Clinical and immunological evaluation of anti-apoptosis protein, survivin-derived peptide vaccine in phase I clinical study for patients with advanced or recurrent breast cancer.

BACKGROUND: We previously reported that survivin-2B, a splicing variant of survivin, was expressed in various types of tumors and that survivin-2B peptide might serve as a potent immunogenic cancer vaccine. The objective of this study was to examine the toxicity of and to clinically and immunologically evaluate survivin-2B peptide in a phase I clinical study for patients with advanced or recurrent breast cancer. METHODS: We set up two protocols. In the first protocol, 10 patients were vaccinated with escalating doses (0.1-1.0 mg) of survivin-2B peptide alone 4 times every 2 weeks. In the second protocol, 4 patients were vaccinated with the peptide at a dose of 1.0 mg mixed with IFA 4 times every 2 weeks. RESULTS: In the first protocol, no adverse events were observed during or after vaccination. In the second protocol, two patients had induration at the injection site. One patient had general malaise (grade 1), and another had general malaise (grade 1) and fever (grade 1). Peptide vaccination was well tolerated in all patients. In the first protocol, tumor marker levels increased in 8 patients, slightly decreased in 1 patient and were within the normal range during this clinical trial in 1 patient. With regard to tumor size, two patients were considered to have stable disease (SD). Immunologically, in 3 of the 10 patients (30%), an increase of the peptide-specific CTL frequency was detected. In the second protocol, an increase of the peptide-specific CTL frequency was detected in all 4 patients (100%), although there were no significant beneficial clinical responses. ELISPOT assay showed peptide-specific IFN-gamma responses in 2 patients in whom the peptide-specific CTL frequency in tetramer staining also was increased in both protocols. CONCLUSION: This phase I clinical study revealed that survivin-2B peptide vaccination was well tolerated. The vaccination with survivin-2B peptide mixed with IFA increased the frequency of peptide-specific CTL more effectively than vaccination with the peptide alone, although neither vaccination could induce efficient clinical responses. Considering the above, the addition of another effectual adjuvant such as a cytokine, heat shock protein, etc. to the vaccination with survivin-2B peptide mixed with IFA might induce improved immunological and clinical responses.

Protective mechanism of beta-SQAG9 liposome, a sulfonoglycolipid extracted from sea urchin intestines, against hepatic ischemia reperfusion injury.

We previously reported that beta-SQAG9 liposome, a sulfonoglycolipid extracted from sea urchin intestines, had a protective effect against hepatic ischemia reperfusion (I/R) injury. In this study, we made a detailed investigation of this protective effect and its mechanism. Rats were pretreated either with beta-SQAG9 liposome (treated group) or with phosphate-buffered saline solution (control group). Thereafter, they were subjected to partial hepatic I/R. The serum levels of aspartate aminotransferase, alanine aminotransferase, and lactate dehydrogenase were measured, and histological damage was evaluated with hematoxylin and eosin staining. To investigate the protective mechanism of beta-SQAG9 liposome on I/R injury, the serum levels and the tissue messenger RNA levels of TNF-alpha and IL-1beta were measured, and polymorphonuclear neutrophil (PMN) infiltration was histologically evaluated by immunohistochemistry. Moreover, to investigate an interaction between beta-SQAG9 liposome and L-selectin on PMNs, flow cytometric analysis and immunofluorescence were performed. beta-SQAG9 liposome reduced the hepatic I/R injury. The pretreatment with beta-SQAG9 liposome reduced the PMN infiltration into the liver parenchyma. On the other hand, there was no apparent difference in the serum levels and the tissue messenger RNA levels of the proinflammatory cytokines between the two groups. Thus, beta-SQAG9 liposome might reduce the hepatic I/R injury by inhibition of the PMN infiltration into the liver parenchyma, which was independent of the regulation of cytokine production. Moreover, we demonstrated that beta-SQAG9 liposome specifically bound to L-selectin on PMN cell surface, which mediated the PMN infiltration. beta-SQAG9 liposome might competitively antagonize L-selectin on PMNs and suppress the subsequent PMN infiltration, resulting in the reduction in I/R injury.

Autoantibodies to survivin in patients with chronic hepatitis and hepatocellular carcinoma.

OBJECTIVES: Autoantibodies to tumor-associated antigens including survivin have been detected in sera from patients with hepatocellular carcinoma (HCC). However, little is known about autoantibody responses to tumor-associated antigens in patients with chronic hepatitis, which strongly predisposes to development of HCC. METHODS: We subjected sera from 57 patients with chronic hepatitis and 29 patients with HCC to an enzyme-linked immunosorbent assay (ELISA) using a full-length recombinant survivin protein. A cutoff value for positivity was determined as the mean absorbance +2SD for sera from healthy volunteers. RESULTS: In patients with chronic viral hepatitis, elevated anti-survivin antibodies were detected in 10 of 57 sera (17.5%); in HCC patients, such elevation were detected in 7 of 29 sera (24.1%). The levels of anti-survivin antibodies in HCC patients with HCV infection were significantly higher than those in the healthy control and HCC patients with HBV infection. However, there were no significant differences in the levels of anti-survivin antibodies between HCV and HCC patients with HCV infection. CONCLUSIONS: We demonstrated that elevated anti-survivin antibodies were detected for the first time in patients with chronic viral hepatitis. The results suggest that the levels of anti-survivin antibodies have no association with the progression of HCV or HBV to HCC.

Detection of autoantibodies to livin and survivin in Sera from lung cancer patients.

Survivin and livin are highly expressed in cancer cells and transformed cells, but show little or no expression in normal differentiated tissues. Although human antibody responses to cancer-associated antigens have been detected, the response to livin has not yet been described in lung cancer patients. We examined prevalence of anti-livin antibodies in such patients with a specific enzyme-linked immunosorbent assay (ELISA) using recombinant protein. Using a cutoff value for positivity determined as the mean absorbance +2S.D. for healthy control samples, 19 of 37 lung cancer patients (51.3%) were positive for anti-livin antibodies. Of 31 samples from the same lung cancer patients, 18 (58.1%) were positive for anti-survivin antibodies. When sera from 31 lung cancer patients were assessed simultaneously by anti-survivin and anti-livin ELISAs. Twenty-one patients (71%) were positive for survivin, livin, or both. Intensity of anti-livin antibody responses did not correlate with intensity of anti-survivin responses. Like anti-survivin antibodies, anti-livin antibodies, thus, can be detected in many lung cancer patients. Testing for both antibodies together may prove useful in detecting lung cancer, but more extensive studies are needed to establish the clinical significance of anti-livin antibodies.

Detection of autoantibodies to survivin and livin in sera from patients with breast cancer.

BACKGROUND: Survivin and livin are highly expressed in cancer cells and transformed cells, but show little or no expression in normal differentiated tissues. Human antibody responses to tumor-associated antigens have been detected, but little is known about the response to survivin and livin in breast cancer patients. METHODS: We examined the prevalence of anti-survivin and livin antibodies in breast cancer patients with a specific enzyme-linked immunosorbent assay (ELISA) using recombinant protein. RESULTS: Using a cutoff value for positivity determined as the mean absorbance +2SD for healthy control samples, sera from 11 of 46 breast cancer patients (23.9%) were positive by the ELISA using recombinant survivin protein. Of 46 samples from the same breast cancer patients, 15 (32.6%) were positive for anti-livin antibodies. In addition, 24 (52.2%) were positive for 1 or both ELISAs using the respective proteins. Intensity of anti-livin antibody responses did not correlate with intensity of anti-survivin responses. CONCLUSIONS: Anti-livin antibodies were detected in sera from breast cancer patients by an anti-livin ELISA using full-length recombinant livin protein. Like survivin, livin may act as a major cancer antigen in breast cancer patients.

Introduction of a survivin gene-specific small inhibitory RNA inhibits growth of pancreatic cancer cells.

BACKGROUND: The anti-apoptotic molecule survivin is expressed in human cancers of various origins. Since this molecule possesses multiple functions, including apoptosis inhibition, cell cycle promotion and enhancement of Fas ligand expression, survivin has attracted growing attention as a target in cancer treatment. A survivin-specific small inhibitory RNA (siRNA) was introduced into pancreatic cancer cells to investigate its effect on cancer cell growth. MATERIALS AND METHODS: Survivin mRNA and protein expression were examined by RT-PCR and Western blotting, respectively. DNA histogram analysis was performed using a flow cytometer. RESULTS: The introduction of survivin-specific siRNA reduced survivin mRNA and protein expression in PANC-1 cells by over 90% and to an undetected amount, respectively, and induced growth inhibition. The siRNA transfectants showed pronounced morphological changes including enlargement of cells and multinucleation. siRNA transfectants did not show cell cycle arrest, but underwent apoptosis. CONCLUSION: Our data suggest that the use of survivin-specific siRNA deserves further investigation as a novel approach to cancer therapy.

TCV-116, an angiotensin II type 1 receptor antagonist, reduces hepatic ischemia-reperfusion injury in rats.

BACKGROUND: In Pringle's maneuver during liver surgery and liver transplantation, ischemia-reperfusion (I/R) is an unavoidable process, and protection against hepatic I/R injury is a major unresolved problem. Therefore, various pharmacologic approaches to prevent hepatic I/R injury are currently under trial. In this study, we investigated whether TCV-116, an angiotensin II type 1 receptor antagonist, can reduce this injury. METHODS: The rats were pretreated either with TCV-116 (group 1) or with the vehicle alone (group 2). The rats in group 3 were not pretreated. Thereafter, they were subjected to partial hepatic I/R. RESULTS: After reperfusion, the mean peak plasma concentrations of aspartate aminotransferase, alanine aminotransferase, lactic dehydrogenase, and creatine kinase were lower in group 1 than in groups 2 and 3. The magnitude of hepatic injury was reduced in group 1 compared with that in groups 2 and 3. The mean peak plasma concentrations of tumor necrosis factor-alpha, cytokine-induced neutrophil chemoattractants-1, and interleukin-6 were lower in group 1 than in groups 2 and 3. The number of neutrophils infiltrating the liver was also lower in group 1 than in groups 2 and 3. The mean peak plasma concentration of hepatocyte growth factor (HGF) was higher in group 1 than in groups 2 and 3. CONCLUSIONS: TCV-116 reduced the hepatic I/R injury by inhibiting inflammatory cytokine production and by enhancing HGF production.

Phase I clinical study of anti-apoptosis protein, survivin-derived peptide vaccine therapy for patients with advanced or recurrent colorectal cancer.

Survivin is a member of the inhibitor of apoptosis protein (IAP) family containing a single baculovirus IAP repeat domain. It is expressed during fetal development but becomes undetectable in terminally differentiated normal adult tissues. We previously reported that survivin and its splicing variant survivin-2B was expressed abundantly in various types of tumor tissues as well as tumor cell lines and was suitable as a target antigen for active-specific anti-cancer immunization. Subsequently, we identified an HLA-A24-restricted antigenic peptide, survivin-2B80-88 (AYACNTSTL) recognized by CD8+ cytotoxic T lymphocytes (CTLs). We, therefore, started a phase I clinical study assessing the efficacy of survivin-2B peptide vaccination in patients with advanced or recurrent colorectal cancer expressing survivin. Vaccinations with survivin-2B peptide were given subcutaneously six times at 14-day intervals. Of 15 patients who finished receiving the vaccination schedule, three suffered slight toxicities, including anemia (grade 2), general malaise (grade 1), and fever (grade 1). No severe adverse events were observed in any patient. In 6 patients, tumor marker levels (CEA and CA19-9) decreased transiently during the period of vaccination. Slight reduction of the tumor volume was observed in one patient, which was considered a minor responder. No changes were noted in three patients while the remaining eleven patients experienced tumor progression. Analysis of peripheral blood lymphocytes of one patient using HLA-A24/peptide tetramers revealed an increase in peptide-specific CTL frequency from 0.09% to 0.35% of CD8+ T cells after 4 vaccinations. This phase I clinical study indicates that survivin-2B peptide-based vaccination is safe and should be further considered for potential immune and clinical efficacy in HLA-A24-expression patients with colorectal cancer.

Expression of survivin mRNA and livin mRNA in non-small-cell lung cancer.

It has been suggested that suppression of apoptosis may contribute to the development and progression of cancer. Anti-apoptotic survivin and livin genes are highly expressed in cancer cells and transformed cells, but show little or no expression in normal differentiated tissues. However, there are no available data concerning livin expression in non-small-cell lung cancer (NSCLC). We therefore measured livin mRNA and survivin mRNA expression in 38 NSCLC cancer samples and 15 paired non-cancerous lung tissue samples using a quantitative reverse transcription-polymerase chain reaction (RT-PCR). While both mRNAs showed higher expression in cancers than non-cancerous tissues (P < 0.001), livin mRNA and survivin mRNA expression did not correlate with one another. When a cut-off value for positivity was set at the mean + S.D. for expression values in non-cancerous tissues, positivity rates for livin mRNA and survivin mRNA expression were 76.3% (29 of 38) and 36.8% (14 of 38) in lung cancers and 6.7% (1 of 15) and 0% (0 of 15), respectively, in paired non-cancerous lung tissue samples. Livin mRNA and survivin mRNA expression in tumors were up-regulated in 23 of 31 (74.2%) early-stage NSCLC patients and 11 of 31 (35.5%), respectively. Expression of both mRNAs in tumors varied independently of tumor histology. These results support the possibility that the livin gene may play a role in NSCLC development and increased expression of livin mRNA may serve as a new target for lung cancer treatment as well as survivin.

Decreased gene expression for telomeric-repeat binding factors and TIN2 in malignant hematopoietic cells.

BACKGROUND: Details of mechanisms regulating telomere length are poorly understood in human hematopoietic cells. MATERIALS AND METHODS: Gene expression for TRFs and TIN2 was studied in hematopoietic cell lines, blood or bone marrow cells from acute leukemia and in normal leukocyte fractions. RESULTS: The telomeres were longest in normal leukocytes, shorter in patient samples and still shorter in malignant hematopoietic cell lines. TRF1 mRNA, TRF2 mRNA and TIN2 mRNA in three myeloblastic cell lines and six lymphoblastic cell lines were significantly less abundant than in the corresponding normal cell types. In patients with acute myeloid leukemia, expression of these gene was also less than in normal cells. In additional studies in culture, HL-60 cells with initially high telomerase activity and low expression of TRF mRNA and TIN2 mRNA differentiated into granulocytic and monocytic cells with low telomerase activity and high expression of these mRNAs. CONCLUSION: In hematopoietic carcinogenesis, gene expression for suppressors of telomerase activity, such as TRF and TIN2, is decreased. These genes might hold promise for gene therapy against cancer.

Down-regulation of TRF1, TRF2 and TIN2 genes is important to maintain telomeric DNA for gastric cancers.

BACKGROUND: The maintenance of telomeres may be required for long-term proliferation of tumors. Activity of telomerase, a ribonucleoprotein complex that elongates telomeres, has been found in almost all human tumors but not in adjacent normal cells. Several factors which regulate telomere length, TRF1 and 2, TIN2, tankyrase and Rap1, have been identified. TRF1, TRF2 and TIN2 are negative regulators of telomere length, while tankyrase and Rap1 act as positive regulators. In this study, we quantitated the mRNA of these five genes in gastric cancers to clarify the mechanism by which cancer cells maintain telomere length. MATERIALS AND METHODS: The expression of these five genes transcription was determined using a quantitative RT-PCR. RESULTS: TRF1, TRF2 and TIN2 mRNAs were significantly down-regulated in cancers compared to non-cancerous mucosa. Neither tankyrase nor Rap1 was upregulated in cancers. CONCLUSION: Down-regulation of TRF1, TRF2 and TIN2 gene expression may be important to maintain telomeres in gastric cancer.

Overexpression of early growth response-1 as a metastasis-regulatory factor in gastric cancer.

BACKGROUND: To investigate the potential role of a nuclear transcription factor, early growth response-1 (Egr-1), in formation and progression of gastric cancer, we compared its expression in gastric cancers with that in non-cancerous tissues. MATERIALS AND METHODS: Egr-1 mRNA expression was measured using TaqMan RT-PCR. The corresponding protein expression was examined immunohistochemically. RESULTS: Egr-1 mRNA expression was significantly higher in gastric cancer tissues than in normal mucosa (p < 0.0005). These differences were also reflected by protein product expression. Moreover, Egr-1 mRNA expression was higher in cases with metastasis to lymph nodes or remote organs. In cultured gastric cancer cells known to have a high metastatic potential, expression of this mRNA was higher than that of parental cells. CONCLUSION: It was suggested that Egr-1 has a significant role in carcinogenesis and in cancer progression, especially metastasis. Measurement of this mRNA should be useful for evaluation of the metastatic potential of gastric cancer.

Enhanced expression of the UROC28 gene in human breast cancer: relationship to ERBB2 gene expression.

BACKGROUND: ERBB2, a highly important oncogene in invasive breast cancer, is not only a prognostic factor but also a predictive marker for response to therapeutic agents. Recently, An et al. (10) identified a novel gene, UROC28, that is also overexpressed in breast cancer. To examine possible interrelationships, we quantitated UROC28 and ERBB2 mRNA in breast cancers. MATERIALS AND METHODS: The expression of UROC28 and ERBB2 mRNA in breast cancer tissues were determined using RT-PCR. The expression was also examined in T-47D breast cancer cells treated with estrogen. RESULTS: UROC28 mRNA expression was greater in cancers than in noncancerous tissues (p < 0.0001), as was ERBB2 mRNA. ERBB2 and UROC28 gene expression was dose-dependently down-regulated in T-47D breast cancer cells treated with estradiol. However UROC28 mRNA and ERBB2 mRNA expression did not correlate with one another. CONCLUSION: These results indicate that UROC28 may be a useful target molecule in breast cancer diagnosis and treatment, complementing ERBB2.

A proapoptotic caspase recruitment domain protein gene, TMS1, is hypermethylated in human breast and gastric cancers.

BACKGROUND: Conway et al. demonstrated that methylation of the proapoptotic gene, TMS1, was observed in breast cancer cell lines and tissues, resulting in decreased TMS1 gene transcription. However, whether the TMS1 gene is hypermethylated in other cancers is uncertain. MATERIALS AND METHODS: The expression of TMS1 mRNA was determined by quantitative RT-PCR. Methylation of the TMS1 gene was detected using methylation-specific PCR followed by bisulfite-modification of DNA. RESULTS: Methylation of the TMS1 gene was observed in breast, gastric and colorectal cancer cells. Down-regulation of TMS1 gene transcription in colorectal cancer cells was restored by treatment with a demethylating agent. Methylation of the TMS1 gene was observed in 2 out of 19 breast cancer specimens and 1 out of 9 gastric cancers, but in none of 13 colorectal cancers. CONCLUSION: These results suggest a direct role for aberrant methylation of the TMS1 gene in the progression of breast and gastric cancer involving down-regulation of the proapoptotic TMS1 gene.

Selection of an internal control gene for quantitation of mRNA in colonic tissues.

BACKGROUND: GAPDH, beta-actin and 18S rRNA are widely employed as internal control genes, with the assumption that they are expressed constitutively to similar degrees in different cells and tissues and under different experimental conditions. In this study, we tested this assumption by assessment of the transcription of these three genes in human colonic tissues using a quantitative RT-PCR. RESULTS: GAPDH transcription was significantly greater in both colonic adenomas and cancers than in normal mucosa. In addition, transcription of beta-actin was significantly increased in cancers. The expression of 18S rRNA was essentially constant among these various tissues. Stable expression of 18S rRNA was observed during the growth of colonic cancer cells stimulated with serum, but both GAPDH and beta-actin transcription were up-regulated, coinciding with cell proliferation. CONCLUSION: These results indicate that 18S rRNA is more reliable than GAPDH and beta-actin as an internal control gene for quantitative comparison of mRNA in colonic cancers.

A novel gene containing PDZ and LIM domains, PCD1, is overexpressed in human colorectal cancer.

BACKGROUND: The process of colorectal cancer development involves accumulated genetic alterations affecting APC, K-ras and p53. A recently identified gene, PCD1, was reported to be up-regulated in human malignancies including colorectal cancers, but relationships between PCD1 gene expression and clinicopathological findings, as well as the timing of genetic alteration of PCD1 in colorectal cancer development, are not clear. To determine whether PCD1 contributes to colorectal cancer progression, we investigated the expression of PCD1 mRNA in human colorectal tissues. MATERIALS AND METHODS: The expression of PCD1 mRNA was determined by quantitative RT-PCR. The mutation of p53 was detected by a PCR-SSCP method. RESULTS: Up-regulation of PCD1 gene transcription was observed not in adenomas but in cancers compared to normal mucosa (p < 0.0001). Primary tumors with a mutation of p53 showed significantly greater PCD1 gene expression than tumors without such a mutation (p = 0.0134). CONCLUSION: The PCD1 gene may play a role in colorectal cancer development from adenomas.

PCD1, a novel gene containing PDZ and LIM domains, is overexpressed in human breast cancer and linked to lymph node metastasis.

BACKGROUND: Despite surgical removal of the primary tumor of breast cancer in patients with apparently localized disease, relapse at local or distant sites may occur because undetectable micrometastases were present at the time of diagnosis. Identification of molecules associated with breast cancer metastasis suggests possible new treatments. PCD1, a gene encoding a new member of the PDZ and LIM domain-containing protein family, recently was identified. We examined the relationships between PCD1 mRNA expression in breast cancers and metastasis. MATERIALS AND METHODS: PCD1 mRNA expression in breast cancer tissues was examined using a quantitative reverse transcription polymerase chain reaction. RESULTS: PCD1 mRNA expression was greater in cancers than in noncancerous tissues (p < 0.0001). In addition, high PCD1 gene expression was more frequent in patients with lymph node metastasis. CONCLUSION: PCD1 appears to contribute to breast cancer progression and nodal metastasis, thus representing a potential target molecule in breast cancer diagnosis and treatment.

Survivin mRNA expression in patients with breast cancer.

We measured survivin mRNA expression in breast cancers using a fluorogenically detected reverse transcription-polymerase chain reaction, seeking relationships between expression and clinical and histological variables. The mean relative expression of survivin mRNA was 0.347+/-0.466 in breast cancers and 0.017+/-0.023 in noncancerous breast tissue resected with the tumors. A cut-off value for positivity was set at the mean + SD for expression in the noncancerous tissues; positivity for survivin mRNA expression was 90.2% (37 out of 41 cases) in breast cancers and 23% (3 out of 13) in adjacent noncancerous breast tissues. Expression in tumors varied independently of clinicopathological factors except for lymphatic invasion. As measured by our sensitive detection system, survivin mRNA is a useful diagnostic marker for breast cancer.