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.

Expression of proline-directed protein kinase, (p34cdc2/p58cyclin A), a novel cell proliferation marker in childhood brain tumors.

The presence of two proteins of the proline-directed protein kinase (PDPK), the catalytic subunit p34cdc2 and the regulatory subunit p58cyclin A was determined in seven primitive neuroectodermal tumors (PNETs), three choroid plexus neoplasms and eleven astroglial tumors. The highest expression was registered in the cellularly undifferentiated PNETs and glioblastoma multiforme from the astroglial malignant group. Rabbit immunoantiserum against the two subunits of PDPK, a cell proliferation marker, was employed to detect proliferation activity in childhood brain tumors. The PDPK activity was present from Gl- to M-phases in 21 childhood brain tumors with different central nervous system (CNS) localization and cellular atypia. Immunocytochemical analysis employed an indirect, alkaline phosphatase conjugated biotin-streptavidin antigen detection technique on frozen and routine, formalin-fixed and paraffin-wax-embedded tissue sections of brain tumors. We compared the proliferation activity in the cells of normal, morphologically changed and neoplastically transformed choroid plexus. The average proliferation activity was low in comparison with other tissues. The results in normal and neoplastically transformed choroid plexus were very similar. The lowest proliferation activity in the astroglial group belonged to pilocytic ASTRs. The use of cell differentiation as a prognostic factor in primary brain tumors has already been established and is strongly suggested by our research group. Further systematic neoplasm studies and regular employment of these two polyclonal antibodies for immunocytochemical screening experiments are necessary to determine their true diagnostic and prognostic significance.

Comparison of long-term outcome of children and adolescents with disseminated non-lymphoblastic non-Hodgkin lymphoma treated with COMP or daunomycin-COMP: A report from the Children's Cancer Group.

BACKGROUND: Early Children's Cancer Group (CCG) trials indicated that the cyclophosphamide, vincristine, methotrexate, and prednisone (COMP) regimen was superior to the LSA2L2 regimen for non-lymphoblastic (NLB) non-Hodgkin lymphoma (NHL). Studies by other groups suggested that addition of anthracyclines to standard therapies could improve outcome. Therefore, in 1983 CCG initiated study CCG-503, a randomized trial of COMP vs. daunomycin-COMP (D-COMP) in children and adolescents with disseminated NLB NHL. PROCEDURES: Between December 1983 and April 1990, 404 eligible patients were entered. Patients without central nervous system (CNS) or marrow involvement were randomized to receive COMP (N = 139) or D-COMP (N = 145). Randomization was stratified by histology and site of disease. Patients with CNS or marrow involvement (stage IV) were non-randomly treated with D-COMP (N = 120). RESULTS: Ten-year event-free survival in COMP and D-COMP patients was similar: 55 +/- 4.3% (Estimate +/- SE) vs. 57 +/- 4.2% (not significant). Stage I-III patients with large-cell (LC) NHL had worse 10-year event-free survival (EFS) (48 +/- 4.9%) than those with small non-cleaved cell (SNCC) NHL disease (61 +/- 3.5%, P < 0.05 in multivariate analysis), but equivalent survival (65 +/- 4.7% vs. 63 +/- 3.5%) due to significantly higher salvage rates in LC patients, especially those failing more than 12 months from diagnosis. Ten-year EFS in stage IV patients was 39 +/- 5.2%. Addition of daunomycin resulted in higher rates of grade 3/4 hematologic toxicity and stomatitis, as well as late cardiac-related deaths. The incidence of second malignant neoplasms was 1.0% at 10 years. CONCLUSIONS: Addition of daunomycin to standard COMP therapy did not improve outcome in pediatric disseminated NLB NHL. Patients with LC disease had a significantly reduced long-term EFS, but were retrieved at a higher rate than patients with SNCC disease, resulting in equivalent long-term survival.

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.

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.

Invasion and metastasis: the expression and significance of matrix metalloproteinases in carcinomas of the lung.

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 lung adenocarcinomas employing an indirect 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]. 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 lung adenocarcinomas, 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). Focal (+), high intensity (A,B) staining could be detected for MMP-2, -9, and -13. Thus, it seems that the stromelysins are involved in the generalized growth and expansion of the neoplastic cell mass, while MMP-2, -9 and -13 are involved in the neoangiogenic and focal clonal selection and expansion phenomena associated with in situ tumor progression, invasion of the microvasculature, and metastasis.

Matrix metalloproteinase expression in malignant melanomas: tumor-extracellular matrix interactions in invasion and metastasis.

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 malignant melanomas (MMs) employing an indirect 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]. 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 MMs, 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). Focal (+), high intensity (A,B) staining could be detected for MMP-2, -9, and -13. Thus, it seems that the stromelysins are involved in the generalized growth and expansion of the neoplastic cell mass, while MMP-2, -9 and -13 are involved in the neoangiogenic and focal clonal selection and expansion phenomena associated with in situ tumor progression, invasion of the microvasculature, and metastasis.

Immunocytochemical detection of matrix metalloproteinase expression in prostate cancer.

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 prostatic carcinomas employing an indirect 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]. 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 lung adenocarcinomas, 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). Focal (+), low to high intensity (C to A) staining could be detected for MMP-2, while no immunoreactivity was observed employing MoABs directed against MMP-9 and -13. Thus, it seems that the stromelysins are involved in the generalized growth and expansion of the neoplastic cell mass, while MMP-2 is involved in the neoangiogenic and focal clonal selection and expansion phenomena associated with in situ tumor progression, invasion of the microvasculature, and metastasis.

Immunocytochemical detection of the expression of members of the matrix metalloproteinase family in adenocarcinomas of the pancreas.

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 pancreatic adenocarcinomas employing an indirect 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]. 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 lung adenocarcinomas, 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). Focal (+), high intensity (A,B) staining could be detected for MMP-2 and -13, while no immunoreactivity was observed employing the anti-MMP-9 MoAB. Thus, it seems that the stromelysins are involved in the generalized growth and expansion of the neoplastic cell mass, while MMP-2 and -13 are involved in the neoangiogenic and focal clonal selection and expansion phenomena associated with in situ tumor progression, invasion of the microvasculature, and metastasis.

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.

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 homeobox B3, B4, and C6 gene product expression in lung carcinomas.

The so-called homebox (HOX) was described as a highly conserved DNA motif of 183 base pairs, encoding the 61 amino acid DNA-binding homeodomain. Numerous HOX genes have subsequently been shown to bind to DNA and regulate the transcription of other genes. In humans the class I HOX genes are placed in four clusters on different chromosomes. The order of the genes within each of these clusters is evolutionarily conserved to a high degree and suggests that such an organization may be essential in the function of these genes during normal embryo- and histogenesis. Re-expression of HOX gene products has been reported in a wide variety of neoplastically transformed cells and it seems very likely that the HOX genes represent yet another class of oncofetal antigens involved in both normal development and cellular carcinogenesis, as well as tumor progression. The expression pattern of three homeobox gene products (HOX-B3, HOX-B4, and HOX-C6), all shown to be involved in lung tissue development, was examined immunocytochemically, in human lung carcinoma (LC) tissues. In all observed LC cases, HOX-C6 was present in over 60% of neoplastic cells (+3) demonstrating a medium grade (B and C) staining intensity. A smaller number of neoplastically transformed epithelial cells also expressed the proteins HOX-B3 and -B4 (10% to 60% or +2 to +3 and a medium grade staining intensity or B and C). The significance of these novel oncofetal antigens in tumor cell biology and as target molecules in the immunotherapy of lung carcinomas should be established by future studies.

Homeobox B3, B4, and C6 gene product expression in osteosarcomas as detected by immunocytochemistry.

Osteosarcoma (OS) is a malignant neoplastic disease of the bone, of mesenchymal origin and with considerable morphologic heterogeneity, consisting of malignant stoma with evidence of malignant osteoid, bone and/or cartilage production. The mammalian homeobox (HOX) represents a highly conserved DNA motif of 183 base pairs, encoding the 61 amino acid DNA-binding homeodomain, through which the HOX gene products regulate the transcription of other genes involved in onto- and histogenesis. Re-expression of HOX proteins has been identified in a wide variety of neoplastically transformed cell types and it seems that the HOX genes represent yet another family of oncofetal antigens involved in both normal development and oncogenesis, as well as tumor tissue progression. During this study, the expression pattern of three HOX gene products (HOX-C6, -B3, and -B4) was examined immunocytochemically in human osteosarcoma (OS) tissues. In all observed (16/16) OS cases, HOX-C6 was present in over 90% of the neoplastically transformed cells (+4), demonstrating a high to medium grade (A to B) staining intensity. Similar results were obtained in OS cells for the other two observed proteins (HOX-B3 and -B4; over 90% or +4 and a high to medium grade staining intensity or A and B). The significance of the expression of class I HOX proteins in the pathobiology, diagnosis and prognostication of human OS should be established by further investigations.

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

Immunocytochemical detection of the homeobox B3, B4, and C6 gene products within the human thymic cellular microenvironment.

The homeobox (HOX) 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. The expression pattern of three homeobox gene products (HOX-B3, HOX-B4, and HOX-C6) was examined immunocytochemically in human thymuses of different ages and developmental stages (prenatal: 16 weeks and postnatal: 3 years, 5 years, and 21 years) employing an indirect alkaline phosphatase conjugated antigen detection technique on formalin-fixed, paraffin-embedded tissue sections. The immunoreactivity was located in the thymic RE cellular network (cortical and medullar), showing different intensity (+3 to +4 or 50% to 90% and over 90% in the prenatal thymic tissue and +2 to +3 or 50% to 90% during the three different postnatal stages). Intense expression was identified in the thymic medulla, including very strong immunoreactivity in the immigrating, committed hematopoietic stem cells (HSCs) present within the interlobular connective tissue (ICT). Strong presence of the HOX-B3 and HOX-B4 proteins was detected in the thymic Hassall's bodies (HBs), suggesting an intensive functional activity of the RE cells present within these unique formations within the thymic medulla. The precise role of these and other HOX gene products in the various steps of intrathymic T lymphopoieis should be elucidated through further basic molecular biological research.

Novel insights into the function of the thymic Hassall's bodies.

During thymic ontogenesis, the HBs appear when lymphopoiesis is already established and the cortex, medulla and the cortico-medullary junction are capable of conducting the positive and negative selection of T lymphocytes undergoing progressive maturation. The HBs are structurally organized from RE cells, which usually undergo hypertrophy prior to their inclusion in the outer cell layer of the corpuscles. The cellular microenvironment of the thymic medulla is composed of networks of cell types, of a variety of origins, and all of them may participate in the construction of growing, progressive HBs. Histochemically, we detected a rich content of basic non-histone proteins, PAS positive substance (glycogen) and acid mucopolysaccharides within the bodies. Employing the histological stain of Pasini and immunocytochemical methods with monoclonal antidodies (MoABs) AE2 and AE3, high molecular weight (56.5 to 67 kD) basic keratins were defined in human HBs. Employing a panel of MoABs developed against thymic RE cell surface antigens, we observed immunoreactivity localized to the outer cell layer of the HBs with MoABs TE8, TE16 and TE19, while the centrally located cells reacted positively with TE15 and TE19. Immunoreactivity in human skin, employing the TE8, TE16 and TE19 MoABs was also observed in the epidermal granulosa cell layer, while TE15 reacted with cells of the stratum corneum. The presence of endocrine, peptide secreting RE cells within the HBs was defined with the use of MoAB A2B5, which binds to the GQ ganglioside. The hypertrophied, physiologically active RE cells of the peripheral cell layer of the HBs reacted positively with medium to strong intensity when stained with MoABs UJ127.11, J1153, A2B5, 215.D11, and 275.G7. We also observed the expression of transforming growth factor-beta type II receptors in HBs. The recently detected expression of the homobox gene products B3, B4, and C6, transcription factors involved in developmental processes related to hematopoiesis within HBs provides further evidence that HBs are important functional components of the RE network of the thymus which provide developing thymocytes with paracrine and juxtacrine signals to ensure their proper functional maturation during intrathymic lymphopoiesis. Our transmission electronmicroscopical (TEM) studies on HBs determined the existence of groups of RE cells connected to one another by desmosomes. We also observed long cytoplasmic processes originating from medullary RE cells and directly contacting thymic T lymphocytes and accessory antigen presenting cells (macrophages, dendritic cells, interdigitating cells, Langerhans cells, etc.) by the use of scanning electronmicroscopy (SEM). Thus, our results indicate that the HBs are unique, antigenically distinct, functionally active, multicellular components of the nonlymphocytic, cellular micro-environment of the thymic medulla, and participate in the physiological activities of the prenatal and adult thymus.

Review of thymic hormones in cancer diagnosis and treatment.

The thymus is an endocrine organ. A unified, physiological concept of humoral regulations of the immune response has emerged in the last three decades. The thymus is the major site of production of immunocompetent T lymphocytes from their hematopoietic stem cells. This complex process required direct cell to cell, receptor based interactions, as well as in situ paracrine information via the numerous cytokines and thymic hormones produced by the cells of thymic microenvironment. Thymic hormones induce in situ T-cell marker differentiation, expression and functions. These polypeptide hormones have also been shown by means of immunocytochemistry to localize in the reticulo-epithelial (RE) cells of the thymic cellular microenvironment. Due to the great complexity of the intrathymic maturation sequence of T lymphocytes and the diverse immunophenotypically unique subpopulations of T lymphocytes, it is quite unlikely that a single thymic humoral factor could control all of the molecular steps and cell populations involved. It is much more likely that an extremely rich and diverse, but genetically determined, milieu is present within the thymus, and that thus the control of intrathymic T lymphocyte maturation and the functional maturation of T cells involves the orchestral interaction of various thymic-specific factors and other molecules during the differentiation process. Thymosin fraction 5 and its constituent peptides influence several properties of lymphocytes including cyclic nucleotide levels, migration inhibitory factor production, T-dependent antibody production, as well as the expression of various cell surface maturation/differentiation markers. Recently, derivatives of thymic hormones, mostly of thymosins, have been detected as products of neoplastically transformed cells and employed in the early diagnosis of neoplasms. In clinical trials, thymic hormones strengthen the effects of immunomodulators in immunodeficiencies, autoimmune diseases, and neoplastic malignancies. Combined chemo-immunotherapeutical anti-cancer treatment seems to be more efficacious than chemotherapy alone, and the significant hematopoietic toxicity associated with most chemotherapeutical clinical trials can be reduced significantly by the addition of immunotherapy.