Survivin in brain tumors: an attractive target for immunotherapy

Summary Survivin, a member of the inhibitor of apoptosis proteins gene family, was recently shown to be expressed by tumors originating from different cell lineages. There are also cumulative evidences that spontaneous immune response against survivin derived epitopes may occur. Here, using RT-PCR, Western-blot analysis and immunohistochemistry, we show that survivin is widely expressed by gliomas, meningiomas and schwannomas, bothin vitro andin vivo. These data indicate that survivin may serve as an attractive target for immunotherapies designed for brain tumors.     

Prognostic significance of the immunohistochemical index of survivin in glioma: a comparative study with the MIB-1 index

Summary Objective: Survivin has been identified as a protein expressed in cancer cells and a member of the inhibitor-of-apoptosis protein family. Recent studies suggest that the expression of survivin increases during the G2/M phase of the cell cycle, and may be used in clinical prognosis. We examined whether survivin expression in human gliomas would be a correlative of prognosis. Methods: We prepared polyclonal anti-survivin serum to establish a survivin index for stained sections, using an immunohistochemical procedure, according to the method used for scoring MIB-1 index, and then stained 29 paraffin-embedded sections from surgical specimens of 29 patients who were classified into three grades of World Health Organization with the mean age of low grade astocytoma (grade II) being 34.7; anaplastic astrocytoma (grade III), 48.8; and glioblastoma multiform (grade IV), 58.4. Results: On staining with the anti-survivin antiserum, all specimens contained positive cells, but the survivin index was heterogeneous among grades. The mean percentage of immunoreactive cells in each specimen was 70.0 (SD 18.2) in grade II, 81.3 (16.5) in grade III, and 85.0 (13.6) in grade IV. Then we compared the survivin index to the MIB-1 index and found that in low-grade gliomas (grade II and III), the difference in survival times between the high and low survivin indexes was significant (P=0.007), whereas that between the high and low MIB-1 indexes was not significant (P=0.092).Conclusion: Survivin is more sensitive marker than MIB-1 for the evaluation of low-grade gliomas in that it helps to predict patient survival. Much larger glioma patient series are needed to validate the findings of our limited study.     

Weapons Ovarian Epithelial Tumors May Use in Immune Escape: An Immunohistochemical Correlational Study

Investigate FasL and survivin expression in a series of primary ovarian surface epithelial tumors, correlate their expression with each other, and characterize the presence of CD3+ T-lymphocytes in studied tumors and determine whether their presence correlates with FasL or survivin expression in malignant cases. FasL and survivin expression was assessed in 54 ovarian epithelial tumors. The results were compared between different tumor types and grades. Correlation between both markers’ expression in all studied tumors was done. Either marker’s expression was compared to the mean CD3+ T-lymphocytes per HPF in the studied malignant tumors. Either FasL or survivin expression was significantly higher in malignant versus benign ovarian epithelial tumors (p < 0.001 for both) and both markers were strongly correlated to each other (r = 0.877 & p < 0.001). Malignant tumors show significantly higher mean CD3+ T-lymphocytes than benign and borderline tumors. The mean CD3+ T-lymphocytes decrease significantly on increasing malignant tumor grade (p = 0.019) and expression of both FasL and survivin (r = −0.729, -0.582, respectively & p < 0.001 for both). The higher expression of FasL and survivin in malignant as compared to benign ovarian tumors suggest that they have a significant role in pathogenesis of ovarian carcinoma. Both markers are strongly correlated to each other and may contribute to immune escape of ovarian carcinoma as their higher expression is associated with decreased number of CD3 + T-lymphocytes.     

Lack ofNF1 expression in a sporadic schwannoma from a patient without neurofibromatosis

Summary The neurofibromatosis type 1 (NF1) gene encodes a tumor suppressor protein, neurofibromin, which is expressed at high levels in Schwann cells and other adult tissues. Loss ofNF1 expression has been reported in Schwann cell tumors (neurofibrosarcomas) from patients with NF1 and its loss is associated with increased proliferation of these cells. In this report, we describe downregulation ofNF1 expression in a single spinal schwannoma from an individual without clinical features of neurofibromatosis type 1 or 2. Barely detectable expression ofNF1 RNA was found in this tumor byin situ hybridization using anNF1-specific riboprobe as well as by Northern blot and reverse-transcribed (RT)-PCR analysis. In Schwann cells cultured from this schwannoma, abundant expression ofNF1 RNA could be detected by Northern blot and RT-PCR analysis. These results suggest that, in some tumors, expression ofNF1 may be downregulated by factors produced within the tumor and may represent a novel mechanism for inactivating these growth suppressing genes and allowing for increased cell proliferation in tumors.     

Nuclear survivin expression predicts poorer prognosis in glioblastoma

Survivin is a member of the inhibitor of apoptosis family, and is expressed in various malignant tumors. Survivin overexpression has been reported to be a poorer prognostic factor in various malignancies. However, the prognostic value of survivin expression in patients with glioblastoma is still controversial. Therefore, in this study the role of survivin as a predictor for survival was investigated in patients with glioblastoma. Tissue specimens were obtained from 66 patients with glioblastoma treated with radiotherapy. Survivin expression was detected by an immunohistochemical method. Nuclear and cytoplasm survivin scores were defined by using the cell positivity and staining intensity. The scores were defined as follows, 0 (no staining), 1 (less than 50% of cell positivity and any staining), 2 (more than 50% of cell positivity and weak to moderate intensity) and 3 (more than 50% of cell positivity and strong intensity). The correlation between survivin scores and the overall survival rate was evaluated. Nuclear and cytoplasm survivin staining were noted in 47 and 58 patients, respectively. The number of patients with nuclear survivin score of 0, 1, 2 and 3, were 19 (28.8%), 26 (39.4%), 9 (13.6%) and 12 (18.2%), respectively. The 3-year overall survival rate of the nuclear survivin score 3 was 0%, significantly lower than the 11.6% of the nuclear survivin score ≤2 (P = 0.0003). Cytoplasm survivin score did not correlate with the prognosis. Nuclear survivin expression may be a useful biomarker for predicting prognosis in patients with glioblastoma.     

Fowlpox‐based survivin vaccination for malignant mesothelioma therapy

Survivin protein is an attractive candidate for cancer immunotherapy since it is abundantly expressed in most common human cancers and mostly absent in normal adult tissues. Malignant mesothelioma (MM) is a deadly cancer associated with asbestos or erionite exposure for which no successful therapies are currently available. In this study, we evaluated the therapeutic efficacy of a novel survivin‐based vaccine by subcutaneous or intraperitoneum injection of BALB/c mice with murine fiber‐induced MM tumor cells followed by vaccination with recombinant Fowlpox virus replicons encoding survivin. Vaccination generated significant immune responses in both models, leading to delayed tumor growth and improved animal survival. Flow cytometry and immunofluorescence analyses of tumors from vaccinated mice showed CD8⁺ T‐cell infiltration, and real‐time PCR demonstrated increased mRNA and protein levels of immunostimulatory cytokines. Analyses of survivin peptide‐pulsed spleen and lymph node cells from vaccinated mice using ELISPOT and intracellular cytokine staining confirmed antigen‐specific, interferon‐γ‐producing CD8⁺ T‐cell responses. In addition pentamer‐based flow cytometry showed that vaccination generated survivin‐specific CD8⁺ T cells. Importantly, vaccination did not affect fertility or induce autoimmune abnormalities in mice. Our results demonstrate that vaccination with recombinant Fowlpox expressing survivin improves T‐cell responses against aggressive MM tumors and may form the basis for promising clinical applications.     

Survivin expression in ganglioglioma

Summary Gangliogliomas are unusual central nervous system (CNS) neoplasms occurring mainly in children and young adults and inducing chronic pharmacoresistant epilepsy. These are usually well differentiated neuroepithelial tumors composed of neurons in association with neoplastic glial cells. Gangliogliomas present with favorable outcome. However, some may recur and/or progress to anaplasia and be associated with a dismal prognosis. Since histopathological features do not consistently correlate with clinical outcome, reliable prognostic factors have yet to be defined in gangliogliomas. Survivin is an anti-apoptotic protein whose expression has been found to be of prognostic significance in many human cancers, including gliomas. The objective of this study was to assess survivin expression using immunohistochemistry in 15 gangliogliomas. Ten lesions were low-grade neoplasms whereas 5 were high-grade tumors. Survivin expression appeared restricted to the neoplastic glial component and was detected in 6/15 gangliogliomas. Two additional tumors expressed survivin upon relapse. Half survivin expressing lesions displayed less than 1% immunoreactive cells. Survivin expression in more than 5% neoplastic glial cells was detected only in malignant and/or recurrent gangliogliomas. Extended lifespan in survivin expressing cells might enhance aggressive behavior in these tumors through accumulation of mutations, thereby allowing progression to malignant phenotypes. Survivin expression may carry a negative prognostic value in gangliogliomas.     

High survivin predicts a poor response to endocrine therapy, but a good response to chemotherapy in advanced breast cancer

Summary Variants of survivin with differing subcellular localizations might mediate the different functions of survivin, i.e. cell-cycle regulation and apoptosis inhibition. Highly proliferative tumors are more sensitive to chemotherapy, whereas apoptosis resistant cells would be refractory to endocrine therapy. Possibly, this explains incongruent data on the association of survivin with prognosis in breast cancer. Survivin levels were measured using ELISA in 800 × g pellets and 100,000 × g supernatants of breast cancer tissues from patients that were treated with either chemotherapy or endocrine therapy for advanced disease. These fractions might be enriched with nuclear or cytoplasmatic located survivin variants. Survivin levels were associated with tumors with poor prognostic clinical characteristics. For the patients treated with endocrine therapy, the patients with high survivin levels exhibited a significantly shorter progression free survival (PFS) than those who had low levels (pellet survivin Hazard Ratio (HR)=2.74, 95% Confidence Interval (CI)=1.31–5.72, p=0.008 and median PFS 5.8 versus 8.6 months, p=0.006, log-rank; cytosolic survivin HR=3.03, 95% CI=1.45–6.35, p=0.003). In contrast, for patients treated with chemotherapy, those with high cytosolic survivin had a significantly longer PFS than those with low levels (median PFS of 6.2 months, versus 4.7 months for patients with low cytosolic concentrations, p=0.024, log-rank). Thus, high levels of survivin are mainly related with a poor response to endocrine therapy, but a good response to chemotherapy. This phenomenon might be related to the different functions of survivin.     

Immunohistochemical analysis of SOX6 expression in human brain tumors

We previously demonstrated that the developmentally regulated gene,SOX6, is strongly expressed in glioma cells and in the fetal brain, but only faintly in the normal adult brain. Recent studies have indicated that brain tumor cells may share antigens, signaling systems, and behavior with neural stem/progenitor cells. To test the validity of this proposition, we analyzed the expression of SOX6 in various human central nervous system (CNS) tumors. Immunohistochemical analysis revealed that astrocytic and oligodendroglial tumors expressed SOX6; neuronal-glial cell tumors (central neurocytoma) and embryonal tumors (medulloblastoma), which arise from multipotential stem cell precursors, also showed a high intensity of SOX6 staining. In contrast, ependymal tumors (ependymoma and subependymoma), meningioma, and schwannoma, which are all well differentiated tumors, showed either no staining or only faint staining for SOX6. These results suggest that SOX6 may be expressed in bipotential or multipotential cells capable of neuronal and glial differentiation, but not in fully differentiated cells. SOX6 may be a useful marker for the diagnosis of tumors arising from immature bipotential cells that may differentiate into neuronal and glial cells.     

Expression of human Krev-1 gene in lungs of transgenic mice and subsequent reduction in multiplicity of ethyl carbamate-induced lung adenomas

Mice of the A/J strain are useful models of lung cancer because they develop tumors spontaneously or after treatment with ethyl carbamate. These tumors are thought to arise from either Clara cells (papillary tumors) or alveolar type 2 cells (alveolar tumors); like many human lung adenocarcinomas, the mouse tumors involve Ki-ras activation. Transformation with Ki-ras can be reversed by coexpression of the Krev-1 gene in tissue culture. To test the tumor suppressor activity of Krev-1 in vivo, we produced transgenic A/J mice expressing Krev-1 under the control of the rabbit uteroglobin promoter, which directs expression of heterologous genes to the lung Clara cells. Krev-1 was expressed specifically in the lungs of transgenic mice. Sixty-six mice (35 transgenic and 31 nontransgenic) from three lines were given ethyl carbamate, and the numbers of resulting lung tumors were compared between transgenic and nontransgenic animals. The mean number (± standard deviation) of ethyl carbamate-induced lung tumors was 21.7 ± 1.3 in transgenic mice and 26.9 ± 1.3 in their nontransgenic littermates (P < 0.01). Sequencing of polymerase chain reaction-amplified ras DNA from 15 transgenic mouse tumors and 16 nontransgenic mouse tumors (controls) detected mutations in codon 61 in 13 tumors from the transgenic group and 11 tumors in the control group, whereas mutations in codon 12 were detected in only one tumor in the transgenic group and in four tumors in the controls. Together, these data demonstrate for the first time the tumor suppressor activity of Krev-1 in vivo and suggest that Krev-1 tumor suppressor activity may be specific for cells harboring mutations in codon 12 of ras. © 1996 Wiley-Liss, Inc.     

Mutations of the p53 Gene and Loss of Heterozygosity at Chromosome 17p13.1 are Associated with Increased Survivin Expression in Breast Cancer

Expression of survivin, a member of the inhibitor-of-apoptosis (IAP) family, is elevated in fetal tissues and in various human cancers originating in the breast, lung, prostate, colon, pancreas, and stomach. Since overexpression of the survivin gene has been linked to poor patient survival in several cancers, survivin may be an important prognostic marker. Mechanisms up-regulating survivin gene expression in cancer are poorly understood. Recently, wild-type p53 was found to repress expression of the survivin gene by binding to the survivin promoter, thereby inhibiting promoter activity. Further, loss of heterozygosity (LOH) at 17p13 distal to the p53 gene is associated with more aggressive behavior of breast cancers. We therefore tested the hypothesis that not only p53 gene mutation but also LOH at 17p13 can up-regulate survivin expression in breast cancer. Survivin mRNA expression was greater in cancers than in uninvolved tissues (p < 0.0001). Mutations of the p53 gene were detected in 5 of 25 tumors; higher survivin gene expression was evident in these. LOH at the D17S938 locus (17p13.1) was found in 10 of 25 tumors, and most of these also showed increased survivin gene expression. Thus expression of survivin may be regulated not only by p53 but additionally by a putative tumor suppressor gene located at 17p13 distal to the p53 gene.     

p53 expression and subcellular survivin localization improve the diagnosis and prognosis of patients with diffuse astrocytic tumors

Purpose

Diffuse astrocytic tumors are the most frequently occurring primary central nervous system (CNS) tumors. Their histological sub-classification into diffuse astrocytoma (DA), anaplastic astrocytoma (AA) and glioblastoma (GB) is challenging and the available prognostic factors are limited to age and tumor subtype. Biomarkers that may improve the histological sub-classification and/or serve as prognostic factors are, therefore, urgently needed. The relationship between survivin and p53 in diffuse astrocytic tumor progression and survival is currently unclear. Here, we aimed to assess the relevance of these proteins in the accuracy of the histological sub-classification of these tumors and their respective treatment responses.

Methods

One hundred and thirty-three formalin-fixed paraffin-embedded diffuse astrocytic tumor samples were included. The tumor samples were histologically reviewed and subsequently assessed for p53 and survivin expression and the presence of the IDH R132H mutation by immunohistochemistry. p53 expression levels and survivin subcellular localization patterns were correlated with histological classification and clinical outcome.

Results

We found that age and histological subtype were the only features with a prognostic impact. In addition, we found that high p53 expression levels and a nuclear survivin localization correlated with the AA subtype, whereas cytoplasmic survivin localization correlated with the GB subtype. We also found that patients carrying tumors with a high cytoplasmic survivin expression, a high nuclear survivin expression or a high p53 expression, and who did not receive radiotherapy, exhibited poorer short-term and long-term overall survival rates.

Conclusions

Our data suggest that subcellular survivin localization and p53 expression may be employed as valuable tools to improve the accuracy of the histological sub-classification of diffuse astrocytic tumors. Patients whose tumors overexpress these proteins may benefit from radiotherapy, irrespective age and/or histological classification.

     

N-cadherin Expression in Breast Cancer: Correlation with an Aggressive Histologic Variant – Invasive Micropapillary Carcinoma

Summary Upregulation of N-cadherin in epithelial tumor cells has been shown to contribute to the invasive/metastatic phenotype. It remains however to be determined whether N-cadherin is increased in human breast cancers with enhanced malignant potential. We examined a large number of invasive breast cancer specimens (n=114) for N- and E-cadherin. These specimens compared invasive duct carcinomas (IDCs) of varying histologic grades with an aggressive subtype, invasive micropapillary carcinoma of the breast (MPAP), which has a high propensity for lymphatic invasion and lymph node metastasis. Staining scores for N- and E-cadherin were compared between non-MPAP and MPAP IDCs, and between the invasive and ductal carcinoma in situ (DCIS) of each IDC using statistical analysis. We found that N-cadherin was expressed in 76% of MPAP and 52% of non-MPAP carcinomas, and E-cadherin in 57% of MPAP and 36% of non-MPAP tumors. More MPAP (25%) compared to non-MPAP (5%) tumors expressed both cadherins. Of the two cadherins, N-cadherin was significantly associated with MPAP tumors (p=0.033) compared to E-cad (p=0.171). Moreover, in the majority of tumors that were positive for N-cadherin, the staining scores were increased in the IDC relative to intraductal components, and this effect was more dramatic in the MPAP carcinomas. This difference for N-cadherin was greater than the corresponding difference for E-cadherin in the MPAP group (p=0.005), whereas such changes were not significant in the non-MPAP group (p=0.10). Thus, N-cadherin is associated with tumor aggressiveness and metastatic potential and may contribute to tumor progression.     

Quantitative PCR analysis of the expression profile of genes related to multiple drug resistance in tumors of the central nervous system

Objectives To evaluate and compare the profile of expression of genes related to drug resistance in brain tumors and to analyze the impact of the increased expression of these genes on overall survival. Methods Eighty microdissected brain tumor samples from 79 patients were analyzed by RQ-PCR for the genes MDR1, MRP1, MRP3, LRP and BCRP. Protein expression was assessed by immunohistochemistry for MRP1 and LRP genes. Pediatric cases (0 to 20 years): 46 (17F:29M, median age 7.3 ± 5.9 years); adult tumors: 33 (17F:16M, median age 46.6 ± 14.5 years). Histological diagnoses: 21 astrocytomas I and II, 28 astrocytomas III and glioblastomas, 17 medulloblastomas, 8 ependymomas, and 6 oligodendrogliomas. Results glial tumors expressed higher MDR1 (P = 0.003) and BCRP (P = 0.03) levels than embryonic tumors. Low-grade astrocytomas expressed high MDR1 (P = 0.001), MRP3 (P = 0.01) and LRP (P = 0.02) levels. The MRP1 gene was preferentially expressed by medulloblastomas (P = 0.04) and ependymomas (P = 0.04); ependymomas also presented an increase of LRP (P = 0.02). The mRNA levels of MRP1 and LRP genes were associated to protein expression. The profile of gene expression of primary pilocytic astrocytomas of the hypothalamus and of the other locations was similar. Increased expression of resistance genes, separately or jointly, was not correlated with shorter overall survival in patients with medulloblastomas/PNET and malignant gliomas. Conclusions Drug resistance genes do not explain the higher sensitivity of gliomas of the hypothalamus/pituitary/optic pathways to chemotherapy. The increased expression of resistance genes had no impact on the overall survival of patients with medulloblastomas/PNET and high grade gliomas. High MDR1, MRP3 and LRP levels may be implicated in the primary resistance of pilocytic astrocytomas to chemotherapy.     

Determination of 35 cell surface antigen levels in malignant pleural effusions identifies CD24 as a marker of disseminated tumor cells

Many targets have been identified in solid tumors for antibody therapy but it is less clear what surface antigens may be most commonly expressed on disseminated tumor cells. Using malignant pleural effusions as a source of disseminated tumor cells, we compared a panel of 35 antigens for their cancer specificity, antigen abundance and functional significance. These antigens have been previously implicated in cancer metastasis and fall into four categories: (i) cancer stem cell, (ii) epithelial‐mesenchymal transition, (iii) metastatic signature of in vivo selection and (iv) tyrosine kinase receptors. We determined the antigen density of all 35 antigens on the cell surface by flow cytometry, which ranges from 3 × 10³–7 × 10⁶ copies per cell. Comparison between the malignant and benign pleural effusions enabled us to determine the antigens specific for cancer. We further chose six antigens and examined the correlation between their expression levels and tumor formation in immunocompromised mice. We concluded that CD24 is one of the few antigens that could simultaneously meet all three criteria of an ideal target. It was specifically and abundantly expressed in malignant pleural effusions; CD24^high tumor cells formed tumors in mice at a faster rate than CD24^low tumor cells, and shRNA‐mediated knockdown of CD24 in HT29 cells confirmed a functional requirement for CD24 in the colonization of the lung. Concomitant consideration of antigen abundance, specificity and functional importance can help identify potentially useful markers for disseminated tumor cells.