Jingfang Gao

Legumain Expression in Relation to Clinicopathologic and Biological Variables in Colorectal Cancer

Purpose: Legumain, a novel asparaginyl endopeptidase, has been observed to be highly expressed in several types of tumors including colorectal cancer. However, there is no study examining the relationship of legumain expression to clinocopathologic and biological variables in colorectal cancers. Experimental Design: We investigated legumain expression in 164 primary colorectal cancers, 34 corresponding distant normal mucosa samples, 89 adjacent normal mucosa samples, and 33 lymph node metastases using immunohistochemistry. We also did Western blotting analysis on three additional colorectal cancers and three colonic cell lines. Results: Legumain expression was increased in primary tumors compared with distant or adjacent normal mucosa (P < 0.05), but there was no significant change between primary tumors and metastases (P > 0.05). Legumain expression was positively related to poorer differentiation/mucinous carcinoma (P = 0.04), higher degree of necrosis (P = 0.03) and apoptosis (P < 0.0001), positive proliferating cell nuclear antigen (P < 0.0001) and p53 expression (P = 0.049), and had a positive tendency towards stromelysin 3 (P = 0.058) and PINCH positivity (P = 0.05). The patients with tumors that showed both weak and lower percentage of the legumain expression, either in tumor (P = 0.01) or in stroma (P = 0.04), had a better prognosis. Conclusions: The legumain expression may be involved in colorectal cancer development and have a prognostic value in the patients.

Polymorphism in the promoter region of the NFKB1 gene increases the risk of sporadic colorectal cancer in Swedish but not in Chinese populations

Objective. An insertion/deletion polymorphism (−94ins/delATTG) in the promoter region of the NFKB1 gene correlates to an increased risk of ulcerative colitis, a known risk factor for colorectal cancer, but this polymorphism has not been studied in colorectal cancer patients. The purpose of this study was to investigate whether this polymorphism is related to colorectal cancer risk and clinicopathological variables. Material and methods. Case samples were taken from four groups of Swedish patients: 193 unselected patients, 90 patients with ≥3 affected 1st-degree relatives, 85 patients with 2 affected 1st-degree relatives, and 109 sporadic cancer patients, and one group of 193 unselected Chinese patients. Controls included 439 Swedish and 458 Chinese healthy individuals. Genotypes were determined by polymerase chain reaction (PCR)-restriction fragment length polymorphism. Results. The deletion increased the risk of colorectal cancer among Swedish unselected patients (OR=3.81, 95% CI: 2.17–6.69, p<0.0001 for heterozygote deletion, and OR=4.65, 95% CI: 2.43–8.89, p<0.0001 for homozygote deletion) and sporadic cancer patients (OR=7.73, 95% CI: 3.06–19.57, p<0.0001 for heterozygote deletion, and OR=6.58, 95% CI: 2.35–18.43, p<0.0001 for homozygote deletion) compared to homozygote insertion (wild-type), but not among the other Swedish or Chinese patients (p>0.05). Similar evidence was seen in age-adjusted analyses (p<0.0001). The polymorphism did not correlate to clinicopathological variables (p>0.05). Conclusions. Deletion of the polymorphism was associated with increased susceptibility to sporadic colorectal cancers in the Swedish population, but not in the Swedish patients with a family history of colorectal cancer or in Chinese patients.

Association of NFKBIA polymorphism with colorectal cancer risk and prognosis in Swedish and Chinese populations

Objective. The inhibitory proteins, IκBs, regulate the activity of nuclear factor kappa-beta (NF-κB), which is implicated in tumorigenesis by regulating expression of a variety of genes involved in cellular transformation, proliferation, invasion, angiogenesis and metastasis. Variants in the genes encoding IκBs may be involved in cancer development through the activation of NF-κB. The objective of this study was to investigate the susceptibility of an A to G variation (rs696) in the 3′ UTR of NFKBIA (encoding IκBα) to colorectal cancer (CRC) and the association of this polymorphism with clinicopathologic variables in CRC patients. Material and methods. A case-control study was carried out on a Swedish (155 CRCs, 438 controls) and a Chinese population (199 CRCs, 577 controls). The genotype of NFKBIA was determined by PCR-restriction fragment length polymorphism. Results. The frequency of the AG genotype was increased in the Chinese patients ≥50 years of age compared with the Chinese controls (odds ratio (OR) = 3.06, 95% confidence interval (CI) = 1.55–6.02, p=0.001), even when adjusted for age (OR = 3.20, 95% CI = 1.61–6.38, p=0.001). The GG genotype of NFKBIA was related to a poorer survival rate in the Swedish patients, independent of gender, age, tumour location, Dukes’ stage and differentiation (hazard ratio = 3.10, 95% Cl = 1.28–7.60, p=0.01). Conclusions. Chinese individuals ≥50 years of age carrying the AG genotype of NFKBIA may be at an increased risk of developing CRC, and the GG genotype of NFKBIA may be considered as a prognostic factor for Swedish CRC patients.

NF-κB p65 phosphorylated at Serine-536 is an independent prognostic factor in Swedish colorectal cancer patients

IntroductionThe NF-κB transcription factor protein family has diverse cellular and biological functions, and posttranslational modification is important to regulate these functions. An important site of phosphorylation of NF-κB p65 subunit is at serine-536 (phospho-Ser536-p65), and this phosphorylation is involved in regulation of transcriptional activity, nuclear localization, and protein stability.Patients and MethodsIn this study, we investigated expression of phospho-Ser536-p65 in colorectal cancers and its relationships with clinicopathological factors. The expression of phospho-Ser536-p65 was examined by immunohistochemistry in 203 primary colorectal cancers, 156 normal mucosa specimens, and 18 metastases in the lymph nodes.ResultsThe expression of phospho-Ser536-p65 increased from normal mucosa to primary tumor (p < 0.0001). Further, the increased expression of phospho-Ser536-p65 in the cytoplasm of the primary tumors correlated with worse survival of the patients independently of gender, age, tumor location, stage, and differentiation (p = 0.04; hazard ratio, 1.89; 95% CI 1.03–3.47).ConclusionThe NF-κB p65 subunit phosphorylated at serine-536 is an independent prognostic factor in colorectal cancer patients.

PINCH is an independent prognostic factor in rectal cancer patients without preoperative radiotherapy - a study in a Swedish rectal cancer trial of preoperative radiotherapy

BackgroundThe clinical significance between particularly interesting new cysteine-histidine rich protein (PINCH) expression and radiotherapy (RT) in tumours is not known. In this study, the expression of PINCH and its relationship to RT, clinical, pathological and biological factors were studied in rectal cancer patients.MethodsPINCH expression determined by immunohistochemistry was analysed at the invasive margin and inner tumour area in 137 primary rectal adenocarcinomas (72 cases without RT and 65 cases with RT). PINCH expression in colon fibroblast cell line (CCD-18 Co) was determined by western blot.ResultsIn patients without RT, strong PINCH expression at the invasive margin of primary tumours was related to worse survival, compared to patients with weak expression, independent of TNM stage and differentiation (P = 0.03). No survival relationship in patients with RT was observed (P = 0.64). Comparing the non-RT with RT subgroup, there was no difference in PINCH expression in primary tumours (invasive margin (P = 0.68)/inner tumour area (P = 0.49). In patients with RT, strong PINCH expression was related to a higher grade of LVD (lymphatic vessel density) (P = 0.01)ConclusionsPINCH expression at the invasive margin was an independent prognostic factor in patients without RT. RT does not seem to directly affect the PINCH expression.

RAD50/MRE11/NBS1 proteins in relation to tumour development and prognosis in patients with microsatellite stable colorectal cancer

RAD50/MRE11/NBS1 complex is essential for DNA double-strand break repair and for maintaining genomic integrity. In this study, we immunohistochemically examined MRE11, NBS1 and RAD50 expression in primary CRCs (n=208), the corresponding distant (n=41) and adjacent normal mucosa (n=130), and lymph node metastases (n=26), and investigated their clinicopathological significance in colorectal cancers (CRCs). We found that the intensity and percentage of MRE11 and NBS1 in primary CRCs were positively correlated with each other and with RAD50 (P<0.0001). Strong expression of MRE11, NBS1 or combined RAD50/MRE11/NBS1 was related to MSS, positive hMLH1 expression, earlier tumour stage (TNM stage I and II) and favourable survival (P<0.05). A high percentage of MRE11 expression was associated with less local recurrence and high apoptotic activity (P<0.05). In MSS CRCs, the expression of MRE11 and NBS1 was stronger than that in normal mucosa (P<0.05), and strong expression of NBS1 in primary tumour was related to favourable survival of patients in TNM stage I and II (univariate analysis: P=0.03; multivariate analysis: P=0.07). In MSI CRCs, neither MRE11 nor NBS1 expression showed differences among normal mucosa, primary tumour and metastasis, or among clinicopathological variables. In conclusion, RAD50/MRE11/NBS1 proteins interacted with each other, which had different clinicopathological significance in MSS and MSI CRCs, and further, each component of the complex might have additional roles. NBS1 might be a prognostic factor for patients with MSS tumour in TNM stage I and II.

The location of lymphangiogenesis is an independent prognostic factor in rectal cancers with or without preoperative radiotherapy

BACKGROUND Lymphangiogenesis and angiogenesis are essential for tumour development and progression. The lymphatic vessel density (LVD) and blood vessel density (BVD) and their relationship to outcome have been studied extensively, however the clinical significance of the location of LVD/BVD in tumour is not known. In the present study, the location and degree of LVD/BVD and their relationship to preoperative radiotherapy (RT), clinicopathological, histopathological and biological factors were studied in rectal cancer patients participating in a Swedish clinical trial of preoperative RT. PATIENTS AND METHODS The location and degree of LVD/BVD were analysed in primary tumours (n = 138/140) and in their subgroups of non-RT (n = 74) and RT (n = 64/66). Further, the degree of LVD/BVD was examined in the corresponding distant normal mucosa (n = 35/31) and adjacent normal mucosa (n = 72/91). All sections were immunohistochemically examined by using D2-40 and CD34 antibodies. RESULTS In the whole series of the patients, a higher LVD at the periphery was related to negative p53 expression (P = 0.03) and favourable survival independent of tumour-node-metastasis stage, differentiation and p53 expression (P = 0.03). LVD was increased in p53-negative tumours after RT (P = 0.01). CONCLUSION LVD at the periphery of the tumour was an independent prognostic factor in rectal cancer patients.

The Different Roles of hRAD50 in Microsatellite Stable and Unstable Colorectal Cancers

RAD50 protein is essential for DNA double-strand break repair and maintaining genomic integrity. In this study, we investigated the clinicopathological significance of hRAD50 expression and mutation in microsatellite stable (MSS) and unstable (MSI) colorectal cancers (CRCs). hRAD50 expression was examined in primary CRC (n=268), the corresponding distant (n=69) and adjacent normal mucosa (n=138), and lymph node metastasis (n=44) by immunohistochemistry. hRAD50 mutation was analyzed in 87 primary CRCs by PCR-SSCP-DNA sequencing. hRAD50 expression was increased in MSS primary CRCs, but not MSI ones, compared with distant/adjacent normal mucosa (p<0.05). There was no difference in the hRAD50 expression between primary and metastatic CRCs. The increased hRAD50 expression in MSS primary CRCs was related (p<0.05) or tended to be related (p=0.05) to early tumor stage, better differentiation, high inflammatory infiltration, p53 overexpression. Frameshift mutations of (A)9 at coding region of hRAD50 were only found in MSI CRCs. Our results suggest that hRAD50 may play different roles in the development of MSS and MSI CRCs: increased hRAD50 expression in MSS CRCs may be a cellular response against tumor from further progression, while hRAD50 mutation may be involved in the development of MSI CRCs.

Abstract 740: PINCH is an independent prognostic factor in rectal cancer patients without preoperative radiotherapy: A study in a Swedish rectal cancer trial of preoperative radiotherapy

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Background: Particularly interesting new cysteine-histidine rich protein (PINCH) connected to integrins of the cell surface, is involved in cell spreading, motility and proliferation. The expression of PINCH and its relationship to outcome have been studied, however, the clinical significance between PINCH and preoperative radiotherapy (RT) in tumors is unknown. In this study, PINCH expression and its relationship to RT, clinical, histological and biological factors were investigated in rectal cancer patients, and in colon cancer and fibroblast cell lines. Material and Methods: PINCH expression was immunohistochemically examined at the invasive margin and inner tumour area in 137 primary rectal adenocarcinomas (72 cases without RT and 65 cases with RT). PINCH expression in colon cancer (KM12C) and fibroblast (CCD18 Co) cell lines was examined by real time PCR. The KM12C and CCD 18 Co cell lines, cultured separately and co-cultured, were analyzed over time at 8h, 24h, 48h and 72h after radiation. Results: In patients without RT, strong PINCH expression at the invasive margin of tumors was related to worse survival, compared to patients with weak expression, independent of both TNM stage and differentiation (p = 0.03). No survival relationship in patients with RT was observed (p = 0.64). Real time PCR analyzes showed that KM12C irradiated cells tended to have a higher expression of PINCH at 24h compared to cells without radiation, while in co-cultured KM12C cells, PINCH tended to increase at 8h. PINCH seemed to increase at 8h in CCD 18 Co cells with radiation compared to cells without radiation, while in co-cultured CCD 18 Co cells, PINCH seemed to be lower at 8h in radiated cells than in non-radiated cells. Conclusions: PINCH expression at the invasive margin was an independent prognostic factor in patients without RT. RT might up-regulate the expression of PINCH, which may help us to select the best-suited patients for RT in the future. The levels of PINCH differ when separately cultured and co-cultured cells were compared, which might indicate that PINCH is involved in the crosstalk between tumor cells and their surrounding fibroblasts. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 740. doi:1538-7445.AM2012-740

MANBA polymorphism was related to increased risk of colorectal cancer in Swedish but not in Chinese populations

β-mannosidase, encoded by MANBA, has been suggested to be implicated in cancers, while genetic variations in the MANBA in relation to colorectal cancer (CRC) risk has not been examined. In this study, we investigated the relationship of a polymorphic CA repeat in MANBA gene with CRC risk in 152 Swedish CRC patients and 441 Swedish controls, and 196 Chinese CRC patients and 577 Chinese controls, as well as the clinicopathologic significance of this polymorphism on CRC patients, by using capillary electrophoresis. The MANBA genotypes were related to CRC risk in the Swedish population (p=0.03), but not in the Chinese population. In the Swedish population, individuals with < 22 CAs/< 22 CAs had significantly increased risk for CRC compared with those with ≥22 CAs/≥ 22 CAs (gender-age-adjusted analysis: OR 1.93, 95% CI 1.06–3.51). There was no relationship between the polymorphism and clinicopathologic variables. These findings suggest the different susceptibilities of this polymorphism to CRC development in the two populations.