Intra-individual changes of active matrix metalloproteinase-9 are associated with clinical in-stent restenosis of bare metal stents.

OBJECTIVES: Increased chronic postprocedural levels of active matrix metalloproteinase-9 (MMP-9) have been associated retrospectively with a history of in-stent restenosis (ISR). This study aimed to determine whether index or post-percutaneous coronary intervention (PCI) plasma levels of active MMP-9 are a predictor of subsequent clinical ISR, in a standard population of patients treated with bare metal coronary stents. METHODS: Four hundred thirty-two patients were prospectively recruited and sampled at index and 3 and 6 months after PCI. Those who developed symptomatic angiographically confirmed ISR were compared to randomly selected, asymptomatic controls, stratified by index presentation in a nested case-control design. Plasma samples were analyzed for the active form of MMP-9. RESULTS: In all, 35 patients (8.1%) developed ISR, and these were compared to 98 controls. The increase in active MMP-9 over 3 months was significantly greater in the ISR group (p = 0.030) and independent of the established risk factors. Index clinical presentation was not associated with acute changes in active MMP-9; however, patients with ST-elevation myocardial infarction had greater increases in active MMP-9 at 3 months. CONCLUSIONS: The change in active MMP-9 over 3 months after bare metal coronary stent placement appears to be independently associated with the development of ISR in a standard PCI population.

Pro-MMP-9/TIMP-1 ratio correlates poorly with a direct assessment of MMP-9 activity.

OBJECTIVE: To determine if the pro-MMP-9/TIMP-1 ratio is an accurate surrogate for endogenously active MMP-9 levels. METHODS: Plasma active MMP-9, pro-MMP-9 and TIMP-1 were measured in 295 patients. RESULTS: There was a weak negative correlation between the pro-MMP-9/TIMP-1 ratio and active MMP-9. TIMP-1 was more closely correlated with active MMP-9 than pro-MMP-9. CONCLUSION: Pro-MMP-9/TIMP-1 ratio measured with ELISA is not a good surrogate measure for active MMP-9, and direct measurements of active MMP-9 are therefore recommended.

Seasonal variation and stability of matrix metalloproteinase-9 activity and tissue inhibitor of matrix metalloproteinase-1 with storage at -80°C.

OBJECTIVE: To determine whether active matrix metalloproteinase (MMP)-9 and tissue inhibitor of matrix metalloproteinase (TIMP)-1 displayed seasonal variation and were stable in storage. METHODS: Plasma active MMP-9 and TIMP-1 were measured at three time-points in 163 individuals. RESULT: There was no evidence for seasonal variation or declining levels for up to three years of storage at -80°C. CONCLUSION: Active MMP-9 and TIMP-1 appear to be stable seasonally, and in storage for at least three years.

Active matrix metalloproteinases 3 and 9 are independently associated with coronary artery in-stent restenosis.

OBJECTIVE: This study aimed to determine whether plasma levels of active matrix metalloproteinases (MMP) are predictors of in-stent restenosis (ISR) in New Zealand patients treated with bare-metal coronary stents. METHODS: A group of 152 patients with a history of ISR were compared with 151 symptom free 1-year post-stenting patients (non-ISR). Demographic and angiographic characteristics were collected. Plasma samples were analyzed for the active forms of MMP-1, -2, -3 and -9 as well as tissue inhibitor of metalloproteinases (TIMP-1) using ELISA-based isoform sensitive assays. RESULTS: Both active MMP-9 and active MMP-3 were independently associated with history of ISR. Elevated levels of both active MMP-3 and -9 had an adjusted odds ratio of 11.8 (95% CI: 4-35, p<0.0001) for association with ISR, with 37% of ISR patients having such levels versus 11% on non-ISR. The addition of both of the MMP biomarkers significantly increased the area under the curve (AUC) of a receiver operator characteristic (ROC) analysis incorporating the significant demographic and angiographic variables (AUC 0.85 versus 0.78, p<0.005). CONCLUSION: Measures of plasma active MMP isoforms appear to be independently associated with ISR, and assessment of multiple MMP markers yields cumulative utility.

Elevated plasma active matrix metalloproteinase-9 level is associated with coronary artery in-stent restenosis.

OBJECTIVE: This study aimed to determine whether the plasma levels of matrix metalloproteinase-9 (MMP-9) or tissue inhibitor of metalloproteinases-1 (TIMP-1) were altered in patients with a history of symptomatic in-stent restenosis (ISR). METHODS AND RESULTS: A group of 158 patients with a history of ISR were compared with 128 symptom-free patients. Plasma samples and a detailed risk factor history were collected. Plasma samples were analyzed for pro-MMP-9 and latent MMP-9 and active MMP-9, latent MMP-3, and TIMP-1. Several variables were associated with ISR, including index coronary disease extent and severity (number of diseased vessels and American College of Cardiology/American Heart Association lesion classification), number, diameter, and total length of stent(s) inserted, and plasma high-density lipoprotein cholesterol. Plasma active MMP-9 (odds ratio, 1.96; 95% CI, 1.43 to 2.69) showed independent risk association with ISR. Patients with multiple sites of ISR had significantly higher levels of active MMP-9 compared with patients with only a single ISR lesion or no ISR. CONCLUSIONS: Plasma active MMP-9 levels may be a useful independent predictor of bare metal stent ISR.

The methylenetetrahydrofolate reductase C677T polymorphism does not associate with susceptibility to abdominal aortic aneurysm.

OBJECTIVES: To test whether the T variant of the C677T polymorphism in the gene for 5,10-methylenetetrahydrofolate reductase (MTHFR) would associate with three distinct forms of vascular disease, abdominal aortic aneurysm (AAA), coronary artery disease (CAD) and peripheral vascular disease (PVD). BACKGROUND: Increases in homocysteine induce elastolytic activity in the arterial wall, a condition which may favour vascular pathogenesis including aneurysm formation. Homozygosity of the common T variant of the C677T polymorphism in the gene for MTHFR has been shown to associate with increased levels of homocysteine. Thus, this functional polymorphism may lead to an increased propensity to develop cardiovascular disease and, in particular, AAA. METHODS: An association study was conducted across 1207 subjects; 428 patients with AAA, 271 CAD patients, 226 PVD patients and 282 controls being genotyped for the C667T variants of MTHFR. RESULTS: There were no significant differences in the frequency of the MTHFR C677T variant between any of the groups examined. AAA patients who were homozygotes for the 677T allele did, however, appear to have significantly larger aneurysms than C allele carriers. CONCLUSION: This study provides no evidence that the T variant of MTHFR is associated with susceptibility to AAA, CAD or PVD. It may, however, be a contributory factor in AAA severity as indicated by aneurysm size.

Skip metastases in colon cancer: assessment by lymph node mapping using molecular detection.

BACKGROUND: Colon cancer has been assumed to spread sequentially through the regional lymphatic bed, with skip metastases occurring in only 1% to 3% of cases. Molecular techniques allow the detection of occult metastases, but to date have not been applied to assess the pattern of regional lymphatic spread of colon cancer. METHODS: Fifty-five tumors from 54 patients with colonic adenocarcinoma were studied. Lymph node mapping was performed on fresh colonic specimens recording the position of each node on an anatomical diagram. Half of each lymph node was submitted for routine histology examination and half assayed for keratin 20 gene expression by reverse transcription-polymerase chain reaction. Logistic regression was used to analyze the distribution of histologic and occult metastases. RESULTS: A total of 1084 lymph nodes were dissected (median, 19 nodes; range, 4-52). Sixty-four lymph nodes from 20 tumors had histologically evident metastases and 76 lymph nodes from 13 tumors had occult metastases. There was no difference in the distribution of either histologic or occult metastases among paracolic, intermediate, and apical node groups. Ten patients had evidence of anatomical skip lesions after lymph node mapping and molecular analysis, only 1 of which was histologically detectable. CONCLUSIONS: This study demonstrates a higher incidence of skip metastases in colon cancer assessed by molecular techniques than has previously been reported, challenging the concept of sequential development of early lymph node metastases.

Familial abdominal aortic aneurysms in the Otago region of New Zealand.

PURPOSE: To investigate the familial incidence and phenotypic characteristics of patients with abdominal aortic aneurysms (AAA) in the Otago region of New Zealand. METHODOLOGY: A retrospective audit based pilot study and a prospective study of patients having abdominal aortic aneurysm repair from September 1988 to September 1999 was performed. RESULTS: 248 probands were enrolled, of which 19.4% had one or more first degree relative affected. The age at diagnosis of the familial (70.2) and non-familial (70.5) patients was similar. The proportion of females was increased in the familial subgroup. Hypercholesterolaemia was the only phenotypic feature to differentiate familial from non-familial patients and was associated with an earlier age of presentation. In the familial families, brothers were the most common relative affected and 77% of the families had two patients with AAA. CONCLUSION: 19.4% of patients operated on in the Otago area for AAA had a familial component to their aneurysm.

Diagnostic use of the sentinel node in colon cancer.

PURPOSE: The aim of this study was to compare the lymphatic drainage of colon cancer with the anatomic distribution of histologic and submicroscopic lymph node metastases. METHODS: Patients attending for colectomy were eligible to enter the study. At the commencement of surgery, 40 MBq of 99mTc colloidal antimony sulfide in 2 ml of Patent Blue dye was injected subserosally around the tumor. Resection was completed in a standard fashion. After resection, specimens were imaged with a gamma camera to determine the site of sentinel lymph nodes, and then dissected, recording the position of the lymph nodes on an anatomic diagram. Recovered lymph nodes were bisected, one-half for routine histology and one-half for assessment by keratin 20 (K20) reverse transcription polymerase chain reaction. The kappa measure of agreement was used to assess concordance between sentinel nodes and histologic and submicroscopic metastases. RESULTS: Four hundred fifty-six lymph nodes were dissected from 26 tumors and evaluated using lymphoscintigraphy and lymph node mapping. Sentinel nodes were evident in 23 tumors (88 percent). The sensitivity of sentinel nodes involvement as a predictor of metastatic disease was 55 percent (95 percent confidence interval, 23-83), with a false negative (nondiagnostic) rate of 45 percent. Sentinel nodes involved the apical group in four tumors, and represented anatomic "skip" lesions in four tumors. CONCLUSIONS: Direct lymphatic drainage to the apical group does occur in colon cancer; however, sentinel node mapping of colon cancer by this technique is of little clinical value because of the poor concordance between lymph node metastases and sentinel nodes.

Keratin 20 is a specific marker of submicroscopic lymph node metastases in colorectal cancer: validation by K-RAS mutations.

Lymph node status has major prognostic importance in colorectal cancer and greater precision in the diagnosis of lymph node metastases should provide better prognostic and therapeutic guidance. Keratin 20 (K20) gene expression has been used as a marker of lymph node metastases, but the evidence for this remains circumstantial. This study has therefore sought to determine K20 specificity and to correlate K20 expression with mutant K-RAS expression, in order to provide direct evidence that K20 expression in lymph nodes of colorectal cancer patients genuinely reflects metastatic disease. Specificity of K20 expression was established against a range of tissue types and 289 lymph nodes from 41 non-cancer control patients. K20 expression was restricted to gastrointestinal epithelia and was only present in one of the 289 control lymph nodes, giving a calculated specificity of 97.6% (95% confidence limits: 87.1-99.9%). Forty-two tumour samples were analysed for the presence of K-RAS codon 12 gene mutations using a RT-PCR mutant allele-specific amplification (MASA) technique. Thirteen tumours (31%) had codon 12 mutations detected by MASA and these were further analysed to determine the exact nature of the mutation. MASA was then used to screen the lymph nodes from these patients for the presence of the tumour-specific K-RAS transcript and the results were compared with K20 RT-PCR and histopathology from the same samples. Whilst K-RAS MASA was not as sensitive as K20 RT-PCR, there was substantial agreement between the assays. There were no K20-negative lymph nodes which were found to be K-RAS MASA-positive, whereas seven nodes in four patients were K20-positive and K-RAS-negative, in keeping with the differences in assay sensitivity. These results further validate K20 as a marker by providing greater certainty that what is being detected represents occult metastatic disease.

Analysis of potential markers for detection of submicroscopic lymph node metastases in breast cancer.

We have developed sensitive assays for cytokeratin (K) 8, 16, 19, stromelysin 3 (ST3), MUC1 and maspin mRNAs using reverse transcription polymerase chain reaction (RT-PCR) and used these to assess lymph node status in patients undergoing surgery for breast cancer. In addition the RT-PCR assays were tested against lymph nodes from non-cancer patients to determine their specificity. Despite high sensitivity RT-PCR assays for K8, K16, K19, ST3 and maspin were not found to be useful as markers of submicroscopic disease as transcripts of these genes were detected in the great majority of control lymph nodes tested. Expression of MUC1 was also not found to be useful as it was both insensitive and non-specific. The importance of assessing potential markers against an adequately sized control population is demonstrated, as failure to do so can lead to erroneous conclusions.

Application of inhibitor typing in a study of the transmission and retention in the human mouth of the bacterium Streptococcus salivarius.

Inhibitor production (P)-typing was used as a strain marker in epidemiological studies of Streptococcus salivarius. 43 per cent of 180 adult subjects had inhibitory Strep. salivarius strains as components of their oral microbiota. Strains of 13 different P-type patterns were detected and strains of different P-types often co-existed in the same subject. Adults from whom inhibitor-producing Strep. salivarius strains had been isolated retained their characteristic bacteriocinogenic strains over a 3-yr period. A specific Strep. salivarius-inhibitor screening method was used to study oral acquisition of Strep. salivarius by 14 newborn babies. Initially the babies were colonized by a wide variety of strains, many of which were not detected in the mothers' mouths. By the fifth day of life, strains with P-types identical with those in the mother had often become established as quantitatively prominent members of the babies' Strep. salivarius population.