The prognostic value of peripheral CD4+CD25+ T lymphocytes among early stage and triple negative breast cancer patients receiving dendritic cells-cytokine induced killer cells infusion.

OBJECTIVE: This study aimed to assess the prognostic value of CD4+CD25+ T lymphocyte in peripheral blood among breast cancer patients treated with adoptive T lymphocytes immunotherapy. METHODS: 217 patients participated in the follow-up study. CD4+CD25+ proportion was measured by flow cytometry in peripheral T cells. The median survival was estimated by Kaplan-Meier curve, Log-rank test and Cox hazard proportion regression model, between groups of CD4+CD25+ proportion more than 5% and less than or equal to 5% in peripheral T cells. RESULTS: Peripheral CD4+CD25+ T lymphocytes had not a relationship with progression-free survival. It was featured that above 5% peripheral CD4+CD25+ proportion of T cells was related with the median overall survival by a shorten of 51 months (p < 0.05) with the HR 1.65 (95%CI 1.04, 2.62). Above 5% CD4+CD25+proportion of T cells produced the HR to be 1.76 (95%CI 1.07, 2.87) In stage 0-II patients, and 3.59 (95%CI 1.05, 12.29) in triple negative breast cancer patients. CONCLUSIONS: Cellular immunity restoration recovered by adoptive T cell infusions which resulted in less proportion of peripheral CD4+CD25+T lymphocytes could be a potential prognostic indicator among early stage and triple negative patients.

HER2-specific T lymphocytes kill both trastuzumab-resistant and trastuzumab-sensitive breast cell lines in vitro.

OBJECTIVE: Although the development of trastuzumab has improved the outlook for women with human epidermal growth factor receptor 2 (HER2)-positive breast cancer, the resistance to anti-HER2 therapy is a growing clinical dilemma. We aim to determine whether HER2-specific T cells generated from dendritic cells (DCs) modified with HER2 gene could effectively kill the HER2-positive breast cancer cells, especially the trastuzumab-resistant cells. METHODS: The peripheral blood mononuclear cells (PBMCs) from healthy donors, whose HLA haplotypes were compatible with the tumor cell lines, were transfected with reconstructive human adeno-association virus (rhAAV/HER2) to obtain the specific killing activities of T cells, and were evaluated by lactate dehydrogenase (LDH) releasing assay. RESULTS: Trastuzumab produced a significant inhibiting effect on SK-BR-3, the IC50 was 100ng/ml. MDA-MB-453 was resistant to trastuzumab even at a concentration of 10,000 ng/ml in vitro. HER2-specific T lymphocytes killed effectively SK-BR-3 [(69.86±13.41)%] and MDA-MB-453 [(78.36±10.68)%] at 40:1 (effector:target ratio, E:T), but had no significant cytotoxicity against HER2-negative breast cancer cell lines MDA-MB-231 or MCF-7 (less than 10%). CONCLUSION: The study showed that HER2-specific T lymphocytes generated from DCs modified by rhAAV/HER2 could kill HER2-positive breast cancer cell lines in a HER2-dependent manner, and result in significantly high inhibition rates on the intrinsic trastuzumab-resistant cell line MDA-MB-453 and the tastuzumab-sensitive cell line SK-BR-3. These results imply that this immunotherapy might be a potential treatment to HER2-positive breast cancer.

Activation of sonic hedgehog signaling pathway is an independent potential prognosis predictor in human hepatocellular carcinoma patients.

OBJECTIVE: The activation of hedgehog (HH) pathway is implicated in the development of human malignancies including hepatocellular carcinoma (HCC). However, the clinical impact of HH activation in HCC patients is still unclear. This study was conducted to confirm whether the expression of HH pathway components was associated with HCC progression and clinical outcome. METHODS: This study was a sample-expanded and prolonged follow up of one of our previous studies. It included 46 HCC patients who underwent surgical treatment from 2002 to 2005. The expression of sonic HH (SHH), patched-1 (PTCH1), smoothened (SMOH) and glioma-associated oncogene-1 (GLI1) genes in tumor and adjacent normal tissues extracted from the patients were examined by reverse transcription-polymerase chain reaction (RT-PCR) to explore the relationship between these genes and the clinical prognosis of HCC. RESULTS: The expression levels of SHH, PTCH1, SMOH and GLI1 in HCC tissues were 60.87%, 50.00%, 32.61% and 54.35%, respectively. The expression levels of SHH-related molecules were relatively intense in cancer tissue, but insignificantly correlated with any clinicopathological factors of tumor. Transcriptional factor GLI1 was the only molecule associated with poor prognosis among the HCC patients. The expression of GLI1 gene in tumor tissues was significantly related with disease-free survival (DFS) (P=0.042) and overall survival (OS) (P=0.030). The simultaneous expression of GLI1 in tumor and adjacent normal liver tissues correlated with DFS (P<0.029) and OS (P<0.025). CONCLUSIONS: HH signaling activation is an important event in the development of human HCC. The expression of GLI1 in SHH pathway is possibly involved in HCC progression, which may be a useful prognostic indicator of HCC.

[Dendritic cells infected by recombinant adeno-associated virus with CEA gene to induce antigen-specific CTL response to CD44(+)CD24(-/low) cells from MCF-7 breast cancer cell line].

OBJECTIVE: To infect dendritic cells (DC) by recombinant human adeno-associated virus (rh-AAV) vector with CEA gene to generate antigen-specific CTL cells in vitro and to assay the CEA specific cytotoxic T lymphocyte(CTL), response to CD44(+)CD24(-/low) breast cancer stem cells. METHODS: Peripheral blood mononuclear cells were induced to generate DCs by cytokines interleukin-4(IL-4), granulocyte-macrophage colony-stimulating factor(GM-CSF)and tumor necrosis factor-alpha(TNF-α) while T lymphocytes were cultured with cytokines interleukin-2(IL-2). DCs were infected by CEA gene containing rh-AAV. After being matured, DCs were co-cultured with T cells to generate CTL cells. CD44(+)CD24(-/low) breast cancer stem cells were sorted out from MCF-7 and MDA-MB-231 cells. CEA specific CTL response to CD44(+)CD24(-/low) breast cancer stem cells was assayed by MTT method. RESULTS: The percentages of CD44(+)CD24(-/low) breast cancer stem cells subsets in MCF-7 and MDA-MB-231 were 5.1% and 76.3% respectively. DCs transfected with CEA gene could induce CEA antigen CTLs response. The killing ratio of MCF-7 cells in CEA gene transfection group was 46.5% plusmn; 15.0% with significant difference (P=0.009) as compared with that of CEA gene untransfection group. As for CD44(+)CD24(-/low) breast cancer stem cells subset from MCF-7 cells, CEA gene transfection group resulted in a higher killing ratio of 44.7% ± 28.2% as compared with that of CEA untransfection group. While the inhibitory rate of non-breast cancer stem cells subset from MCF-7 cells in CEA gene transfection group was 50.6% ± 22.2% (P=0.05). CTL cells generated by DCs transfected with CEA gene did not show inhibitory activity to MDA-MB-231 breast cancer cells (without CEA expression) as compared with CEA gene untransfection group. It was the same in CD44(+)CD24(-/low) breast cancer stem cells subset and non-breast cancer stem cells subset from MDA-MB-231 breast cancer cells (P>0.05). CONCLUSION: DCs infected by rh-AAV with CEA gene could induce antigen-specific CTL response to kill CEA expressing breast cancer cells involved in CD44(+)CD24(-/low) breast cancer stem cells subset. It suggests that immune therapy might be a potential treatment of breast cancer stem cells.

MUC1-positive circulating tumor cells and MUC1 protein predict chemotherapeutic efficacy in the treatment of metastatic breast cancer.

Chemotherapy plays an important role in the treatment of metastatic breast cancer. It is important to monitor chemotherapeutic efficacy, to find a simple and efficient tool to guide treatment, and to predict the efficacy of treatment in a timely and accurate manner. This study aimed to detect mucin-1 (MUC1)-positive circulating tumor cells and MUC1 protein in the peripheral blood of patients with metastatic breast cancer and to investigate their relationship to chemotherapeutic efficacy. MUC1 mRNA was detected in the peripheral blood of 34 patients with newly diagnosed metastatic breast cancer by reverse transcription-polymerase chain reaction. The positive rates of MUC1 mRNA were 88.2% before chemotherapy and 70.6% after chemotherapy, without a significant difference (P=0.564); MUC1 mRNA expression before chemotherapy had no correlation with treatment effectiveness (P=0.281). The response rate of MUC1 mRNA-negative patients after first-cycle chemotherapy was significantly higher (P=0.009) and the progression-free survival (PFS) was clearly longer than those of MUC1 mRNA-positive patients (P=0.095). MUC1 protein in peripheral blood plasma was detected by an ELISA competitive inhibition assay. The patients with decreased MUC1 protein after chemotherapy had a significantly longer PFS than those with elevated MUC1 protein (P=0.044). These results indicate that the outcomes of MUC1 mRNA-negative patients after chemotherapy are better than those of MUC1 mRNA-positive patients. In addition, patients with decreased expression of MUC1 protein have a better PFS.

[Expression of genes related to Sonic Hedgehog signaling in human hepatocellular carcinomas].

OBJECTIVE: To investigate the expression status of Sonic Hedgehog signaling genes and molecules in human hepatocellular carcinomas(HCC), and to explore the relationship between these genes and clinical prognosis. METHODS: HCC tissue and adjacent normal tissue from 29 HCC patients were assayed for the expression of hedgehog signaling genes by reverse transcription-polymerase chain reaction chain reaction (RT-PCR) techniques and for the expression of hedgehog signaling molecules by immunohistochemistry. The expressions of Shh, Ptch, Smoh, Gli-1 mRNA were assayed as well as Shh, Ptch proteins in 29 cases of HCC and in 29 liver tissues adjacent to the tumor. RESULTS: Expression of Shh mRNA was detectable in about 51% of HCCs examined. Consistent with this, hedgehog target genes Ptch, Smoh and Gli-1 mRNA were expressed in over 68%, 48% and 62% of the tumors, respectively, and the expressions of Shh and Ptch proteins in HCC tumor tissues correlated with those of Shh and Ptch mRNA in tumor tissues (P=0.041 and P=0.035). This suggested that the hedgehog pathway was frequently activated in HCCs. The simultaneous expression of Gli-1 in HCC and liver tissues adjacent to the tumor had significantly relationship with poor prognosis. CONCLUSION: Hedgehog signaling activation is an important event for development of human HCCs. It also suggests that markers for hedgehog signaling activation may be useful for the determination of prognosis.

[Pilot study on the correlation between high incidence of CD44+/CD24 -/low/ABCG2- cells and poor prognosis in breast cancer].

OBJECTIVE: To explore whether the CD44+/CD24(-/low)/ABCG2(-) (ATP binding cassette superfamily G member 2) cells are associated with prognosis and clinical response in breast cancer patients. METHODS: We investigated the paraffin-embedded tissues of 43 breast cancer patients with (23 cases) and without (20 cases) recurrences. Double-staining immunohistochemistry (IHC) was applied for the detection of CD44+/CD24(-/low) cells and single-staining IHC for ABCG2. Flow cytometry was used to analyze the CD45(-)/CD44+/CD24(-/low)/ABCG2(-) cells in 4 mL peripheral blood of patients with metastasis breast cancer and 11 healthy female volunteers as controls. RESULTS: The positive rate of ABCG2 in recurrence-group was higher but with no difference compared with controls (78.3 % vs 60.0%, P = 0.32). Double-staining IHC revealed that the percentage of CD44(+)/CD24(-/low) cells was higher in recurrence-group than non-recurrence group(65.2% vs 35.0%, P = 0.048)and higher percentage of CD44+/CD24(-/low) cells was significant associated with poor overall survival(P = 0.031). Patients with higher percentage of CD44+/CD24(-/low) cells have shorter disease free survival (DFS), but have no statistical significance. Flow cytometry revealed that the CD45(-)/CD44+/CD24(- /low)/ABCG2(-) cells were higher in breast cancer patients than those of the volunteers (median 679/10(5) cells vs 12/10(5) cells). The cell number of this subset was affected by chemotherapy but was not statistically consistent with clinical response. CONCLUSION: Our study suggests that CD44+/CD24(-/low) breast cancer stem cells in tumor tissue may be associated with poor prognosis. The incidence of CD44+/CD24(-/low)/ABCG2(-) cells in peripheral blood is more frequent in breast cancer patients but further investigation should be made to explore the relationship of this subset and disease prognosis.

Identification and expression analysis of novel LAGE-1 alleles with single nucleotide polymorphisms in cancer patients.

OBJECTIVE: The purpose of this study is to identify single nucleotide polymorphisms (SNPs) in the coding region alleles of the X chromosomal LAGE-1 gene, and investigate the frequency of such SNPs in both cancer patients and healthy controls, and thus determine the potential significance of these SNPs with respect to cancer vaccine therapy. METHODS: In this study, different mRNAs transcribed from the LAGE-1 gene were identified by RT-PCR from healthy donors and cancer patients samples. RESULTS: A new LAGE-1 allele containing three coding region SNPs (69A/G, 317C/G, and 397T/G) were identified. The allele is highly expressed as the LAGE-1a mRNA variant AY679089 in some of the cancer patients. The three SNPs altered the LAGE-1 gene sequence to that of NYESO-1 at both the nucleotide and amino acid level. CONCLUSION: There is a high frequency of the LAGE-1 gene allele with SNPs in coding regions in cancer patients. There was a clear relationship between the variant AY679089 and gastric cancer. The SNPs may lead to accelerated progress of poorly differentiated gastric cancer. The SNPs found in these alleles may also alter the immunological characteristics of LAGE-1a and should be taken into account if this antigen is adopted as a cancer vaccine component.

[Identification and expression of non-coding RNAs NC28 and NC119 in human tumors].

OBJECTIVE: To explore and identify the non-coding RNAs related to tumors. METHODS: We used RT-PCR and Northern blot to analyze non-coding RNAs in tumor tissues and cell lines. RESULTS: Two predicted non-coding RNAs were confirmed to be expressed in cancer tissues and cell lines by RT-PCR and DNA sequencing. We detected the expression of two non-coding RNA transcripts by Northern blot. The length of NC28 was about 1800 nt, and that of NC119 was about 1200nt. CONCLUSIONS: NC28 and NC119 have a tumor-associated expression pattern. The non-coding RNAs may play a role in the development of tumors.

Expression profile of cancer-testis genes in 121 human colorectal cancer tissue and adjacent normal tissue.

PURPOSE: Among tumor antigens identified to date, cancer-testis (CT) antigens, which are coded by CT genes, are identified as a group of highly attractive targets for cancer vaccines. This study is the first to analyze the mRNA expression and possible correlation with pathologic characteristics of multiple CT genes in a large cohort of colorectal cancer (CRC) patients. EXPERIMENTAL DESIGN: The expression of 10 individual CT genes in 121 CRC and adjacent tissues were analyzed by RT-PCR method. The presence of autologous antibodies against NY-ESO-1 was examined in serum samples by ELISA. To confirm the protein expression, immunohistochemistry was done for detecting the NY-ESO-1 antigen in mRNA-positive CRC tissues. RESULTS: The CT genes were detected with various frequencies in CRC tissue, SCP-1, 1.7%; SSX-2, 2.5%; SSX-4, 2.5%; SSX-1, 5.0%; CT10, 6.6%; NY-ESO-1, 9.9%; MAGE-1, 11.6%; LAGE-1, 15.7%; MAGE-4, 22.3%; and MAGE-3, 27.3%. In 56.2% of tumor tissues examined in this study, at least one CT gene was detected. In contrast, no CT gene expression was found in cancer adjacent tissues. Among 10 CT genes investigated, NY-ESO-1 and LAGE-1 are of particular interest because their mRNA expression in CRC was rarely reported before. In our study, NY-ESO-1 mRNA was found to express in 9.9% of the samples, and also correlated significantly with stages (P = 0.041) and local lymph node metastasis (P = 0.002). In addition, we also identified one NY-ESO-1 antibody-positive serum sample. MAGE-4 mRNA was expressed at a high frequency in tumor tissues with vessel emboli samples (P = 0.025). CONCLUSIONS: These results suggested that CT genes, especially NY-ESO-1 and LAGE-1, do express in CRC. More than 50% of the CRC patients in this study express at least one CT gene, making them eligible for CT vaccination. NY-ESO-1 gene may serve as a marker for local metastasis and advanced disease. MAGE-4 gene is significantly associated with the vessel emboli.

[The expression of 11 cancer/testis (CT) antigen genes in esophageal carcinoma].

OBJECTIVE: To investigate the expression status of 11 different cancer/testis (CT) antigen genes in esophageal carcinoma. METHODS: Esophageal carcinoma tissue and adjacent normal esophageal mucosa taken from 35 esophageal carcinoma patients were assayed for the expression of 11 different CT antigen genes by RT-PCR techniques. RESULTS: Of the 11 CT antigen genes analyzed, none of them was expressed in normal esophageal mucosa. MAGE-3 was found to be the most frequently expressed in esophageal carcinoma tissues (62.9%), followed, in the order of expression frequency, by MAGE4 (31.4%), LAGE-1 (28.6%), MAGE-1 (25.7%), CT10 (20.0%), NY-ESO-1 (20.0%), CT7 (5.7%) and SCP1 (2.9%). No expression of SSX-1, SSX-2 and SSX-4 was found. Among the 35 cases, 28 (80.0%) expressed at least one CT antigen gene, 21 (60.0%) expressed more than 2 CT antigen genes, and 4 of the 21 (19.0%) expressed more than 4 CT antigens, which accounted for 11.4% of total number of patients (4/35). No CT antigen expression was found in the tumor tissue in 7 cases, including 5 cases in stage II and 1 case each in stage I and IV, respectively. Of the 11 CT genes examined, expression of 5 genes (NY-ESO-1, LAGE-1, MAGE-1, MAGE-3 and MAGE-4) was correlated with tumor progression. SCP-1 and CT10 expression was found more frequently in early stage patients. With progression of the disease, the frequency of co-expression of multiple CT antigen genes was significantly increased reaching 28.6% in stage III patients. CONCLUSION: Of the 11 different CT antigen genes examined by RT-PCR in esophageal carcinoma, 8 genes were detected in various frequencies in 28 of the 35 esophageal cancer patients studied. They are candidate tumor-associated antigens in the preparation of tumor vaccines for immunotherapy in esophageal cancer patients.

Dominant B cell epitope from NY-ESO-1 recognized by sera from a wide spectrum of cancer patients: implications as a potential biomarker.

Monitoring the spontaneous antibody (Ab) response against a panel of relevant tumor-associated antigens (TAA) in cancer patients may provide useful information regarding the clinical status of cancer. However, current Ab detection approaches require the purification of recombinant proteins, which is often difficult to achieve. In order to bypass the purification of recombinant proteins, we identified a dominant B-cell epitope from a shared tumor antigen NY-ESO-1. A synthetic peptide of the epitope, ESO:1-40, was as sensitive as the recombinant protein for detecting Ab against NY-ESO-1 in most patients. NY-ESO-1 specific Ab present in the sera of patients with melanoma, prostate cancer, nonsmall cell lung cancer, esophageal cancer, gastric cancer and hepatocellular carcinoma reacted with the dominant peptide at a similar frequency as the recombinant protein. To our knowledge, ESO:1-40 is the first peptide epitope recognized by sera from a wide spectrum of cancer patients but not healthy donors. This simple and straightforward approach may allow the investigation of the clinical significance of spontaneous Ab responses against multiple TAA and their correlation with the clinical course of malignant diseases in the future.

Cancer/testis antigen expression and autologous humoral immunity to NY-ESO-1 in gastric cancer.

Gastric cancer has the highest mortality rate and the second-highest morbidity rate of all malignant tumors in China. Since cancer/testis (CT) antigens are expressed in various types of human tumors but generally not in normal tissue except for testis, they are promising antigens for cancer immunotherapy. NY-ESO-1, in particular, is the most immunogenic of the CT antigens. To study the feasibility of developing a CT antigen vaccine for gastric cancer, 101 gastric cancer samples were analyzed for the presence of NY-ESO-1 mRNA and that of 10 other CT antigen genes. Twelve out of 101 samples (11.9%) were found to be NY-ESO-1 mRNA-positive, 11 of them from advanced stage patients. In 7 of the 12 NY-ESO-1 mRNA-positive samples, the NY-ESO-1 protein was also detected by immunohistochemistry. An autologous humoral immune response to NY-ESO-1 was detected in 6 of 12 advanced stage NY-ESO-1 mRNA-positive patients, indicating that NY-ESO-1 is immunogenic in advanced stage gastric cancer. The serum from a patient with an NY-ESO-1 negative but LAGE-1 positive tumor was also found to be NY-ESO-1 antibody positive, possibly due to cross-reactivity between NY-ESO-1 and LAGE-1. All NY-ESO-1 mRNA-positive gastric cancer samples also expressed one to seven additional CT genes, revealing a tendency toward a clustered expression pattern, regardless of disease stage. About 74% of the samples expressed at least one CT antigen, most frequently MAGE-3 (41.6%). NY-ESO-1 and MAGE-3 are thus potential targets for a multivalent CT antigen vaccine.

[Expression of multiple cancer-testis antigen genes in non-small cell lung cancer treated by chemotherapy prior surgery].

OBJECTIVE: To investigate the possibility of utilizing cancer-testi (CT) antigens as targets for immunotherapy of non-small cell lung cancer (NSCLC) with vaccines. METHODS: Tissues from 51 NSCLC patients who had chemotherapy prior surgery were assayed for the expression of 11 different CT antigens by RT-PCR. RESULTS: Of the 11 CT antigens analyzed, MAGE-3 was found to be expressed most frequently in NSCLC tissues and CT7 the least frequently. The frequencies of CT antigen expression was: MAGE-3 (38%), NY-ESO-1 (21%), CT10 (17%), LAGE-1 (15%), MAGE-4 (13%), SCP-1, SSX1 and SSX4 (12%), MAGE-1 (10%), SSX2 (6%), and CT7 (2%). Among these cases, 34 (67%) expressed at least one CT antigen gene. 13 of the 17 cases in which no CT antigen expression was found in the tumor tissue, the tumors were classified as at the stage I. MAGE-3 and CT10 were found to be expressed more frequently in tissues from patients with late stage diseases while SCP-1 was found more frequently in earlier stages of NSCLC. CT expression was more frequently found in squamous cell carcinoma than in adenocarcinoma. CONCLUSIONS: (1) Cancer vaccines with CT antigens including MAGE-3, NY-ESO-1, LAGE-1, etc, are suitable for immunotherapy of NSCLC after chemotherapy and surgery. (2) NSCLC patients at different stages of disease may be treated with vaccines of different CT antigen composition. (3) CT antigen vaccines are most attractive for patients with late stage NSCLC and/or squamous cell carcinoma of NSCLC.

[Expression of NY-ESO-1/LAGE-1 genes in hepatocellular carcinoma and autologous humoral responses induced thereby].

OBJECTIVE: To investigate the potential of utilizing NY-ESO-1/LAGE-1 antigens in hepatocellular carcinoma (HCC) vaccines. METHODS: RT-PCR method was used to detect the expression of NY-ESO-1/LAGE-1 mRNA in the cancerous tissues and adjacent tissues resected from 34 patients with HCC. ELISA assay was adopted to analyze the NY-ESO-1 specific antibodies in 37 serum samples of HCC patients, 1 positive control sample, and 8 samples of normal persons. RESULTS: Nine (26.5%) out of the 34 HCC samples were NY-ESO-1 mRNA positive, while 12 (35.3%) were LAGE-1 positive. Among them, seven HCC samples expressed both genes, and 14 (41.6%) expressed at least one of the genes. Among the 37 serum samples tested six contained high titer of anti-NY-ESO-1 antibodies. Five of the samples were from stage III or later stage HCC patients; one was from a stage II patient. CONCLUSION: NY-ESO-1/LAGE-1 mRNA is expressed in a high frequency in HCC tissue samples, and induces autologous humoral responses in HCC patients. Both of the antigens can be considered as candidates for HCC vaccines.