O rig in a l P a p e r llivmo«Ui»i« David Green Kochurani Maliekel Elena Sushko Rasheed Akhtar Gerald A. Soff Haemostasis 1997;27:112-118 Received: January 28,1997 Accepted in revised form: February 6. 1997 Activated-Protein-C Resistance in Cancer Patients Key W ords Abstract Protein C Cancer Thrombosis Factor V Leiden Hypercoagulability Background: Resistance to activated protein C (aPC) is usually linked to KARGER E-Mail email@example.com Fax + 41 61 306 12 34 http://ww\v. karger, ch factor V Leiden, but may occur in other disorders associated with hyper coagulability. In this study, we investigated the frequency of resistance to aPC in patients with advanced cancer and examined the relationship of aPC resistance to other markers of coagulation activation. Methods: Patients (n = 39) had an established diagnosis of advanced cancer; controls (n = 20) were healthy persons. aPC resistance was measured as the ratio of activated partial thromboplastin times with and without aPC (aPC-sensitivity ratio, aPC-SR). The factor V Leiden mutation was detected by a polymerase-chain-reaction based technique. Other assays were performed by standard laboratory methods. Data were analyzed using t tests and the Pearson correlation. Results: aPC-SR was below 2 SD for 5 of the cancer patients (13%), but none of the controls; only 1 of the 5 had the factor V Leiden mutation. aPC-SR was inversely correlated (p < 0.01) with fac tor VIII and fibrinogen in patients and with prothrombin activation frag ment 1.2 (FI.2) in controls. Patient factorVIII, von Willebrand factor, (vWF), fibrinogen, F I.2 and D dimer were all significantly increased (p < 0.01); anlithrombin III, protein C and proteins were similar to controls. Factor VIII correlated with vWF (p < 0.001) and FI.2 with d-dimer (p < 0.001). Other associations (p < 0.05) were observed between factor V and protein C, fibrinogen and protein C, factor V and antithrombin III and protein C and antithrombin III. Four cancer patients had a history of thromboembolism; their aPC-SR was similar to that of patients without thrombosis. Of the several coagulation measures examined, only vWF was higher in the patients with thrombosis (p = 0.01). Interpretation: Cancer patients have evidence of intravascular coagulation and increases in pro coagulants and may have aPC resistance. The aPC resistance is not due to factor V Leiden, but is rather associated with elevated levels of factor VIII and fibrinogen, and in itself does not predict thrombosis. €> 1997 S. Karger AG, Basel 0301-0147/97/0273-0112/12.00/0 David Green, MD. PhD 345 E Superior Street, Room 1407 Chicago, IL 60611 (USA) Tel. (312) 908 4701 Downloaded by: Kings's College London 18.104.22.168 - 10/27/2017 9:07:49 AM Division of Hematology/Oncology, Department of Medicine, Northwestern University Medical School, Chicago, 111., USA PC Resistance in Cancer Patients Haemostasis 1997;27:112-118 Activated protein C (aPC) resistance was originally described by Dahlback et al. [ 1] as an impaired responsiveness to aPC. This was measured by examining the ratio of activated partial thromboplastin times (aPTT) with and without aPC (aPC-sensitivity ratio, aPC-SR). Subsequently, resistance to aPC was found in most instances to be due to a mutation in fac tor V (Arg506Gln; factor V Leiden), rendering it partially resistant to proteolysis by aPC , However, not all patients with aPC resistance have the factor V Leiden mutation [3-5]. To investigate this phenomenon, we examined the APC-SR, as well as other hemostatic fac tors, in persons anticipated to have enhanced coagulability, i.e. patients with cancer . M aterials and M ethods Study Subjects Patients (n = 39) with an established diagnosis of cancer attending an oncology clinic were the subjects of this study. Their ages ranged from 42 to 85 years (mean 65); 33 were men. The cancers included lung (10), prostate (10), colorectal (6), head and neck (6), stomach/pancreas (4) and other cancers (3). All pa tients had advanced metastatic cancer, and most were receiving chemotherapy, radiation or hormonal thera py. A careful history was recorded for each patient, and information regarding previous episodes of thrombo embolism was noted. Control subjects (n = 20) were healthy persons between the ages of 26 and 55 years (mean 35), equally divided between men and women. The study was approved by the Institutional Review Board, and all subjects provided written informed con sent. 113 Downloaded by: Kings's College London 22.214.171.124 - 10/27/2017 9:07:49 AM Coagulation Studies Fibrinogen was assessed by the method of Clauss using reagents from Dade Division, Baxter Healthcare Corporation, Deerfield, 111., USA , The assay was calibrated with standard normal plasma (SNP reagent; Dade), and the results were calculated using the data management system of the MLA-Electra 800 clot tim er. The technical error of the assay, expressed as a per centage of the mean, was 5.6 ± 2.4%. Factor V and factor VIII coagulant activities were assayed by a one-stage system using reagents from Pacific Hemostasis, Huntersville, N.C. and George King Biomedical, Overland Park, Kans., USA. The standard curve was prepared using Universal Refer ence Plasma from Curtin Matheson Scientific, Woodale, 111., USA, and the results were calculated as a per centage of the standard with the data management sys tem of the MLA-Electra 800. The technical error of the assay was 6.0 ± 2.3%. von Willebrand factor (vWF) antigen was mea sured by an enyzme-linked immunosorbent assay (ELISA)  obtained from American Bioproducts, Parsippany, N.J., USA. In brief, a plastic support coated with specific rabbit anti-human vWF anti bodies binds the factor in the test plasma. Rabbit antivWF antibody coupled with peroxidase binds to the remaining free antigenic determinants of the factor, and the bound peroxidase is revealed by its activity on o-phenylenediamine in the presence of hydrogen per oxide. A standard curve was prepared using Universal Reference Plasma, and the results were reported as a percentage of the standard. The technical error of the assay was 7.6 ± 1.5%. Prothrombin activation fragment 1.2 (F1.2) was measured by an ELISA  using the Enzygnost F 1+2 kit from Behring, Marburg, Germany, with an interas say coefficient of variation of 5-7.5%. D dimer was measured using the Asserachrom D-Di (Diagnostica Stago, Asnières, France) ELISA method. Antithrom bin III was examined using a synthetic chromogenic substrate method (Stachrom AT III, Diagnostica Sta go) and the PAP-4C instrument (Bio/Data, Hatboro, Pa., USA,). Functional protein C was determined us ing the activator extracted from A. contortrix venom and the ST4 instrument (Staclot Protein C, Diagnosti ca Stago). The functional activity of free protein S was assayed by a method based on the inhibition of factor Va, using Staclot Protein S and the ST4 instrument (Diagnostica Stago). The aPC resistance assay was performed by a mod ification of the aPTT, as described by Dahlback et al. , using the ST4 instrument and reagents distributed by Pharmacia, Franklin, Ohio, USA. Comparison of the aPTT obtained for a particular sample in the pres ence of aPC with the results obtained without aPC yields an aPC-dependent sensitivity ratio (aPC-SR). A decreased aPC-SR is observed with aPC resistance. The technical error of the assay was 7.7%. The factor V Leiden mutation was detected by the method of Bcrtina et al. , The polymerase chain reaction was used to amplify a 267-bp segment of the Introduction Table 1 . Coagulation assay results in cancer patients and healthy controls (means ± SD) aPC-SR Factor V ,% Factor VIII, % vWF, % Fibrinogen, mg/dl F1.2,nM /l D dimer, ng/ml AT III, % Protein C, % Protein S ,% p value Controls (n = 20) Patients (n) 3.2r0.7 (39) 89 ±20 (39) 161 ±63 (39) 211 ±89 (39) 438 ± 152 (39) 1.55 ±0.99 (24) 1,674± 1,237 (24) 105 ± 19 (24) 95 ±31 (24) 120 ±31 (24) 3.4±0.4 81 ± 11 119 ± 32 92 ± 27 270 ±5 0.79±0.17 240 ±96 101 ± 13 114 ± 17 134 ±27 n.s. n.s. <0.01 <0.01 <0.01 <0.01 <0.01 n.s. n.s. n.s. AT III = Antithrombin III. factor V gene, which includes the G to A mutation at nucleotide 1691, from leukocyte DNA. The primers PR-6967 and PR-990 were from Bertina et al. , and the PCR product was digested with Mnl 1 restriction enzyme (New England Biolabs, Beverly, Mass., USA). The 1691 <G-A>point mutation results in the loss of an Mnl 1 restriction site, and the wild-type restriction pat tern thus yields 37-, 67- and 163-bp fragments, while the 1691(G_A) mutant yields 67- and 200-bp bands. Statistical Analysis Data were expressed as the mean ± SD. Compari sons between groups were made with a t test for inde pendent samples. Pearson correlation coefficients were calculated between clotting factors and the aPC-SR. The statistical software was True Epistat, version 3.1 (Epistat Services, Richardson, Tex., USA). A p value (two tailed) of <0.05 was considered significant. Table 2. Pearson correlation coefficients for the aPC resistance ratio Cancer patients Factor V Factor VIII von Willebrand factor Fibrinogen F 1.2 D dimer Antithrombin III Protein C Protein S -0.14 -0.37* -0.04 -0.36* 0.17 0.08 -0.31 -0.25 0.29 Controls -0.42 0.18 0.06 -0.00 -0.69** - 0.1 -0.11 -0.32 0.33 *p = 0.02;**p< 0.001. Table 1 shows the results of the coagula tion assays in the patients and controls. The values for factor VIII, vWF, fibrinogen, F 1.2 and D dimer were significantly higher in the patients. The increase in factor V was of bor derline significance (p = 0.06), and antithrom bin III, protein C and protein S were similar to control values. In the patients, strong corre 114 Haemostasis 1997;27:112-118 lations (p < 0.001) were observed between fac tor VIII and vWF and F 1.2 and D dimer. Other associations (p < 0.05) were observed between factor V and protein C, factor V and antithrombin III, protein C and antithrombin III and protein C and fibrinogen. aPC-SR did not differ significantly be tween cancer patients and controls. The 2 SD Green/Maliekel/Sushko/Akhtar/Soff Downloaded by: Kings's College London 126.96.36.199 - 10/27/2017 9:07:49 AM Results 6 .0 0 n Fig. 1. Correlation between the aPC-SR and factor VIII in patients with advanced malignancy. Pear son correlation; r = -0.37; p = 1 - 00-1 ------------------------1----------------------- 1------------------------1------------------------1----------------------- 1----------------------1— 0.0 50.0 100.0 150.0 200.0 250.0 300.0 Factor VIII (%) 0 .02. Fig. 2. Correlation between the aPC-SR and fibrinogen in patients with advanced malignancy. Pear son correlation: r = -0.36; p = Fibrinogen (mg/dl x 100) cut-off value for aPC-SR was determined to be 2.6 for the controls; none of the 20 controls, but 5 of the 39 cancer patients, had ratios below this value. Thirty-eight patients were tested for the factor V Leiden mutation; only the patient with the lowest aPC-SR (1.7) was found to have this mutation. To determine the factors responsible for the low aPC-SR in some of the cancer pa tients, correlation coefficients between the aPC-SR and the hemostatic factors were ex amined. Significant inverse correlations were observed for factor VIII and fibrinogen in the cancer patients (table 2, fig. 1, 2). Correlation PC Resistance in Cancer Patients Haemostasis 1997:27:112-118 115 Downloaded by: Kings's College London 188.8.131.52 - 10/27/2017 9:07:49 AM 0 .02 . Discussion An association between cancer and throm bosis has been recognized since the work of Trousseau in 1865. As many as 90% of cancer patients will have elevation of plasma-clotting factor levels (fibrinogen, factors V, VIII, IX and XI) and thrombocytosis . In addition, many will have evidence of activation of coag ulation, with elevated levels of D dimer, fibrinopeptide A, F 1.2 and thrombin-antithrom bin complexes [11, 12], Decreases in clotting inhibitors have also been reported. Flowever, the frequency of resistance to aPC and its rela tionship to thrombosis in the cancer patient has thus far received little attention. Dahlback  has calculated that while a normal aPC-SR almost always (99%) predicts the absence of the factor V gene mutation, the predictive value of a low aPC-SR is only about 69%. Thus, there are other influences 116 Haemostasis 1997:27:112—118 on the test for aPC resistance. Technical con siderations are important since the test is based on the aPTT and may be affected by the aPTT reagent, the preparation of aPC select ed and the instrument  used to record the clotting times. All of these were standardized in our study: the same instrument (ST4) and reagent batch were used for all aPTT tests. The method of collection and storage of pa tient samples may also affect aPTT results; for example, freezing of samples decreases the aPC-SR [ 15], perhaps by lysing residual plate lets . To assure accurate aPC-SR results when frozen stored samples are examined, the pooled normal control plasma should also be frozen and thawed, as was done in our investi gation. The duration of storage (up to 12 months) does not appear to influence the assay [15, 16], In most series, the lowest values for aPCSR occur in individuals with the factor V Leiden mutation [ 17]; this was also true in our study. However, there are other reasons for an abnormal aPC-SR, such as heparin, the lupus anticoagulant [18, 19], oral anticoagulant therapy  and vitamin K deficiency . These are suspected if the patients’ baseline aPTT is prolonged. The aPTT was slightly prolonged in 3 patients, but their aPC-SR were within the normal range, and none was receiving warfarin. Since the aPC-SR test depends, in part, on the aPTT and the inhibitory activity of the protein C/S system, factors affecting these measurements could alter the aPC-SR. The aPTT is very sensitive to factor VIII levels , and it is therefore not surprising that we observed an inverse correlation between fac tor VIII and the aPC-SR. It was also of inter est that a similar correlation was noted be tween the fibrinogen concentration and the aPC-SR. Both factor VIII and fibrinogen in crease two-fold during pregnancy , and there is a significant reduction in aPC-SR , Grcen/Maliekel/Sushko/Akhtar/Soff Downloaded by: Kings's College London 184.108.40.206 - 10/27/2017 9:07:49 AM coefficients with factor V and vWF were not significant in the cancer patients; in the con trols, the correlation with factor V was of bor derline significance (p = 0.07). Four cancer patients had a history of thromboembolism; their aPC-SR levels were similar to those of patients without thrombo sis (p = 0.5). None of the 3 who were tested had the factor V Leiden mutation. The levels of factor VIII, vWF, fibrinogen, F 1.2, and D dimer were also compared with those of pa tients without thrombosis. Only vWF was sig nificantly increased (315 vs. 199%; p = 0.01) in the subpopulation with thrombosis. Nine teen patients received treatment that included chemotherapy and/or radiation; their levels of aPc-SR (3.0 ± 0.7), factor VIII (176 ± 66) and fibrinogen (447 ± 184) did not differ sig nificantly from those who did not receive these therapies (aPC-SR: 3.3 ± 0.7; factor VIII: 147 ± 57; fibrinogen; 429 ± 118). suggesting that the levels of these factors af fect the aPC-SR. With regard to the protein C/S system, neither the levels of factor V (within the range of 12.5-100%) nor protein S (above 20%) have been found to influence the test [23, 24], The aPC-SR is a marker for thrombophilia , While we did not observe a direct asso ciation between aPC-SR and thrombosis, we did note that our cancer patients had signifi cant increases in F 1.2 and D dimer, indica tors of coagulation activation. Thus, aPC-SR should be added to the spectrum of hyper coagulability measures characteristic of ad vanced malignancy. Acknow ledgm ents We thank D. CundilT, N. Reynolds and H. Kohl for expert technical assistance. E.S. was a Thrombosis and VascularTraining Center Fellow under the auspices of the Council on Thrombosis of the International Soci ety and Federation of Cardiology and the International Society on Thrombosis and Haemostasis. Grant sup port was received from the Feinberg Cardiovascular Research Institute. References PC Resistance in Cancer Patients 6 Rickies FR, Levine M, Edwards RL: Hemostatic alterations in cancer pa tients. Cancer Metastasis Rev 1992: 11:237-248. 7 Sirridgc MS, Shannon R: Laborato ry evaluation of hemostasis and thrombosis, ed 3. Philadelphia, Lea & Febiger, 1983, pp 164-165. 8 Batlle J, Lopez-Fernandez MF: Lab oratory assays for von Willebrand factor; in Zimmerman TS, Ruggeri ZM (ed): Coagulation and bleeding disorders. New York, Dekker, 1989, pp 330-331. 9 Pelzer H, Schwarz A, StuberW: De termination of human prothrombin activation fragment 1+2 in plasma with an antibody against a synthetic peptide. Thromb Haemost 1991 ;65: 153-159. 10 Edwards RL, Rickies FR: Thrombo sis and cancer; in Hull R, Pinco GF (eds): Disorders of Thrombosis. Philadelphia, Saunders, 1996, pp 374-382. 11 Myers TJ, Rickies FR, Barb C, Cronlund M: Activation of blood coagulation in acute leukemia-fibrinopeptide A (FPA) generation as an indicator of disease activity. Blood 1981;57:518-525. 12 Iversen LH, Okholm M, ThoriaciusUssing O: Pre- and postoperative state of coagulation and fibrinolysis in plasma of patients with benign and malignant colorectal disease - a preliminary study. Thromb Hae most 1996;76:523-528. 13 Dahlback B: Resistance to activated protein C, the Arg506 to Gin muta tion in the factor V gene, and venous thrombosis. Thromb Haemost 1995;73:739-742. 14 Baker R, Thorn J, van BockxmeerF: Diagnosis of activated protein C re sistance (factor V Leiden) (letter). Lancet 1994;344:1162. 15 Trossaert M, Conard J, Horellou MH. Samama MM: Influence of storage conditions on activated pro tein C resistance assay (letter). Thromb Haemost 1995;73:163— 164. 16 Legnani C, Palareti G, Biagi R, Coccheri S: Evaluation of activated pro tein C resistance in stored plasma (letter). Lancet 1994;343:1289. 17 Rosendaal FR, Bertina RM, Reitsma PH: Evaluation of activated pro tein C resistance in stored plasma. Lancet 1994;343:1289-1290. Haemostasis 1997;27:l 12—118 117 Downloaded by: Kings's College London 220.127.116.11 - 10/27/2017 9:07:49 AM 1 Dahlback B, Carlsson M, Svensson PJ: Familial thrombophilia due to a previously unrecognized mecha nism characterized by poor antico agulant response to activated pro tein C: Prediction of a cofactor to activated protein C. Proc Natl Acad SciUSA 1993;90:1004-1008. 2 Bertina RM, Koeleman BPC, Ros ter T, Rosendaal FR. Dirven RJ, Ronde H, van dcr Velden PA, Reitsma PW: Mutation in blood coagula tion factor V associated with resis tance to activated protein C. Nature 1994;369:64-67. 3 Zoller B, Svensson PJ, He X, Dahl back B: Identification of the same factor V gene mutation in 47 out of 50 thrombosis-prone families with inherited resistance to activated protein C. J Clin Invest 1994;94: 2521-2524. 4 Cumming AM, Tait RC, Fildcs S, Yoong A, Keeney S, Hay CR: Devel opment of resistance to activated protein C during pregnancy. Br J Haematol 1995;90:725-727.' 5 Fisher M, Fernandez JA, Ameriso SF, Xie D, Gruber A, Paganini-Hill A, Griffin JH: Activated protein C resistance in ischemic stroke not due to factor V argininc506 'glutamine mutation. Stroke 1996;27:1163— 1166. 118 21 Edson JR, Krivit W, White JG: Kaolin partial thromboplastin time: High levels of procoagulants pro ducing short clotting times or mask ing deficiencies of other procoagu lants or low concentrations of anti coagulants. J Lab Clin Med 1967;70: 463-470. 22 Lctsky EA, de Swiet M: Maternal hemostasis: Coagulation problems of pregnancy: in Loscalzo L, Schafer AI (eds): Thrombosis and Hemor rhage. Boston, Blackwell, 1994, pp 965-998. Haemostasis 1997,27:112-118 23 Colucci M, Ciavarclla N, Giliberti -MG, Semcraro N: Resistance to ac tivated protein C (APC): Influence of factor V levels (letter). Thromb Haemost 1994;72:987-988. 24 de Ronde H, Bertina RM: Laborato ry diagnosis of APC-resistance: A critical evaluation of the test and the development of diagnostic criteria. Thromb Haemost 1994,72:880- 886. 25 Dahlback B: Inherited thrombophil ia: Resistance to activated protein C as a pathogenic factor of venous thromboembolism. Blood 1995:85: 607-614. Green/Maliekel/Sushko/Akhtar/Soff Downloaded by: Kings's College London 18.104.22.168 - 10/27/2017 9:07:49 AM 18 Martorell JR, Munoz-Castillo A, Gil JL: False positive activated protein C resistance test due to anti-phos pholipid antibodies is corrected by platelet extract (letter). Thromb Haemost 1995;74:796-797. 19 Ehrenforth S, Radtke KP, Scharrer I: Acquired activated protein C-resistance in patients with lupus anti coagulants (letter). Thromb Hae most 1995;74:797-798. 20 Trossaert M, Conard J, Horellou MH, Samama MM, Ireland H, Bayston TA, Lane DA: Modified APC resistance assay for patients on oral anticoagulants (letter). Lancet 1994;344:1709.