Background: NY-ESO-1 is a human gene that codes for antigens that are expressed in malignancies of various histological types, but not in normal tissues, except the testes. The expression of NY-ESO-1 in intracranial brain tumors including astrocytomas (ASTRs) and medulloblastomas (MEDs)/primitive neuroectodermal tumors (PNETs) was examined since the expression of NY-ESO-1 has only previously been explored in depth in neuroblastomas. Materials and Methods: During our immunohistochemical study, a sensitive, four-step, alkaline phosphatase-conjugated antigen detection technique was employed. The expression of NY-ESO-1 was thereby examined in 6 cases of MED/PNET and 14 cases of ASTR. Results: All 6 MED/PNET cases demonstrated high levels of immunoreactivity (overexpression) with the highest immunostaining intensity grades A and B. In the astrocytic tumors of various subtypes examined, the level of NY-ESO-1 expression was not as strong as that in MEDs/PNETs. However, there was a significant increase in expression level when comparing low-grade pilocytic ASTRs to high-grade anaplastic ASTRs and glioblastomas. Conclusion: As evidenced by our results, NY-ESO-1 overexpression increases as the malignancy grade of the astrocytic tumors increases. These data suggest that antigen-directed immunotherapy of primary brain tumors could target cancer/testis antigens (CTAs), especially those expressed at higher frequency such as NY-ESO-1.

5308 The cancer-testis (CT) family of antigens is expressed in a variety of malignant neoplasms and is silent in normal tissues, except for the testis. These antigens might therefore represent potential targets for specific antigen-directed immunotherapy. Expression of two members of this family, MAGE-1 and NY-ESO-1, has been described in various cancers, including melanomas, serous ovarian neoplasms, certain carcinomas and sarcomas, and lung cancer, to name a few. The objective of this study was to evaluate the extent and prognostic significance of CT antigen expression in childhood astrocytomas. During our immunohistochemical study, a four-step, alkaline phosphatase conjugated antigen detection technique was employed. The results of the study demonstrate immunocytochemical presence and cellular localization of the MAGE-1 CT-antigen only in anaplastic, high-grade ASTRs (100%), including glioblastomas. NY-ESO-1 expression levels were more diversified across the grades of ASTRS, but demonstrated significantly higher expression in high grade anaplastic ASTRs and glioblastomas when compared to low grade pilocytic ASTRs. Our results demonstrate that CT antigen expression in astrocytomas correlates directly with the degree of the malignancy because both MAGE-1 and NY-ESO-1 overexpression increased as the grade of the astrocytic tumor increased. These data suggest that CT antigen expression could be used for prognostic purposes and that immunotherapy of primary brain tumors could possibly target CT antigens, especially those expressed at higher frequency such as MAGE-1 and NY-ESO-1. Future observations will guide the use of CT antigens in brain tumor immunotherapy.

The reticulo-epithelial (RE) cellular network of the thymic stromal cellular microenvironment plays a vital role in neuroendocrine regulation and lymphoid cell homing and development. Transmission electronmicroscopic observations have confirmed that there are four functional subtypes of medullar RE cells: undifferentiated; squamous; villous; and cystic. Immunocytochemical observations have shown that the secreted thymic hormones, thymosin alpha1 and thymopoietin (and its short form, thymopentin or TP5), are both produced by RE cells. Thymic RE cells also produce numerous cytokines, including IL-1 and -6, G-CSF, macrophage-CSF and GM-CSF that likely are important during the various stages of thymocyte activation and differentiation. The coexistence of pituitary hormone and neuropeptide secretion, such as growth hormone, prolactin, adrenocorticotopic hormone and thyroid-stimulating hormone, among many others, and the production of a number of interleukins and growth factors, as well as the expression of receptors for all, by the same RE cell, is an unique molecular biological phenomenon. The thymic RE cell network represents an important cellular and humoral microenvironment in the neuroendocrine homeopathic regulatory mechanisms of the multicellular organism.

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.

Dendritic cells (DCs) are present in essentially every mammalian tissue, where they operate at the interface of innate and acquired immunity by recognizing pathogens and presenting pathogen-derived peptides to T lymphocytes. According to the research group of Shortman, experimental results suggest a "dual" DC differentiation model, demonstrating the existence of both myeloid-derived (with characteristic IF: CD11b+, CD11c+, CD8alpha- and DEC205+) and lymphoid-derived DCs (showing CD11b- CD11c-, CD8alpha+ and DEC205+ IF). DCs, including interdigitating cells (IDCs) and Langerhans cells (LCs), are characterized by dendritic morphology, high migratory mobility and are the most effective, "professional" cells for antigen presentation in primary immune responses. Most of the DCs express immunocytochemically detectable antigens like: S-100, CD1a, CD40 receptor, adhesion molecules (ICAM-1 or CD54, LFA-1 and LFA-3), integrins (CD11a, CD11c and CD18), CD45, CD54, co-stimulatory molecules (B7-1 or CD80, B7-2 or CD86), F418, MHC class I and II and DEC-205, multilectin receptor, immunostimulatory cytokine (IL-12) and, of course, Fc and complement receptors. Following recognition and uptake of antigens, mature dendritic cells (DCs) migrate to the T lymphocyte rich area of draining lymph nodes, display an array of antigen-derived peptides on the surface of major histocompatibility complex (MHC) molecules and acquire the cellular specialization to select and activate naive antigen-specific T lymphocytes. Immunotherapeutic ideas are based on the ability of the mammalian immune system to recognize neoplastically transformed cells. Immunotherapy of human neoplasms has always represented a very attractive fourth-modality therapeutic approach, especially in light of the many shortcomings of conventional surgical, radiation and chemotherapies in the management of neoplastically transformed cells. The cancer vaccine approach to therapy is based on the notion that the immune system could possibly mount a rejection strength response against the neoplastic cell conglomerate. The efficiency of DCs for T lymphocyte stimulation moved a number of research groups to develop DC- based immunotherapy approaches. The failure of cancer vaccines may be attributed to the relationship between host and neoplasm: through a natural selection process, the host facilitates the selective enrichment of clones with highly aggressive neoplastically transformed cells, being in various stages of differentiation and only during certain stages express neoplastic cell specific molecules.

(ROR1), on B-CLL cells. ROR1 protein is expressed only by CD5+CD19+ cells, and not by other cells in the peripheral blood of all B-CLL patients tested (n=15). ROR1 protein expression was observed in Ig-mutated and Ig-unmutated B-CLL cells at highly consistent levels, and we found no difference in the expression of ROR1 protein among primary B-CLL cells obtained from different anatomical niches such as peripheral blood, lymph node, and bone marrow. Normal B cells from healthy donors and lymphoblastoid cell lines established from them do not express ROR1 protein. Activation of B cells with CD40 ligand resulted in up-regulation of a number of co-stimulatory molecules in both normal B cells and B-CLL cells; nonetheless, neither did this activation induce ROR1 protein expression on normal B cells nor alter ROR1 protein expression on B-CLL cells, further demonstrating that ROR1 protein is constitutively expressed in B-CLL. Collectively, our ROR1 protein expression analysis in human B-CLL suggests that, based on its highly restricted, consistent, and constitutive expression, ROR1 protein may be a relevant target antigen for novel immunotherapeutic strategies that employ or induce B-CLL-specific antibodies or T cells or both.

Vascular endothelial growth factor (VEGF) is a homodimeric, disulfide-linked glycoprotein which exhibits endothelial cell-specific mitogenic properties. VEGF is also a potent inducer of vascular permeability. There is considerable experimental evidence that VEGF isoforms are strongly involved in provoking neoangiogenesis of neoplastic cells and, consequently, the growth and progression of primary neoplasms (i.e., astrocytic gliomas), including the formation of an invasive and metastatic immunophenotype (IP). During this immunohistochemical study, the presence and tissue localization of VEGF(121) was observed in anaplastic, high-grade astrocytomas (AAs) and in glioblastoma multiforme (GBMs) employing the specific monoclonal antibody against it. A sensitive, four-step, alkaline phosphatase-conjugated antigen detection technique was used. The immunoreactivity demonstrated a cytoplasmic, cell surface and extracellular matrix localization pattern in more than 90% of the tumor cells, with high intensity immunoreactivity (++++, A,B) in every high-grade astrocytic glioma tissue. VEGF(121) expression was identified mostly within the cytoplasm of tumor cells, suggesting an embryonic, undifferentiated and more malignant cellular IP of high-grade gliomas. Tumor-related neo-angiogenesis and endothelial cell proliferation were also present. The great majority of high-grade astrocytic gliomas are incurable with the three classic therapeutic modalities. In the future, the development of targeted anti-neoplastic treatment strategies, adapted to individual patients, will require molecular identification of the different classes of neoplasm (including subtypes of astrocytomas) according to their stages, biology, prognosis and therapeutic options.

The design of new antineoplastic agents that can halt the progression of human malignancies with minimal systemic damage has been emerging in recent years, with 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), we carried out an immunohistochemical (IHC) analysis of 22 primary human EACAs and 14 precursor lesions. Relevant clinico-pathologic 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 yr. (range 51-93). All 22 EACAs were of endometrioid type, of which 10 (45%) were grade I, 8 (36%) grade II and 4 (18%) were grade III. Overall, 4 of 9 (44%) EHs, 4 of 5 (80%) ACHs, and 18 of 22 (88%) EACAs were COX-2 positive. 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 of 22 (77%) adenocarcinomas as compared to 2 of 14 (14%) of precursor lesions (EH and ACH). Areas of adenomyosis were COX-2 +, 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 in EACAs. 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 basic immunphenotyping (IP) of brain tumors has essentially been completed. Advances in immunobiology have yielded a plethora of highly specific poly- and monoclonal antibodies useful in the IP assessment of normal and neoplastic cells. In our extensive studies, we employed a carefully selected panel of over 90 monoclonal antibodies directed against various tumor-associated antigens and utilized a four-step, alkaline phosphatase conjugated antigen detection technique, modified by us. Results: MED/PNETs characteristically expressed Synaptophysin and Chromogranin A; HLA-A,B,C, HLA-DR; Vimentin, TE4, TE7, TE8, TE15; AE1 and AE3; TE3; CLA and UJ 308; Len 2/a and Leu 3a/3b, NF-H and MAP-1, MAP-2, MAP-5; GFAP; p53; HOX-B3, -B4, -C6; c-erb-2 (HER-2), c-erb-3 (HER-3), c-erb-4 (HER-4); Caspase-3; FasR; Survivin; CD105 (Endoglin); NIMP-2, -3, -9, -10, -13; and COX-2. ASTRs expressed GFAP, Vimentin, Tenascin, HLA-A,B,C, HLA-DR; CLA and UJ 308; UJ 167.11, A2B5, and Thy-1; Chromogranin A; NF-H, TE7; Leu-2/a and Leu 3a/3b; p53; Caspase-3, -6, -8, -9; FasR; CD105 (Endoglin), NINIP-3, -10; MAGE-1; VEGF121.; and COX-2. In conclusion, PNETs and ASTRs display a heterogeneous mixed IP; both differentiated and immature neurofilament proteins are present in the great majority of cases; neuronal differentiation is always present; COX-2 inhibitors may allow some level of immunoprevention; these markers and combinations thereof will allow more precise differential diagnosis and prognostication; and future observations will aid in the development of individualized, fourth modality, immunotherapeutical approaches for primary childhood brain tumors specifically targeted against combinations of cell-surface localized antigens attacking the primary tumor through various pathways.

The overexpression of COX enzymes has been demonstrated in human neoplasms at various sites, including the colon, gastrointestinal tract, lung, skin and recently in brain tumors. In this study, COX-2 receptor overexpression in primary childhood brain tumors was determined and the distribution pattern of COX-2 receptors was examined. A sensitive, 4-step, alkaline phosphatase conjugated antigen detection technique was used and a specific monoclonal antibody for medulloblastomas/ primitive neuroectodermal tumors (MEDs/PNETs), anaplastic, high-grade astrocytomas (ASTRs) and in glioblastoma multiformes (GMs) was employed. All of the 14 MEDs/PNETs observed demonstrated high levels of immunoreactivity (overexpression), with the highest immunostaining intensity (grades A and B). However, of the 14 subtypes of astrocytic tumors examined, the COX-2 receptor expression level did not even approach those of the MEDs/PNETs levels. However, significant differences were found when comparing low grade pilocytic ASTRs to high grade anaplastic ASTRs and glioblastomas. In two low grade pilocytic ASTRs, the expression level never exceeded 20%, while in high grade glial tumors (6 anaplastic ASTRs and 6 GMs) 30 to 50% of the tumor cells overexpressed COX-2 receptors, documenting an increase in COX-2 receptor overexpression with the increasing grade of the astrocytic tumor. In view of these findings, it would appear likely that COX-2 inhibitors may represent a chemo-preventive tool in treating childhood brain tumors, which are the leading cause of solid tumor cancer death in children under the age of 20.

Overactivation of epidermal growth factor receptor (EGFR) signaling has been recognized as an important step in the pathogenesis and progression of multiple forms of cancer of epithelial origin. Reports regarding EGFR family members in brain tumors are sparse and, thus, the significance of EGFR expression in childhood brain tumors is unclear. In this study, the expression of the EGFR family members was analyzed in 22 medulloblastomas. During the immunohistochemical study, a sensitive, four-step, alkaline phosphatase conjugated antigen detection technique was employed. The results demonstrated the presence of c-erbB-2 (HER-2) and c-erbB-4 (HER-4) in 10 to 50% of the neoplastic cells of high-grade glial tumors with high immunoreactivity, while c-erbB-3 (HER-3) was only detected in less than 10% of the neoplastically-transformed cells. In a follow-up, 70% of children, usually under 4 years of age, with c-erbB-2 (HER-2)-positive MEDs/PNETs, succumbed to the cancer. The Kaplan-Meier estimation revealed a significant correlation between c-erbB-2 expression and survival (p = 0.002), suggesting that c-erbB-2 (HER-2) is probably a prognostic marker for limited survival. Medulloblastoma is the most common malignant brain tumor that occurs during childhood. Multimodality treatment regimens have substantially improved survival in this disease; however, the tumor is incurable in about one-third of patients with medulloblastoma, and the current treatment has a detrimental effect on long-term survivors. As such, the results of this study further support the idea that targeting EGFR alone, or in combination with its downstream mediators, represents a promising new approach for the management of childhood brain tumors. Moreover, c-erbB-2 (HER-2) expression may also be of use in better classifying brain tumors.

Neoplastic transformation evolves over a period of time involving the progression of the cellular immunophenotype (IP) from normal to hyperplastic to dysplastic, and finally, to fully malignant IPs. Superimposed on these changes is the interaction of the initiated cell with its microenvironment, whereby the neoplastically transformed cells, through the regulation or dysregulation of cytoskeletal, integrin, protease and adhesion molecules, develop a novel manner of relation with their surrounding microenvironment. Studies of the neuroendocrine-immune network revealed that the hormonal and cytokine milieu plays an important role impacting the growth and dedifferentiation capabilities of neoplastic cells. This is further affected by the tumour cells themselves determining the constitution of this hormonal microenvironment, allowing the most aggressive and invasive of neoplastically transformed cell clones to promote their own growth and dissemination. The elucidation of the steps of the progression of cancer from premalignant to metastatic and invasive forms is of utmost importance in the differential diagnosis of neoplasms and in the establishment of more efficacious therapeutic regimens. These regimens will certainly begin to take on a more individualised form. The functional characterisation of various human malignancies as to the neoplastically transformed cells’ IP, the bases of their interaction with tissue stromal elements, and the molecules involved in the humoral microenvironment of the particular stage of tumour will certainly allow for the better diagnosis, staging, prognostication and treatment of cancers in the future. This paper reviews carcinogenesis from nutritional, genetic and molecular, and humoral aspects, and discusses the importance of tumour markers in the diagnosis and therapeutic management of human cancer.

This application claims the isolation of a cancer-specific antigen of similar to 150 kDa molecular weight present in human breast cancer, colon cancer, lung cancer and other cancers. The inventors suggest that 'the antibodies have tumour specificity and are useful for therapy, diagnosis, monitoring, detecting and imaging of cancers. The antibody-recognised cancer-specific surface antigens can serve as targets for detecting, diagnosing, inhibiting or killing neoplastic cells.' The cancer-specific antigen is noted to be absent from human breast, colon, oesophagus, liver, lung and ovary tissue cells.' The inventors also discuss a number of possible applications of antibodies directed against the purportedly cancer-specific antigen, utilising methods that include antibody humanisation, immunoconjugation, preparation of chimeric antibodies, and other molecular research and diagnostic and therapeutic techniques.

s: Abstracts for the 20th Annual Scientific Meeting of the International Society for Biological Therapy of Cancer (Primary Authors are Italicized): NOVEL APPROACHES TO MONOCLONAL ANTIBODIES

During the process of programmed cell death (PCD), the cell disintegrates into small, membrane-bound apoptotic bodies. Caspase-3 is ubiquitously expressed in normal and neoplastically-transformed human cells and serves as an executioner in the apoptotic or PCD pathway. During our immunocytochemical study, a sensitive, four-step, alkaline phosphatase-conjugated antigen detection technique was employed. The results demonstrated the presence of apoptotic activity within the cellular microenvironment of childhood medulloblastoma/primitive neuroectodermal tumor. The observations identified the cytoplasmic presence of caspase-3 in more than 20% of neoplastic cells. The immunocytochemical expression pattern demonstrated a translocation tendency from the cytoplasm to the cell nuclei in the apoptotic cells in about 5% of the tumor cells. Caspase-3 presence was also detected in the tumor infiltrating lymphocytes (TILs), representing the host's immune, mostly CD8+, cytotoxic, tumor-associated antigen (TAA)-directed effector cells. This phenomenon may play an important role in these tumors' maintenance of immune privilege and evasion of immune attacks. We suggest that the grade and intensity of apoptosis may not only have diagnostic and prognostic significance, but could also play a leading role in the biological (fourth modality) antineoplastic treatment of these highly malignant, neuroectodermal brain tumors.

"Flat" colorectal adenomas and adenocarcinomas are well documented in the Japanese literature but only sporadically reported in the English literature. The present study involved systematic morphological analysis of a large series of colorectal carcinomas (CRCs) to determine the frequency of these "flat" CRCs (FCRCs) and analyze their pathological characteristics and associated patient survival. The study group comprised 47 patients (19 females and 28 males) with primary CRC who underwent colorectal resection at the H. Lee Moffitt Cancer Center between 1997 and 2002. These cases were selected based on the gross appearance of the tumors and after review of all of the hematoxylin and eosin-stained tumor sections in a series of 190 consecutive colorectal resections for CRCs. Application of strict morphological criteria classified 22 tumors as FCRCs. For comparison, 25 "polypoid" CRCs (PCRCs) were also identified. Cases of ulcerative fungating annular CRCs and CRCs with mixed gross appearance were excluded from this analysis. Clinicopathologic data, including patient survival, were compared for FCRCS and PCRCs. Statistical analyses were carried out using the chi(2) or Fisher's exact test and log-rank tests. Overall, 22 of 190 CRCs (11%) were found to meet the morphological criteria of FCRCs. Mean patient age was 70.6 years (range, 55 to 87) for FCRCs versus 68.5 years (range, 54 to 91) for PCRCs, and mean tumor size was 4.7 cm (range, 1.6 to 9) for FCRCs versus 4.4 cm (range, 0.5 to 10) for PCRCs. None of the 22 FCRCs and only 1 of 25 (4%) PCRCs were well differentiated; 17 of 22 (77%) FCRCs and 23 of 25 (92%) PCRCs were moderately differentiated; and 5 of 22 (22%) FCRCs and 1 of 25 (4%) PCRCs were poorly differentiated (P = 0.0087). FCRC cases were staged as 0 stage T1, 3 (14%) stage T2, and 19 (86%) stage T3; PCRC cases, as 4 (16%) stage T1, 14 (56%) stage T2, and 7 (28%) stage 3 (P = 0.000031). Similarly, angiolymphatic invasion was identified in 12 of 22 (54%) FCRCs versus 4 of 25 (16%) PCRCs (P = 0.0123). Although some differences between FCRCs and PCRCs were observed on resection in terms of nodal status (N), presence of metastases (M), and perineural invasion, these differences were not statistically significant. In comparison with PCRCs, FCRCs were associated with significantly shorter postresection patient survival at 1 to 5 years (P = 0.028). We have demonstrated in this report that a proportion of primary CRCs resected at our institution were indeed "flat." Furthermore, these FCRCs exhibited higher histological grades, higher T stage, more frequent angiolymphatic invasion, and shorter patient survival compared with PCRCs. Based on these data, FCRC appears to be a worse subtype of colon cancer than PCRC. Further appraisal of FCRCs and additional studies to further elucidate the molecular mechanisms underlying their morphogenesis are warranted.

During the physiological process of PCD, the cell initiates a sequence of events culminating in the disintegration of the cell into small, membrane-bound apoptotic bodies. The intrinsic part of the PCD program arises from the mitochondria when it releases cytochrome c from the mitochondrial intermembrane space into the cytosol, forming the caspase-activating complex or apoptosome. The family of caspases is involved in the execution of genetically controlled PCD. Caspase-3 is expressed in normal and neoplastically transformed human cells and, like other caspases, is synthesized as an inactive, 32kDa proenzyme. Caspase-6 cleaves nuclear mitotic apparatus protein (NuMA) and mediates the shrinkage and fragmentation of cell nuclei. Caspase-8 is an initiation caspase that activates the caspase cascade during apoptosis, while caspase-9 is the initiator caspase in the caspase cascade in apoptotic normal and neoplastically transformed cells. During our immunocytochemical study, a sensitive, four-step, alkaline phosphatase conjugated antigen detection technique was employed. The results did in fact demonstrate the presence of high apoptotic activity within the cellular microenvironment of high-grade astrocytomas and glioblastomas. The observations identified cytoplasmic expression of caspase-3 and caspase-6 in more than 50 per cent of tumor cells, caspase-8 and caspase-9 in more than 10 per cent of tumor cells in high-grade anaplastic ASTR and glioblastoma. The immunocytochemical expression pattern in about 10 per cent of the tumor cells for caspase-3 and caspase-6 and about 1 to 5 per cent of the tumor cells for caspase-8 and caspase-9 demonstrated a translocation tendency from the cytoplasm to the cell nuclei in the apoptotic cells. This phenomenon may play an important role in these tumors' maintenance of immune privilege and evasion of immune attacks. We suggest that caspase-3, -6, -8 and -9 immunocytochemistry could have prognostic and immunotherapeutic significance in the treatment of these highly malignant glial tumors.

4955 Apoptosis (programmed cell death, PCD) serves an important role in the normal morphogenesis, immunoregulation, and homeostatic mechanisms in both normal and neoplastically transformed cells. The balance between cell proliferation and cell death determines the growth and differentiation rate and the basic phenomenon of cellular homeostasis within every complex multicellular tissue. During the physiological process of PCD, the cell initiates a sequence of events culminating in the fragmentation of its DNA, nuclear collapse, and finally disintegration of the cell into small, membrane-bound apoptotic bodies. The intrinsic part of the PCD program arises from the mitochondria, the so-called “killer organelles” releasing cytochrome c from the mitochondrial intermembrane space into the cytosol, which forms the caspase-activating complex or apoptosome. The family of caspases is involved in the execution of genetically controlled PCD. Caspase-3 is ubiquitously expressed in normal and neoplastically transformed human cells and like other caspases is synthesized as an inactive, 32kDa proenzyme. Caspase-8 is an initiation caspase that activates the caspase cascade during apoptosis. Caspase-9 is the initiator caspase in the caspase cascade in apoptotic normal and neoplastically transformed cells. During our immunocytochemical study, a sensitive, four step, alkaline phosphatase conjugated antigen detection technique was employed. This study is one of the first studies to explore the question of caspase-3, -8, and -9 in astrocytomas. The results did in fact demonstrate the presence of high apoptotic activity within the cellular microenvironment of high-grade astrocytomas and glioblastomas. The observations identified cytoplasmic expression of caspase-3 in more than 50 per cent of tumor cells, caspase-8 in more than 10 per cent of tumor cells, and caspase-9 in more than 10 per cent of tumor cells in high grade anaplastic ASTR and glioblastoma. The immunocytochemical expression pattern in about 10 per cent of the tumor cells for caspase-3, about 1 to 5 per cent of the tumor cells for caspase-8, and about 1 to 5 per cent of the tumor cells for caspase-9 demonstrated a translocation tendency from the cytoplasm to the cell nuclei in the apoptotic cells. Caspase-3, -8, and -9 presence was also detected in the TILs, representing the host’s immune, mostly CD8+, cytotoxic, tumor associated antigen (TAA) directed effector cells. This phenomenon may play an important role in these tumors’ maintenance of immune privilege and evasion of immune attacks. We suggest that caspase-3, -8, and -9 immunocytochemistry could have prognostic and immunotherapeautic significance in the treatment of these highly malignant glial tumors.