Altered expression of cell cycle regulatory proteins in benign and malignant bone and soft tissue neoplasms.

BACKGROUND: The binding of cyclins to cyclin-dependent kinases regulates cell proliferation. Overexpression of cyclins is believed to deregulate the cell cycle in human tumors. Here the expression of G1 cyclins D1 and D3, and of Ki-67 in a variety of bone and soft tissue sarcomas was assessed as compared to adjacent normal tissue and to a subset of leiomyomas. MATERIALS AND METHODS: Twenty-nine human bone and soft tissue sarcomas were evaluated. Tissue sections from each case were subjected to immunostaining for cyclin D1, cyclin D3 and Ki-67 using the avidin-biotin complex method. RESULTS: Cyclin D1 nuclear positivity was detected in 28% of sarcomas and in none of the leiomyomas. Cyclin D3 nuclear positivity was present in 62% of sarcomas and in none of the leiomyomas. Ki-67 nuclear staining was positive in 86% of sarcomas but in only 16% of leiomyomas. In addition, upregulation of cyclin D1 was observed in leiomyosarcomas, pleomorphic sarcomas and gastrointestinal stromal tumors, but not in liposarcomas or osteosarcomas. Cyclin D3, however, was expressed in all of the sarcoma types including 2 out of 5 liposarcomas and 1 out of 4 osteosarcomas. The normal soft tissue adjacent to the tumors when present (10 cases) was negative for cyclin D1 and D3, and expressed Ki-67 in 5% of the cell nuclei. The expression of cyclin D3 was also noted in human sarcoma cell lines (SKLMS, MG63, SaOS-2 and HT1080) by Western blot. CONCLUSION: The higher expression of cyclin D1 and D3 and of Ki-67 in bone and soft tissue sarcomas, as compared to leiomyomas and peritumoral normal soft tissue, suggests that high cyclin expression may contribute to deregulation of the cell cycle in bone and soft tissue tumors. These data suggest a role of cyclins in the process of human sarcomagenesis.

Cyclooxygenase-2 (COX-2) expression in human endometrial carcinoma and precursor lesions and its possible use in cancer chemoprevention and therapy.

In recent years, the design of new antineoplastic agents that can halt the progression of human malignancies with minimal systemic damage has been at the forefront of cancer research, with cyclooxygenase-2 (COX-2) as a major target molecule. With an aim to demonstrate the expression and role of COX-2, the principal putative target of COX-2 inhibitor therapy, in endometrial adenocarcinoma (EACA) and precursor lesions, atypical complex hyperplasia (ACH) and endometrial hyperplasia (EH), an immunohistochemical (IHC) analysis of 22 primary human EACAs and 14 precursor lesions was carried out. Relevant clinicopathological data were tabulated from a random computer-generated sample of 22 primary EACA patients, treated by hysterectomy at our institution. Representative tumor sections including adjacent precursor lesions and normal endometrium (NE) were immunostained with human monoclonal anti-COX-2. Qualitative and semi-quantitative COX-2 IHC staining scores were determined based on the proportion of immunoreactive cells and the intensity of cytoplasmic COX-2 expression. Fisher's exact test and the Wilcoxon Rank Sum test were used for statistical analysis. Mean patient age was 68 years (range 51-93). All 22 EACAs were of endometrioid type, of which ten (45%) were grade I, eight (36%) grade II and four (18%) were grade III. Overall, four out of nine (44%) EHs, four out of five (80%) ACHs, and 18 out of 22 (88%) EACAs were COX-2 positive. The mean COX-2 IHC scores for EH and EACAs were 33 (SD 24.11) and 76 (SD 54.57), respectively (p = 0.022). Strong or moderate COX-2 expression was observed in 17 out of 22 (77%) adenocarcinomas as compared to two out of 14 (14%) of the precursor lesions (EH and ACH). The areas of adenomyosis were COX-2 positive, while myometrial smooth muscle and normal fallopian tube tissues stained negative for COX-2. The demonstration of frequent and strong expression of COX-2 in human EACAs supports a possible role for COX-2 inhibitors. Furthermore, an increasing expression of COX-2 from EH to invasive EACAs suggests potential usefulness of COX-2 inhibition to halt the progression of precursor lesions to invasive endometrial cancers.

The respiratory epithelium. III. Histogenesis of epidermoid metaplasia and carcinoma in situ in the human.

The histogenesis of epidermoid metaplasia and carcinoma in situ was analyzed in human bronchial epithelium. The conclusion is that epidermoid metaplasia and carcinoma in situ can result from conversion of mucous cells. This implies the direct transformation of one type of fully differentiated cell to another. The study therefore emphasizes the differentiation potentialities of the mucous cells that can divide and undergo goblet cell hyperplasia and epidermoid metaplasia. Epidermoid metaplasia is a common reaction to injury in the bronchus. In our series of cases it was especially frequent in patients without neoplastic disease who had undergone intratracheal intubation or tracheostomy and who had been maintained on a respirator in the Shock Trauma Unit, University of Maryland. Future studies will be required to distinguish the difference, if any, between epidermoid metaplasia destined to become malignant carcinoma and that which is not. One difference noted in this study was the absence of overt cornification in epidermoid metaplasia in patients without neoplastic disease.

Immunocytochemical detection of leukocyte-associated and apoptosis-related antigen expression in childhood brain tumors.

During systematic cell-surface antigen expression profile analyses of 76 primary childhood brain tumors [34 medulloblastomas (MED)/primitive neuroectodermal tumors (PNETs) and 42 astrocytomas (ASTR)], a library of monoclonal antibodies (MoABs) directed against various leukocyte-associated, lymphocyte cell-line differentiation antigens in childhood brain tumors was utilized. The antigens were detected employing an indirect, biotin-streptavidin conjugated alkaline phosphatase (AP) immunocytochemical technique. Major histocompatibility complex (MHC) class I restricted, tumor-associated antigen (TAA) specific, CD8(+) cytotoxic T lymphocytes (CTL) were identified in 58/76 (76.32%) brain tumors, and usually represented 1-10% of all cells, but in some cases 30-44% of the cells were CD8(+). CD4(+), MHC class II restricted helper lymphocytes were present in 65/76 (85.53%) brain tumors, and accounted for 1-10% of the observed cells. Macrophages were present in 74/76 (97.37%) brain tumors, and their number also represented 1-10% of all observed cells in the brain tumor frozen sections. Leukocyte common antigen (LCA) expression was detected in all 76 (100%) brain tumors studied. MoAB UJ 308 detected the presence of premyelocytes and mature granulocytes in 60/76 (78.95%) brain tumors. Natural killer (NK) cells were not defined in the observed brain tumors. The great majority of childhood glial tumors, particularly ASTRs express Fas (APO-1/CD95) receptor whereas normal cells in the central nervous system (CNS) do not. FasR is a transmembrane glycoprotein which belongs to the nerve growth factor/tumor necrosis factor (NGF/TNF) receptor superfamily. As part of our screening, the 42 childhood ASTRs were also investigated for expression of CD95. We detected strong expression (strong intensity of staining, number of stained cells 50-100%) of FasR, employing formalin fixed, paraffin-wax embedded tissue slides. Brain tumors and melanomas have been shown to produce their autocrine FasL, and are even capable of switching CD95-related signal transduction from the PCD pathway to a proliferative pathway. In view of our results, we conclude that: (1) the tumor infiltrating leukocytes in MEDs/PNETs and ASTRs represent a very diverse population and are present in a great majority of the cases studied; (2) the strong expression of FasR in ASTRs provides a manner in which T lymphocytes may exert their anti-tumor effects, but may also represent yet another way that tumors may evade the immune response; and (3) further observations of the expression of various antigens involved in juxtacrine, in situ growth control are necessary for the refinement of cellular immunotherapeutical approaches in the treatment of human malignancies.

Genetically engineered monoclonal antibodies for direct anti-neoplastic treatment and cancer cell specific delivery of chemotherapeutic agents.

Classical therapeutic modalities such as surgery, radiation, and chemotherapy not only fail to cure the great majority of malignant tumors, but their employment often leads to severe and debilitating side effects. The severe cancer related morbidity is also in direct correlation with the use of x-radiation and chemotherapy, making them less than ideal forms of therapy. The development of hybridoma technology and the advances in monoclonal antibody (MoAB) production have revitalized the initial concept of Ehrlich concerning the existence of cancer cell-targeted, specific "magic bullets". Entirely new approaches to cancer therapy that are neoplastic cell-directed, and specifically lethal to malignant cells and less toxic to normal tissues are being observed and developed, adhering to the old prayer: "Destroy the diseased tissues, preserve the normal." Immunotherapy as a fourth modality of cancer therapy has already been developed and proven to be quite effective. Strategies for the employment of antibodies for anti-cancer immunotherapy include: 1) Immune reaction directed destruction of cancer cells; 2) Interference with the growth and differentiation of malignant cells; 3) Antigen epitope directed transport of anti-cancer agents to malignant cells; 4) Anti-idiotype vaccines; and 5) Development of engineered (humanized) mouse monoclonals for anti-cancer therapy. In addition, a variety of different agents (e.g. toxins, radionuclides, chemotherapeutic drugs) have been conjugated to mouse and human MoABs for selective delivery to cancer cells. Preclinical observations in athymic, nude mice using xenografted human cancers and mouse, anti-human MoABs were more than impressive and have lead to the development of clinical trials. Phase I studies established the safety of employing immunoconjugates in humans, but the in vivo therapeutic results were less impressive. The clinical use of mouse MoABs in humans is limited due to the development of a foreign anti-globulin immune response by the human host. Genetically engineered chimeric human-mouse MoABs have been developed by replacing the mouse Fc region with the human constant region. Moreover, the framework regions of variable domains of rodent immunoglobulins were also experimentally replaced by their human equivalents. These antibodies can also be designed to have specificities and effector functions determined by researchers, which may not appear in nature. The development of antibodies with two binding ends (bispecific antibodies) provided a great improvement in targeting cancer cells. The existing inadequacies of MoABs in immunotherapy may also be improved by increasing their efficiency with chemical coupling to various agents such as bacterial or plant toxins, radionuclides or cytotoxic drugs. The astonishing immunophenotypic (IP) heterogeneity of neoplastically transformed cells, the different cytotoxic activity associated with the moiety linked to given MoABs, and mostly the impressive genetic modulation capabilities of cancer cells still remain as yet unsolved difficulties in the present immunotherapy of human cancer. In writing this review article, one of our main goals is to encourage further clinical research with the use of genetically engineered rodent MoABs and various immunoconjugates in the treatment of human cancer, as well as the combination of such immunotherapy with the three conventional modalities of therapy. Finally, we propose that MoAB-based immunotherapy be accepted as a conventional form of therapy and employed not only in terminal cancer patients but also, for instance, during and following surgical resection.

Independent prognostic role of p16 expression in lung cancer.

OBJECTIVES: The cyclin-dependent kinase p16 (also known as Ink4A, Mts1, Cdkn2, and Cdkn4i) has been proposed as a tumor suppressor gene mapped on chromosome segment 9p21. This study evaluated p16 protein expression in 135 lung cancer specimens and investigated potential genetic alterations occurring in this gene. RESULTS: We found altered p16 immunohistochemical expression to be a frequent event in lung cancer and to be independent of either the histologic type or any other clinical-pathologic feature. Western blot analyses performed on about one third of the specimens correlated highly with these results. In addition, we found p16 immunohistochemical expression to be a favorable prognostic factor in lung cancer in that its reduction or loss correlated with a worse outcome for the patients. Polymerase chain reaction amplification and direct sequencing of p16 exons 1 and 2 revealed no mutations, indicating that p16-altered expression in lung cancer is not necessarily linked to mutational events of these genes. CONCLUSIONS: We conclude that p16-altered expression is both an independent and frequent event in lung cancer and may have an important role in tumorigenesis and in malignant progression of a significant proportion of these cancers. However, the actual incidence and relevance of p16 mutations in this neoplasm continues to be debated, and its analysis seems inconclusive. Our results suggest a prognostic role for the immunodetection of this protein on formalin-fixed and paraffin-embedded specimens. They further suggest its routine use in the evaluation of the frequently unpredictable behavior of lung cancer.

Cancer growth and progression in the framework of comparative oncology. A new approach to cancer therapy.

The influence of comparative oncology is most timely in view of the fact that there are significant intra- and interspecies differences in tumor development, tumor behavior and tumor reaction to various types of treatment, chemotherapy, radiotherapy, immunotherapy and multimodality-therapy. A comparison of the diverse processes which occur under normal, physiological, neoplastic and therapeutic conditions will result in a clearer understanding of tumor growth and progression. Similarly, it can be applied for selection of suitable treatment by enriching our knowledge about the progression of neoplastic diseases. Such a course will also enhance a general acceptance of the value of comparative oncology.

Immunocytochemical detection of prostate specific antigen expression in human breast carcinoma cells.

To date, no true tissue specific antigen has been discovered. Prostate-specific antigen (PSA) was initially reported to be a tissue specific protein, detected in the seminal fluid and produced by normal and abnormal epithelial cells of the prostate gland. PSA is a 33 kD glycoprotein, with serine protease activity, and it is produced by several different tissues in the human body. Its expression levels may be elevated during benign and neoplastic cell growth in the prostate, and in a number of other human malignancies. The detection of PSA is also useful in monitoring the efficacy of anticancer treatment in malignant prostatic adenocarcinoma. In the present immunocytochemical study, PSA expression was examined employing a biotin-streptavidin based, alkaline phosphatase conjugated antigen detection technique in 16 routine, neutral formalin fixed, paraffin-wax embedded, primary BC tissue sections. Human postnatal thymic tissue, among others, was used as a negative tissue control, while normal prostate and prostate carcinomas (PCs) were included in the collection of antigen positive tissues. We observed the presence of PSA in all 16 BC cases, and this expression was independent of estrogen receptor status. The intensity of the staining was moderate to high (B to A) and localized to 20% to 40% of the total BC cell population, with cells of similar immunoreactivity being clustered in groups within the tumor microenvironment. This result directly contradicts the previous opinion concerning the prostate epithelium specificity of PSA expression and production. The immunophenotype (IP) heterogeneity of BC cells is further substantiated by their PSA positivity and its association with the presence of steroid hormone receptors. The establishment of the clinical significance of these findings necessitates further in vivo and in vitro research in BCs. The prognostic significance of PSA in BCs may lie in the identification of a subset of estrogen receptor negative BC patients who have malignancies associated with a good prognosis. PSA related, novel antineoplastic immunotherapy may also be recommended.

Immunocytochemical detection of prostate specific antigen expression in human primary and metastatic melanomas.

Prostate-specific antigen (PSA), a 33 kD glycoprotein, was initially reported to be a tissue specific protein, detected in the seminal fluid and produced by normal and abnormal epithelial cells of the prostate gland, as well as other tissues in the human body. The expression of PSA has been described to be elevated during benign and neoplastic cell growth in the prostate, and in a number of other human malignancies. The presence and production of PSA in human primary cutaneous malignant melanomas (CMMs) and metastatic malignant melanomas (MMMs) has not been reported prior to the present study. We examined the expression of PSA employing a biotin-streptavidin based, alkaline phosphatase conjugated antigen detection technique in routine, neutral formalin fixed, paraffin-wax embedded, 3-4 microns thick tissue sections of 30 CMMs and 10 MMMs. Human postnatal thymic tissue, among others, was used as a negative tissue control, while normal prostate and prostate carcinomas (PCs) were included in the collection of antigen positive tissues. We observed the presence of PSA in 16/30 CMMs and 6/10 MMMs. The intensity of the staining was moderate (C to B) and localized to between 20% and 30% of the total tumor cell population in both CMMs and MMMs, with cells of similar immunoreactivity being clustered in groups within the tumor microenvironment. This result directly contradicts the previous opinion concerning the prostate epithelium specificity of PSA expression and production. The immunophenotype (IP) heterogeneity of malignant melanoma cells in further substantiated by the pattern of their PSA immunoreactivity. The establishment of the clinical significance of these findings necessitates further in vivo and in vitro research in malignant melanomas. PSA related, novel antineoplastic immunotherapy may also be recommended in the treatment of both CMMs and MMMs.

Immunophenotypically varied cell subpopulations in primary and metastatic human melanomas. Monoclonal antibodies for diagnosis, detection of neoplastic progression and receptor directed immunotherapy.

During a systematic, immunocytochemical screening of 40 human cutaneous melanomas (30 primary and 10 metastatic) for immunophenotype (IP) heterogeneity, we employed a library of 20 well characterized, commercially available mono- and polyclonal antibodies. The use of the sensitive, indirect, four to six step immunoperoxidase or alkaline phosphatase conjugated streptavidin-biotin antigen detection technique provided excellent results. The immunocytochemically most characteristic IP for primary cutaneous melanoma, as detected by us was: HMB45+, S-100+, CEA+, vimentin+, cytokeratin 19+, p53+, Rbgene+, nm23+, HLA-DR+, HL.A-DP+, c-erbB3/HER-3+/-, cytokeratin 10/13+/-, HLA-DQ-, cytokeratin 5/8-, EMA-, c-myc-, and actin-. During melanoma progression, a tendency toward poor differentiation (dedifferentiation) and an increase in c-myc expression have both been observed, the latter downregulating HLA-A,B,C expression and consequently diminishing the possibility of melanoma cell Iysis by powerful CD8+, cytotoxic T lymphocytes (CTL) or other cytotoxic cells which requires HLA class I antigens. The development of the metastatic potential in melanomas caused an increase in CEA expression, eliminated the presence of nm23, and prompted the appearance of actin among the intermediate filaments, composing the cytoskeleton of these malignant tumor cells. The most characteristic IP for MMs, identified by this study was HMB45+, S-100+, CEA+, EMA+, vimentin+, HLA-DR+, HLA-DP+, cytokeratin 19+, actin-, c-erbB3/HER-3+, p53+, cytokeratin 10/13+/-, c-myc+/-, c-erbB2/HER-2+/-, HLA-DQ-, cytokeratin 5/8-, Rb gene-, nm23-. It has been observed that adhesion molecules and integrins play a significant role in the complex process of melanoma metastasis and thus we propose a blocking of these de novo expressed molecules with the appropriate antibodies as a form of immunotherapy of PMs and early stages of MMs.

Human cancer detection and immunotherapy with conjugated and non-conjugated monoclonal antibodies.

Classical therapeutic modalities such as surgery, radiation and chemotherapy not only fail to cure the majority of neoplastic disease, but their employment also leads to severe and debilitating side effects. The severe cancer related morbidity is often associated with the use of radiation and chemotherapy, making them less than ideal forms of therapy. Entirely new approaches to cancer therapy that are tumor cell directed, and specifically lethal to malignant cells and less toxic to normal tissues are being observed and developed, adhering to the old prayer "Destroy the diseased tissues, preserve the normal." Following the initial advances of Ehrlich, immunotherapy as a fourth modality of cancer therapy has already been developed and proven to be quite effective. Unfortunately, the cancer cell population is not a static entity, but rather a continually changing one. Considerable variations have been determined between individual malignant cells. Our strong belief is that it is necessary for present-day clinical oncologists to become aware of the existence of immunotherapy and learn how to employ it in order to improve the efficacy and decrease the side effects of modern cancer therapy. The development of hybridoma technology and the advances in monoclonal antibody (MoAB) production have revitalized the concept concerning the existence of cancer cell-targeted, specific "magic bullets". In addition, a variety of different agents (e.g. toxins, radionuclides, chemotherapeutic drugs) have been conjugated to mouse and human MoABs for selective delivery to cancer cells. Preclinical observations in athymic, nude mice using xenografted human cancers and mouse, anti-human MoABs were more than impressive and have lead to several clinical trials. Strategies for the employment of MoABs for cancer immunotherapy include: a) Immune reaction directed destruction of cancer cells; b) Interference with the growth and differentiation of malignant cells; c) Antigen epitope directed transport of anti-cancer agents to malignant cells; d) Anti-idiotype vaccines. Phase I studies have established the safety of employing immunoconjugates in humans, but the therapeutic results were less impressive. The clinical use of mouse MoABs in humans is limited due to the development of an anti-globulin immune response to the non-human immunoglobulins by the human host. Genetically engineered chimeric human-mouse MoABs have been developed by replacing the mouse Fc region with the human constant region. Moreover, the framework regions of variable domains of rodent immunoglobulins were also experimentally replaced by their human equivalents. These antibodies can also be designed to have specificities and effector functions determined by researchers, which may not appear in nature. The astonishing immunophenotypic (IP) heterogeneity of cancer cells, the different cytotoxic activity associated with the moiety linked to given MoABs, and mostly the impressive genetic modulation capabilities of cancer cells still remain as yet unsolved difficulties in the present immunotherapy of human cancer. Antibodies with two binding ends (bispecific antibodies) provide a great improvement in targeting cancer cells. The existing inadequacies of MoABs in immunotherapy may also be improved by increasing their efficiency with chemical coupling to various agents such as bacterial or plant toxins, radionuclides or cytotoxic drugs. In writing this review article, one of our main goals is to encourage further clinical research with the use of genetically engineered rodent MoABs and various immunoconjugates in the treatment of human cancer, as well as the combination of such immunotherapy with the three conventional modalities of therapy. Finally, we propose that MoAB-based immuno-therapy be accepted as a conventional form of therapy and employed not only in terminal cancer patients, but also, for instance, during and following surgical resection.

Reversal of multidrug resistance of tumor cells.

Drug resistance to chemotherapy is rapidly emerging. Resistance to one drug carries over resistance to unrelated anticancer drugs leading to multidrug resistance (MDR). A major factor of MDR is P-glycoprotein (P-gp) mediated ABC transport found in many eukaryotic cells. P-gp acts as a drug eMux pump. The mdr1 gene involved in P-gp 170 protein production is localized in the human chromosome 7 band p2 1.0-21.1. Point mutations after cross-resistance patterns. A variety of stimuli increase the expression of the mdr1 gene: lowered extracellular pH, heat shock, arsenite, cytotoxic agents, anticancer drugs, transfection with oncogenes, HIV-I, and UV-irradiation. An alternative hypothesis to the efflux pump claims that P-gp modifies the intracellular environment to reduce accumulation of anticancer drugs in cancer cells by creating ionic or proton gradients. Chemosensitizers that block P-gp drug extrusion are generally lipid-soluble at physiological pH, possess a basic nitrogen atom and at least two co-planar rings. P-gp blocking does not depend on drug chirality. This opens the way of treating P-gp related MDR with chiral versions of drugs relatively harmless in terms of side-effects. We believe that resistance modifiers combined with cytostatics will chemotherapeutically be more effective for cancer patients.

Controversies on the prognostic significance of tumor infiltrating leukocytes in solid human tumors.

We have performed immunophenotypical (IP) analyses of tumor infiltrating leukocytes (TIL) in both childhood brain tumors (medulloblastomas[MEDs]/primitive neuroectodermal tumors [PNETs] and astrocytomas [ASTRs]) and malignant melanomas (both primary and metastatic) employing a well-characterized library of monoclonal antibodies (MoABs) directed against leukocyte differentiation/activation associated antigens. The antigens were detected by an indirect, biotinstreptavidin conjugated alkaline phosphatase (AP) immunocytochemical technique. Our systematic cell-surface antigen expression profile analysis of 76 primary childhood brain tumors (34 MEDs/PNETs and 42 ASTRs) identified CD8+ CTL in 58/76 brain tumors. CD4+, MHC class II restricted helper lymphocytes were present in 65/76 brain tumors and represented 1-10% of the observed cells. Macrophages were present in 74/76 childhood brain tumor cases observed by us. Leukocyte common antigen (LCA) expression was demonstrated in all 76 brain tumors studied. MoAB UJ 308 detected the presence of premyelocytes and mature granulocytes in 60/76 brain tumors. They were localized perivascularly, within the tumor tissue, or close to necrotic regions. Natural killer (NK) cells were not defined in the childhood brain tumors observed in this study. The IP characteristics of the heterogeneous leukocytic infiltrate of 30 primary (PMs) and 10 metastatic melanomas (MMs) was also investigated by us. We established the presence of some type of melanoma infiltrating host's immunological effector cells in all 40 observed melanoma cases. More specifically, we found NK cells, macrophages and granulocytes in 30/30 PMs and 10/10 MMs. These effector cells represented the vast majority (> 80%) of the melanoma infiltrating immunocompetent cells. T lymphocytes were observed in 20/30 PMs and 6/10 MMs, but their numbers represented only between 5% to 10% of the heterogeneous leukocytic infiltrate. B cells were found in 22/30 PMs and 8/10 MMs, their numbers representing less than 5%. Presence of cells of the dendritic reticulum, involved in antigen presentation was not determined in any of the observed PMs and MMs. The notion that infiltration of the neoplastically transformed mass of cells by TIL is always a prognostically positive phenomenon has changed in recent years as research on extracellular matrix remodeling and angiogenesis have identified numerous secreted factors which are common to both neoplastically transformed cells and infiltrating leukocytes.

Immunocytochemical detection of the homeobox B3, B4, and C6 gene products in childhood medulloblastomas/primitive neuroectodermal tumors.

The homeobox was originally described as a conserved DNA motif of about 180 base pairs. The protein domain encoded by the homeobox, the homeodomain, is thus about 60 amino acids long. The homeodomain is a DNA-binding domain, and many homeobox genes have now been shown to bind to DNA and regulate the transcription of other genes. Thus homeodomain proteins are basically transcription factors, most of which play a role in development. The homeobox genes seem to represent another class of oncofetal antigens involved in both normal development and carcinogenesis, as well as tumor progression. It has been shown that HOX-B3 and HOX-B4 are preferentially expressed in primitive CD34+, lineage-committed hematopoietic stem cells (HSCs) in human bone marrow. HOX-B3 overexpression in HSCs causes defective lymphoid development and progressive myeloproliferation, while HOX-B4 leads to selective expansion of HSCs without altering their differentiation. The HOX-C6 gene product leads to cell differentiation in neuroblastomas, while also being associated with the neoplastically transformed mammary cell phenotype and progression in primary cutaneous lymphomas. The expression pattern of these three homeobox gene products (HOX-B3, HOX-B4, and HOX-C6) was examined immunocytochemically in childhood MEDs/PNETs employing an indirect alkaline phosphatase conjugated technique on formalin-fixed, paraffin-embedded tissue sections. Strong staining intensity (A, B) of HOX-B3 and HOX-B4 was registered in all MEDs/PNETs, with immunoreactivity in between 50% and 90% (+3), but usually over 90% (+4) of the tumor cells. HOX-C6 was detected at medium intensity (mostly B) in 50% to 90% (+3) of the MED/PNET cells. This report is the first to describe the expression of these three homeobox gene products in MEDs/PNETs, and provides further evidence for the role of these proteins in the progression of human malignancies. The value of these genes and proteins in the early diagnosis and possible treatment of various human neoplasms, including childhood brain tumors, should be assessed in further immunocytochemical and molecular biological experiments.

The role of the reticulo-epithelial (RE) cell network in the immuno-neuroendocrine regulation of intrathymic lymphopoiesis.

The thyrnus provides an optimal cellular and humoral microenvironment for the development of immunocompetent T lymphocytes. Although yolk sac derived pre-T, committed hematopoietic stem cells enter the thymus using a homing receptor, the immigration process also requires secretion of a peptide, called thymotaxin by the cells of the reticulo-epithelial (RE) network of the thymic cellular microenvironment. The thymic RE cells are functionally specialized based on their location within the thymic microenvironment. Thus, although subcapsular, cortical, and medullary RE cells are derived from a common, endodermal in origin epithelial precursor cell, their unique location within the gland causes their specialization in terms of their immunophenotypical and in situ physiological properties. The subcapsular, endocrine, RE cell layer (giant or nurse cells) is comprised of cells filled with PAS positive granules, which also express A2B5/TE4 cell surface antigens and MHC Class I (HLA A, B, C) molecules. In contrast to the medullary RE cells, these subcapsular nurse cells also produce thymosins beta 3 and beta 4. The thymic nurse cells (TNCs) display a neuroendocrine cell specific immunophenotype (IP): Thy-1+, A2B5+, TT+, TE4+, UJ13/A+, UJ127.11+, UJ167.11+, UJ181.4+, and presence of common leukocyte antigen (CLA+). Medullar RE cells display MHC Class II (HLA-DP, HLA-DQ, HLA- DR) molecule restriction. These cells also contain transforming growth factor (TGF)-beta type II receptors and are involved in the positive selection of T cells. Transmission electronmicroscopic (TEM) observations have defined four, functional subtypes of medullary RE cells: undifferentiated squamous, villous and cystic. All subtypes were connected with desmosomes. The secreted thy nic hormones, thymulin, thymosin-alpha 1 and thymopoietin (its short form, thymopentin or TP5) were detected immunocytochemically to be produced by RE cells. Thymic RE cells also produce numerous cytokines including IL-1, IL-6, G-CSF, M-CSF, and GM-CSF molecules that likely are important in various stages of thymocyte activation and differentiation. The co-existence of pituitary hormone and neuropeptide secretion [growth hormone (GH), prolactin (PRL), adrenocorticotropic hormone (ACTH), thyroid stimulating hormone (TSH), triiodothyronine (T3), somatostatin, oxytocin (OT), follicle stimulating hormone (FSH), luteinizing hormone (LH), arginine vasopressin (AVP), growth hormone releasing hormone (GHRH), corticotropin releasing hormone (CRH), nerve growth factor (NGF), vasoactive intestinal peptide (VIP), pro-enkephalin (pro-enk), and beta-endorphin (beta-end)], as well as production of a number of interleukins and growth factors and expression of receptors for all, by RE cells is an unique molecular biological phenomenon. The thymic RE cell network is most probably comprised of cells organized into sub-networks--functional units composed of RE cells with differing hormone production/hormone receptor expression profiles, involved in the various stages of T lymphocyte maturation. Furthermore, it is quite possible that even on the level of individual RE cells, the numerous projections associated with a single cell, which engulf developing lymphocytes, nurturing and guiding them in their maturation, may differ in their hormone production and/or hormone receptor expression profile, thus allowing a single cell to be involved in distinct, separate steps of the T cell maturation process. Based on our systematic observations of the thymus in humans and other mammalian species, we suggest that the thymic RE cells represent an extremely important cellular and humoral network within the thymic microenvironment and are involved in the homeopathic regulation mechanisms of the multicellular organism, in addition to the presentation of various antigens to developing lymphocytes, and providing growth regulatory signals which may range from stimulatory to apoptotic signaling within the thymus. (ABSTRACT TRUNCA

Matrix metalloproteinases in neoplasm-induced extracellular matrix remodeling in breast carcinomas.

Structural changes in the extracellular matrix (ECM) are necessary for cell migration during normal and pathologic tissue remodeling and neoplastic cell invasion. The matrix metalloproteinases (MMPs) and their inhibitors have been identified to be critical modulators of ECM composition and are thus, crucial in neoplastic cell progression, invasion and metastasis. Expression of MMP-2, -3, -9, -10, and -13 was investigated in human breast carcinomas (BCs) employing an indirect, biotin-streptavidin based, alkaline phosphatase conjugated immunocytochemical technique. Evaluation of the results was based on (a) the percent of neoplastically transformed cells/surrounding stroma that reacted positively and (b) a measure of staining intensity [graded from A (highest) to D (negative)]. The two forms of stromelysin, MMP-3 and -10, share 82% sequence homology, but exhibit differences in cellular synthesis and inducibility by cytokines and growth factors in vitro. Strong overall expression of MMP-3 and -10 was found in BCs, especially in the ECM adjacent to blood vessels. Positive immunoreactivity could be seen for these two MMPs in the ECM surrounding over 90% of the neoplastically transformed cells (++++), and the staining intensity was also the strongest possible (A). High intensity immunoreactivity (A,B) but focal was detected employing a MoAB targeted against the MMP-9 enzyme. No presence of MMP-2 or -13 could be established in the BC cases observed by us. Based on these results we propose that MMP-3 and -10 are implicated in the pathogenesis of BC, while MMP-9 is possibly involved in neo-angiogenic events also closely associated with growth and expansion of the neoplastically transformed cell mass, as well as metastasis of individual, extremely aggressive, expressing dedifferentiated cellular immunophenotype (IP) cell clones selected during the microevolution of the BC.

Failure of cancer vaccines: the significant limitations of this approach to immunotherapy.

Immunotherapy has always represented a very attractive fourth-modality therapeutic approach, especially in light of the many shortcomings of conventional surgery, radiation, and chemotherapies in the management of cancer. Subsets of neoplastically transformed cells have been shown to (re-)express on their surface molecules which are not typically present on the surface of neighboring normal cells. In some instances, especially in malignant melanomas, cytotoxic T lymphocytes (CTLs) directed against such tumor associated antigens (TAAs) have been isolated. The cancer vaccine approach to therapy is based on the notion that the immune system could possibly mount a rejection strength response against the neoplastically transformed cell conglomerate. However, due to the low immunogenicity of TAAs, downregulation of MHC molecules, the lack of adequate costimulatory molecule expression, secretion of immunoinhibitory cytokines, etc., such expectations are rarely fulfilled. Various approaches have been explored ranging from the use of irradiation inactivated whole-cell vaccines derived from both autologous and allogeneic tumors (even tumor cell lines), and genetically modified versions of such cellular vaccines which aim at correcting costimulatory dysfunction or altering the in situ humoral milieu to aid immune recognition and activation. Anti-idiotype vaccines, based on cancer cell associated idiotypes, have also been explored which aim at increasing immunogenicity through in vivo generation of vigorous immune responses. Dendritic cell (DC) vaccines seek to improve the presentation of TAAs to naive T lymphocytes. Unfortunately, there is always the possibility of faulty antigen presentation which could result in tolerance induction to the antigens contained within the vaccine, and subsequent rapid tumor progression. The theoretical basis for all of these approaches is very well founded. Animal models, albeit highly artificial, have yielded promising results. Clinical trials in humans, however, have been somewhat disappointing. Although general immune activation directed against the target antigens contained within the cancer vaccine has been documented in most cases, reduction in tumor load has not been frequently observed, and tumor progression and metastasis usually ensue, possibly following a slightly extended period of remission. The failure of cancer vaccines to fulfill their promise is due to the very relationship between host and tumor: through a natural selection process the host leads to the selective enrichment of clones of highly aggressive neoplastically transformed cells, which apparently are so dedifferentiated that they no longer express cancer cell specific molecules. Specific activation of the immune system in such cases only leads to lysis of the remaining cells expressing the particular TAAs in the context of the particular human leukocyte antigen (HLA) subclass and the necessary costimulatory molecules. The most dangerous clones of tumor cells however lack these features and thus the cancer vaccine is of little use. The use of cancer vaccines seems, at present, destined to remain limited to their employment as adjuvants to both traditional therapies and in the management of minimal residual disease following surgical resection of the primary cancer mass.

Immunocytochemical detection of MMP-3 and -10 expression in hepatocellular carcinomas.

Structural changes in the extracellular matrix (ECM) are necessary for cell migration during tissue remodeling and tumor invasion. The matrix metalloproteinases (MMPs) and their inhibitors have been shown to be critical modulators of ECM composition and are thus crucial in neoplastic cell invasion and metastasis. Expression of MMP-2, -3, -9, -10, and -13 was investigated in human hepatocellular carcinomas (HCCs) employing an indirect alkaline phosphatase conjugated immunocytochemical technique. Evaluation of the results was based on (a) the persent of neoplastically transformed cells/surrounding stroma that reacted positively and (b) a measure of staining intensity [graded from A (highest) to D]. The two forms of stromelysin, MMP-3 and -10, share 82% sequence homology, but exhibit differences in cellular synthesis and inducibility by cytokines and growth factors in vitro. Strong overall expression of MMP-3 and -10 was found in HCCs, especially in the ECM adjacent to blood vessels. Positive immunoreactivity could be seen for these two MMPs in the ECM surrounding over 90% of the neoplastically transformed cells (++++), and the staining intensity was also the strongest possible (A,B). No immunoreactivity was detected using antibodies directed against MMP-2, -9, and -13.

Matrix metalloproteinase expression in childhood astrocytomas.

Structural changes in the extracellular matrix (ECM) are necessary for cell migration during tissue remodeling and local neoplastic cell invasion. The matrix metalloproteinases (MMPs) and their inhibitors have been shown to be critical modulators of ECM composition and are thus, crucial in tumor cell invasion and metastasis. The immunocytochemical profile of MMP-2, -3, -9, -10, and -13 expression was observed in 24 primary human childhood astrocytomas (ASTRs) employing an indirect alkaline phosphatase conjugated antigen detection technique. Evaluation of the results was based on (a) the percent of neoplastically transformed cells that reacted positively and (b) a measure of staining intensity [graded from A (highest) to D (negative)]. The two forms of stromelysin, MMP-3 and -10, share 82% sequence homology, but exhibit differences in cellular synthesis and inducibility by cytokines and growth factors in vitro. Strong overall expression of MMP-3 and -10 was found in ASTRs, especially in the ECM adjacent to blood vessels. Positive immunoreactivity could be seen for these two MMPs in the ECM surrounding over 90% of the neoplastically transformed cells (+4) and the staining intensity was also the strongest possible (A,B). No immunoreactivity was detected using antibodies directed against MMP-2, -9, and -13. Based on these results, MMP-3 and -10 are implicated in the pathogenesis of pediatric ASTRs. Further characterization of the expression and utilization of MMPs and their inhibitors in the progression of ASTRs may establish differential regulation and utilization of the various MMPs during the progression of glial tumors, from low-grade pilocytic ASTR to high-grade glioblastoma multiforme.

Immunocytochemical detection of the homeobox B3, B4, and C6 gene products in breast carcinomas.

Breast cancer (BC) represents the most frequent neoplasm in women with a risk of incidence between 10% and 12%. The detection of tumor associated and oncofetal antigen re-expression in a variety of neoplastically transformed cell types has aided in the more precise diagnosis and prognostication of human cancers. The homeobox (HOX) genes encode proteins which contain a 61 amino acid DNA-binding homeodomain and are involved in the transcriptional regulation of other genes during normal onto- and histogenesis. The class I HOX genes are organized in four clusters on different chromosomes in humans, with a high conservation in the order of the genes within each of these clusters. Re-expression of HOX gene products has been reported in a wide variety of neoplastically transformed cells and it seems quite likely that the HOX genes represent yet another class of oncofetal antigens involved in both normal development and carcinogenesis, as well as tumor progression. The expression pattern of three HOX gene products (HOX-B3, -B4, and -C6) was examined immunocytochemically in 11 human breast carcinoma (BC) tissues. In all observed BC cases, HOX-C6 was present in over 90% of the neoplastically transformed cells (+4) demonstrating a high grade (A and B) staining intensity. The same expression pattern was defined for the other two observed proteins (HOX-B3 and -B4; over 90% or +4 and a high grade staining intensity or A and B). Current treatment of BC encompasses the three "classic" modalities of therapy: surgical resection, radiotherapy, and chemotherapy. Although advances have been made, we still face great difficulties in the treatment of this deadly human neoplasm. Therefore, we are always seeking novel tumor associated antigens (TAAs), including oncofetal antigens, to use as molecular targets in cancer cell directed fourth modality immunotherapy.