Molecular targets for diagnostic and intraoperative imaging of pancreatic ductal adenocarcinoma after neoadjuvant FOLFIRINOX treatment.

Neoadjuvant systemic treatment is increasingly being integrated in the standard treatment of pancreatic ductal adenocarcinoma (PDAC) patients to improve oncological outcomes. Current available imaging techniques remain unreliable in assessing response to therapies, as they cannot distinguish between (vital) tumor tissue and therapy induced fibrosis (TIF). Consequently, resections with tumor positive margins and subsequent early post-operative recurrences occur and patients eligible for potential radical resection could be missed. To optimize patient selection and monitor results of neoadjuvant treatment, PDAC-specific diagnostic and intraoperative molecular imaging methods are required. This study aims to evaluate molecular imaging targets for PDAC after neoadjuvant FOLFIRINOX treatment. Expression of integrin αvß6, carcinoembryonic antigen cell adhesion molecule 5 (CEACAM5), mesothelin, prostate-specific membrane antigen (PSMA), urokinase-type plasminogen activator receptor, fibroblast activating receptor, integrin α5 subunit and epidermal growth factor receptor was evaluated using immunohistochemistry. Immunoreactivity was determined using the semiquantitative H-score. Resection specimens from patients after neoadjuvant FOLFIRINOX treatment containing PDAC (n = 32), tumor associated pancreatitis (TAP) and TIF (n = 15), normal pancreas parenchyma (NPP) (n = 32) and tumor positive (n = 24) and negative (n = 56) lymph nodes were included. Integrin αvß6, CEACAM5, mesothelin and PSMA stainings showed significantly higher expression in PDAC compared to TAP and NPP. No expression of αvß6, CEACAM5 and mesothelin was observed in TIF. Integrin αvß6 and CEACAM5 allow for accurate metastatic lymph node detection. Targeting integrin αvß6, CEA, mesothelin and PSMA has the potential to distinguish vital PDAC from fibrotic tissue after neoadjuvant FOLFIRINOX treatment. Integrin αvß6 and CEACAM5 detect primary tumors and tumor positive lymph nodes.

Molecular imaging of the urokinase plasminogen activator receptor: opportunities beyond cancer.

The urokinase plasminogen activator receptor (uPAR) plays a multifaceted role in almost any process where migration of cells and tissue-remodeling is involved such as inflammation, but also in diseases as arthritis and cancer. Normally, uPAR is absent in healthy tissues. By its carefully orchestrated interaction with the protease urokinase plasminogen activator and its inhibitor (plasminogen activator inhibitor-1), uPAR localizes a cascade of proteolytic activities, enabling (patho)physiologic cell migration. Moreover, via the interaction with a broad range of cell membrane proteins, like vitronectin and various integrins, uPAR plays a significant, but not yet completely understood, role in differentiation and proliferation of cells, affecting also disease progression. The implications of these processes, either for diagnostics or therapeutics, have received much attention in oncology, but only limited beyond. Nonetheless, the role of uPAR in different diseases provides ample opportunity to exploit new applications for targeting. Especially in the fields of oncology, cardiology, rheumatology, neurology, and infectious diseases, uPAR-targeted molecular imaging could offer insights for new directions in diagnosis, surveillance, or treatment options.

Identifying Biomarkers in Lymph Node Metastases of Esophageal Adenocarcinoma for Tumor-Targeted Imaging.

INTRODUCTION: Tumor-targeted imaging is a promising technique for the detection of lymph node metastases (LNM) and primary tumors. It remains unclear which biomarker is the most suitable target to distinguish malignant from healthy tissue in esophageal adenocarcinoma (EAC). OBJECTIVE: We performed an immunohistochemistry study to identify viable tumor markers for tumor-targeted imaging of EAC. METHODS: We used samples from 72 patients with EAC to determine the immunohistochemical expression of ten potential tumor biomarkers for EAC (carbonic anhydrase IX [CA-IX], carcinoembryonic antigen [CEA], hepatic growth factor receptor, epidermal growth factor receptor, epithelial membrane antigen [EMA], epithelial cell adhesion molecule [EpCAM], human epidermal growth factor receptor 2 [HER-2], urokinase plasminogen activator receptor, vascular endothelial growth factor-A [VEGF-A], and VEGF receptor 2). Immunohistochemistry was performed on tissue microarrays of LNM (n = 48), primary EACs (n = 62), fibrotic tissues (n = 11), nonmalignant lymph nodes (n = 24), and normal esophageal and gastric tissues (n = 40). Tumor marker staining was scored on intensity and percentage of positive cells. RESULTS: EMA and EpCAM showed strong expression in LNM (> 95%) and primary EACs (> 95%). Significant expression was also observed for LNM and EAC using VEGF-A (85 and 92%), CEA (68 and 54%), and CA-IX (4 and 34%). The other tumor biomarkers showed expression of 0-15% for LNM and primary EAC. Except for VEGF-A, nonmalignant lymph node staining was scored as slight or absent. CONCLUSIONS: High expression rates and correlation between LNM in EAC combined with low expression rates in healthy lymph nodes and esophagus tissues were observed for EpCAM and CEA, meaning these are promising targets for tumor-targeted imaging approaches for lymph nodes in patients with EAC.

Potential targets for tumor-specific imaging of vulvar squamous cell carcinoma: A systematic review of candidate biomarkers.

INTRODUCTION: Vulvar squamous cell carcinoma (VSCC) is a rare malignancy with an increasing incidence, especially in young women. Surgical treatment of VSCC is associated with significant morbidity and high recurrence rates, which is related to the limited ability to distinguish (pre)malignant from healthy tissue. There is a need for new tools for specific real-time detection of occult tumor lesions and localization of cancer margins in patients with VSCC. Several tumor-specific imaging techniques are developed to recognize malignant tissue by targeting tumor markers. We present a systematic review to identify, evaluate, and summarize potential markers for tumor-specific imaging of VSCC. METHODS: Relevant papers were identified by a systematic cross-database literature search developed with assistance of an experienced librarian. Data were extracted from eligible papers and reported based on the Preferred Reporting Items for Systematic reviews and Meta-Analyses (PRISMA) guidelines. VSCC-specific tumor markers were valued based on a weighted scoring system, in which each biomarker was granted points based on ranked eligibility criteria: I) percentage expression, II) sample size, and III) in vivo application. RESULTS: In total 627 papers were included of which 22 articles met the eligibility criteria. Twelve VSCC-specific tumor markers were identified and of these 7 biomarkers were considered most promising: EGFR, CD44v6, GLUT1, MRP1, MUC1, CXCR-4 and VEGF-A. DISCUSSION: This overview identified 7 potential biomarkers that can be used in the development of VSCC-specific tracers for real-time and precise localization of tumor tissue before, during, and after treatment. These biomarkers were identified in a small number of samples, without discriminating for VSCC-specific hallmarks such as HPV-status. Before clinical development, experimental studies should first aim at validation of these biomarkers using immunohistochemistry and cell line-based examination, discriminating for HPV-status and the expression rate in lymph nodes and precursor lesions.

Correlation Between Preoperative Serum Carcinoembryonic Antigen Levels and Expression on Pancreatic and Rectal Cancer Tissue.

Carcinoembryonic antigen (CEA)-targeted imaging and therapeutic agents are being tested in clinical trials. If CEA overexpression in malignant tissue corresponds with elevated serum CEA, serum CEA could assist in selecting patients who may benefit from CEA-targeted agents. This study aims to assess the relationship between serum CEA and CEA expression in pancreatic (n = 20) and rectal cancer tissues (n = 35) using histopathology. According to local laboratory standards, a serum CEA >3 ng/mL was considered elevated. In pancreatic cancer patients a significant correlation between serum CEA and percentage of CEA-expressing tumor cells was observed (P = .04, ρ = .47). All 6 patients with homogeneous CEA expression in the tumor had a serum CEA >3 ng/mL. Most rectal cancer tissues (32/35) showed homogeneous CEA expression, independent of serum CEA levels. This study suggests that selection of pancreatic cancer patients for CEA-targeted agents via serum CEA appears adequate. For selection of rectal cancer patients, serum CEA levels are not informative.

Preclinical uPAR-targeted multimodal imaging of locoregional oral cancer.

OBJECTIVES: Establishing adequate resection margins and lymphatic mapping are crucial for the prognosis of oral cancer patients. Novel targeted imaging modalities are needed, enabling pre- and intraoperative detection of tumour cells, in combination with improved post-surgical examination by the pathologist. The urokinase-receptor (uPAR) is overexpressed in head and neck cancer, where it is associated with tumour progression and metastasis. MATERIAL AND METHODS: To determine suitability of uPAR for molecular imaging of oral cancer surgery, human head and neck tumours were sectioned and stained for uPAR to evaluate the expression pattern compared to normal mucosa. Furthermore, metastatic oral squamous carcinoma cell line OSC-19 was used for targeting uPAR in in vivo mouse models. Using anti-uPAR antibody ATN-658, equipped with a multimodal label, the in vivo specificity was investigated and the optimal dose and time-window were evaluated. RESULTS: All human oral cancer tissues expressed uPAR in epithelial and stromal cells. Hybrid ATN-658 clearly visualized tongue tumours in mice using either NIRF or SPECT imaging. Mean fluorescent TBRs over time were 4.3±0.7 with the specific tracer versus 1.7±0.1 with a control antibody. A significant difference in TBRs could be seen between 1nmol (150µg) and 0.34nmol (50µg) dose groups (n=4, p<0.05). Co-expression between BLI, GFP and the NIR fluorescent signals were seen in the tongue tumour, whereas human cytokeratin staining confirmed presence of malignant cells in the positive cervical lymph nodes. CONCLUSION: This study shows the applicability of an uPAR specific multimodal tracer in an oral cancer model, combining SPECT with intraoperative guidance.

EpCAM as multi-tumour target for near-infrared fluorescence guided surgery.

BACKGROUND: Evaluation of resection margins during cancer surgery can be challenging, often resulting in incomplete tumour removal. Fluorescence-guided surgery (FGS) aims to aid the surgeon to visualize tumours and resection margins during surgery. FGS relies on a clinically applicable imaging system in combination with a specific tumour-targeting contrast agent. In this study EpCAM (epithelial cell adhesion molecule) is evaluated as target for FGS in combination with the novel Artemis imaging system. METHODS: The NIR fluorophore IRDye800CW was conjugated to the well-established EpCAM specific monoclonal antibody 323/A3 and an isotype IgG1 as control. The anti-EpCAM/800CW conjugate was stable in serum and showed preserved binding capacity as evaluated on EpCAM positive and negative cell lines, using flow cytometry and cell-based plate assays. Four clinically relevant orthotopic tumour models, i.e. colorectal cancer, breast cancer, head and neck cancer, and peritonitis carcinomatosa, were used to evaluate the performance of the anti-EpCAM agent with the clinically validated Artemis imaging system. The Pearl Impulse small animal imaging system was used as reference. The specificity of the NIRF signal was confirmed using bioluminescence imaging and green-fluorescent protein. RESULTS: All tumour types could clearly be delineated and resected 72 h after injection of the imaging agent. Using NIRF imaging millimetre sized tumour nodules were detected that were invisible for the naked eye. Fluorescence microscopy demonstrated the distribution and tumour specificity of the anti-EpCAM agent. CONCLUSIONS: This study shows the potential of an EpCAM specific NIR-fluorescent agent in combination with a clinically validated intraoperative imaging system to visualize various tumours during surgery.

Endoglin targeting inhibits tumor angiogenesis and metastatic spread in breast cancer.

Endoglin, a transforming growth factor-ß co-receptor, is highly expressed on angiogenic endothelial cells in solid tumors. Therefore, targeting endoglin is currently being explored in clinical trials for anti-angiogenic therapy. In this project, the redundancy between endoglin and vascular endothelial growth factor (VEGF) signaling in angiogenesis and the effects of targeting both pathways on breast cancer metastasis were explored. In patient samples, increased endoglin signaling after VEGF inhibition was observed. In vitro TRC105, an endoglin-neutralizing antibody, increased VEGF signaling in endothelial cells. Moreover, combined targeting of the endoglin and VEGF pathway, with the VEGF receptor kinase inhibitor SU5416, increased antiangiogenic effects in vitro and in a zebrafish angiogenesis model. Next, in a mouse model for invasive lobular breast cancer, the effects of TRC105 and SU5416 on tumor growth and metastasis were explored. Although TRC105 and SU5416 decreased tumor vascular density, tumor volume was unaffected. Strikingly, in mice treated with TRC105, or TRC105 and SU5416 combined, a strong inhibition in the number of metastases was seen. Moreover, upon resection of the primary tumor, strong inhibition of metastatic spread by TRC105 was observed in an adjuvant setting. To confirm these data, we assessed the effects of endoglin-Fc (an endoglin ligand trap) on metastasis formation. Similar to treatment with TRC105 in the resection model, endoglin-Fc-expressing tumors showed strong inhibition of distant metastases. These results show, for the first time, that targeting endoglin, either with neutralizing antibodies or a ligand trap, strongly inhibits metastatic spread of breast cancer in vivo.

Clinical prognostic value of combined analysis of Aldh1, Survivin, and EpCAM expression in colorectal cancer.

BACKGROUND: Tumour aggressiveness might be related to the degree of main cancer hallmark acquirement of tumour cells, reflected by expression levels of specific biomarkers. We investigated the expression of Aldh1, Survivin, and EpCAM, together reflecting main cancer hallmarks, in relation to clinical outcome of colorectal cancer (CRC) patients. METHODS: Immunohistochemistry was performed using a tumour tissue microarray of TNM (Tumour, Node, Metastasis)-stage I-IV CRC tissues. Single-marker expression or their combination was assessed for associations with the clinical outcome of CRC patients (N=309). RESULTS: Increased expression of Aldh1 or Survivin, or decreased expression of EpCAM was each associated with poor clinical outcome, and was therefore identified as clinically unfavourable expression. Analyses of the combination of all three markers showed worse clinical outcome, specifically in colon cancer patients, with an increasing number of markers showing unfavourable expression. Hazard ratios ranged up to 8.3 for overall survival (P<0.001), 36.6 for disease-specific survival (P<0.001), and 27.1 for distant recurrence-free survival (P<0.001). CONCLUSIONS: Our data identified combined expression levels of Aldh1, Survivin, and EpCAM as strong independent prognostic factors, with high hazard ratios, for survival and tumour recurrence in colon cancer patients, and therefore reflect tumour aggressiveness.

Interaction with colon cancer cells hyperactivates TGF-ß signaling in cancer-associated fibroblasts.

The interaction between epithelial cancer cells and cancer-associated fibroblasts (CAFs) has a major role in cancer progression and eventually in metastasis. In colorectal cancer (CRC), CAFs are present in high abundance, but their origin and functional interaction with epithelial tumor cells has not been elucidated. In this study we observed strong activation of the transforming growth factor-ß (TGF-ß)/Smad signaling pathway in CRC CAFs, accompanied by decreased signaling in epithelial tumor cells. We evaluated the TGF-ß1 response and the expression of target genes including matrix metalloproteinases (MMPs) and plasminogen activator inhibitor (PAI)-1 of various epithelial CRC cell lines and primary CAFs in vitro. TGF-ß1 stimulation caused high upregulation of MMPs, PAI-1 and TGF-ß1 itself. Next we showed that incubation of CAFs with conditioned medium (CM) from epithelial cancer cells led to hyperactivation of the TGF-ß signaling pathway, enhanced expression of target genes like PAI-1, and the expression of α-smooth muscle actin (α-SMA). We propose that the interaction of tumor cells with resident fibroblasts results in hyperactivated TGF-ß1 signaling and subsequent transdifferentiation of the fibroblasts into α-SMA-positive CAFs. In turn this leads to cumulative production of TGF-ß and proteinases within the tumor microenvironment, creating a cancer-promoting feedback loop.

Bone healing and mannose-binding lectin.

Non-union of a fracture is a phenomenon that may complicate bone healing. Consolidation of a fracture can be divided into three phases: inflammation, reconstruction, and remodeling. Both the complement system and the coagulation cascade interact at various steps throughout these phases. Several complement components are specifically associated with the inflammation phase of bone healing. However, in which way complement components influence the remodeling phase has not been established yet. Mannose-Binding Lectin (MBL) and its associated serine protease MASP-2 (Mannanbinding lectin serine protease-2) are important initiating proteins of the complement system and have also been implicated in coagulation. With respect to the characteristics and interactions of MBL, it is likely to assume a considerable influence of MBL in the remodeling phase of bone healing. A deficiency in MBL then, caused by a genetic variation, may disturb this particular process during bone healing, due to either an accumulation of apoptotic cells or to a diminished scaffold of fibrin molecules. The next step would be early identification of patients with a deficiency of MBL, allowing for early therapeutic intervention or even non-union preventive measures. This review aims to discuss the true and hypothesized role of MBL in bone healing and the consequences of a depletion of the protein in the etiology of fracture non-union.

MMP-2 and MMP-9 in normal mucosa are independently associated with outcome of colorectal cancer patients.

BACKGROUND: Upregulation of the matrix metalloproteinases MMP-2 and MMP-9 in various cancers has been associated with worse survival of the patients. METHODS: We assessed MMP-2 and MMP-9 levels in normal colorectal mucosa from colorectal cancer patients in relation to the course of the disease. RESULTS: A high protein expression of MMP-2 as well as MMP-9 in normal mucosa was found to be correlated with worse 5-year survival. The combination of both parameters was an even stronger prognostic factor. These protein levels were found not to be related to the corresponding single nucleotide polymorphisms of MMP-2 (-1306C>T) and MMP-9 (-1562C>T). Multivariate analyses indicated that the MMP-2 and MMP-9 levels in normal mucosa are prognostic for survival, independent of TNM classification. CONCLUSION: MMP-2 and MMP-9 levels in normal mucosa are indicative of the course of disease in colorectal cancer patients.

Implant removal associated complications in children with limb fractures due to trauma.

PURPOSE: The purpose of this study was to analyze the number and type of complications that occurred after fracture implant removal and to investigate whether implant removal should be performed routinely in children. METHODS: In a retrospective study, patient records were used for the analyses of patient characteristics, surgery reports, and complications. Children under the age of 16 years with a limb fracture due to trauma, treated with either Kirschner wires (K-wires), elastic stable intramedullary nails (ESIN), or screw fixation between 2000 and 2007, were included. Exclusion criteria were as follows: refracture, pathological fracture, fracture of the hands and feet, or polytrauma patients (Injury Severity Score [ISS] > 15). RESULTS: Three-hundred and nine fractures were analyzed. All K-wires (173) and ESIN (96) were removed as per standard procedure, resulting in 17/173 and 7/96 complications after removal, respectively. In 19/40 patients with screw fixation treatments, it was decided to remove the material after fracture consolidation, resulting in 4/19 complications. The decision in 21 treatments to leave the screw in situ led to four complications. No significant difference in complication rates could be found for the three groups after removal surgery (17/173, 7/96, and 4/19) or between hardware removal (4/19) and retention (4/21) in the case of screw fixation. CONCLUSIONS: The removal of K-wires, ESIN, and screws is considered to be a safe procedure in children and is, by definition, indicated for K-wires and ESIN after fracture healing.

Clinical significance of stromal apoptosis in colorectal cancer.

BACKGROUND: Epithelial and stromal cells play an important role in the development of colorectal cancer (CRC). We aimed to determine the prognostic significance of both epithelial and stromal cell apoptosis in CRC. METHODS: Total apoptosis was determined by caspase-3 activity measurements in protein homogenates of CRC specimens and adjacent normal mucosa of 211 CRC patients. Epithelial apoptosis was determined by an ELISA specific for a caspase-3-degraded cytokeratin 18 product, the M30 antigen. Stromal apoptosis was determined from the ratio between total and epithelial apoptosis. RESULTS: Epithelial and stromal apoptosis, as well as total apoptosis, were significantly higher in CRC compared with corresponding adjacent normal mucosa. Low total tumour apoptosis (< or = median caspase-3 activity) was associated with a significantly worse disease recurrence (hazard ratio (HR), 95% confidence interval (95% CI): 1.77 (1.05-3.01)), independent of clinocopathological parameters. Epithelial apoptosis was not associated with clinical outcome. In contrast, low stromal apoptosis (< or = median caspase-3/M30) was found to be an independent prognostic factor for overall survival, disease-free survival and disease recurrence, with HRs (95% CI) of 1.66 (1.17-2.35), 1.62 (1.15-2.29) and 1.69 (1.01-2.85), respectively. INTERPRETATION: Stromal apoptosis, in contrast to epithelial apoptosis, is an important factor with respect to survival and disease-recurrence in CRC.

MMP-2 geno-phenotype is prognostic for colorectal cancer survival, whereas MMP-9 is not.

The prognostic significance of single-nucleotide polymorphisms (SNPs) and tumour protein levels of MMP-2 and MMP-9 was evaluated in 215 colorectal cancer patients. Single-nucleotide polymorphism MMP-2(-1306T) and high MMP-2 levels were significantly associated with worse survival. Extreme tumour MMP-9 levels were associated with poor prognosis but SNP MMP-9(-1562C>T) was not. Tumour MMP levels were not determined by their SNP genotypes.

Tissue level, activation and cellular localisation of TGF-beta1 and association with survival in gastric cancer patients.

Transforming growth factor-beta1 (TGF-beta1), a tumour suppressing as well as tumour-promoting cytokine, is stored as an extracellular matrix-bound latent complex. We examined TGF-beta1 activation and localisation of TGF-beta1 activity in gastric cancer. Gastric tumours showed increased stromal and epithelial total TGF-beta1 staining by immunohistochemistry. Active TGF-beta1 was present in malignant epithelial cells, but most strongly in smooth muscle actin expressing fibroblasts. Normal gastric mucosa from the same patient showed some staining for total, and little for active TGF-beta1. Active TGF-beta1 levels were determined by ELISA on tissue homogenates, confirming a strong increase in active TGF-beta1 in tumours compared to corresponding normal mucosa. Moreover, high tumour TGF-beta1 activity levels were significantly associated with clinical parameters, including worse survival of the patients. Total and active TGF-beta1 levels were not correlated, suggesting a specific activation process. Of the different proteases tested, active TGF-beta1 levels were only correlated with urokinase activity levels. The correlation with urokinase activity suggests a role for plasmin in TGF-beta1 activation in the tumour microenvironment, resulting in transformation of resident fibroblasts to tumour promoting myofibroblasts. In conclusion we have shown localisation and clinical relevance of TGF-beta1 activity levels in gastric cancer.

Increased mucosal matrix metalloproteinase-1, -2, -3 and -9 activity in patients with inflammatory bowel disease and the relation with Crohn's disease phenotype.

BACKGROUND AND OBJECTIVE: Matrix metalloproteinases are associated with matrix turnover in both physiological and pathological conditions. We postulate an association between aberrant matrix metalloproteinases proteolytic activity and the intestinal tissue destruction, seen in patients with Crohn's disease and/or ulcerative colitis. MATERIALS AND METHODS: Surgically resected inflamed and non-inflamed ileum and colon with/without extensive fibrosis from 122 Crohn's disease, 20 ulcerative colitis and 62 control patients were homogenized. Protein levels of matrix metalloproteinases and tissue inhibitor of metalloproteinases were measured by enzyme-linked immunosorbent assays (ELISA), while matrix metalloproteinases and myeloperoxidase activity were measured by specific activity assays. RESULTS: Expression of total levels of matrix metalloproteinases-1, -2, -3 and -9 relative to tissue inhibitor of metalloproteinases-1 and -2 was increased in inflamed inflammatory bowel disease compared to non-inflamed inflammatory bowel disease and control intestinal mucosa. Also, net matrix metalloproteinases-1, -2, -3 and -9 activity in inflamed inflammatory bowel disease was increased, with similar expression profiles in Crohn's disease and ulcerative colitis. Within inflamed inflammatory bowel disease, a close correlation of matrix metalloproteinases with myeloperoxidase was observed. The expression of matrix metalloproteinases and tissue inhibitor of metalloproteinases was similar in inflamed Crohn's disease tissue with or without extensive fibrosis and not related to fistulizing disease. CONCLUSIONS: We have shown increased net matrix metalloproteinases activity in intestinal inflammatory bowel disease tissue, likely to contribute to the tissue damage and remodelling seen in inflammatory bowel disease.

Clinical impact of MMP and TIMP gene polymorphisms in gastric cancer.

Gastric cancers express enhanced levels of matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs). Single-nucleotide polymorphisms (SNPs) in MMP and TIMP genes may be associated with disease susceptibility and might also affect their antigen expression. We studied the genotype distribution and allele frequencies of SNPs of MMP-2, -7, -8 and -9 and TIMP-1 and -2 in gastric cancer patients in relation to tumour progression, patient survival and tissue antigen expression. The genotype distribution and allele frequencies were similar in gastric cancer patients and controls, except for MMP-7(-181A>G). In addition, the genotype distribution of MMP-7(-181A>G) was associated with Helicobacter pylori status (chi(2) 7.8, P=0.005) and tumour-related survival of the patients. Single-nucleotide polymorphism TIMP-2(303C>T) correlated significantly with the WHO classification (chi(2) 5.9, P=0.03) and also strongly with tumour-related survival (log rank 11.74, P=0.0006). Single-nucleotide polymorphisms of MMP-2, -8, -9 and TIMP-1 were not associated with tumour-related survival. Only the gene promoter MMP-2(-1306C>T) polymorphism correlated significantly with the protein level within the tumours. First-order dendrogram cluster analysis combined with Cox analysis identified the MMP-7(-181A>G) and TIMP-2(303C>T) polymorphism combination to have a major impact on patients survival outcome. We conclude that MMP-related SNPs, especially MMP-7(-181A>G) and TIMP-2(303C>T), may be helpful in identifying gastric cancer patients with a poor clinical outcome.

Matrix metalloproteinase-2 is a consistent prognostic factor in gastric cancer.

In a pioneer study, we showed 10 years ago that enhanced tissue levels of the matrix metalloproteinases (MMPs) MMP-2 and MMP-9 in gastric cancers, as determined by zymography, were related with worse overall survival of the patients. To corroborate these observations, we now assessed MMP-2 and MMP-9 with new techniques in an expanded group of gastric cancer patients (n = 81) and included for comparison MMP-7, MMP-8 and the tissue inhibitors of MMPs, TIMP-1 and -2. All MMPs and TIMP-1 were significantly increased in tumour tissue compared to normal gastric mucosa. Matrix metalloproteinase-7, -8 and -9, and the TIMPs showed some correlations with the clinicopathologic parameters TNM, WHO and Laurén classification, but their levels were not related with survival. Regardless of the determination method used, that is, enzyme-linked immunosorbent assay or bioactivity assay, an enhanced tumour MMP-2 level did not show a significant correlation with any of the clinicopathological parameters, but was confirmed to be an independent prognostic factor in gastric cancer.