[The Mutated Gene Sequenced by NGS and the Correlation of Their Coexistent Mutual Exclusiveness in NPM1 Mutated Acute Myeloid Leukemia].

OBJECTIVE: To analyze the clinicobiological heterogeneity of NPM1 mutated (NPM1mut) acute myeloid leukemia (AML) detected by next generation sequencing (NGS) and their coexistence and mutual exclusivity relationship in the AML subtype. METHODS: The NGS data based on 112 genes related to blood disease in 238 newly diagnosed patients with NPM1mut were collected. The χ2 test and non-parametric test were used to analyze the distribution correlation between the genes in the mutational spectrum. RESULTS: Among all the patients, at least one co-mutation was detected out. The median number per case of the mutated genes, including NPM1mut was 4.5 (range 2－14), among them, there were 5.0 (range 2－10) for NPM1mut/FLT3-ITD+ and 4.0 (range 2－14) for NPM1mut/FLT3-ITD- cases, but it was no significant difference between the two groups (P=0.378). A total of 240 NPM1 mutational events were detected out in entire 238 NPM1mut patients, of which 10 (4.2%) were missense mutations, and were all found in NPM1mut/FLT3-ITD- patients. Most (9/10, 90%) of these NPM1 missense mutations were accompanied by AML subtype-defining cytogenetic or molecular abnormalities, of which 7 patients were in low risk or 2 in high risk. The most common NPM1mut coexisting mutations were DNMT3A (104, 43.7%), followed were FLT3-ITD (95, 39.9%) and FAT1 (57, 23.9%), FLT3-ITD and DNMT3A showed significant coexistence (P=0.005). FLT3-ITD showed significantly reciprocal exclusivity with FLT3-nonITD (P<0.001), NRAS (P<0.001), PTPN11 (P=0.017) and IDH1 (P=0.005), and showed an exclusivity inclination with KRAS (P=0.073). In addition, FLT3-nonITD along with KRAS (P=0.035), NRAS along with KRAS (P=0.008) and PTPN11 (P=0.039) coexisted significantly. CONCLUSION: Prognoses of AML involving less common NPM1 missense mutations should be stated on a case by case basis. The mutational landscape and co-occurrence and mutual exclusivity correlations of NPM1mut AML provide a mechanism explaining biological diversity and clinical heterogeneity in this AML subset.

[Prognostic Significance of the Percentage of Blasts with CD34+/CD38low/-/CD123+ Phenotype in Acute Myeloid Leukemias].

OBJECTIVE: To investigate the percentage of blasts with the CD34+/CD38low/-/CD123+ phenotype in de novo acute myeloid leukemia (AML) patients and analyse its correlation with prognosis. METHODS: The percentage of CD34+/CD38low/-/CD123+ cells in the blast population of 148 newly diagnosed patients with AML was determined by using flow cytometry and its correlation with complete response, disease-free survival and overall survival were evaluated. RESULTS: The median percentage of CD34+/CD38low/-/CD123+ cells in newly diagnosed patients was 2.8% (ranged from 0.01 to 67%). The high expression of CD34+/CD38low/-/CD123+ in AML patients positively correlated with the NPM1 wild-type (χ2=5.194,P<0.05), but did not relate with the positive FLT3-ITD mutations (χ2=0.418,P>0.05). Further multivariable analysis showed that the higher expression of the CD34+/CD38low/-/CD123+ was associated with lower complete remission (P<0.05), worse disease-free survival(P<0.01) and shorter overall survival(P<0.01) in AML patients. CONCLUSION: The percentage of CD34+/CD38low/-/CD123+ cells at diagnosis significantly correlates with the response to treatment and survival. This prognostic marker may be used to rapidly identify the risk of treatment failure in clinical practice.

Decitabine enhances bortezomib treatment in RPMI 8226 multiple myeloma cells.

The present study investigated the interactions between decitabine (DAC) and bortezomib (BTZ) in RPMI 8226 multiple myeloma (MM) cells. Cells were exposed to DAC alone and in combination with BTZ for 48 h. A Cell Counting Kit­8 assay was performed to assess the rate of proliferation inhibition in the cells. Cell apoptosis was investigated by Annexin V-fluorescein isothiocyanate and propidium iodide staining. Flow cytometry was used to detect the different cell cycle stages. Western blotting was performed to analyze the protein expression levels of poly(ADP­ribose) polymerase 1 (PARP­1), caspase­3, ­9 and DNA (cytosine­5­)­methyltransferase 1 (DNMT1). Reverse transcription­quantitative polymerase chain reaction was used to assess DNMT1 gene expression. The combination of DAC and BTZ increased the proliferation inhibition, apoptotic rate and G0­G1 arrest compared with use of a single therapeutic agent. In addition, the combination treatment enhanced PARP­1 cleavage, caspase­3 and caspase­9 activation and downregulated the protein and mRNA expression levels of DNMT1. Therefore, the current study determined that the combination of BTZ and the epigenetic agent DAC may be a novel therapeutic strategy to improve the efficacy of BTZ in patients with MM.

Overexpression of HOXA10 promotes gastric cancer cells proliferation and HOXA10(+)/CD44(+) is potential prognostic biomarker for gastric cancer.

Gastric cancer (GC) is a malignant cancer with poor prognosis. This study aims to investigate the roles of homeobox A10 (HOXA10) in GC and the correlations between HOXA10/CD44 expression and GC prognosis. Based on qRT-PCR and Western Blot analyses in 50 pairs of fresh GC samples and adjacent normal samples, it is identified that HOXA10 was significantly up-regulated in GC tissues at mRNA and protein levels. Cell proliferation, migration, and invasion were enhanced in GC cells with overexpressed HOXA10, while inhibited in cells with silenced HOXA10. Through IPA software, HOXA10 was predicted to interact with CD44 via MSN, which was preliminarily confirmed by using Western Blot. Through immunohistochemistry and tissue microarray (N=264), it is found that HOXA10 expression was significantly correlated with tumor size (P=0.011) and CD44 expression (P<0.001), while CD44 expression was significantly correlated with tumor size (P<0.001), depth of tumor invasion (P<0.001), lymph node metastasis (P<0.001), distant metastasis (P=0.001), UICC stage (P<0.001), histological differentiation (P<0.001), and HOXA10 expression (P<0.001). Additionally, the over-all survival and disease-free survival of HOXA10(+)/CD44(+) patients were dramatically decreased in comparison with that of HOXA10(+)/CD44(-), HOXA10(-)/CD44(+), or HOXA10(-)/CD44(-) patients (P<0.001), suggesting that the combinatory expression of HOXA10 and CD44 was correlated with poor GC prognosis. In conclusion, HOXA10 and CD44 might play roles in GC tumorigenesis, metastasis, and invasion. HOXA10(+)/CD44(+) expression might serve as a prognostic biomarker for GC, which needs more studies to validate.

[MicroRNA-10a expression in FAB different subtype of acute myeloid leukemia and its relationship with drug resistance].

OBJECTIVE: This study was to investigate the expression of miR-10a in the different FAB subtype of acute myeloid leukemia (AML) and its relationship with drug resistance. METHODS: Forty de novo patients with AML, 16 patients with non-malignant hematologic disease and three AML cell lines HL-60, U937 and HL-60/ADR were enrolled in this study, the MiR-10a expression in bone marrow mononuclear cells of above-mentioned patients and 3 AML cell lines was detected by TaqMan RT-PCR. The correlation of miR-10a with clinicopathological factors of AML patients was analyzed. RESULTS: The miR-10a expression level in HL-60 cell line was higher than that in U937 cell line (P = 0.039). And its expression level in de novo AML patients was higher than that in patients with non-malignant hematologic disease (P < 0.01). FAB-AML-M3 patients exhibited higher expression of miR-10a than that in M1, M2 and M4 (P < 0.05); HL-60/ADR cell line showed higher miR-10a expression than that in HL-60 cell line (P < 0.01) . Except M3, the patients without CR (non-CR) after the first cycle of chemotherapy showed a higher level of miR-10a as compared with CR patients (P < 0.01). CONCLUSION: The high expression of miR-10a may be closely related to over-proliferation of promyelocyte and drug resistance of acute myeloid leukemia cells, except M3.

Allogeneic hemopietic stem cell transplants for the treatment of B cell acute lymphocytic leukemia.

OBJECTIVE: Explore the feasibility of allo- hemopietic stem cell transplants in treating patients with B cell acute lymphocytic leukemia. METHODS: Between september 2006 and February 2011, fifteen patients with B cell acute lymphocytic leukemia (ALL) were treated by allo-hemopietic stem cell transplants (HSCT). Stem cell sources were peripheral blood. Six patients were conditioned by busulfan (BU) and cyclophosphamide (CY) and nine patients were conditioned with TBI and cyclophosphamide (CY). Graft versus host disease (GVHD) prophylaxis regimen consisted of cyclosporine A (CSA), methotrex ate (MTX) and mycophenolatemofetil (MMF). RESULTS: Patients received a median of 7.98×108·kg⁻¹ (5.36-12.30×108·kg⁻¹) mononuclear cells (MNC). The median time of ANC>0.5×109/L was day 12 (10-15), and PLT>20.0×109/L was day 13 (11-16). Extensive acute GVHD occurred in 6 (40.0%) patients, and extensive chronic GVHD was recorded in 6 (40.0%) patients. Nine patients were alive after 2.5-65 months follow-up. CONCLUSION: Allogeneic stem cell transplant could be effective in treating patients with B cell acute lymphocytic leukemia.

Long non-coding RNA HOTAIR is a powerful predictor of metastasis and poor prognosis and is associated with epithelial-mesenchymal transition in colon cancer.

Colon cancer is one of the most frequently diagnosed cancer and the third most fatal malignancy worldwide. HOTAIR, a cancer-associated long non-coding RNA (lncRNA), is a powerful biomarker of metastasis and poor prognosis in a diverse group of cancers. Nevertheless, an understanding of how HOTAIR is involved in colon cancer progression is limited. In the present study, we hypothesized that HOTAIR plays a crucial role in colon cancer development. We evaluated the expression of HOTAIR in 120 colon cancer samples, matched adjacent non-tumor mucosa and 32 lymph node metastasis tissues by real-time PCR. Increased HOTAIR expression was significantly correlated with the depth of tumor invasion, lymph node metastasis, organ metastasis, histological differentiation, vascular invasion and advanced tumor stage. Patients with high HOTAIR expression had higher recurrence rates and reduced metastasis-free and overall survival than patients with low HOTAIR expression. Moreover, our findings revealed that HOTAIR had a limited effect on cell proliferation but significantly promoted colon cancer cell migration and invasion in vitro. Depletion of HOTAIR increased the expression of E-cadherin while concomitantly decreasing expression of vimentin and MMP9. Hence, HOTAIR may be another pleiotropic modulator participating in epithelial-mesenchymal transition (EMT). These results indicate that HOTAIR may also be a valuable predictor for colon cancer management; furthermore, this lncRNA may be a potential target for cancer prevention and treatment.

Increased expression of TBX2 is a novel independent prognostic biomarker of a worse outcome in colorectal cancer patients after curative surgery and a potential therapeutic target.

T-box2 (TBX2) plays a critical role in embryonic development. Recently, deregulated expression of TBX2 has been implicated in several malignancies. However, the expression and the role of TBX2 in colorectal cancer (CRC) remain unclear. In this study, we found that TBX2 was obviously up-regulated in CRC in comparison with the corresponding normal mucosa at transcriptional and protein level. Up-expression of TBX2 was significantly associated with depth of tumor invasion (P = 0.006), distant metastasis (P = 0.038), advanced AJCC stage (P = 0.008), and relapse (P = 0.003). TBX2 was a significantly prognostic factor for decreased survival and increased disease recurrence independent of tumor stage(II, III stage) and functioned as a biomarker to identify prognosis of patients with CRC (OS: HR 2.154; 95% CI 1.019-4.551; P = 0.044, DFS: HR 2.253; 95% CI 1.109-4.575; P = 0.025). Furthermore, TBX2 could serve as a potential target of cancer drug therapy.

Identification and validation that up-expression of HOXA13 is a novel independent prognostic marker of a worse outcome in gastric cancer based on immunohistochemistry.

Homeobox (HOX) gene family is known to be classic examples of the intimate relationship between embryogenesis and tumorigenesis. However, less is known about the involvement of HOX gene family with gastric cancerogenesis. Here, we screened the expression of HOX gene family in gastric cancers and explored the relationships between them by cDNA microarray. We found several differentially expressed HOX genes in gastric cancers, especially HOXA10 (11/12) and HOXA13 (11/12) with significantly higher expression in the cancerous tissues. Furthermore, we validated HOXA13 as a novel prognostic marker in gastric cancer based on immunohistochemistry and statistical analysis. HOXA13 expression was significantly up-regulated in cancerous tissues compared with the corresponding non-cancerous mucosa (P < 0.001). Up-expression of HOXA13 was significantly correlated with T stage (P = 0.002), M stage (P = 0.024), advanced UICC stage (P < 0.001), histological differentiation (P = 0.005), and relapse (P = 0.001). Patients with positive HOXA13 expression had a obviously lower overall survival (OS) and disease-free survival (DFS) rate than patients with negative HOXA13 expression (HR 3.331, 95 % CI 1.722-6.442, P < 0.001; HR 3.289, 95 % CI 1.703-6.351, P < 0.001, respectively). Univariate and multivariate Cox analysis confirmed that HOXA13 could serve as a significant independent prognostic factor for DFS and OS. Therefore, our results indicated that several HOX genes might be closely involved in the process of the gastric tumorigenesis. Furthermore, up-expression of HOXA13 might be associated with highly aggressive phenotype of gastric cancer. HOXA13 was a significant independent prognostic factor and could serve as a putative biomarker for diagnosis and prognosis of gastric cancer.

[Influence of 1,25(OH)(2) vitamin D(3) on maturation of human dendritic cells and DC-mediated immune tolerance].

This study was aimed to investigate the effect of 1,25(OH)(2) vitamin D(3) [1,25(OH)(2) Vit D(3)] on the differentiation, maturation and function of human dendritic cells (DC) in vitro and its mechanism. Human peripheral blood mononuclear cells were induced to differentiate to DC in vitro. The DC in test group were cultured with 1,25(OH)(2) Vit D(3) 1 nmol/L for 9 d, while the DC in control group were cultured with the equivalent of absolute alcohol. The expression of co-stimulatory molecules on DC were analyzed by flow cytometry. T cell proliferation induced by DC was assessed by MTT method. The expression of indoleamine 2, 3-dioxygenase (IDO) protein was determined by Western blot. The results showed that compared with the control group, the expression of CD80, CD83 and CD86 on DC in test group was significantly down-regulated (P < 0.05), while the CD1a was up-regulated (P < 0.05). The expression rate of CD80, CD83, CD86, CD1a in test group were (40.43 ± 9.83)%, (20.04 ± 4.73)%, (14.45 ± 5.38)%, (58.48 ± 10.72)% respectively, while in control group were (29.36 ± 13.34)%, (35.91 ± 10.19)%, (27.15 ± 11.64)%, (72.20 ± 12.79)% respectively. Compared with the control group, 1,25(OH)(2) Vit D(3)-treated DC exhibited a markedly reduced ability to stimulate allogenic T cell proliferation and up-regulated IDO protein expression.It is concluded that 1,25(OH)(2) Vit D(3) efficiently inhibits the maturation of DC and DC-mediated T cell proliferation, which may be related to the up-regulation of IDO protein expression.

[Comparison of allelic frequencies of 15 short tandem repeat loci between chronic myeloid leukemia patients and non-related healthy individuals].

OBJECTIVE: To compare allelic frequencies of 15 short tandem repeat (STR) loci (D8S1179, D21S11, D7S820, CSF1PO, D3S1358, TH01, D13S317, D16S539, D2S1338, D19S433, vWA, TPOX, D18S51, D5S818 and FGA) between chronic myeloid leukemia (CML) patients and non-related healthy individuals from Changzhou region in order to predict genes related with the CML. METHODS: Blood samples were collected from 745 healthy subjects and 132 CML patients with complete remission. Genotypes were determined with gene scan technology and multiplex PCR with fluorescence-labeled primers. Allelic polymorphisms of 15 STR loci were compared between the two groups. Potential genes related with CML were predicted with statistical analysis of differences in allelic frequencies. RESULTS: Allelic frequencies of 3 loci, including CSF1PO, vWA and TPOX, showed a significant difference (P<0.05) between the two groups. CONCLUSION: CSF1PO, vWA and TPOX loci may be related with CML, albeit that the exact biologic mechanisms is unclear.

[Effects of simvastatin on PI3K/AKT signaling pathway in human acute monocytic leukemia cell line SHI-1].

To investigate the effects of 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor simvastatin (SV) on proliferation, apoptosis and the PI3K/AKT signaling pathway in human acute monocytic leukemia cell line SHI-1. SHI-1 cells were incubated with different concentrations of SV (5, 10, 15 µmol/L). Otherwise, SHI-1 cells without any treatment were used as control. Cells in different groups were collected at 24, 48 and 72 h after incubation for further detection. MTT method was used to assay the growth inhibition rate and flow cytometry was used to detect the early stage apoptosis ratio. The human PI3K-AKT Signaling Pathway RT(2) Profiler(TM) PCR Array was used to detect the expression of 84 genes involved in PI3K-AKT signaling. The results indicated that the SV inhibited the proliferation and inducted the apoptosis of SHI-1 cells in time- and dose-dependent manners significantly. The growth inhibition rates of SHI-1 cells treated with 15 µmol/L SV for 24, 48 and 72 h were 26.82, 47.09 and 63.92, respectively; and their early stage apoptosis ratios were 5.75, 13.25 and 15.59, respectively. Compared with the control group, expression levels of 39 genes were changed in the group of 15 µmol/L SV at 48 h, among them 26 genes were down-regulated and 13 genes were up-regulated. It is concluded that the SV can inhibit proliferation and induce apoptosis of SHI-1 cells, and the mechanism may be associated with the changes of gene expression level in PI3K-AKT signaling pathway regulated by SV.

Identification of recurrence-related microRNAs in hepatocellular carcinoma following liver transplantation.

Tumor recurrence-related microRNAs (miRNAs) in hepatocellular carcinoma (HCC) following orthotopic liver transplantation (OLT) are not clear yet. This study was designed to determine whether altered miRNA expression is associated with HCC recurrence and prognosis following OLT. 18 miRNAs, including 6 up-regulated and 12 down-regulated miRNAs were identified by microarray in primary HCC samples of patients who had developed HCC recurrence (n = 5) compared to those with non-recurrence (n = 5) following OLT by using p < 0.05 as cutoff value. The six most significantly altered miRNAs (fold change ≥ 2: miR-19a, miR-886-5p, miR-126, miR-223, miR-24 and miR-147) were further confirmed by qRT-PCR in the remaining 105 HCC samples. In receiver-operating characteristic curve analysis, this six miRNAs were of high sensitivity and specificity in predicting HCC recurrence. Using Cox regression and risk score analysis, we built a six-miRNA signature based on their qRT-PCR readings for the prediction of outcome of HCC following OLT. Kaplan-Meier and Cox proportional regression revealed this six-miRNA signature was a significant independent predictor of overall survival (log-rank p = 0.020) and recurrence-free survival (log-rank p < 0.001). Finally, the data were further reconfirmed in an independent cohort of 50 patients from another transplant center. In addition, bioinformatics Gene Ontology and pathway analysis were also performed to better understand the critical roles of these miRNAs in HCC recurrence. Our study, in addition to suggesting a different miRNA expression pattern between HCC samples of patients with recurrence and those with non-recurrence, proposes that this six-miRNA signature may serve as biomarker for prognosis of HCC patients following OLT.

miR-203 expression predicts outcome after liver transplantation for hepatocellular carcinoma in cirrhotic liver.

Many recent studies have shown the utility of microRNAs (miRs) as cancer-related biomarkers. The aim of the present study was to evaluate the correlation between miR-203 expression and prognosis of patients with hepatocellular carcinoma (HCC) and cirrhosis after liver transplantation (LT). Sixty-six HCC samples from patients who had undergone LT were examined for miR-203 expression using quantitative reverse transcription-polymerase chain reaction. The data were correlated with clinicopathological parameters and prognosis. Patient survival was analyzed by the Kaplan-Meier method and log-rank test. Cox regression was used for multivariate analysis of prognostic factors. We found that miR-203 expression was low in tumor tissues of patients (n = 16) with post-LT HCC recurrence in comparison with those in patients with non-recurrence (n = 50) (P = 0.003). Patients with higher miR-203 expression had significantly better recurrence-free survival (RFS) and overall survival (OS) (P = 0.016 for RFS; P = 0.014 for OS). Multivariate analysis revealed that high-miR-203 expression was an independent predictor of good prognosis (HR 0.202, P = 0.006 for RFS; HR 0.332, P = 0.013 for OS). Our results suggest that miR-203 could be a novel prognostic marker in HCC patients who have undergone LT and might also be a potential therapeutic target.

[Effects of simvastatin plus all-trans retinoic acid on WT1/hDMP1 gene expression profiles of human promyelocytic leukemia cell line NB4].

OBJECTIVE: To investigate the effects of simvastatin (SV) plus all-trans retinoic acid (ATRA) on the proliferation, differentiation, apoptosis and WT1/hDMP1 gene expression profiles of human promyelocytic leukemia cell line NB4. METHODS: The NB4 cell was incubated with simvastatin and ATRA alone or in combination. And the NB4 cell without any treatment was adopted as a normal control. The cells of different groups were collected at 24, 48 and 72 h post-incubation. Their morphological changes were observed after Wright staining. The method of MTT was employed to assay the growth inhibition rate and flow cytometry was used to detect the early-stage ratios of apoptosis and cell necrosis. Real-time quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) was used to detect the WT1/hDMP1 gene expression levels. RESULTS: The cell inhibition rates increased gradually (F = 7.15, P = 0.000) at 15, 10 and 5 µmol/L SV respectively. And so did the expression levels of CD11b (F = 3.41, P = 0.014) and Annexin-V (F = 43.38, P = 0.000). However the expression levels of WT1 decreased gradually (F = 5.35, P = 0.001) reversely with the elevated levels of hDMP1 (F = 22.61, P = 0.000). Furthermore the NB4 cell exhibited the most significant changes at 15 µmol/L SV. After a 72-hour incubation, the expression levels of CD11b (89.46% ± 9.13%)and hDMP1 (626.9 ± 56.9) in NB4 cells at 15 µmol/L SV plus 0.5 µmol/L ATRA were significantly higher than those with ATRA(71.27% ± 7.27%, P = 0.000 and 421.8 ± 38.3, P = 0.003 in each) and SV alone(62.41% ± 6.37%, P = 0.003 and 241.4 ± 21.9, P = 0.003 in each). A combination of 15 µmol/L SV with 0.5 µmol/L ATRA displayed obvious interactions with the expressions of CD11b and hDMP1 (F = 4.09, P = 0.025 and F = 29.58, P = 0.000 in each). And there was no significant interaction for cell inhibition rates and Annexin-V expression. CONCLUSION: Simvastatin in vitro inhibits the proliferation of NB4 cell, induces its differentiation and promotes its apoptosis. And the lowered expression of WT1 has a dose-dependent correlation with the elevated expression of hDMP1. It indicates that simvastatin has the synergistic in vitro anti-promyelocytic potency.

Biglycan expression correlates with aggressiveness and poor prognosis of gastric cancer.

Biglycan, a member of the small leucine-rich proteoglycan family, has been implicated in the development and progression of human cancers. However, the clinical significance of biglycan expression in gastric cancer has not been determined. In the present study, biglycan mRNA and protein concentrations were analyzed using quantitative realtime reverse transcription polymerase chain reaction and Western blot in 69 gastric cancer and adjacent non-tumorous tissues, respectively. Biglycan expression was further assessed using immunohistochemistry in tissue microarrays that contained 264 cases of gastric cancer, and others containing normal or metastasized lymph node tumor tissues. Biglycan was upregulated at the transcriptional and translational levels and there was a correlation between the expression of biglycan mRNA and protein (P = 0.000, κ = 0.769). Over-expression of biglycan was strongly associated with lymph node metastasis, tumor (T) classification, metastasis (M) classification, vascular invasion and Union for International Cancer Control (UICC) stage. Patients with biglycan-positive tumors had a significantly higher disease recurrence rate and poorer survival than patients with biglycan-negative tumors after the radical surgery. Multivariate analysis revealed that biglycan expression is an independent prognostic indicator for survival of patients with gastric cancer. The data from the current study demonstrate that elevated expression of biglycan may play an important role in the development and progression of gastric cancer, and could be further evaluated as a biomarker for predication of a poor clinical outcome.

Identification and validation of Kallikrein-ralated peptidase 11 as a novel prognostic marker of gastric cancer based on immunohistochemistry.

BACKGROUND AND OBJECTIVES: It is important to identify and validate the differentially expressed genes in gastric cancer to screen diagnostic and/or prognostic tumor markers. METHODS: cDNA expression microarray, gene set enrichment analysis, and bioinformatics approaches were used to screen the differentially expressed genes between gastric cancer tissues and adjacent non-cancerous mucosa. A novel candidate prognostic marker, Kallikrein-related peptidase 11 (KLK11), was validated in 400 Chinese gastric cancer patients. KLK11 expression in gastric cancer tissues was detected using real-time PCR and Western blot. KLK11 protein expression was further analyzed by immunostaining on tissue microarray, followed with clinicopathological significance and survival analysis. RESULTS: KLK11 expression was significantly decreased in gastric cancer compared with that in normal gastric mucosa (P<0.001). Furthermore, KLK11 expression was much lower in poorly differentiated cancer samples than that in well-differentiated group (P<0.01). Survival analysis showed that negative KLK11 expression was associated with nearly fivefold increased risk of distant metastasis after curative gastrectomy (HR 4.65, P<0.01). Multivariate Cox regression analysis showed that KLK11 expression emerged as a significant independent prognostic factor for disease-free survival and overall survival (P<0.05). CONCLUSIONS: The results indicated that KLK11 expression was decreased in gastric cancer and might serve as a novel independent prognostic marker.

Mutation analysis of tumor suppressor gene PTEN in patients with gastric carcinomas and its impact on PI3K/AKT pathway.

The aim of this study was to clarify the participation of PTEN mutation in gastric carcinogenesis and its impact on PI3K/AKT pathway. All nine exons of PTEN were screened for mutations by direct sequencing in 144 patients with pathologically proven gastric carcinoma and their corresponding normal mucosae, followed by Western blotting to detect the changes in PI3K/AKT pathway. Direct sequencing indicated there were 27 cases with mutations among 144 patients consisting of 15 cases (55.6%) of missense mutation, nine nonsense mutations (33.3%), two 1-bp deletion (7.4%), and a mutation within intron 6 (3.7%). The mutation hot spots at codons 36, 75, 232 and 393 had not been observed previously, and the mutation sites in exons 3, 5, 6 and 8 were not found, suggesting that there might be some unique characteristic of PTEN inactivation mechanism in the Shanghai population. The PTEN mutation rate was significantly higher at pTMN stages III and IV than that at stages I and II (P<0.005), and it was higher in poorly differentiated gastric cancer than in well or moderately differentiated types (P<0.05). PTEN and E-cadherin protein expression in gastric cancer was significantly down-regulated comparing with that in paracancerous tissues, while the PI3K, AKT, MMP-2, MMP-9 and NF-kappaBp65 protein were overexpressed in cancer tissues. Our results implicated that the mutations of PTEN did not occur at a significant rate in gastric carcinoma in Shanghai, but might play a role in tumorigenesis. The mutation status of PTEN was significantly relevant to pTNM staging and degree of cell differentiation, hinting that PTEN might be a prognostic biomarker of gastric cancer. The decreased expression of PTEN and E-cadherin, together with the overexpression of PI3K, AKT, MMP-2, MMP-9 and NF-kappaBp65, contributed cooperatively to the accelerated progress of gastric cancer.

[Application of 1D wavelet analysis in detecting species of harmful algae blooms with absorption spectra of phytoplankton].

In the present study, the feasibility of using wavelet analysis to extract the eigen spectra from the absorption spectra of phytoplankton for species detection was investigated. Thirteen absorption spectra taken from single species cultures, representing four divisions (Dinophyta, Bacillariophyta, Haptophyta, and Chlorophyta), six genus (Gymnodinium, Prorocentrum, Skeletonema, Guinardia, Phaeocystis, and Prasinophyte) and seven species (Karenia mikimotoi, Karenia brevis, Prorocentrum minimum, Skeletonema costatuma, Guinardia delicatula, Phaeocystis globosa, and Pyramimonas parkeae), were used. First, the 1D wavelet analysis with five levels was applied to the thirteen absorption spectra, so each spectrum was decomposed with 5 levels. The 5th level component of low frequency corresponds to the background without information for species detection, and 1st and 2nd level component of high frequency is the random noise. The other levels (3rd to 5th) of high frequency are the useful information, and the sum of levels (3rd to 5th) of high frequency was retained as the eigen spectra for species detection. Second, the clustering analysis was used to the eigen spectra for examining the performance of the wavelet analysis in extracting species information. The clustering results were compared with the known species class information, and the results show that the 13 absorption spectra are correctly classified at the level of division, genus and species. This means that the wavelet analysis has good performance in extracting the eigen spectra for species detection. However, the above results were obtained with only limited species, and the more species data are required to identify the extensive validity of the conclusion.

[Gene KIR in match with HLA-Cw impacts on NK cell cytotoxicity].

The aim of this study was to investigate how the killer immune globulin-like inhibition receptor (KIR) in match with HLA-Cw impacts on NK cell activity. Mononuclear cells were isolated in 20 ml peripheral blood from 27 healthy persons by Ficoll-Hypaque and purified by NK cell isolation kit. Target cells were mononuclear cells isolated from bone marrow of 30 de novo AML patients. The KIR expression were detected by flow cytometry with antibodies against CD158a, CD158b. The 2 ml of peripheral blood from healthy persons and AML patients were collected, the DNA was extracted by using PROTRANS method, the HLA-Cw and KIR gene were detected by PCR-SSP typing with sequence specific primers. The NK cell cytotoxicity against AML cells was determined by MTT after combination of KIR with HLA-Cw gene. The results indicated that the purity of NK cells was (90.8 +/- 6.08)%. The less the KIR/HLA-Cw matched, the more activity was shown in NK cells. When no match of NK cell/target cell (KIR/HLA-Cw) there was, the cytotoxicity was (50.66 +/- 8.40)%, 1 or 2 matches showed cytotoxicity of (38.28 +/- 6.71)% and (19.74 +/- 4.15)% (p < 0.001). Expression level of KIRs on NK cells also was related with cytotoxicity level (p < 0.001). It is concluded that the interaction between inhibitory KIR and HLA ligands, and also expression level of KIRs on NK cells both impact significantly on NK cell function, that the less match of KIR/HLA-Cw, and the less expression of KIRs on NK cells result in the stronger NK cell cytotoxicity.