Distinct biomarkers for different bones in osteoporosis with rheumatoid arthritis.

BACKGROUND: Rheumatoid arthritis (RA) is known to cause secondary osteoporosis and fragility fractures. This study aimed to identify biomarkers predictive of bone mineral density (BMD) change at three anatomical sites in patients with RA. METHODS: We conducted a prospective longitudinal study in patients with RA. In 2012, we recruited 379 patients from an RA cohort, 329 of whom underwent evaluation of blood and urine biomarkers together with measurement of BMD in the lumbar spine, proximal femur, and distal forearm. The BMD in these three regions was reassessed in 2014. We performed multivariate linear regression analysis to identify those factors associated with BMD change. RESULTS: The averages of age, body mass index, and disease activity score in 28 joints (DAS28) at baseline were 63.2 (minimum to maximum, 32-85), 21.3 (12.3-30.0), and 3.2 (0.1-5.9), respectively. Univariate analysis showed that the annual BMD change was significantly associated with the use of steroid, bisphosphonate (BP) or vitamin D (VitD), and serum homocysteine in the lumber spine; DAS28, the use of BP or VitD, CRP, and anti-cyclic citrullinated peptide antibody (ACPA) in the proximal femur; and the dosage of MTX, the use of BP or VitD, and serum tartrate-resistant acid phosphatase 5b (TRACP-5b) in the distal forearm, respectively. CONCLUSIONS: Predictive biomarkers for BMD change in RA patients differ at each anatomical site. Practitioners should treat each anatomical site with different markers and prescribe osteoporosis drugs to prevent fractures for RA patients.

Interleukin-18 is associated with the presence of interstitial lung disease in rheumatoid arthritis: a cross-sectional study.

OBJECTIVE: Serum interleukin-18 (IL-18) levels are increased in patients with interstitial lung disease (ILD). In addition, IL-18 levels are increased in patients with rheumatoid arthritis (RA) and are associated with arthritis activity. We determined whether increased IL-18 levels are associated with ILD in RA. METHOD: RA patients were enrolled using an RA cohort database. Plasma IL-18 levels were measured by enzyme-linked immunosorbent assay. ILD was determined by a pulmonologist and a radiologist based on chest radiography and computed tomography findings. IL-18 levels for RA with ILD and RA without ILD were compared. Associations between ILD and various markers including IL-18 and confounding factors (e.g. smoking history) were investigated by logistic regression analysis. Diagnostic values of IL-18 for the presence of ILD were investigated using receiver operating characteristics curve analysis. RESULTS: ILD was complicated in 8.2% (n = 26) of the study population (N = 312). Plasma IL-18 levels were higher for RA patients with ILD than for RA patients without ILD (721.0 ± 481.4 vs 436.8 ± 438.9 pg/mL, p < 0.001). IL-18, Krebs von den Lungen-6, and anti-cyclic citrullinated peptide antibody titre and glucocorticoid doses were independently associated with the presence of ILD during multivariate logistic regression analysis. Sensitivity and specificity of IL-18 levels for the detection of ILD in RA patients were 65.3% and 76.3%, respectively (area under the curve = 0.73). CONCLUSION: Plasma IL-18 levels were higher for RA patients with ILD than for those without ILD. Increased IL-18 levels were associated with the presence of ILD.

Impact of respiratory function on the progression from metabolically healthy non-overweight to metabolically abnormal phenotype.

BACKGROUND AND AIMS: Recent studies identified that metabolically abnormal non-overweight phenotype is a risk factor for cardiovascular diseases. However, only little is known about risk factors for the progression from metabolically healthy non-overweight (MHNO) to metabolically abnormal phenotype. In this study, we investigated the impact of respiratory function on the progression from MHNO to metabolically abnormal phenotype. METHODS AND RESULTS: In this retrospective cohort study, 8949 (3872 men and 5077 women) individuals with MHNO, who participated in a health-checkup program from 2004 to 2015, were enrolled. Four metabolic factors (high-normal blood pressure or hypertension, impaired fasting glucose or diabetes, hypertriglyceridemia, and low HDL cholesterol concentration) were used to define metabolically healthy (less than two factors) or metabolically abnormal (two or more factors) phenotypes. Respiratory function was measured by spirometry. Over a median 4.0 years of follow-up, 927 participants progressed to metabolically abnormal phenotype. The percentage of FVC for predicted values (HR 0.98, 95% CI 0.93-1.03, p = 0.418) was not associated with the progression to metabolically abnormal phenotype after adjusting for covariates, including age, sex, alcohol consumption, exercise, smoking status, and body mass index, whereas the percentage of FEV1 for predicted values (%FEV1) (HR 0.87, 95% CI 0.84-0.91, p < 0.001) and the FEV1/FVC ratio (HR 0.86, 95% CI 0.78-0.95, p = 0.004) were associated with the progression to metabolically abnormal phenotype. CONCLUSION: Decrease in respiratory function in terms of %FEV1 and the FEV1/FVC ratio is associated with the progression to metabolically abnormal phenotype in individuals with MHNO.

Prolyl hydroxylase regulates axonal rewiring and motor recovery after traumatic brain injury.

Prolyl 4-hydroxylases (PHDs; PHD1, PHD2, and PHD3) are a component of cellular oxygen sensors that regulate the adaptive response depending on the oxygen concentration stabilized by hypoxia/stress-regulated genes transcription. In normoxic condition, PHD2 is required to stabilize hypoxia inducible factors. Silencing of PHD2 leads to the activation of intracellular signaling including RhoA and Rho-associated protein kinase (ROCK), which are key regulators of neurite growth. In this study, we determined that genetic or pharmacological inhibition of PHD2 in cultured cortical neurons prevents neurite elongation through a ROCK-dependent mechanism. We then explored the role of PHDs in axonal reorganization following a traumatic brain injury in adult mice. Unilateral destruction of motor cortex resulted in behavioral deficits due to disruption of the corticospinal tract (CST), a part of the descending motor pathway. In the spinal cord, sprouting of fibers from the intact side of the CST into the denervated side is thought to contribute to the recovery process following an injury. Intracortical infusion of PHD inhibitors into the intact side of the motor cortex abrogated spontaneous formation of CST collaterals and functional recovery after damage to the sensorimotor cortex. These findings suggest PHDs have an important role in the formation of compensatory axonal networks following an injury and may represent a new molecular target for the central nervous system disorders.

Heart rate-corrected QT interval is a novel risk marker for the progression of albuminuria in people with Type 2 diabetes.

AIMS: A close association between heart rate-corrected QT interval (QTc) and albuminuria in people with Type 2 diabetes has been reported in cross sectional studies. The aim of this study was to evaluate the relationship between QTc and change in urine albumin excretion (UAE) or progression of albuminuria in people with Type 2 diabetes. METHODS: We measured QTc in 251 consecutive people at baseline. We performed a 5-year follow-up cohort study to assess the relationship between QTc and change in UAE, defined as an increase of UAE/follow-up duration (year), or progression of albuminuria, defined as an increase in the category of diabetic nephropathy. RESULTS: During follow-up, 23 of 151 people with normoalbuminuria and 13 of 73 people with microalbuminuria at baseline had progression of albuminuria. Multiple regression analysis demonstrated that QTc was independently associated with change in UAE (ß = 0.176, P = 0.0104). Logistic regression analyses showed that QTc was a risk marker for progression of albuminuria [odds ratio per 0.01-s increase in QTc 1.35, 95% confidence interval (CI) 1.11-1.66, P = 0.0024] after adjusting for confounders. According to the receiver operator characteristic (ROC) analysis, the optimal cut-off point of QTc for progression of albuminuria was 0.418 s [area under the ROC curve 0.75 (95% CI 0.66-0.82), sensitivity = 0.86, specificity = 0.56, P < 0.0001]. CONCLUSIONS: Heart rate-corrected QT interval could be a novel risk marker for progression of albuminuria in people with Type 2 diabetes.

Factors affecting variability in home blood pressure in patients with type 2 diabetes: post hoc analysis of a cross-sectional multicenter study.

Recent studies have shown that variability in home blood pressure has an important role in the progression of organ damage. The objective of this study was to investigate the factors that affect variability in home blood pressure in patients with type 2 diabetes. We assessed the relationship between home blood pressure variability, defined as coefficient of variation of mean of triplicate morning and evening blood pressure for 14 consecutive days, and various factors using univariate and multivariate linear regression analyses in 1114 patients with type 2 diabetes. Age (ß=0.149, P<0.001), female sex (ß=0.125, P=0.010), duration of diabetes mellitus (ß=0.103, P=0.005), heart rate (ß=0.136, P<0.001), current smoker (ß=0.118, P=0.005), white-coat hypertension (ß=0.136, P=0.002) and treatment with calcium channel blockers (ß=-0.094, P=0.024) were independently associated with coefficient of variation of morning systolic blood pressure. Our findings implicate that factors that might be intervened such as heart rate, smoking status, use of antihypertensive medication in addition to age, sex and duration of diabetes mellitus are associated with variability in home blood pressure in patients with type 2 diabetes.

Expression of Tn and sialyl Tn antigens in synovial tissues in rheumatoid arthritis.

OBJECTIVES: The carbohydrate chains represented by mucins (MUCs) are expressed by a variety of normal and malignant secretory epithelial cells and induce a variety of immunoreactions. Tn and sialyl Tn antigens are tumour-associated carbohydrate antigens which are borne on the core proteins of mucins. The purpose of this study is to investigate the existence of tumour-associated carbohydrate antigens in rheumatoid arthritis (RA). METHODS: . We examined the expression of Tn and sialyl Tn antigens in synovial tissues from RA and osteoarthritis (OA) patients by immunohistochemistry. In addition, mucins from synovial fluid (SF) from RA patients are purified by gel filtration and density gradient ultracentrifugation and the existence of these antigens examined by dot and Western blotting. RESULTS: We found that Tn and sialyl Tn antigens were strongly expressed in synovial cells and infiltrating mononuclear cells on the sublining layer and lymphoid follicles in synovial tissues in RA compared with those in osteoarthritis. Tn and sialyl Tn antigens were detected in purified mucins of SF from RA patients. CONCLUSIONS: Tumour-like synovial hyperplasia cells expressed Tn and sialyl Tn antigens. This finding suggests that the mucins exhibiting with abnormal glycosylation may be in part responsible for synovial hyperplasia, leading to the joint destruction in the pathogenesis of RA.

Metabolic syndrome as a risk factor for high-ocular tension.

OBJECTIVE: To investigate the relationship between the metabolic syndrome and intraocular pressure (IOP). METHODS: An observational study was conducted in a medical health checkup program at a general hospital. This study involved 14 003 apparently healthy Japanese men and women, 18-83 years of age, with a mean IOP of 14.8 (3.0) mm Hg. IOP was examined by noncontact tonometer. High-ocular tension was defined as IOP >21 mm Hg without optic-disc abnormalities or history of receiving any anti-glaucoma therapy. Modified criteria of the revised National Cholesterol Education Program Adult Treatment Panel III (rATPIII), the new International Diabetes Federation definition, and the Japan Society for The Study of Obesity definition were used to characterize the metabolic syndrome. Air temperature was assessed from the Gifu Meteorological Observatory, Gifu, Japan. RESULTS: In the male and female subjects, mean IOP and the prevalence of high-ocular tension became high in direct correlation with the increased number of metabolic syndrome components. To analyze by logistic regression, the metabolic syndrome defined by rATPIII was positively and maximum temperature was negatively correlated with high-ocular tension in males (adjusted odds ratio: 2.0 [95% confidence interval, CI, 1.43-2.78] and 0.63 [95% CI, 0.54-0.73], respectively) and in females (adjusted odds ratio: 7.09 [95% CI, 3.74-13.43] and 0.67 [95% CI, 0.53-0.87], respectively). Three of five metabolic syndrome components (fasting plasma glucose, blood pressure, and triglycerides) were related to high-ocular tension. CONCLUSION: The metabolic syndrome is a risk factor for high-ocular tension.

Gene expression profiling of the response of esophageal carcinoma cells to cisplatin.

Cisplatin is the most common chemotherapeutic agent used in esophageal cancer. However, sensitivity to cisplatin varies greatly between patients. It is important to identify the gene(s) that are related to the sensitivity to cisplatin in esophageal cancer patients. The IC50 for cisplatin was measured for 15 esophageal cancer cell lines (TE1-5, TE8-15, KYSE140, and KYSE150). RNA was extracted from each of these cell lines and a normal esophageal epithelial cell line, namely, Het1A, and gene expression profiles were analyzed using an oligonucleotide microarray consisting of 34 594 genes. TE4 was highly resistant and TE12, 14, and 15 were sensitive to cisplatin. Thirty-seven genes were differentially expressed in the cisplatin-resistant esophageal cancer cell line. Our investigation provides a list of candidate genes that may be associated with resistance to cisplatin in esophageal cancer cells, which may serve as a basis for additional functional studies.

Clinical characteristics of chronic graft-versus-host disease following umbilical cord blood transplantation for adults.

Chronic GVHD is a significant complication following allogeneic hematopoietic stem cell transplantation; however, the clinical characteristics of chronic GVHD following cord blood transplantation (CBT) in adults have not been well described. Between March 2001 and November 2005, a total of 77 patients underwent CBT at eight transplantation centers of the Nagoya Blood and Marrow Transplantation Group. Of 77 patients, 29 survived without graft failure or progression of underlying diseases for at least 100 days after transplantation. The median age of the 29 patients was 42 years (range, 18-67 years). Seven patients developed chronic GVHD (extensive, n=4; limited, n=3) disease. The cumulative incidence of chronic GVHD 1 year after day 100 was 24% (95% confidence interval (CI), 11-41%), and the organs involved were the skin (n=6), oral cavity (n=4), liver (n=1) and gastrointestinal tract (n=1). In three patients, chronic GVHD was resolved with supportive care. The remaining four were successfully treated with additional immunosuppressive therapy. Event-free survival rates of the 29 patients with and without chronic GVHD 3 years after day 100 were 83 (95% CI, 27-97%) and 36% (95% CI, 17-56%), respectively (P=0.047). These results suggest that chronic GVHD following CBT is mild and has a graft-versus-malignancy effect.

A novel role of phospho-beta-catenin in microtubule regrowth at centrosome.

Beta-catenin is a biologically important molecule playing critical roles in both cell adhesion and transcriptional regulation in the Wnt pathway. Here, we show that phospho-beta-catenin (phosphorylated at Ser33/37/Thr41), which is reported to be degraded immediately after its phosphorylation, accumulated in the centrosome. Whereas phospho-mimicking mutant, S33/37/T41E-beta-catenin, could localize to the centrosome, S33A-beta-catenin that lacks the phosphorylation site lost its localization to the centrosome. Phospho-beta-catenin localized mainly to mother centrosome during the interphase and was recruited to daughter centrosome in M-phase. Depletion of beta-catenin with small interfering RNA or inhibition of its phosphorylation by LiCl treatment caused disruption of radial microtubule (MT) array and retardation of the MT regrowth during the recovery from nocodazole treatment. In addition, these treatments increased the frequency of mono-astral MT reorganization. Furthermore, overexpression of the nonphosphorylatable beta-catenin, but not the phospho-mimicking beta-catenin, markedly disrupted radial MT array and repressed the MT regrowth. In contrast, phospho-mimicking beta-catenin localized to both of the duplicated centrosomes with aberrant larger and denser radial MTs array formation. In addition, some of the cells overexpressing phospho-mimicking beta-catenin had multiple centrosomes. Taken together, this study demonstrates a novel role of phospho-beta-catenin in MT organization at the centrosomes.

Significance of additional high-dose cytarabine in combination with cyclophosphamide plus total body irradiation regimen for allogeneic stem cell transplantation.

The combination of cyclophosphamide (CY) and total body irradiation (TBI) has been used as a standard conditioning regimen for allogeneic transplantation. Several studies showed an advantage of adding high-dose cytarabine (HDCA) to this regimen. To clarify the significance of additional HDCA, we conducted a retrospective multicenter study and compared the clinical results of these two regimens. From June 1985 to March 2003, 219 patients with hematological malignancies underwent allogeneic transplantation after conditioning with CY+TBI 12Gy (n=73) or CA+CY+TBI 12Gy (n=146). Engraftment, overall survival, transplant-related mortality (TRM), relapse rate and incidence of graft-versus-host disease (GVHD) were compared according to risks and donors. Addition of HDCA had no impact on the relapse rate in all subgroups, and it was associated with lower TRM among standard-risk patients after related transplantation, and with higher TRM and worse survival among standard-risk patients after unrelated transplantation. The incidence of acute GVHD was not significantly different between the two regimens, and HDCA resulted in a higher incidence of chronic GVHD among standard-risk patients after related transplantation. In summary, addition of HDCA is not beneficial for high-risk patients, and is not recommended for standard-risk patients receiving unrelated transplantation.

Short-term methotrexate could reduce early immune reactions and improve outcomes in umbilical cord blood transplantation for adults.

Post transplant immune disorders are problematic in cord blood transplantation (CBT) for adult patients, and optimal prophylaxis has not been established. We investigated whether intensive graft-versus-host disease (GVHD) prophylaxis using short-term methotrexate (MTX) has a prognostic impact on CBT. Post-CBT immune reactions were classified according to time course as pre-engraftment immune reaction (PIR), engraftment syndrome (ES) or acute GVHD. Between March 2001 and November 2005, a total of 77 patients underwent CBT at eight transplantation centers. Median age was 48 years (range, 18-69 years). Preparative regimens comprised myeloablative (n=31) or reduced-intensity (n=46). Acute GVHD prophylaxis included cyclosporine alone (n=23), tacrolimus alone (n=12), cyclosporine plus MTX (n=17), tacrolimus plus short-term MTX (n=23) or cyclosporine plus methylprednisolone (n=2). Cumulative incidences of PIR, ES and grade II-IV GVHD were 36, 12 and 23%, respectively. Short-term MTX exerted significant favorable effects on post-CBT immune reactions (hazard ratio, 0.55; 95% confidence interval (95% CI), 0.31-0.98; P=0.04) in multivariate analysis. Overall survival rates for patients with and without short-term MTX at day 180 were 59% (95% CI, 42-73%) and 16% (95% CI, 6.6-30%) (P=0.0001), respectively. Short-term MTX could offer one optimal regimen to reduce immune reactions and improve outcomes in CBT.

Stat5 expression predicts response to endocrine therapy and improves survival in estrogen receptor-positive breast cancer.

Constitutively activated signal transducers and activators of transcription (Stats), in particular Stat3 and Stat5, have been demonstrated to directly contribute to oncogenesis by stimulating cell proliferation and preventing apoptosis in various cancers. Stat3 is essential in mammary gland epithelial cell apoptosis and involution, whereas Stat5 is well established as a key factor in mammary epithelial cell growth and differentiation. Crosstalk between Stats and estrogen receptor (ER) has been demonstrated by several laboratories and we have focused on the role of Stat5 in ER-positive breast cancer. Using immunohistochemical techniques, we examined the expression of Stat3 and Stat5 in 517 human breast cancer tissues and analyzed their significance for prognosis and prediction of response to endocrine therapy. Stat5 expression was significantly correlated with histological grade (P<0.0001), ER (P=0.02), and progesterone receptor (P=0.026) expression. There was no difference between Stat3 expression and clinicopathological factors. In 346 patients with ER-positive breast cancer, patients with Stat5 positive tumors had significantly increased overall survival (P=0.0009) in multivariate analysis. There were 70 patients who received endocrine therapy as first-line treatment for metastatic breast cancer at relapse. The patients whose primary breast tumors were Stat5 positive, had significantly better response to endocrine therapy (P=0.04), and longer survival after relapse (P=0.0003), than those whose tumors were Stat5 negative. The present study demonstrates for the first time that Stat5 is a predictive factor for endocrine therapy response and a strong prognostic molecular marker in ER-positive breast cancer. Our data suggest that the expression of Stat5 is helpful in selecting patients who may benefit from endocrine therapy.

In vitro characterization of missense mutations associated with quantitative protein S deficiency.

OBJECTIVE: To elucidate the molecular consequences of hereditary protein S (PS) deficiency, we investigated the in vitro synthesis of the PS missense mutants in COS-1 cells and their activated protein C (APC) cofactor activities. PATIENTS: Four patients with quantitative PS deficiency suffering from venous thrombosis were examined. RESULTS: We identified three distinct novel missense mutations, R275C, P375Q and D455Y, and two previously reported missense mutations, C80Y and R314H. The P375Q and D455Y mutations were found in one patient and observed to be in linkage on the same allele. The R314H mutant showed the lowest level of expression (32.7%), and the C80Y, P375Q + D455Y, and R275C mutants exhibited a moderate impairment of expression, that is, 43.8%, 49.5%, and 72.3% of the wild type, respectively. Furthermore, pulse-chase experiments demonstrated that all mutants showed impaired secretion and longer half-lives in the cells than the wild type PS. In the APC cofactor assays, the C80Y mutant showed no cofactor activity, and the R275C mutant showed reduced activity, 62.3% of the wild type PS, whereas the R314H and P375Q + D455Y mutants exhibited normal cofactor activity. CONCLUSION: These data indicate that the C80Y and R275C mutations affect the secretion and function of the PS molecule, and that the R314H and P375Q + D455Y mutations are responsible for only secretion defects, causing the phenotype of quantitative PS deficiency observed in the patients.

A case of coagulation factor V deficiency caused by compound heterozygous mutations in the factor V gene.

We investigated the molecular basis of a severe factor V (FV) deficiency in a Japanese female, and identified two distinct mutations in the FV gene, a novel cytosine insertion (1943insC) and a previously reported point mutation (A5279G). We expected the patient to be a compound heterozygote for those mutations, as a 1943insC, but not an A5279G, was found in the mother and a sibling. The 1943insC will cause a frame-shift after 590Gln, resulting in amino acid substitutions with two abnormal residues followed by a stop codon in the FV A2 domain (FS592X). The A5279G will cause an amino acid alteration in the FV A3 domain (Y1702C), which has been observed in several ethnic groups. We found that both mutant mRNAs were detected by reverse transcriptase polymerase chain reaction (RT-PCR) in the patient's platelets, whereas no FV antigen and activity were detected in plasma. On the one hand, the RT-PCR signal from the FS592X-FV mutant mRNA was markedly reduced, suggesting that the RNA surveillance system would eliminate most of the abnormal FS592X-FV transcripts with a premature termination. On the other hand, expression analyses revealed that only small amounts of Y1702C-FV with a low specific activity were secreted, and that the FS592X-FV was not detected in cultured media. These data indicated that both mutant FV molecules would be impaired, at least in part, during the post-transcriptional process of protein synthesis and/or in secretion. Taken together, it seems to suggest that each gene mutation could be separately responsible for severe FV deficiency, while this phenotype is due to the in-trans combination of the two defects.

Risk and prognostic factors for Japanese patients with chronic graft-versus-host disease after bone marrow transplantation.

The incidence and prognostic factors for chronic graft-versus-host disease (cGVHD) were evaluated for 255 Japanese patients who survived more than 100 days after bone marrow transplantation, and of whom 119 (47%) developed cGVHD. Prior acute GVHD (grade 2-4) and use of an unrelated donor were significantly associated with the onset of cGVHD. Presence of cGVHD did not have an impact on mortality (hazard ratio (HR) = 0.89; 95% confidence interval (CI), 0.59-1.3). Three factors at diagnosis were associated with cGVHD-specific survival: presence of infection (HR = 4.1; 95% CI, 1.6-10.3), continuing use of corticosteroids at the onset of cGVHD (HR = 3.9; 95% CI, 1.7-9.1), and a Karnofsky performance score <80 (HR = 4.7; 95% CI, 2.0-11.3). The probability of cGVHD-specific survival at 4 years was 79% (95% CI, 70-86%). The severity and death rate of Japanese patients with cGVHD was lower than those for populations in Western countries, which might be the result of greater genetic homogeneity of Japanese ethnics. Our patients could not be accurately classified when the proposed prognostic models from Western countries were used, thus indicating the need for a different model to identify high-risk patients.

Expression of matrix metalloproteinases in benign and malignant follicular thyroid lesions.

AIMS: To examine expression of matrix metalloproteinases (MMPs) and related proteins in follicular thyroid lesions (FTLs) and to determine their usefulness for differential diagnosis of FTLs, particularly between minimally invasive carcinoma and adenoma. METHODS AND RESULTS: Six widely invasive follicular carcinomas (WIFCs), 15 minimally invasive follicular carcinomas (MIFCs), 19 follicular adenomas (FAs) and 10 adenomatous goitres (AGs) were analysed immunohistochemically for MMP-1, MMP-2, MMP-7, MMP-9, membrane-type 1-MMP (MT1-MMP) and tissue inhibitor of matrix metalloproteinase-2 (TIMP-2). MMP-1 was positive in all FTLs. MMP-2 and MMP-7 were positive in more than 80% of WIFC and MIFC cases, whereas they were negative in all FA and AG cases except one MMP-2+ FA (P < 0.001). MMP-9 stained positive significantly more in MIFC than FA or AG cases (P < 0.05, respectively). The positivity of MT1-MMP and TIMP-2 was different among some of the FTLs, but with no significant difference between MIFC and FA cases. In-situ hybridization of MMP-2 and MMP-7 mRNA in selected cases demonstrated the expression of these enzymes in the tumour cells as well as in some stromal cells. CONCLUSIONS: Our results confirm MMP expression mainly in malignant FTLs and suggest that MMP-2 and MMP-7 may be useful markers to distinguish MIFC from FA.