Results from the UNITED study: a multicenter study validating the prognostic effect of the tumor-stroma ratio in colon cancer.

BACKGROUND: The TNM (tumor-node-metastasis) Evaluation Committee of Union for International Cancer Control (UICC) and College of American Pathologists (CAP) recommended to prospectively validate the cost-effective and robust tumor-stroma ratio (TSR) as an independent prognostic parameter, since high intratumor stromal percentages have previously predicted poor patient-related outcomes. PATIENTS AND METHODS: The 'Uniform Noting for International application of Tumor-stroma ratio as Easy Diagnostic tool' (UNITED) study enrolled patients in 27 participating centers in 12 countries worldwide. The TSR, categorized as stroma-high (>50%) or stroma-low (≤50%), was scored through standardized microscopic assessment by certified pathologists, and effect on disease-free survival (DFS) was evaluated with 3-year median follow-up. Secondary endpoints were benefit assessment of adjuvant chemotherapy (ACT) and overall survival (OS). RESULTS: A total of 1537 patients were included, with 1388 eligible stage II/III patients curatively operated between 2015 and 2021. DFS was significantly shorter in stroma-high (n = 428) than in stroma-low patients (n = 960) (3-year rates 70% versus 83%; P < 0.001). In multivariate analysis, TSR remained an independent prognosticator for DFS (P < 0.001, hazard ratio 1.49, 95% confidence interval 1.17-1.90). As secondary outcome, DFS was also worse in stage II and III stroma-high patients despite adjuvant treatment (3-year rates stage II 73% versus 92% and stage III 66% versus 80%; P = 0.008 and P = 0.011, respectively). In stage II patients not receiving ACT (n = 322), the TSR outperformed the American Society of Clinical Oncology (ASCO) criteria in identifying patients at risk of events (event rate 21% versus 9%), with a higher discriminatory 3-year DFS rate (stroma-high 80% versus ASCO high risk 91%). A trend toward worse 5-year OS in stroma-high was noticeable (74% versus 83% stroma-low; P = 0.102). CONCLUSION: The multicenter UNITED study unequivocally validates the TSR as an independent prognosticator, confirming worse outcomes in stroma-high patients. The TSR improved current selection criteria for patients at risk of events, and stroma-high patients potentially experienced chemotherapy resistance. TSR implementation in pathology diagnostics and international guidelines is highly recommended as aid in personalized treatment.

Scoring the tumor-stroma ratio in colon cancer: procedure and recommendations.

The tumor-stroma ratio (TSR) has been reported as a strong, independent prognostic parameter in colon cancer as well as in other epithelial cancer types, and may be implemented to routine pathology diagnostics. The TSR is an easy technique, based on routine hematoxylin and eosin stained histological sections, estimating the amount of stroma present in the primary tumor. It links tumors with high stromal content to poor prognosis. The analysis time is less than 2 min with a low inter-observer variation. Scoring of the TSR has been validated in a number of independent international studies. In this manuscript, we provide a detailed technical description of estimating the TSR in colon cancer, including examples, pitfalls, and recommendations.

A new regulatory function of activated factor V: inhibition of the activation by tissue factor/factor VII(a) of factor X.

BACKGROUND: We observed that minute amounts of thrombin or the enzyme Russell's viper venom activating factor V (RVV-V) added to plasma strongly diminish the potential of that plasma to generate thrombin after being triggered by tissue factor. OBJECTIVE: To find the mechanism behind this phenomenon. METHODS AND RESULTS: Thrombin generation (TG) initiated by tissue factor (TF) is strongly and dose-dependently inhibited by addition of activated factor V (FVa) or by addition of a factor V activator (thrombin or RVV-V). No inhibition is seen when TG is triggered via the intrinsic pathway or by direct activation of factor X. The effect is independent of proteins C and S and tissue factor pathway inhibitor (TFPI). In factor VII-deficient plasma the effect is seen when it is spiked with recombinant factor VII (FVII) and to a much lesser extent when spiked with recombinant FVIIa. In a purified system, FVa also dose-dependently inhibits the activation of FX by TF/FVII(a). The inhibitory effect is neutralized by antibodies against the light chain of FVa but not by antibodies against the heavy chain. CONCLUSIONS: Our observations can be explained by assuming that FVa, via its light chain, binds to the complex TF/FVII(a) and prevents it from activating FX. We assume that this mechanism reduces the possibility that thrombin and factor Xa escaping from a wound area into the circulation, together with blood-borne tissue factor, would trigger intravascular coagulation.

Fixed dosage of low-molecular-weight heparins causes large individual variation in coagulability, only partly correlated to body weight.

BACKGROUND: Low-molecular-weight heparins (LMWHs) are routinely given without the control of their effect on coagulation. The endogenous thrombin potential (ETP) is a sensitive detector of the heparin effect. QUESTION: What is the interindividual variation in TG after a fixed dose of LMWH in normal volunteers, is it explained by variation in weight? METHODS: Subcutaneous (s.c.) injection, in 12 healthy volunteers, of 9000 aXa-units of unfractionated heparin (UFH) and of three heparins with narrow MW distribution around 10.5, 6.0 and 4.5 kD. Measurement of anti-thrombin (aIIa) and antifactor Xa (aXa)-activities and ETP at 11 time points over 24 h. RESULTS: The coefficient of variation (CV) of the AUCs of aXa- and aIIa-activities is 50% for UFH and 22-37% for LMWHs. Because of the hyperbolic form of the dose-response curve, the CV of the inhibition of the ETP is lower: 32% for UFH and 13-21% for the LMWHs. Fixed dosage of LMWH caused under-dosage in 10-13% of the samples and over-dosage in 5-11%. High or low response is an individual property independent of the type of heparin injected and only partially explained by variation in body weight. CONCLUSION: Optimized individual dosage of LMWH is possible through recognition of high and low responders, which requires one measurement of the heparin concentration or, preferably, the heparin effect on the ETP, 2-5 h after a first injection.

Thrombin generation for the control of heparin treatment, comparison with the activated partial thromboplastin time.

Heparin can be quantified with antifactor Xa and IIa tests (aXa, aIIa) but the anticoagulant power of heparin depends upon plasma properties as well as upon heparin concentrations and thus differs between subjects. Measuring the effect, as with the activated partial thromboplastin time (APTT) therefore is clinically more relevant. Here we investigate the use of the endogenous thrombin potential (ETP) for this purpose. In 12 volunteers 9000 IU of four heparins of different mol. wt distributions were injected. Samples were taken at 11 time points between 0 and 24 h. With the exception of the 0 and 24-h time points, heparin could be demonstrated by its aIIa and aXa activity in virtually all samples. The APTT showed the effect of this heparin in 34% of the samples; the ETP in 80%. This is partly due to the wide margins of the normal values, caused by large interindividual variation [coefficient of variation (CV) approximately 12% for the APTT, approximately 17% for the ETP]. The intraindividual variation is much smaller (CV approximately 4% for the APTT, approximately 5% for the ETP). Relative to the baseline value of the individual, the heparin effect was recognized by the APTT in 55% of the cases and by the ETP in 98%. There were no large differences between the different types of heparin.

Fibrin polymerization is crucial for thrombin generation in platelet-rich plasma in a VWF-GPIb-dependent process, defective in Bernard-Soulier syndrome.

Defective prothrombin consumption has been reported in the proband case of Bernard-Soulier syndrome (BSS). There is no consensus, however, on whether the formation of platelet procoagulant activity (PPA) is impaired in BSS and, if so, whether this is due to the lack of GPIb-V-IX-dependent binding of thrombin or of von Willebrand factor (VWF). We show thrombin generation (TG) in platelet-rich plasma of BSS (BSS-PRP) to be defective provided that fibrin remains present in the reaction mixture and that the giant platelets are not damaged by frequent subsampling. In BSS-PRP addition of (thrombin-free) fibrin did not increase TG as in normal PRP, supporting our previous hypothesis that the interaction of fibrin, VWF and GPIb triggers PPA development. Fibrin formed during the lag phase of TG by a snake venom enzyme which only removed fibrinopeptide A induced an immediate burst of TG, that was inhibited by a monoclonal antibody against GPIb (6D1) that abolishes ristocetin-induced binding of VWF to platelets. Inversely, inhibition of polymerization decreased TG and the residual activity was insensitive to 6D1. We conclude that polymerizing fibrin interacts with VWF so as to activate GPIb.

The ionic contrast medium ioxaglate interferes with thrombin-mediated feedback activation of factor V, factor VIII and platelets.

Clinical observation shows that radiographic contrast media (CM) may influence thrombus formation. In the search for the underlying mechanism, we have shown that the ionic CM ioxaglate is a potent inhibitor of thrombin generation in platelet-poor and platelet-rich plasma, whereas the influence of the non-ionic contrast medium iodixanol is minimal. Ioxaglate boosts the inhibitory effect of the platelet GPIIb/IIIa antagonist abciximab and the effects of ioxaglate and heparin are additive. Ioxaglate inhibits the clotting of fibrinogen and the activation of factors V and VIII, and of platelets by thrombin. It does not inhibit hydrolysis of small chromogenic thrombin substrates, nor does it influence the heparin-catalyzed inactivation of thrombin by antithrombin. We assume therefore that ioxaglate interferes with the binding of macromolecular substrates to the anionic exosite I of thrombin. The biological correlation to the observed antithrombotic effect of ioxaglate is then to be found in the inhibition of thrombin generation via inhibition of thrombin-mediated feedback activations.

The inhibition of blood coagulation by heparins of different molecular weight is caused by a common functional motif--the C-domain.

BACKGROUND: Heparins in clinical use differ considerably as to mode of preparation, molecular weight distribution and pharmacodynamic properties. OBJECTIVES: Find a common basis for their anticoagulant action. METHODS: In 50 fractions of virtually single molecular weight (Mr), prepared from unfractionated heparin (UFH) and four low-molecular-weight heparins (LMWH), we determined: (i) the molar concentration of material (HAM) containing the antithrombin binding pentasaccharide (A-domain); (ii) the specific catalytic activity in thrombin and factor Xa inactivation; (iii) the capacity to inhibit thrombin generation (TG) and prolong the activated partial thromboplastin time (APTT). We also calculated the molar concentration of A-domain with 12 sugar units at its non-reducing end, i.e. the structure that carries antithrombin activity (C-domain). RESULTS: The antithrombin activity and the effects on TG and APTT are primarily determined by the concentration of C-domain and independent of the source material (UFH or LMWH) or Mr. High Mr fractions (>15 000) are less active, probably through interaction with non-antithrombin plasma proteins. Anti-factor Xa activity is proportional to the concentration of A-domain, it is Ca2+- and Mr-dependent and does not determine the effect on TG and APTT. CONCLUSION: For any type of heparin, the capacity to inhibit the coagulation process in plasma is primarily determined by the concentration of C-domain, i.e. the AT-binding pentasaccharide with 12 or more sugar units at its non-reducing end.

Conjectures and refutations on the mode of action of heparins. The limited importance of anti-factor xa activity as a pharmaceutical mechanism and a yardstick for therapy.

Low-molecular-weight heparins (LMWHs), like unfractionated heparin (UFH), exert their action primarily by accelerating the interaction between antithrombin (AT) and thrombin. At the levels of aXa activity that are attained in human pharmacology, it does not cause significant (>15%) inhibition of the clotting system. The essential differences between LMWHs and UFH are: (a) LMWHs attain higher plasma concentrations after subcutaneous injection (high bioavailability), and (b) in contrast to LMWHs, UFH contains very large heparin molecules with a putative hemorrhagic action. The reputedly higher aXa activity of LMWH can be shown to be largely due to the absence of Ca(2+) using the current laboratory methods to estimate this activity. Via this artifact the apparently high aXa activity of LMWHs is correlated but not related to their favorable pharmacokinetic properties. Consequently dosage guidelines for the use of different LMWHs cannot be based upon their aXa activity. Until better laboratory methods are available, clinical results are the only reliable guideline to heparin dosage.

Laboratory assessment of antithrombotic therapy: what tests and if so why?

A critical review is given of the tests available for the assessment of the action of anticoagulants, such as heparins, oral anticoagulants and direct thrombin inhibitors, in patients under antithrombotic therapy. The principle of action and the performance of the thromboplastin time (PT), the activated partial thromboplastin time (aPTT), the whole blood clotting time, the thrombin time, the ecarin clotting time and the endogenous thrombin potential (ETP) is discussed, as well as the evidence behind the accepted therapeutic ranges. The two most common tests, PT and aPTT, respond in an essentially different way to clinically effective anticoagulation with heparin and with oral anticoagulants. This means that they covariate with, but do not themselves represent the essential parameter influenced by anticoagulation. The experimental basis for the widely accepted two times prolongation of the aPTT as an indicator for adequate anticoagulation is shown to be meagre in the case of unfractionated heparin and lacking for the other anticoagulants. Common sources for error in the interpretation of anti-factor Xa- and anti-thrombin activity of heparins are indicated. Extensive experience with new tests like the ecarin clotting time and the ETP is still lacking. On the basis of preliminary data and in view of the importance of the enzymatic action of thrombin in the pathogenesis of thrombosis, the ETP is considered a possible candidate for a common parameter to assess different types of anticoagulants.