Increased incidence of neoplasia of the digestive tract in men with persistent activation of the coagulant pathway.

BACKGROUND: Thrombin promotes angiogenesis and cell proliferation in cancer. Whether thrombin turnover influences cancer incidence is unknown. OBJECTIVES: To explore the relation between the status of the coagulant pathway and cancer incidence by population survey. METHODS: Of 4,009 middle-aged men clinically free of malignancy, 3052 (76.1%) were recruited. Measurements of hemostatic status were made annually for 4 years, and follow-up for morbidity and mortality was maintained thereafter. Persistent activation of the coagulant pathway was diagnosed when prothrombin fragment 1+2 and fibrinopeptide A concentrations exceeded the upper quartiles of the population distribution in two consecutive annual examinations. Cancer incidence rates in men developing persistent activation (taking the time of onset of activation as baseline) were compared with those in men remaining free of this condition. RESULTS: Persistent activation of the hemostatic pathway was a distinct entity found in 111 men [43 expected by chance alone (P <0.001)], and associated with activation throughout the coagulation pathway. Total mortality (/1000 person-years) was higher in those with persistent activation than in others (17.1 and 9.7, respectively, P=0.015), owing to a higher mortality from all cancers (11.3 and 5.1, respectively, P=0.01), due in turn largely to a higher mortality from cancers of the digestive tract (6.3 and 1.9, respectively, P=0.004). Trends were similar for non-fatal cancers. CONCLUSIONS: Persistent activation of the coagulant pathway plays a role in the preclinical phase of cancer and is associated with an increased incidence of clinical malignancy, especially of the digestive tract.

A modified Kleihauer technique for the quantification of foetomaternal haemorrhage.

The Kleihauer technique, based on acid elution of maternal red cells, is the mostly widely used technique in the UK to screen for, and estimate the volume of, foetomaternal haemorrhage (FMH) and for determining the need for additional doses of anti-D immunoglobulin to prevent maternal alloimmunization. However, technicians often report difficulties in identifying and accurately counting maternal red cells in the blood film, leading to imprecision in the calculated FMH. In this report, we describe a simple modification of the standard Kleihauer technique, based on performing acid elution of only half of the film. Compared to the standard method, the modified technique showed improved accuracy and reduced interobserver variability across a range of simulated FMH volumes. There was a high degree of correlation between the new technique and FMH estimated by flow cytometry (r2 = 0.916, P < 0.001). Technicians found the new technique easy to incorporate into routine practice in a busy teaching hospital laboratory and were impressed by the relative ease of counting maternal ghost cells. The modified technique has been used routinely in our laboratory for 3 years, during which time our performance in the UK National External Quality Assurance Scheme for FMH has been uniformly satisfactory.

Does inflammatory proteolytic activity contribute to the increased factor IX activation peptide in men at high risk of coronary heart disease? A preliminary study.

In the Second Northwick Park Heart Study, the activation peptides of factor IX (FIXpep) and factor X (FXpep) were measured in 1261 middle-aged men by double-antibody radioimmunoassay. During follow-up 147 men who had a first coronary heart disease (CHD) event were found to have had an increased FIXpep (p = 0.003) and a reduced FXpep (p = 0.05) at baseline compared with those remaining CHD-free (controls). Plasma FIXpep and FXpep were positively associated, but the rate of rise in FIXpep with increasing FXpep was higher in cases than controls (p for interaction = 0.01). In a sample of 87 controls, FIXpep was positively and independently related to the concentrations of a polymorphonuclear-specific fibrinogen degradation product (p = 0.036) and FXpep (p = 0.004), but in larger samples no statistically significant associations were found either with C-reactive protein or with fibrinogen concentration. The findings suggested that the increased FIXpep in men at high CHD-risk may have been partly due to the generation of factor IX inactivation peptides by inflammatory proteolysis and their recognition together with true FIXpep in the radioimmunoassay. Direct evidence for this hypothesis requires development of assays for human elastase-specific factor IX inactivation peptides.

Factor VII activation, apolipoprotein A-I and reverse cholesterol transport: possible relevance for postprandial lipaemia.

Postprandial lipaemia is associated with activation of factor VII (FVII) and efflux of cholesterol from tissues to nascent plasma high density lipoproteins (HDL) containing apolipoprotein A-I (apo A-I). To determine whether FVII activation and cholesterol efflux occur together in other situations, the responses to intravenous infusion of HDL-like apo A-I/phosphatidylcholine discs were measured in 10 healthy men. Disc infusion (40 mg apo A-I/kg body weight) over 4 h was followed by increases in HDL cholesteryl ester and plasma apo A-I (p <0.0001). Significant activation of FVII was apparent during infusion in fasting subjects (p = 0.03), activated FVII averaging 123% of baseline value by 12 h (p <0.0001). Plasma thrombin-antithrombin (TAT) complex increased to 156% of baseline level by 12 h (p >0.05) but individual responses differed considerably. Peak TAT post-infusion was associated inversely with peak HDL triglyceride concentration (p = 0.004). The coagulation responses to disc-infusion may be due to transfer of phosphatidylserine to cell surfaces during cholesterol efflux.

The spectrum of human kallikrein 6 (zyme/protease M/neurosin) expression in human tissues as assessed by immunohistochemistry.

The KLK6 gene is a new member of the human kallikrein gene family and encodes for a secreted protease, human kallikrein 6 (hK6; also known as zyme/protease M/neurosin). No study has as yet reported detailed immunohistochemical localization of hK6 in human tissues. Our purpose was to examine the expression of hK6 in human tissues by immunohistochemistry. We have analyzed 199 paraffin blocks from archival, current, and autopsy material prepared from almost every normal human tissue. We employed an hK6-specific polyclonal rabbit antibody and avidin-biotin to localize hK6 by IHC. The staining pattern, the distribution of the immunostaining, and its intensity were studied in detail. The IHC expression of zyme was generally cytoplasmic. Various normal human tissues expressed the protein abundantly. Glandular epithelia constituted the main immunoexpression sites, with representative organs being the breast, prostate, kidney, endometrium, colon, appendix, salivary glands, bile ducts, and gallbladder. The small intestine, stomach, endocervix, Fallopian tube, epididymis, bronchus, and upper respiratory tract showed a focal expression as well. Choroid plexus epithelium, peripheral nerves, and some neuroendocrine cells (including the islets of Langerhans, cells in the anterior pituitary gland, and adrenal medulla) expressed the protein strongly and diffusely. A characteristic immunostaining was observed in the Hassall's corpuscles of the thymus, the oxyphilic cells of the thyroid and parathyroid glands, the primordial follicles of the ovary, dendritic cells mainly in the spleen, and in various cells of the placenta.

Prognostic value of human kallikrein 10 expression in epithelial ovarian carcinoma.

PURPOSE: Human kallikrein 10 (hK10; also known as the normal epithelial cell-specific 1 gene and protein) is a secreted serine protease, which belongs to the human kallikrein family. It has been reported that hK10 is down-regulated in breast and prostate cancer cell lines and that it may function as a tumor suppressor. Recently, we developed a highly sensitive and specific immunoassay for hK10 and found that this protein is abundantly expressed in ovarian tissue. In this study, we measured quantitatively hK10 levels in ovarian cancer cytosolic extracts and evaluated the prognostic value of this biomarker in ovarian cancer. EXPERIMENTAL DESIGN: Specimens from eight normal ovarian tissues, eight ovarian tissues with benign disease, and 182 ovarian tumors were investigated. RESULTS: hK10 concentration in ovarian tumor cytosols ranged from 0 to 84 ng/mg of total protein, with a median of 2.6. This median was highly elevated in comparison with normal and benign ovarian tissues (P < 0.001). A cutoff of 1.35 ng/mg was selected to categorize tumors as hK10 high and hK10 low. With chi(2) test and Fisher's exact test, high concentration hK10 was found to be associated with advanced disease stage, serous histological type, suboptimal debulking, and large residual tumor (>1 cm; all P < 0.05). hK10 status was additionally correlated with clinical outcome, including progression-free (PFS) and overall survival (OS) using the Cox model. In univariate analysis, we found that patients with hK10 high tumors were more likely to die and relapse, in comparison with patients with hK10 low tumors (hazards ratios for PFS and OS were 1.93 and 2.42, respectively; P < 0.05). Although this correlation disappeared after the entire patient population was subjected to multivariate analysis, it remained significant in the subgroup of patients with stage III/IV ovarian cancer (hazards ratios for PFS and OS were 1.98 and 2.12, respectively; P < 0.05). CONCLUSIONS: Our results indicate that hK10 is a new, independent, unfavorable prognostic marker, especially for late-stage ovarian cancer.

Immunofluorometric assay of human kallikrein 10 and its identification in biological fluids and tissues.

BACKGROUND: The human kallikrein 10 gene [KLK10, also known as normal epithelial cell-specific 1 gene (NES1)] is a member of the human kallikrein gene family. The KLK10 gene encodes for a secreted serine protease (hK10). We hypothesize that hK10 is secreted into various biological fluids and that its concentration changes in some disease states. The aim of this study was to develop a sensitive and specific immunoassay for hK10. METHODS: Recombinant hK10 protein was produced and purified using a Pichia pastoris yeast expression system. The protein was used as an immunogen to generate mouse and rabbit polyclonal anti-hK10 antisera. A sandwich-type immunofluorometric assay was then developed using these antibodies. RESULTS: The hK10 immunoassay has a detection limit of 0.05 microg/L. The assay is specific for hK10 and has no detectable cross-reactivity with other homologous kallikrein proteins, such as prostate-specific antigen (hK3), human glandular kallikrein 2 (hK2), and human kallikrein 6 (hK6). The assay was linear from 0 to 20 microg/L with within- and between-run CVs <10%. hK10 is expressed in many tissues, including the salivary glands, skin, and colon and is also detectable in biological fluids, including breast milk, seminal plasma, cerebrospinal fluid, amniotic fluid, and serum. CONCLUSIONS: We report development of the first immunofluorometric assay for hK10 and describe the distribution of hK10 in biological fluids and tissue extracts. This assay can be used to examine the value of hK10 as a disease biomarker.

Serum and urinary prostate-specific antigen and urinary human glandular kallikrein concentrations are significantly increased after testosterone administration in female-to-male transsexuals.

BACKGROUND: The genes that encode prostate-specific antigen (PSA) and human glandular kallikrein (hK2) are up-regulated by androgens and progestins in cultured cells, but no published studies have described the effect of androgen administration in women on serum and urinary PSA or hK2. METHODS: We measured serum and urinary PSA and hK2 before, and 4 and 12 months post testosterone treatment by immunofluorometric methods in 32 female-to-male transsexuals. RESULTS: Mean serum PSA increased from 1.1 ng/L to 11.1 ng/L and then to 22 ng/L by 4 and 12 months post treatment, respectively; the corresponding mean values in urine were 17, 1420, and 18 130 ng/L, respectively. Serum hK2, another kallikrein closely related to PSA, remained undetectable at the three time points. However, urinary hK2 concentration rose from below the detection limit (<6 ng/L) before treatment to 18 and 179 ng/L by the 4th and the 12th month of treatment, respectively. All changes were statistically significant (P <0.001) at 4 months. CONCLUSIONS: Testosterone administration increases serum and urinary PSA and urinary hK2 in women. These measurements may be useful as indicators of androgenic stimulation in women.

Haemostatic and lipid determinants of prothrombin fragment F1.2 and D-dimer in plasma.

The determinants of plasma levels of prothrombin fragment F1.2 (F1.2) and D-dimer in different populations are unclear and this may complicate their interpretation as predictors of thrombotic risk, particularly in the case of D-dimer. We therefore measured F1.2 and D-dimer levels together with a number of other haemostatic and lipid variables in a cross-sectional community-based study of 150 healthy adults (73 male, 77 female), age range 23-80 years, identified from the list of a general practice by stratified random sampling within sex and decade of age. Plasma F1.2 was significantly higher in females than males and was independently and positively associated with age, factor VII activity (FVIIc) and C1 inhibitor, and inversely associated with high density lipoprotein (HDL) cholesterol. Plasma D-dimer showed a quadratic association with age (p <0.0001). In those < or =55 years D-dimer was inversely associated with dilute clot lysis time (DCLT) and activated protein C (APC) ratio. In those >55 years it was significantly higher in females than males and associated positively with age, fibrinogen and, in males, activated factor XII (FXIIa). In a multiple-linear model which combined both age groups, F1.2 and D-dimer were independently associated with each other (r = 0.22, p = 0.03). Thus, thrombin generation and fibrin turnover/fibrinolysis are associated in healthy subjects. HDL cholesterol (inversely) and FVIIc are associated with basal thrombin generation (i.e. F1.2). Determinants of D-dimer differ according to age and interpretation of the biological significance of D-dimer levels in epidemiological studies may therefore not be straightforward.

Haemostatic factors in human peripheral afferent lymph.

Peripheral afferent lymph was obtained by cannulation of a collecting vessel in 17 healthy men (mean age 26 years). Lymph/plasma ratios of all vitamin K-dependent factors were lower than expected from molecular weight. Factor VII, factor IX and tissue factor pathway inhibitor (TFPI) lymph/plasma activity ratios were higher than antigen ratios. Activated factor VII (FVIIa) and TFPI-Xa complex concentrations were higher in lymph than plasma, and the raised FVIIa did not appear to be due to cannulation. The fibrinogen lymph/plasma activity (Clauss) ratio averaged about 20% of the antigen ratio. The result of an ELISA for D-dimer was higher in lymph than plasma, often more than five-fold. This high level in lymph was not explored but may indicate proteolysis of fibrinogen and fibrin with release of D-like and D-dimer-like fragments in interstitial fluid.

Comparison of novel hemostatic factors and conventional risk factors for prediction of coronary heart disease.

BACKGROUND: This study sought to assess whether novel markers of hemostatic activity are predictive of coronary heart disease (CHD) and improve risk assessment. METHODS AND RESULTS: Conventional CHD risk factors, the activation peptides of factor IX and factor X, factor VII activity and antigen, activated factor XII, prothrombin fragment 1+2, fibrinopeptide A, and fibrinogen were measured in 1153 men aged 50 to 61 years who were free of myocardial infarction at recruitment. Activated factor VII (VIIa) was measured in 829 men. During 7.8 years of follow-up, 104 had a CHD event. Baseline status was related to outcome by logistic regression by using a modified nested case-control design. Screening performance was judged from receiver operating characteristic curves. A high activated factor XII was associated with increased CHD risk, but low levels were not protective. Plasma VIIa and factor X activation peptide were independently and inversely related to risk. Plasma factor IX activation peptide and fibrinogen were positively associated with risk, but the relations were no longer statistically significant after adjustment for other factors, including VIIa and apoA-I. Other hemostatic markers were not associated with CHD risk. CONCLUSIONS: Hemostatic status did not add significant predictive power to that provided by conventional CHD risk factors yet was able to substitute effectively for these factors.

Associations of protein C and protein S with serum lipid concentrations.

Difficulties in the laboratory measurement of protein C and protein S levels cause problems in the diagnosis of deficiency states in individual patients and may complicate estimation of the prevalence of these states in the general population. Some difficulties may be due to unappreciated influences affecting the measured levels of proteins C and S. We measured protein C activity and antigen, total and free protein S antigen, and serum total cholesterol, high-density cholesterol and triglyceride in a community-based study of 150 adults (73 male, 77 female), age range 23-80 years. Participants were identified from the list of a single general practice by stratified random sampling within sex and decade of age. Protein C activity and antigen were strongly associated with serum lipids, mean levels increasing by approximately 0.25 u/ml as total cholesterol and triglyceride concentration each rose from the 5th to 95th centile. Total protein S antigen concentration was associated with total cholesterol, the mean rising by over 0.1 u/ml as total cholesterol increased from the 5th to the 95th centile, whilst a similar rise in triglyceride was associated with an increase in mean free protein S of more than 0.3 u/ml. Overall, physiological variation in total cholesterol and triglyceride concentration was associated with significant variation in protein C and protein S levels, independent of age and sex, suggesting that it is important to take serum lipids into account when investigating patients for protein C or protein S deficiency. Failure to do so may be misleading in some circumstances.

Very low activated factor VII and reduced factor VII antigen in familial abetalipoproteinaemia.

Abetalipoproteinaemia is a rare disorder of apolipoprotein B metabolism associated with extremely low plasma concentrations of triglyceride. To discover whether the general positive association between factor VII and triglyceride levels extends to this condition, 5 patients were compared with 18 controls. All patients had a triglyceride below 100 micromol/l. Plasma unesterified fatty acid concentration was normal. Although factor IX activity was only slightly reduced (mean 88% standard) and factor IX antigen was normal, mean activated factor VII in patients was strikingly reduced to 34% of that in controls, a level similar to that found in haemophilia B. The patients' mean factor VII activity and factor VII antigen were also significantly reduced to 54% and 63% of those in controls, respectively. Mean factor XI activity and tissue factor pathway inhibitor activity were reduced in patients to 70% and 75% of control values respectively, while factor XII, factor XI antigen, factor X, prothrombin and protein C were normal.

MR imaging of primary lymphoma of bone: variability of T2-weighted signal intensity.

OBJECTIVE: The purpose of this study was to investigate the MR imaging characteristics of primary lymphoma of bone. MATERIALS AND METHODS: Records of 27 patients with histologic and clinical evidence of primary lymphoma of bone were retrospectively reviewed. Nineteen of these patients underwent MR imaging before surgical biopsy and initiation of therapy. Fifteen of the 19 patients underwent conventional spin-echo T1- and T2-weighted imaging, and the other four patients underwent T1-weighted and fast spin-echo T2-weighted imaging with fat saturation. MR images were separately and independently reviewed by two observers for signal intensity characteristics and homogeneity on T1- and T2-weighted sequences. Signal intensity characteristics were correlated with semiquantitative histopathologic assessments of tumor fibrosis, maturity of fibrosis, and vascularity. RESULTS: T1-weighted signal intensity ranged from isointense to hypointense relative to muscle for all lesions. Twelve of 19 cases showed heterogeneity of signal intensity on T2-weighted images. Predominant tumor T2-weighted signal intensities relative to fat for the 19 patients were assessed as hypointense (observer 1, n = 3; observer 2, n = 1), isointense (observer 1, n = 10; observer 2, n = 11), and hyperintense (observer 1, n = 6; observer 2, n = 7). No correlation among intralesional fibrosis, maturity of fibrosis, or intralesional vascularity and T2-weighted signal intensity characteristics was found. CONCLUSION: T2-weighted MR imaging characteristics of primary lymphoma of bone vary and do not seem to be a simple reflection of histologic findings of intralesional vascularity or fibrosis.

Sex differences in the determinants of fibrinolytic activity.

Impaired whole blood fibrinolytic activity (FA), measured by the dilute clot lysis time (DCLT), is associated with first episodes of ischaemic heart disease (IHD) in the Northwick Park Heart Study in men, especially under 55 years, and in women. In a community-based study to investigate possible determinants of the DCLT, and therefore to assess which fibrinolytic components might be predictors of first IHD events, we measured fibrinolytic variables in a sub-sample of 150 healthy adults (73 males, 77 females) randomly selected from a single general practice. Most of the variance in DCLT (68% in men, 63% in women) was explained by tissue-type plasminogen activator (t-PA) and plasminogen activator inhibitor type-1 (PAI-1) activities. In multiple regression analysis there was a significant difference in the strength of the association of t-PA activity with DCLT in men compared to women (test for interaction p = 0.05), the association of t-PA activity with DCLT being significant in males but not in females. Plasma PAI-1 activity was strongly associated with DCLT in both sexes. There was no independent association of DCLT with plasma fibrinogen, t-PA antigen, other fibrinolytic inhibitors, body mass index, serum lipids or C-reactive protein. Plasma PAI-1 activity in females and both t-PA and PAI-1 activities in males are the main determinants of whole blood FA measured by DCLT. It is therefore likely that these modulators of the plasma fibrinolytic system are associated with the onset of first clinical episodes of IHD. Elevated levels of t-PA antigen were positively associated with DCLT after adjustment for age and sex and therefore indicate impaired rather than enhanced FA. Further studies of the association of FA with risk of IHD should include not only "global" measures but also assessment of t-PA and PAI-1 activities, particularly as our results suggest that their associations with IHD may differ in men and women.

Immunohistochemical localization of prostate-specific antigen in benign and malignant breast tissues.

Prostate-specific antigen (PSA), a glycoprotein initially thought to be produced only by the epithelial cells of the prostate, has recently been found in 30% of female breast tumours using immunofluorometry. Our aim was to localize PSA immunohistochemically in a selected group of 27 paraffin-embedded breast tissues. A scoring system was developed for the histological assessment of PSA positivity within the breast tissue. One pathologist (DH) scored, classified and graded all tumours. Site-specific PSA staining was noted in the histology slides. Intense staining was identified in apocrine metaplasia and within the lining ductal epithelium of cystically dilated ducts. The epithelium in lesions of sclerosing adenosis was also frequently positive for PSA staining. Hyperplastic ductal epithelium (especially of mild degree) occasionally stained positive, as did normal breast ducts. Better differentiated tumours showed PSA staining [e.g. mucinous carcinoma (colloid)]. If an infiltrating duct carcinoma showed staining for PSA, adjacent intraductal carcinoma was also noted to stain positively, if present.

Orbital cyst of lacrimal sac derivation.

Orbital cyst from lacrimal sac epithelium is rare and thought to originate from diverticulum of the lacrimal sac. The cyst may have direct communication with the sac, or the two structures may be anatomically separate. Since these lesions most commonly present as a medial canthal mass, the differential diagnosis should include other disorders that may cause a mass in this region. A 42 year old woman presented with a two-year history of a mass in the right medial canthal region. Nasolacrimal system was patent on irrigation. Sophisticated radiological investigation utilizing dacryocystography and computed tomography combined with dacryocystography accurately localized the lesion and demonstrated its relationship to the nasolacrimal system. This tremendously facilitated surgical removal. The lesion was excised completely via anterior orbitotomy and submitted for histopathologic examination. Haematoxylin-eosin stains revealed a cystic cavity lined by pseudostratified columnar and cuboidal epithelium with interspersed goblet cells consistent with a cyst derived from lacrimal sac epithelium, located separately, but in close proximity to the lacrimal sac. The Authors present a patient with a mass in the righ medial canthal area histologically diagnosed as an orbital cyst of lacrimal sac epithelium derivation. Sophisticated radiological lacrimal investigation helps in detection, localization and in planning surgery.

Activation of factor VII during alimentary lipemia occurs in healthy adults and patients with congenital factor XII or factor XI deficiency, but not in patients with factor IX deficiency.

Factor VII activity (FVIIc), a risk marker for coronary heart disease, is increased during postprandial lipemia. Factor VII activation accompanies lipolysis of triglyceride-rich lipoproteins, but the nature of this association and whether it is causal remain uncertain. To explore this issue, four patients with homozygous factor XII deficiency, four with complete factor XI deficiency, six with factor IX deficiency, and their respective age- and sex-matched controls were given two isocaloric dietary regimens, one providing on average 136 g fat and the other 19 g fat. Blood was taken before breakfast, immediately before lunch at 195 minutes, and at completion of the study at 390 minutes. All samples for each subject and matched control were assayed as one batch for FVIIc, activated factor VII, and factor VII antigen (FVIIag). Activation of factor VII was observed with the high-fat regimen but not with the low-fat regimen in all controls, factor XII-deficient patients, and factor XI-deficient patients. No factor VII activation was observed during either regimen in factor IX-deficient patients, but a normal postprandial responsiveness of factor VII to dietary fat was restored in one patient who replicated the study after factor IX therapy. Plasma FVIIag was not altered postprandially in either regimen in any group of patients or controls. Factor IX apparently plays an obligatory role in the postprandial activation of factor VII, although the mechanism remains to be determined.