regulate or maintain EGFR overexpression in primary brain tumors by directly interacting with the receptor. This novel HIP1-EGFR interaction may work with or independent of HIP1 modulation of EGFR degradation via clathrin-mediated membrane trafficking pathways. Further investigation of HIP1 function in brain cancer biology and validation of its use as a prognostic or predictive brain tumor marker are now warranted.     

LMP1 signaling and activation of NF-kappaB in LMP1 transgenic mice

Transgenic mice expressing Epstein-Barr virus (EBV) latent membrane protein 1 (LMP1) under the control of an immunoglobulin heavy-chain promoter and enhancer develop lymphoma at a threefold higher incidence than LMP1-negative mice. In vitro, LMP1 activates numerous signaling pathways including p38, c-Jun N terminal kinase (JNK), phosphatidylinositol 3 kinase (PI3K)/Akt, and NF-kappaB through interactions with tumor necrosis receptor-associated factors (TRAFs). These pathways are frequently activated in EBV-associated malignancies, although their activation cannot be definitively linked to LMP1 expression in vivo. In this study, interactions between LMP1 and TRAFs and the activation of PI3K/Akt, JNK, p38, and NF-kappaB were examined in LMP1 transgenic mice. LMP1 co-immunoprecipitated with TRAFs 1, 2, and 3. Akt, JNK, and p38 were activated in LMP1-positive and -negative splenocytes as well as LMP1-positive and -negative lymphomas. Multiple forms of NF-kappaB were activated in healthy splenocytes from LMP1 transgenic mice, in contrast to healthy splenocytes from LMP1-negative mice. However, in both LMP1-positive and -negative lymphomas, only the oncogenic NF-kappaB c-Rel, was specifically activated. Similarly to EBV-associated malignancies, p53 protein was detected at high levels in the transgenic lymphomas, although mutations were not detected in the p53 gene. These data indicate that NF-kappaB is activated in LMP1-positive healthy splenocytes; however, NF-kappaB c-Rel is specifically activated in both the transgenic lymphomas and in the rare lymphomas that develop in negative mice. The LMP1-mediated activation of NF-kappaB may contribute to the specific activation of c-Rel and lead to the increased development of lymphoma in the LMP1 transgenic mice.     

The c-Rel transcription factor and B-cell proliferation: a deal with the devil

Activation of the Rel/NF-kappaB signal transduction pathway has been associated with a variety of animal and human malignancies. However, among the Rel/NF-kappaB family members, only c-Rel has been consistently shown to be able to malignantly transform cells in culture. In addition, c-rel has been activated by a retroviral promoter insertion in an avian B-cell lymphoma, and amplifications of REL (human c-rel) are frequently seen in Hodgkin's lymphomas and diffuse large B-cell lymphomas, and in some follicular and mediastinal B-cell lymphomas. Phenotypic analysis of c-rel knockout mice demonstrates that c-Rel has a normal role in B-cell proliferation and survival; moreover, c-Rel nuclear activity is required for B-cell development. Few mammalian model systems are available to study the role of c-Rel in oncogenesis, and it is still not clear what features of c-Rel endow it with its unique oncogenic activity among the Rel/NF-kappaB family. In any event, REL may provide an appropriate therapeutic target for certain human lymphoid cell malignancies.     

T-cell receptor antibodies in the immunohistochemical studies of normal and malignant lymphoid cells

Three anti-T Cell receptor (TCR) antibodies, BF1, BF2, and WT31 were studied for their specificity and usefulness as immunohistochemical reagents. These antibodies were all satisfactory in the staining of normal peripheral lymphoid tissues and cortical thymic lymphocytes were reactive with BF1 and BF2 but not with WT31. Hassall's corpuscles in two of three thymuses studied reacted with all three antibodies. BF1 was superior to the other two antibodies, especially for lymphoid cells in cytospin preparations. Fifty-five lymphomas, 24 nonlymphoid malignancies, seven established cell lines, and five cases of large granular lymphocyte (LGL) proliferation with neutropenia were studied. The majority of non-T-cell lesions did not react with the antibodies. An occasional case showed weak reactivity which could be easily distinguished from the usual strong reaction with T-cells. Tumor cells from over 90% of snap frozen peripheral T-cell lymphomas reacted with BF1 and BF2. BF1 was the preferred antibody since it gave a stronger and more consistent reaction. It was also the antibody of choice in identifying T-lymphoblastic lymphomas. Michel's solution fixed tissue showed markedly diminished or absent reactivity with BF2 and WT31, but BF1 reactivity was less affected. Some unusual T-cell phenotypes, with respect to the pattern of expression of BF1 antigen and CD3, were observed. BF1 and anti-CD3, in combination, would be useful in identifying T-cell lesions with aberrant or unusual TCR-CD3 phenotypes.     

Nodular lymphocytic lymphoma eventuating into diffuse histiocytic lymphoma: immunoperoxidase demonstration of monoclonality.

The patient described here had a nodular, poorly differentiated lymphocytic lymphoma associated with a serum monoclonal protein, IgG lambda. Following a three year period of radiation-induced clinical remission she developed generalized diffuse histiocytic lymphoma. Direct immunoperoxidase staining of the tissue sections demonstrated that the neoplastic cells of each biopsy only contained IgG lambda immunoglobulin, identical to the serum monoclonal protein. This is presumptive evidence that these two histopathologically distinctive malignant lymphomas, occurring consecutively in the same patient, were responsible for the synthesis and secretion of the same serum M component. This strongly suggests that both lymphoid neoplasms arose from the same malignant clone. The results 1) confirm the light microscopic observation that nodular lymphocytic lymphoma may progress to diffuse histiocytic lymphoma and 2) offer further evidence that histiocytic lymphomas arising in patients with previous B cell malignancies are most probably related to the original B cell proliferation and do not represent the emergence of a second, separate malignant clone.     

Campath-1H monoclonal antibody therapy

Monoclonal antibodies are receiving ever-increasing utilization in the treatment of hematologic malignancies. Campath-1 antibodies are directed against the surface antigen CD52 that is expressed on virtually all lymphocytes and monocytes. Murine forms, Campath-1G and Campath-1M, have been utilized extensively in allogeneic bone marrow transplants in order to purge the allograft of lymphocytes. The humanized form, Campath-1H, is currently the focus of many clinical trials in hematologic malignancies and autoimmune diseases. The genetically engineered Campath-1H has been utilized in the treatment of lymphomas and lymphoid leukemias with impressive results. T-cell prolymphocytic leukemia, chronic lymphocytic leukemia, and non-Hodgkin lymphomas appear to be particularly good targets for this agent. Campath-1H may be administered intravenously or subcutaneously. Infectious complications are the most significant side effect associated with its usage, with fevers, chills, nausea, and vomiting most common. Antibiotic prophylaxis has made the infectious morbidity associated with Campath-1H more manageable. The efficacy demonstrated in clinical trials and manageable toxicities make Campath-1H an appealing agent in the treatment of hematologic malignancies.     

Epstein-Barr virus, lymphomas and Hodgkin's disease.

Epstein-Barr virus (EBV) is implicated in the pathogenesis of a number of human lymphoid malignancies, including the immunoblastic B lymphomas that arise in immunocompromised individuals, Burkitt's lymphoma, Hodgkin's disease, and certain T cell lymphomas. The immunoblastic lymphomas are most likely a direct consequence of EBV-driven B cell lymphoproliferation that would in normal circumstances be eliminated by virus-specific cell-mediated immune responses. The other EBV-associated malignancies arise in individuals with more or less intact cellular immune responses and appear to have a complex multi-step pathogenesis. Throughout the EBV-positive lymphoid malignancies there is an intimate association between tumour cell phenotype and virus latent gene expression, with each of the three forms of latency seen in in vitro models being exemplified in vivo. Tumours with these different forms of virus latency will differ in their susceptibility to EBV-specific immune T cell control.     

CDKN2 (MTS1/p16INK4A) Gene Alterations in Hematological Malignancies

Cyclin dependent kinases (CDKs) make complexes with cyclins, and regulate cell cycle progression by their serine/threonine kinase activities. CDK inhibitors (CDKIs) arrest the inappropriate progression of the cell cycle by combining with CDKs. Because the functional loss of CDKIs may permit unlimited cell growth, their disruptions are thought to be associated with tumorigenesis. Recently, one CDKI, p16, was found, and its gene, CDKN2 (MTS1/p16^INK4A), was identified on chromosome 9p21. Intensive investigations of the CDKN2 gene in various tumors have shown that alterations frequently occur in this gene, thus suggesting that the CDKN2 gene is a tumor suppressor gene. In hematological malignancies, CDKN2 gene alterations may be limited to lymphoid malignancies, especially T-cell type acute lymphocytic leukemias, in which frequent chromosomal abnormalities in the 9p21 region have been reported. The CDKN2 gene is also inactivated in some patients with non-Hodgkin's lymphomas, adult T-cell leukemias, and lymphoid blastic crisis of chronic myelogenous leukemias. The main mechanism of CDKN2 gene inactivation is thought to be homozygous deletion, but point mutations may also inactivate it in some cases. The CDKN2 gene appears to be the major tumor suppressor gene on chromosome 9p21, and it is thought to be involved in the tumorigenesis of various lymphoid malignancies.     

Use of Monoclonal Antibodies for the Diagnosis of T-cell Malignancies: Applications and Limitations

Biopsy samples from 136 peripheral T-cell lymphomas have been examined and compared with benign inflammatory T-cell infiltrates in an attempt to establish whether immunohistological methods may help to improve the distinction between these conditions. The results confirm and extend previous reports and indicate that the aberrant T-cell phenotypes constitute the single most reliable criterion for the distinction between benign and malignant T-cell infiltrates. These phenotypes are expressed frequently in T-cell malignancies in. lymphoid organs and are also seen in a substantial number of biopsy samples from advanced cutaneous T-cell lymphomas (CTCL). In contrast, early CTCL do not express aberrant T-cell phenotypes and are indistinguishable from benign cutaneous conditions in terms of their immunophenotypic properties. It is concluded that immunophenotypic techniques form a valuable supplement to routine histological methods for the diagnosis of T-cell lymphomas in lymphoid organs. The methods may also help to improve the diagnosis of advanced CTCL, but are of no or only limited help for the recognition of the early stages.     

Loss of p27(Kip1) cooperates with cyclin E in T-cell lymphomagenesis

Cyclin E and p27(Kip1) are key regulators for cyclin-dependent kinases (Cdks) acting at the G1-/S-phase transition of the cell cycle. Whereas cyclin E is required for the activation of Cdk2, p27(Kip1) is a specific Cdk inhibitor and can block cell division. High levels of cyclin E and low levels of p27(Kip1) expression have been associated with malignant lymphomas in humans; the level of p27(Kip1) is even considered of prognostic significance. However, mice that lack p27(Kip1) do not develop any malignant lymphomas despite a pronounced lymphoid hyperplasia in thymus and spleen. We have previously described transgenic mice that carry a construct in which the cyclin E cDNA is under the control of the CD2 promoter/enhancer region and thus express high levels of cyclin E in the T-cell compartment (CD2-cyclin E). These animals are not predisposed for T-cell lymphomas in the absence of other cooperating events. Here we show that T-cells from CD2-cyclin E mice that at the same time are deficient for p27(Kip1) show a significantly higher Cdk2 activity than cells from wild-type or single mutant animals. Accordingly, a higher percentage of T cells in S/G2/M phase is found in CD2-cyclin E/p27(Kip1-/-) mice. After a long latency period of over 200 days, these animals develop spontaneous monoclonal T cell lymphoma whereas none of the single CD2-cyclin E transgenic or the p27(Kip1)-deficient mice showed any sign of lymphoid malignancies. Our findings demonstrate that a deregulation of control mechanisms at the G1/S transition by the combination of high cyclin E levels in the absence of p27(Kip1) is sufficient to predispose mice to develop lymphoid malignancies and further support a role of p27(Kip1) as a tumor suppressor and of cyclin E as a dominant oncogene.     

Surface marker studies on lymphoma and leukemia cells with microcytotoxicity test using the UCLA Monoclonal Antibody Tray 5

Surface marker studies of lymphoid malignancies are important in predicting the prognosis. We have investigated surface marker studies on lymphoma and leukemia cells with a new method of a microcytotoxicity test using monoclonal antibodies. The microcytotoxicity test (UCLA Monoclonal Antibody Tray 5) has the advantages of being performed in a few hours with an easy technique. The results showed that T-cell lymphomas and B-cell lymphomas could be clearly differentiated by monoclonal antibodies specific to T or B cells, respectively. Leukemia cells of ALL and AML were also tested for several monoclonal antibodies. However, the problem that most of the monoclonal antibodies used in this tray have not been characterized for specificity has yet to be solved.     

Myeloid,B and T lymphoid and mixed lineage thymic lymphomas in the irradiated mouse

Thymic lymphoma is a very common spontaneous and/or induced malignancy in both inbred mice and in transgenic mouse models of human cancer. Although a thymic lymphoma is defined as thymus-dependent T-cell malignancy, diagnostic criteria vary between studies and considerable heterogeneity has been reported. To define and classify the thymic lymphomas that arose in our study of X-irradiated (CBA/HxC57BL/6)F1, F1 backcross and F1 intercross mice, 66 thymic lymphomas were immunogenotyped for immunoglobulin heavy chain (IgH) and T-cell receptor beta (TCRbeta) gene rearrangements, and/or analysed for expression of lineage-specific markers and allelic loss on chromosome 4. The data indicate that 33% of the thymic lymphomas are very similar to mouse radiation-induced acute myeloid (AML) and mixed lineage (IgH(R), TCRbeta(G)) pre-B lympho-myeloid (L-MLs) leukaemias, 33% are mixed lineage (IgH(R), TCRbeta(R)) B/T lymphoid and <33% can be described as single lineage (IgH(G), TCRbeta(R)) T-cell malignancies. As the myeloid and L-ML leukaemias are not thymus-dependent this suggests that a malignant myeloid or pre-B lympho-myeloid cell can colonize the spleen to give an AML or L-ML leukaemia, or can colonize the thymus where TCRbeta gene rearrangement(s) may be induced to give the mixed lineage thymic lymphomas. Thus, assuming the single lineage T-cell thymic lymphomas fulfil the criteria of a thymus-dependent T-cell malignancy, thymic lymphomas are comprised of at least three distinct malignancies.     

Nasopharyngeal lymphoid hyperplasia after therapy for childhood lymphomas

Although diffuse thymic hyperplasia after therapy has been reported in lymphomas, no report is found in the medical literature about hyperplasia of other lymphoid tissues following chemotherapy in malignancies as far as we could reach. So, we planned to investigate the nasopharyngeal region of the lymphoma cases during their follow-up while at remission. Children who were in follow-up after cessation of oncological therapy with the diagnosis of lymphoma were evaluated for their nasopharyngeal lymphoid tissues by means of computed tomography. When a mass lesion was diagnosed, biopsies were taken. Among 23 lymphoma cases in follow-up, at a median of eight months (range 1-13 months), eight patients (six Hodgkin's and two Non-Hodgkin's malignant lymphoma) developed nasopharyngeal lymphoid hyperplasia (34.78%) that were proven to be benign by means of biopsies. Two of the patients also developed thymic hyperplasia. Nasopharyngeal hyperplasia regressed in four patients at a median of four months. Nasopharyngeal lymphoid hyperplasia may be diagnosed after the cessation of therapy in lymphomas and whatever the cause, it seems to be a benign process.     

Reactivity of monoclonal antibodies Leu 1 and OKT1 with malignant human lymphoid cells. Correlation with conventional cell markers

This study delineated the distribution of reactivity of malignant human lymphoid cells with monoclonal antibodies Leu 1 and OKT1, and correlated this expression with that of conventional lymphoid cell markers. The presence of Leu 1 on benign lymph nodal T-cells and its absence from benign lymph nodal B-cells was confirmed. Twenty-two T-cell neoplasms, expressing a variety of intrathymic and mature peripheral phenotypes, expressed Leu 1, but this expression was heterogeneous with respect to percent-positive cells and antigenic density, and appeared to correlate with stages of T-cell differentiation. This study demonstrated the expression of Leu 1 by 33 of 36 cases of B-CLL, by 10 of 15 cases of the closely allied small lymphocytic cell lymphoma, and by 9 of 29 follicular center-cell lymphomas. This included B-cell malignancies of each surface immunoglobulin isotype, and some cases associated with a monoclonal protein spike. Leu 1 was not expressed by myeloma plasma cells, and was absent from non-B, non-T acute lymphoblastic leukemia cells in each of 15 cases studied. Finally, Leu 1 and OKT1 were expressed in parallel, with respect to percent-positive cells and staining intensity, on benign and malignant T-cells, and on malignant B-cells, wherever studied. Possible explanations for this shared antigen are the existence of a minor Leu 1+ B-cell subset, a transformation-associated event, or glycosylation.     

Mice deficient in MIM expression are predisposed to lymphomagenesis

Missing in metastasis (MIM) is a member of newly emerged inverse Bin-Amphiphysin-Rvs (BAR) domain protein family and a putative metastasis suppressor. Although reduced MIM expression has been associated with bladder, breast and gastric cancers, evidence for the role of MIM in tumor progression remains scarce and controversial. Herein we characterized a MIM knockout mouse strain and observed that MIM-deficient mice often developed enlarged spleens. Autopsy and histological analysis revealed that nearly 78% of MIM(-/-) mice developed tumors with features similar to diffuse large B lymphoma during a period from 1 to 2 years. MIM(-/-) mice also exhibited abnormal distribution of B cells in lymphoid organs with decrease in the spleen but increase in the bone marrow and the peripheral blood. Furthermore, the bone marrow of MIM(-/-) mice contained a higher percentage of pre-B2 cells but fewer immature B-cells than wild-type mice. In response to CXCL13, a B-cell chemokine released from splenic stromal cells, MIM-deficient B-cells did not undergo chemotaxis or morphological changes in response to the chemokine and also did not internalize CXCR5, the receptor of CXCL13. Microarray analyses demonstrated that MIM is the only member of the I-BAR domain family that was highly expressed in human B cells. However, low or absent MIM expression was common in either primary B-cell malignancies or established B-cell acute lymphocytic leukemia or lymphomas. Thus, our data demonstrate for the first time an important role for MIM in B-cell development and suggest that predisposition of MIM-null mice to lymphomagenesis may involve aberrant interactions between B lineage cells and the lymphoid microenvironment.     

Induction of CD44 expression by the epstein-barr virus latent membrane protein LMP1 is associated with lymphoma dissemination

Epstein-Barr Virus (EBV) is implicated in the pathogenesis of endemic Burkitt's lymphoma (BL), B-cell lymphomas occuring under immunosuppression, nasopharyngeal carcinoma and Hodgkin's disease. Two distinct patterns of latent EBV gene expression occur in EBV-associated lymphomas. BLs typically display expression of the nuclear antigen EBNAI only, whereas EBV-associated, non-Burkitt B-cell lymphomas express at least 9 latent viral genes (6 EBNAs and 3 latent membrane proteins), reminiscent of in vitro EBV-immortalized lymphoblastoid cell lines (LCL). BLs are characterized by local, extra-nodal growth, whereas EBV-associated B-cell lymphomas often disseminate to peripheral lymphoid tissue. We show here that BL cells forming local tumors after xenotransplantation into SCID mice disseminate to lymphoid tissue following introduction of the latent membrane protein 1 (LMP1) gene. Introduction of LMP1 into BL cells induced expression of CD44 on the cell surface, a molecule implicated in enhanced lymphoid tumor growth and dissemination. Introduction of CD44 into LMP1-/CD44-BL cells was observed to confer the disseminated tumor growth pattern associated with LMP1 expression. Taken together our results show that expression of LMP1 may regulate expression of CD44 and play an important role in the behavior of EBV-based lymphomas. © 1995 Wiley-Liss, Inc.     

Immunohistochemical detection of multidrug resistance associated P-glycoprotein in tumour and stromal cells of human cancers

The distribution of Gp 170, a multidrug resistance (MDR) associated glycoprotein, also called P-glycoprotein (P-gp), was examined by immunohistochemistry, using C219 and MRK16 monoclonal antibodies. Sixty-five tumour tissues were studied which included 40 non-lymphoid tumours, 15 chemoresistant non-Hodgkin's lymphomas and 10 Hodgkin's disease. The study was performed on both cryostat and special fixation processed and paraplast embedded (ModAMeX) sections. The latter method preserves fixation-sensitive antigens such as P-gp and allows a more precise morphological identification of neoplastic and non-neoplastic cell populations in contrast to cryostat sections. Immunohistochemical expression of P-gp was expected and confirmed in many non-lymphoid tumours, but stromal macrophages and endothelial cells were also frequently stained in these cases. In non-Hodgkin's lymphomas, cells that were stained with both C219 and MRK16 monoclonal antibodies on cryostat sections were identified as macrophages and endothelial cells and not neoplastic lymphoid cells, by the ModAMeX technique. These findings suggest that the quantitative assessment of MDR RNA by Northern blotting performed on fresh homogenates overestimates the MDR content of neoplastic cells in a number of lymphoid and non-lymphoid tumours. In addition, the mechanism of chemoresistance in non-Hodgkin's lymphomas is less likely to be associated with P-gp expression.