- Email: [email protected]
Association between protein C levels and mortality in patients with advanced prostate, lung and pancreatic cancer
Thrombosis Research 154 (2017) 1–6
Contents lists available at ScienceDirect
Thrombosis Research journal homepage: www.elsevier.com/locate/thromres
Full Length Article
Association between protein C levels and mortality in patients with advanced prostate, lung and pancreatic cancer☆ I.T. Wilts a,⁎, B.A. Hutten b, J.C.M. Meijers c,d, C.A. Spek e, H.R. Büller g, P.W. Kamphuisen a,f a
Department of Vascular Medicine, University Medical Center Groningen, University of Groningen, Groningen, The Netherlands Department of Clinical Epidemiology, Biostatistics and Bioinformatics, Academic Medical Center, Amsterdam, The Netherlands Department of Experimental Vascular Medicine, Academic Medical Center, University of Amsterdam, The Netherlands d Department of Plasma Proteins, Sanquin Research, Amsterdam, The Netherlands e Center for Experimental and Molecular Medicine, Academic Medical Center, Amsterdam, The Netherlands f Tergooi Hospital, Hilversum, The Netherlands g Department of Vascular Medicine, Academic Medical Center, University of Amsterdam, The Netherlands b c
a r t i c l e
i n f o
Article history: Received 7 December 2016 Received in revised form 8 February 2017 Accepted 6 March 2017 Available online 07 March 2017 Keywords: Cancer Coagulation Protein C Biomarker
a b s t r a c t Introduction: Procoagulant factors promote cancer progression and metastasis. Protein C is involved in hemostasis, inflammation and signal transduction, and has a protective effect on the endothelial barrier. In mice, administration of activated protein C reduced experimental metastasis. We assessed the association between protein C and mortality in patients with three types of cancer. Methods: The study population consisted of patients with advanced prostate, non-small cell lung or pancreatic cancer, who participated in the INPACT trial (NCT00312013). The trial evaluated the addition of nadroparin to chemotherapy in patients with advanced malignancy. Patients were divided into tertiles based on protein C at baseline. The association between protein C levels and mortality was evaluated with Cox proportional hazard models. Results: We analysed 477 patients (protein C tertiles: b 97, 97–121 and ≥121%). Mean age was 65 ± 9 years; 390 (82%) were male; 191 patients (40%) had prostate cancer, 161 (34%) had lung cancer, and 125 (26%) pancreatic cancer. During a median follow-up of 10.4 months, 291 patients (61%) died. Median protein C level was 107% (IQR 92–129). In the lowest tertile, 75 patients per 100 patient-years died, as compared to 60 and 54 in the middle and high tertile, respectively. Lower levels of protein C were associated with increased mortality (in tertiles: HR for trend 1.18, 95%CI 1.02–1.36, adjusted for age, sex and nadroparin use; as a continuous variable: HR 1.004, 95%CI 1.00–1.008, p = 0.07). Conclusion: Protein C seems inversely associated with mortality in patients with advanced prostate, lung and pancreatic cancer. Further research should validate protein C as a biomarker for mortality, and explore the effects of protein C on progression of cancer. © 2017 Elsevier Ltd. All rights reserved.
1. Introduction Cancer can induce a hypercoagulable state, leading to a greater occurrence of venous thrombosis and pulmonary embolism [1,2]. About 4–10% of patients with unprovoked venous thromboembolism (VTE) are diagnosed with malignant disease in the following 12 months [3, 4]. The prevalence of VTE increases with more advanced stages of
☆ Financial support was received from Aspen Pharma. ⁎ Corresponding author at: University Medical Center Groningen, Department of Vascular Medicine, Hanzeplein 1, 9713 GZ Groningen, The Netherlands. E-mail addresses: [email protected] (I.T. Wilts), [email protected] (B.A. Hutten), [email protected] (J.C.M. Meijers), [email protected] (C.A. Spek), [email protected] (H.R. Büller), [email protected] (P.W. Kamphuisen).
http://dx.doi.org/10.1016/j.thromres.2017.03.001 0049-3848/© 2017 Elsevier Ltd. All rights reserved.
malignancy [1,2]. Moreover, VTE is a predictor for mortality in cancer patients, with an inhospital mortality of 16.3% for patients with VTE versus 6.3% for patients without VTE [5]. Several biomarkers that are related to coagulation have predictive potential in the prognosis of cancer, such as D-dimer and platelet microparticles [6,7]. Further evidence on the close relation between coagulation and cancer is found in the association between use of anticoagulant drugs in patients with cancer and increased survival, although evidence on this is inconsistent [8–11]. This has led to the hypothesis that a hypercoagulable state contributes to cancer progression in terms of tumour growth and metastatic processes, and that endogenous anticoagulant activity could inhibit cancer progression [12,13]. An example of an endogenous anticoagulant is protein C. Its anticoagulant effects derive from the inactivation of factors Va and VIIIa. In
2
I.T. Wilts et al. / Thrombosis Research 154 (2017) 1–6
addition, activated protein C influences inflammatory responses and affects the endothelial barrier function. Over the past years, there has been increasing awareness of the anti-inflammatory and barrier-protective functions of the protein C pathway in a variety of diseases, including sepsis, myocardial infarction and cancer [14]. In cancer, cross-activation of the sphingosine-1-phosphate receptor1 (S1P1) by activated protein C leads to greater stability of cell-to-cell junctions, thereby decreasing extravasation of cancer cells [15]. Indeed, in vitro addition of activated protein C decreased adhesion and transmigration of melanoma cells through endothelium [16]. This possible role for activated protein C in cancer progression has been further investigated in mouse models, where infusion of activated protein C was associated with a reduction in experimental metastasis of melanoma [16]. The non-activated form of protein C, zymogen protein C, also has the ability to influence cancer progression. Overexpression of zymogen protein C in a mouse model with melanoma cells was associated with reduced metastases in a dose-dependent manner, independent of the anticoagulant function [17]. In this case, zymogen protein C was more effective than activated protein C in reducing metastasis, without an increase in bleeding risk [18]. Due to the potential role of inflammatory processes and the coagulation system in cancer progression, it is necessary to gain a better understanding of the role of protein C in the pathophysiology of human cancer patients. In other diseases with an inflammatory component, such as sepsis and myocardial infarction, lower protein C levels were associated with mortality [19,20]. Therefore, we wanted to analyse the association between protein C and mortality in patients with advanced cancer. We hypothesized that lower protein C levels are associated with increased mortality in these patients.
2. Patients and methods 2.1. Study population and design Our study population was derived from the INPACT trial (NCT00312013). In this trial, the addition of the anticoagulant nadroparin to chemotherapeutic treatment was evaluated in patients with one of three different types of advanced malignancies, i.e. cytologically or histologically documented prostate carcinoma within 6 months after diagnosing hormone-refractory state, non-small cell lung cancer (NSCLC) without clinically significant pleural effusion within 3 months after diagnosing stage IIIb and locally advanced pancreatic cancer within 3 months after diagnosis [11]. Exclusion criteria comprised a life expectancy b3 months, a Karnofsky score b 60, presence of a condition requiring anticoagulant treatment, high risk of bleeding, low platelet count (b50,000), renal failure (eGFR b 30 ml min−1), brain metastases and possible or confirmed pregnancy. The study was approved by the respective institutional review boards. All included patients provided written informed consent. Patients were randomized to receive either nadroparin or no nadroparin according to a specific dosage schedule, based on a previous study [21]. The schedule consisted of two weeks of nadroparin at a therapeutic dose and four weeks at a half-therapeutic dose. Patients could receive additional cycles of two weeks therapeutic nadroparin followed by four weeks of washout, with a maximum of 6 cycles. Minimum planned follow-up was 46 weeks. Visits were scheduled at week six and ten, and thereafter patients were contacted at six-week intervals. The study did not show a statistically significant difference in survival between treatment arms. In addition, there was no difference in time to disease progression and the incidence of major bleeding [11]. For the current analysis, all participants of the INPACT trial were included, irrespective of treatment arm. We excluded patients with missing information on type of cancer or missing protein C measurement at baseline.
2.2. Biochemical analysis Protein C was measured using the Coamatic protein C activity kit from Chromogenix (Mölndal, Sweden) and calibrated to the WHO 2nd International Standard (02/342) for protein C. The reference range for this assay is 70–120%. The interassay variation was 7.4% for a sample with approximately 25% activity and 5.9% for a sample with approximately 100% activity. Blood samples for the measurements of protein C were drawn at baseline, frozen after centrifugation and subsequently shipped to a central laboratory for analysis. 2.3. Outcome measures Our primary outcome was all-cause mortality. The secondary outcome was an objectively documented VTE (deep venous thrombosis and/or pulmonary embolism) or an arterial thromboembolic event (myocardial infarction, ischemic stroke, systemic embolism). All potential outcome events were reviewed by an independent adjudication committee blinded to treatment assignment. 2.4. Statistical analysis Patients were categorized into tertiles based on protein C levels at baseline (low, b97%; medium, 97–121%; and high, ≥121%). Differences in demographic and clinical characteristics among the three predefined groups were evaluated using one-way ANOVA (continuous variables) and chi square test (dichotomous variables). Patients were followed from randomisation to the occurrence of a study outcome or censoring, whichever came first. The association between protein C and study outcome (i.e., mortality, thromboembolic events) was first explored by means of KaplanMeier curves and compared using log-rank tests. Hazard ratios (HR) and 95% confidence intervals (95% CI) for this association were calculated by using Cox proportional hazard models. HRs were adjusted for potential confounders such as age, gender and use of nadroparin. Variables with skewed distribution were log-transformed before analysis. A 2sided p value b 0.05 was considered statistically significant. All analyses were performed with the use of SAS software (version 9.3, SAS Institute, INC., Cary, North Carolina). 3. Results 3.1. Characteristics of the study population The population of the INPACT trial consisted of 503 patients. In 24 patients, baseline protein C level was not obtained. In two patients, there was no information on type of cancer. Therefore, our study population consisted of 477 patients. Baseline characteristics did not differ significantly between the current study population and the excluded patients, except for sex (377 out of 477 included patients (82%) were male in the included patients vs. 15 out of 30 excluded patients (50%), p b 0.0001). Patient characteristics of the study population are shown in Table 1. Overall, mean (±standard deviation (SD)) age was 65 (±9) years, and 390 patients (81.8%) were male. There were 191 patients (40%) with prostate cancer, of which 154 (80.6%) had metastatic disease, 161 patients (34%) with lung cancer (no metastasis), and 125 patients (26%) with pancreatic cancer, of which 7 (5.6%) had metastatic disease. Prior to enrolment, 73 patients (15.1%) had already received chemotherapeutic treatment. Cancer surgery was performed in 173 patients (36.3%) and 48 patients (10.1%) had undergone radiotherapy. Median follow-up was 10.4 months (interquartile range (IQR) 5.6–16.9 months) for the total study group. Overall, median protein C level was 107% (IQR 92–129%). The tertiles for the entire study population were defined as low (b 97%), medium (97–121%) and high (≥ 121%). In the patients with pancreatic cancer,
I.T. Wilts et al. / Thrombosis Research 154 (2017) 1–6 Table 1 Baseline characteristics of the study population according to protein C levels.
Age (years) Male gender Body mass index (kg/m2) Creatinin clearance (mL/min) Type of cancer Hormone-refractory prostate Non-small cell lung stage IIIB Locally advanced pancreatic Metastasis Hormone-refractory prostate Locally advanced pancreatic Time since primary cancer diagnosis (years) Cancer treatment before study entry Chemotherapy Radiotherapy Surgery
Lowest tertile b97% N = 155
Mid tertile 97–121% N = 161
Highest tertile ≥121% N = 161
p-Value
66.3 ± 10.3 128 (82.6) 25.0 ± 4.4 85.9 ± 34.6
64.5 ± 8.9 133 (82.6) 24.9 ± 4.2 90.1 ± 31.2
64.8 ± 9.7 129 (80.1) 25.7 ± 4.4 88.9 ± 30.6
0.22 0.80 0.20 0.50 0.38
57 (36.7) 50 (32.3) 48 (31.0)
63 (39.1) 55 (34.2) 43 (26.7)
71 (44.1) 56 (34.8) 34 (21.1)
42 (73.7) 5 (10.4) 0.16 [0.08–1.51]
53 (84.1) 2 (4.7) 0.19 [0.07–2.31]
59 (84.3) 0 0.19 [0.08–2.80]
25 (16.1) 12 (7.7) 55 (35.5)
17 (10.6) 16 (9.9) 64 (39.8)
31 (19.4) 20 (12.5) 54 (33.5)
0.45
0.38
0.09 0.37 0.50
Data are expressed as mean ± standard deviation, median [interquartile range], or no. (%).
median protein C level was 101% (IQR 88–122%). This was significantly lower as compared to the median levels in patients with prostate cancer and lung cancer, (111% (IQR 93–134%), p = 0.006 and 108% (IQR 95– 129%), p = 0.006, respectively). The mean and median protein C levels in patients treated with chemotherapy, radiotherapy and surgery prior to enrolment are available in the Supplement. There were no statistically significant differences between treated and untreated patients. 3.2. Association between protein C and mortality Among the 477 patients, 291 (61%) died during 466 patient-years (62 per 100 patient-years). In the lowest tertile of protein C, 102 patients died during 136 patient-years (75 per 100 patient-years). In the middle tertile, 100 patients per 165 patient-years (60 per 100 patientyears) and in the highest tertile 90 patients per 165 patient-years (54 per 100 patient-years) died (Table 2). Fig. 1 provides Kaplan Meier curves for event-free survival for the patients in the low, middle and high tertile of protein C level. Median survival times were 10.4 months (6.5–19.5), 12.7 months (7.8–26.6) and 14.8 months (7.0–25.8), respectively. As shown in Table 3, patients in the lowest tertile of protein C had a significantly increased mortality risk as compared to the highest tertile (HR 1.39, 95% CI 1.05–1.85, adjusted for sex, age and use of nadroparin). As compared to patients in the middle tertile, we observed no statistically significant difference in risk of mortality (adjusted HR 1.10, 95% CI 0.83–1.46). Overall, lower levels of protein C were associated with increased mortality (HR for trend 1.18, 95% CI 1.02–1.36, adjusted for age, sex and nadroparin use). Table 2 Mortality and thromboembolic events according to protein C levels.
Death – no./patient-years Mortality rate per 100 patient-years Thromboembolic events – no/patient-years Rate of thromboembolic events per 100 patient-years a b c d
Low tertile b97% N = 155
Medium tertile 97–121% N = 161
High tertile ≥121% N = 161
102/136 75 11/130 8
99/165a 60 11/160c 7
90/165b 54 17/158d 11
p-Value = 0.114 as compared to the lowest tertile. p-Value = 0.026 as compared to the lowest tertile. p-Value = 0.636 as compared to the lowest tertile. p-Value = 0.524 as compared to the lowest tertile.
3
In the model with protein C as a continuous variable, lower protein C levels were associated with higher mortality, but this association was not statistically significant (adjusted HR 1.004, 95% CI 1.00–1.008, p = 0.07). Median protein C levels were 105% (IQR 91–127%) for the patients that died during follow up and 112% (IQR 94–129) for the patients who were alive at the end of follow up. In Fig. 2a–c, Kaplan-Meier curves show the cumulative survival for patients with low, medium and high protein C levels, stratified for cancer type. For each of the cancer types, the patients in the lowest tertile of protein C had the shortest median survival time. However, the association between protein C tertiles and mortality did not reach statistical significance for any of the cancer types (adjusted HR for trend in prostate cancer 1.03 (95% CI 0.80–1.33), in lung cancer 1.25 (95% CI 0.96– 1.61), and in pancreatic cancer 1.14 (95% CI 0.88–1.51)). The influence of cancer type on the association between protein C levels and mortality was further explored by redefining the tertiles per cancer type (Supplement). The results of this analysis were similar to the results presented here. When stratifying the association between mortality and protein C for occurrence of thromboembolic events, a clear association was found in the 438 patients without thromboembolic events during follow up (HR for trend 1.22, 95% CI 1.05–1.41, p = 0.01). In the 39 patients with thromboembolic events during follow up, this association was not found (HR for trend 0.88, 95% CI 0.55–1.43) (data not shown). 3.3. Association between protein C and thromboembolic events Overall, there were 39 thromboembolic events during 449 patientyears (9 per 100 patient-years), 11 per 131 patient-years (8 per 100 patient-years) in the lowest tertile group, 11 per 160 patient-years (7 per 100 patient-years) in the mid tertile and 17 per 158 patient-years (11 per 100 patient-years) in the highest tertile of protein C activity (Table 2). There were no statistically significant differences between the tertiles of protein C in the occurrence of thromboembolic events during follow-up. The association between protein C tertiles and thromboembolic events was not statistically significant for any of the cancer types, although there was a trend for prostate cancer (adjusted HR for trend: in prostate cancer 1.94 (95% CI 0.94–4.01), in lung cancer 1.09 (95% CI 0.45–2.63), and in pancreatic cancer 0.99 (95% CI 0.49–1.99)). 4. Discussion This study suggests that protein C levels were inversely associated with mortality in patients with advanced prostate, lung and pancreatic cancer. This association seems not to be affected by nadroparin use, and seemed most pronounced in patients with lung cancer. Our present findings are in line with the recent publication of Tafur et al. [22]. They evaluated protein C as a predictor of cancer-associated thrombosis and mortality in 241 patients with solid tumours of any type and stage, who were scheduled to receive chemotherapy. In this study, patients did not receive anticoagulant drugs. Most patients had gynaecological, breast or pancreatic cancer, and 32% of patients had metastatic disease. Average follow-up was 10.4 months, and during this follow-up 37 patients (15%) died. The authors found that the lowest quartile of protein C activity (≤118%) was associated with higher mortality as compared to all other patients (HR 2.8 adjusted for age, ECOG score and metastatic disease at inclusion). Noteworthy is that the mortality in our population was much higher, 61% versus 15%. Also, the protein C levels found by Tafur et al. were higher overall, with a median of 137.5% as compared to 107% in our population. This could be due to differences in patient characteristics, such as type of cancer and chemotherapeutic regimen, and could also be influenced by the method used for determining protein C. Tafur et al. also found that low protein C was associated with increased incidence of VTE. We did not see this latter association in our population. The overall incidence of VTE during
4
I.T. Wilts et al. / Thrombosis Research 154 (2017) 1–6
Fig. 1. Kaplan-Meier curve of the survival in months according to protein C level. The numbers of patients at risk are given at the bottom of the chart. The low tertile of protein C levels (b97%) is represented in blue, the mid tertile (97–121%) in red and the high tertile (≥121%) in green.
follow up was 13% in the study by Tafur, as compared to only 8.2% in our population. Another recent study, by Sun et al. [23], evaluated the prognostic value of different coagulation parameters in 139 patients with pancreatic cancer and 40 age- and sex-matched healthy controls. Laboratory measurements including protein C were performed before the start of anti-cancer treatment. They found that protein C was significantly lower in patients with cancer as compared to the healthy controls with a median of 103.4% (IQR 89.8–118.2) vs. 115.8% (IQR 107.0– 124.6), respectively. However, there was no association between protein C levels and survival in their study. In the patients with a protein C level below the median, the 1-year survival rate was 34.1%. In the patients with protein C levels equal to or above the median, on the other hand, the 1-year survival rate was 50.4%. This difference was not statistically significant, with a p-value of 0.099. When comparing these results to our study, it should be noted that the study by Sun et al. included patients with various stages of cancer, whereas our analysis only included patients with advanced cancer. This is reflected in the higher survival rates in the previous study. The study by Sun et al. confirms the presence of a hypercoagulable state in patients with pancreatic cancer, and also shows that coagulation parameters can have prognostic value in cancer patients. An association between coagulation and cancer has been demonstrated by several research groups [7,8,22,23]. However, the nature and extent of this relationship is still poorly understood. The underlying mechanism for the association between protein C and mortality may be due to the effects of activated protein C on cancer cell migration, as stated in the introduction [15]. Hypothetically, higher levels of protein C in cancer patients could lead to increased generation of activated protein C
Table 3 Association between protein C levels and mortality.
High (≥121%) Medium (97–121%) Low (b97%)
Unadjusted HR
Adjusted HR
1 (reference) 1.09 (0.82–1.45) 1.39 (1.05–1.85)
1 (reference) 1.10 (0.83–1.46) 1.36 (1.05–1.85)
HR for trend
1.18 (1.02–1.37) Data are expressed as HR (95% CI). The highest tertile of protein C is used as a reference category. Adjustments were made for age, sex and use of nadroparin.
around tumour cells, thereby reducing procoagulant activity and metastasis. In contrast, in vitro research showed that activated protein C increased cancer cell invasion and chemotaxis in a concentration dependent manner in the presence of EPCR and PAR-1 [24,25]. This could indicate that there are so far unknown co-factors or mechanisms involved in the interaction between protein C and cancer in vivo. In line with this hypothesis, Crudele et al. found that the anti-metastatic properties of zymogen protein C in vivo were independent of both its anticoagulant function and EPCR, PAR-1 and PAR-4 mediation [26]. This observation might also explain why the association between protein C activity and mortality in our study was not influenced by use of nadroparin. In conclusion, the role of protein C in cancer progression is complex and so far not completely understood. It is possible that protein C is a mere reflection of overall health status, in which the patients with lower protein C levels represent the patients with the worst prognosis. In our study, the patients with pancreatic cancer probably have the worst prognosis. Protein C levels were indeed lower in these patients. However, as shown in Fig. 2a–c, the association between low protein C and mortality could be found in all three cancer types. The association between protein C and mortality was also found in animal studies, where the mice probably had comparable health status [17]. Malnutrition in cancer patients could theoretically also lead to low levels of protein C, because production of protein C is vitamin K dependent. However, in all three tertiles of our study population, mean BMI was between 24.9 and 25.7 kg/m2. Besides, all patients had a Karnofsky score N 60, indicating they should be capable of sufficient selfcare and nutrition. This makes malnutrition an unlikely explanation for the differences in protein C activity. We had no information on liver function disorders or the presence of liver metastases, therefore we could not analyse possible effects of these conditions on protein C levels. The strength of our study is a relatively large sample size and a nearcomplete follow-up. There are some limitations we should address. First, this was a secondary analysis of the INPACT study. This means the study was not designed to test our hypothesis, and we did not include possible confounders such as other factors or markers of the coagulation system. However, the analysis was part of the initial study design as an exploratory analysis, and the collection of both blood and
I.T. Wilts et al. / Thrombosis Research 154 (2017) 1–6
5
Fig. 2. a–c: Kaplan-Meier curves for the types of cancer. The numbers of patients at risk are given at the bottom of the charts. Fig. 2a: patients with prostate cancer. Fig. 2b: patients with non-small cell lung cancer. Fig. 2c: patients with pancreatic cancer. The differences in survival are not statistically significant for any cancer type. The low tertile of protein C levels (b97%) is represented in blue, the mid tertile (97–121%) in red and the high tertile (≥121%) in green.
6
I.T. Wilts et al. / Thrombosis Research 154 (2017) 1–6
data were performed prospectively. Furthermore, our patients had three different types of advanced cancer. Variations in the biochemical mechanisms of these types of cancer could lead to different effects of protein C levels on prognosis. We compensated for this by performing the analysis for the groups separated according to cancer type, but this led to smaller patient groups and loss of power. Redefining the tertiles of protein C per cancer type did not change the results (Supplement). We separated our cohort into three groups based on the tertiles of protein C levels at baseline. The upper limit of the lowest group was well within the range of what is considered to be normal protein C activity in the general population. However, the ranges for normal protein C levels were established with regard to haemostatic disorders. Probably, different cut-off levels should be used to identify cancer patients with higher risk of mortality. This requires analysis of other cancer cohorts with different cancer types. A better understanding of the interaction between protein C and cancer progression could lead to new therapeutic interventions using some form of protein C. Protein C concentrates are available for the treatment of severe congenital protein C deficiency. In conclusion, this study shows that protein C is associated with mortality in patients with advanced cancer. If these results are confirmed in other cancer cohorts, protein C may be a valuable tool for prognosis in cancer and may even lead to the development of new treatment forms for cancer. Acknowledgments We thank Marian Weijne, Wil Kopatz and Lucy Leverink for their technical assistance. This analysis was supported by Aspen Pharma with an unrestricted grant (previously GSK). There are no personal conflicts of interest for any of the authors. Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.thromres.2017.03.001. References [1] H.K. Chew, T. Wun, D. Harvey, H. Zhou, R.H. White, Incidence of venous thromboembolism and its effect on survival among patients with common cancers, Arch. Intern. Med. 166 (4) (2006 Feb 27) 458–464. [2] J.W. Blom, C.J. Doggen, S. Osanto, F.R. Rosendaal, Malignancies, prothrombotic mutations, and the risk of venous thrombosis, JAMA 293 (6) (2005 Feb 9) 715–722. [3] M. Carrier, G. Le Gal, P.S. Wells, D. Fergusson, T. Ramsay, M.A. Rodger, Systematic review: the Trousseau syndrome revisited: should we screen extensively for cancer in patients with venous thromboembolism? Ann. Intern. Med. 149 (5) (2008 Sep 2) 323–333. [4] M. Carrier, A. Lazo-Langner, S. Shivakumar, V. Tagalakis, R. Zarychanski, S. Solymoss, et al., Screening for occult cancer in unprovoked venous thromboembolism, N. Engl. J. Med. 373 (8) (2015 Aug 20) 697–704. [5] A.A. Khorana, C.W. Francis, E. Culakova, N.M. Kuderer, G.H. Lyman, Frequency, risk factors, and trends for venous thromboembolism among hospitalized cancer patients, Cancer 110 (10) (2007 Nov 15) 2339–2346.
[6] C. Ay, D. Dunkler, R. Pirker, J. Thaler, P. Quehenberger, O. Wagner, et al., High Ddimer levels are associated with poor prognosis in cancer patients, Haematologica 97 (8) (2012 Aug) 1158–1164. [7] J. Thaler, C. Ay, N. Mackman, R.M. Bertina, A. Kaider, C. Marosi, et al., Microparticleassociated tissue factor activity, venous thromboembolism and mortality in pancreatic, gastric, colorectal and brain cancer patients, J. Thromb. Haemost. 10 (7) (2012 Jul) 1363–1370. [8] A.Y. Lee, F.R. Rickles, J.A. Julian, M. Gent, R.I. Baker, C. Bowden, et al., Randomized comparison of low molecular weight heparin and coumarin derivatives on the survival of patients with cancer and venous thromboembolism, J. Clin. Oncol. 23 (10) (2005 Apr 1) 2123–2129. [9] M. Altinbas, H.S. Coskun, O. Er, M. Ozkan, B. Eser, A. Unal, et al., A randomized clinical trial of combination chemotherapy with and without low-molecular-weight heparin in small cell lung cancer, J. Thromb. Haemost. 2 (8) (2004 Aug) 1266–1271. [10] A.K. Kakkar, M.N. Levine, Z. Kadziola, N.R. Lemoine, V. Low, H.K. Patel, et al., Low molecular weight heparin, therapy with dalteparin, and survival in advanced cancer: the fragmin advanced malignancy outcome study (FAMOUS), J. Clin. Oncol. 22 (10) (2004 May 15) 1944–1948. [11] F.F. van Doormaal, M. Di Nisio, H.M. Otten, D.J. Richel, M. Prins, H.R. Buller, Randomized trial of the effect of the low molecular weight heparin nadroparin on survival in patients with cancer, J. Clin. Oncol. 29 (15) (2011 May 20) 2071–2076. [12] F.R. Rickles, A. Falanga, Molecular basis for the relationship between thrombosis and cancer, Thromb. Res. 102 (6) (2001) V215–V224. [13] C.A. Spek, V.R. Arruda, The protein C pathway in cancer metastasis, Thromb. Res. 129 (Suppl. 1) (2012 Apr) S80–S84. [14] J.H. Griffin, B.V. Zlokovic, L.O. Mosnier, Activated protein C: biased for translation, Blood 125 (19) (2015 May 7) 2898–2907. [15] G.L. Van Sluis, T.M. Niers, C.T. Esmon, W. Tigchelaar, D.J. Richel, H.R. Buller, et al., Endogenous activated protein C limits cancer cell extravasation through sphingosine1-phosphate receptor 1-mediated vascular endothelial barrier enhancement, Blood 114 (9) (2009 Aug 27) 1968–1973. [16] M. Bezuhly, R. Cullen, C.T. Esmon, S.F. Morris, K.A. West, B. Johnston, et al., Role of activated protein C and its receptor in inhibition of tumor metastasis, Blood 113 (14) (2009 Apr 2) 3371–3374. [17] J.M. Crudele, G.L. Van Sluis, P. Margaritis, J.I. Siner, M. Sliozberg, J. Maurer, et al., Insights into the mechanism of zymogen protein C protection against cancer progression, Blood 120 (21) (2012 Nov 16). [18] J.M. Crudele, G.L. Van Sluis, P. Margaritis, J.I. Siner, M. Sliozberg, J. Maurer, et al., Modulating cancer progression with zymogen protein C in a murine model, Mol. Ther. (2012 May 20) S266–S267. [19] J.A. Lorente, L.J. Garcia-Frade, L. Landin, R. de Pablo, C. Torrado, E. Renes, et al., Time course of hemostatic abnormalities in sepsis and its relation to outcome, Chest 103 (5) (1993 May) 1536–1542. [20] B. Fellner, M. Rohla, R. Jarai, P. Smetana, M.K. Freynhofer, F. Egger, et al., Activated protein C levels and outcome in patients with cardiogenic shock complicating acute myocardial infarction, Eur. Heart J. Acute Cardiovasc. Care (2016 Mar 2) http://dx.doi.org/10.1177/2048872616637036. [21] C.P. Klerk, S.M. Smorenburg, H.M. Otten, A.W. Lensing, M.H. Prins, F. Piovella, et al., The effect of low molecular weight heparin on survival in patients with advanced malignancy, J. Clin. Oncol. 23 (10) (2005 Apr 1) 2130–2135. [22] A.J. Tafur, G. Dale, M. Cherry, J.D. Wren, A.S. Mansfield, P. Comp, et al., Prospective evaluation of protein C and factor VIII in prediction of cancer-associated thrombosis, Thromb. Res. 136 (6) (2015 Oct 8) 1120–1125. [23] W. Sun, H. Ren, C.T. Gao, W.D. Ma, L. Luo, Y. Liu, et al., Clinical and prognostic significance of coagulation assays in pancreatic cancer patients with absence of venous thromboembolism, Am. J. Clin. Oncol. 38 (6) (2015 Dec) 550–556. [24] L.M. Beaulieu, F.C. Church, Activated protein C promotes breast cancer cell migration through interactions with EPCR and PAR-1, Exp. Cell Res. 313 (4) (2007 Feb 15) 677–687. [25] H. Althawadi, H. Alfarsi, S. Besbes, S. Mirshahi, E. Ducros, A. Rafii, et al., Activated protein C upregulates ovarian cancer cell migration and promotes unclottability of the cancer cell microenvironment, Oncol. Rep. 34 (2) (2015 Aug) 603–609. [26] J.M. Crudele, G.L. Van Sluis, P. Margaritis, J.I. Siner, J. Maurer, S. Zhou, et al., Mechanistic insights into zymogen protein C protection against cancer progression, J. Thromb. Haemost. 11 (2013 Jul) 51–52.
Contents lists available at ScienceDirect
Thrombosis Research journal homepage: www.elsevier.com/locate/thromres
Full Length Article
Association between protein C levels and mortality in patients with advanced prostate, lung and pancreatic cancer☆ I.T. Wilts a,⁎, B.A. Hutten b, J.C.M. Meijers c,d, C.A. Spek e, H.R. Büller g, P.W. Kamphuisen a,f a
Department of Vascular Medicine, University Medical Center Groningen, University of Groningen, Groningen, The Netherlands Department of Clinical Epidemiology, Biostatistics and Bioinformatics, Academic Medical Center, Amsterdam, The Netherlands Department of Experimental Vascular Medicine, Academic Medical Center, University of Amsterdam, The Netherlands d Department of Plasma Proteins, Sanquin Research, Amsterdam, The Netherlands e Center for Experimental and Molecular Medicine, Academic Medical Center, Amsterdam, The Netherlands f Tergooi Hospital, Hilversum, The Netherlands g Department of Vascular Medicine, Academic Medical Center, University of Amsterdam, The Netherlands b c
a r t i c l e
i n f o
Article history: Received 7 December 2016 Received in revised form 8 February 2017 Accepted 6 March 2017 Available online 07 March 2017 Keywords: Cancer Coagulation Protein C Biomarker
a b s t r a c t Introduction: Procoagulant factors promote cancer progression and metastasis. Protein C is involved in hemostasis, inflammation and signal transduction, and has a protective effect on the endothelial barrier. In mice, administration of activated protein C reduced experimental metastasis. We assessed the association between protein C and mortality in patients with three types of cancer. Methods: The study population consisted of patients with advanced prostate, non-small cell lung or pancreatic cancer, who participated in the INPACT trial (NCT00312013). The trial evaluated the addition of nadroparin to chemotherapy in patients with advanced malignancy. Patients were divided into tertiles based on protein C at baseline. The association between protein C levels and mortality was evaluated with Cox proportional hazard models. Results: We analysed 477 patients (protein C tertiles: b 97, 97–121 and ≥121%). Mean age was 65 ± 9 years; 390 (82%) were male; 191 patients (40%) had prostate cancer, 161 (34%) had lung cancer, and 125 (26%) pancreatic cancer. During a median follow-up of 10.4 months, 291 patients (61%) died. Median protein C level was 107% (IQR 92–129). In the lowest tertile, 75 patients per 100 patient-years died, as compared to 60 and 54 in the middle and high tertile, respectively. Lower levels of protein C were associated with increased mortality (in tertiles: HR for trend 1.18, 95%CI 1.02–1.36, adjusted for age, sex and nadroparin use; as a continuous variable: HR 1.004, 95%CI 1.00–1.008, p = 0.07). Conclusion: Protein C seems inversely associated with mortality in patients with advanced prostate, lung and pancreatic cancer. Further research should validate protein C as a biomarker for mortality, and explore the effects of protein C on progression of cancer. © 2017 Elsevier Ltd. All rights reserved.
1. Introduction Cancer can induce a hypercoagulable state, leading to a greater occurrence of venous thrombosis and pulmonary embolism [1,2]. About 4–10% of patients with unprovoked venous thromboembolism (VTE) are diagnosed with malignant disease in the following 12 months [3, 4]. The prevalence of VTE increases with more advanced stages of
☆ Financial support was received from Aspen Pharma. ⁎ Corresponding author at: University Medical Center Groningen, Department of Vascular Medicine, Hanzeplein 1, 9713 GZ Groningen, The Netherlands. E-mail addresses: [email protected] (I.T. Wilts), [email protected] (B.A. Hutten), [email protected] (J.C.M. Meijers), [email protected] (C.A. Spek), [email protected] (H.R. Büller), [email protected] (P.W. Kamphuisen).
http://dx.doi.org/10.1016/j.thromres.2017.03.001 0049-3848/© 2017 Elsevier Ltd. All rights reserved.
malignancy [1,2]. Moreover, VTE is a predictor for mortality in cancer patients, with an inhospital mortality of 16.3% for patients with VTE versus 6.3% for patients without VTE [5]. Several biomarkers that are related to coagulation have predictive potential in the prognosis of cancer, such as D-dimer and platelet microparticles [6,7]. Further evidence on the close relation between coagulation and cancer is found in the association between use of anticoagulant drugs in patients with cancer and increased survival, although evidence on this is inconsistent [8–11]. This has led to the hypothesis that a hypercoagulable state contributes to cancer progression in terms of tumour growth and metastatic processes, and that endogenous anticoagulant activity could inhibit cancer progression [12,13]. An example of an endogenous anticoagulant is protein C. Its anticoagulant effects derive from the inactivation of factors Va and VIIIa. In
2
I.T. Wilts et al. / Thrombosis Research 154 (2017) 1–6
addition, activated protein C influences inflammatory responses and affects the endothelial barrier function. Over the past years, there has been increasing awareness of the anti-inflammatory and barrier-protective functions of the protein C pathway in a variety of diseases, including sepsis, myocardial infarction and cancer [14]. In cancer, cross-activation of the sphingosine-1-phosphate receptor1 (S1P1) by activated protein C leads to greater stability of cell-to-cell junctions, thereby decreasing extravasation of cancer cells [15]. Indeed, in vitro addition of activated protein C decreased adhesion and transmigration of melanoma cells through endothelium [16]. This possible role for activated protein C in cancer progression has been further investigated in mouse models, where infusion of activated protein C was associated with a reduction in experimental metastasis of melanoma [16]. The non-activated form of protein C, zymogen protein C, also has the ability to influence cancer progression. Overexpression of zymogen protein C in a mouse model with melanoma cells was associated with reduced metastases in a dose-dependent manner, independent of the anticoagulant function [17]. In this case, zymogen protein C was more effective than activated protein C in reducing metastasis, without an increase in bleeding risk [18]. Due to the potential role of inflammatory processes and the coagulation system in cancer progression, it is necessary to gain a better understanding of the role of protein C in the pathophysiology of human cancer patients. In other diseases with an inflammatory component, such as sepsis and myocardial infarction, lower protein C levels were associated with mortality [19,20]. Therefore, we wanted to analyse the association between protein C and mortality in patients with advanced cancer. We hypothesized that lower protein C levels are associated with increased mortality in these patients.
2. Patients and methods 2.1. Study population and design Our study population was derived from the INPACT trial (NCT00312013). In this trial, the addition of the anticoagulant nadroparin to chemotherapeutic treatment was evaluated in patients with one of three different types of advanced malignancies, i.e. cytologically or histologically documented prostate carcinoma within 6 months after diagnosing hormone-refractory state, non-small cell lung cancer (NSCLC) without clinically significant pleural effusion within 3 months after diagnosing stage IIIb and locally advanced pancreatic cancer within 3 months after diagnosis [11]. Exclusion criteria comprised a life expectancy b3 months, a Karnofsky score b 60, presence of a condition requiring anticoagulant treatment, high risk of bleeding, low platelet count (b50,000), renal failure (eGFR b 30 ml min−1), brain metastases and possible or confirmed pregnancy. The study was approved by the respective institutional review boards. All included patients provided written informed consent. Patients were randomized to receive either nadroparin or no nadroparin according to a specific dosage schedule, based on a previous study [21]. The schedule consisted of two weeks of nadroparin at a therapeutic dose and four weeks at a half-therapeutic dose. Patients could receive additional cycles of two weeks therapeutic nadroparin followed by four weeks of washout, with a maximum of 6 cycles. Minimum planned follow-up was 46 weeks. Visits were scheduled at week six and ten, and thereafter patients were contacted at six-week intervals. The study did not show a statistically significant difference in survival between treatment arms. In addition, there was no difference in time to disease progression and the incidence of major bleeding [11]. For the current analysis, all participants of the INPACT trial were included, irrespective of treatment arm. We excluded patients with missing information on type of cancer or missing protein C measurement at baseline.
2.2. Biochemical analysis Protein C was measured using the Coamatic protein C activity kit from Chromogenix (Mölndal, Sweden) and calibrated to the WHO 2nd International Standard (02/342) for protein C. The reference range for this assay is 70–120%. The interassay variation was 7.4% for a sample with approximately 25% activity and 5.9% for a sample with approximately 100% activity. Blood samples for the measurements of protein C were drawn at baseline, frozen after centrifugation and subsequently shipped to a central laboratory for analysis. 2.3. Outcome measures Our primary outcome was all-cause mortality. The secondary outcome was an objectively documented VTE (deep venous thrombosis and/or pulmonary embolism) or an arterial thromboembolic event (myocardial infarction, ischemic stroke, systemic embolism). All potential outcome events were reviewed by an independent adjudication committee blinded to treatment assignment. 2.4. Statistical analysis Patients were categorized into tertiles based on protein C levels at baseline (low, b97%; medium, 97–121%; and high, ≥121%). Differences in demographic and clinical characteristics among the three predefined groups were evaluated using one-way ANOVA (continuous variables) and chi square test (dichotomous variables). Patients were followed from randomisation to the occurrence of a study outcome or censoring, whichever came first. The association between protein C and study outcome (i.e., mortality, thromboembolic events) was first explored by means of KaplanMeier curves and compared using log-rank tests. Hazard ratios (HR) and 95% confidence intervals (95% CI) for this association were calculated by using Cox proportional hazard models. HRs were adjusted for potential confounders such as age, gender and use of nadroparin. Variables with skewed distribution were log-transformed before analysis. A 2sided p value b 0.05 was considered statistically significant. All analyses were performed with the use of SAS software (version 9.3, SAS Institute, INC., Cary, North Carolina). 3. Results 3.1. Characteristics of the study population The population of the INPACT trial consisted of 503 patients. In 24 patients, baseline protein C level was not obtained. In two patients, there was no information on type of cancer. Therefore, our study population consisted of 477 patients. Baseline characteristics did not differ significantly between the current study population and the excluded patients, except for sex (377 out of 477 included patients (82%) were male in the included patients vs. 15 out of 30 excluded patients (50%), p b 0.0001). Patient characteristics of the study population are shown in Table 1. Overall, mean (±standard deviation (SD)) age was 65 (±9) years, and 390 patients (81.8%) were male. There were 191 patients (40%) with prostate cancer, of which 154 (80.6%) had metastatic disease, 161 patients (34%) with lung cancer (no metastasis), and 125 patients (26%) with pancreatic cancer, of which 7 (5.6%) had metastatic disease. Prior to enrolment, 73 patients (15.1%) had already received chemotherapeutic treatment. Cancer surgery was performed in 173 patients (36.3%) and 48 patients (10.1%) had undergone radiotherapy. Median follow-up was 10.4 months (interquartile range (IQR) 5.6–16.9 months) for the total study group. Overall, median protein C level was 107% (IQR 92–129%). The tertiles for the entire study population were defined as low (b 97%), medium (97–121%) and high (≥ 121%). In the patients with pancreatic cancer,
I.T. Wilts et al. / Thrombosis Research 154 (2017) 1–6 Table 1 Baseline characteristics of the study population according to protein C levels.
Age (years) Male gender Body mass index (kg/m2) Creatinin clearance (mL/min) Type of cancer Hormone-refractory prostate Non-small cell lung stage IIIB Locally advanced pancreatic Metastasis Hormone-refractory prostate Locally advanced pancreatic Time since primary cancer diagnosis (years) Cancer treatment before study entry Chemotherapy Radiotherapy Surgery
Lowest tertile b97% N = 155
Mid tertile 97–121% N = 161
Highest tertile ≥121% N = 161
p-Value
66.3 ± 10.3 128 (82.6) 25.0 ± 4.4 85.9 ± 34.6
64.5 ± 8.9 133 (82.6) 24.9 ± 4.2 90.1 ± 31.2
64.8 ± 9.7 129 (80.1) 25.7 ± 4.4 88.9 ± 30.6
0.22 0.80 0.20 0.50 0.38
57 (36.7) 50 (32.3) 48 (31.0)
63 (39.1) 55 (34.2) 43 (26.7)
71 (44.1) 56 (34.8) 34 (21.1)
42 (73.7) 5 (10.4) 0.16 [0.08–1.51]
53 (84.1) 2 (4.7) 0.19 [0.07–2.31]
59 (84.3) 0 0.19 [0.08–2.80]
25 (16.1) 12 (7.7) 55 (35.5)
17 (10.6) 16 (9.9) 64 (39.8)
31 (19.4) 20 (12.5) 54 (33.5)
0.45
0.38
0.09 0.37 0.50
Data are expressed as mean ± standard deviation, median [interquartile range], or no. (%).
median protein C level was 101% (IQR 88–122%). This was significantly lower as compared to the median levels in patients with prostate cancer and lung cancer, (111% (IQR 93–134%), p = 0.006 and 108% (IQR 95– 129%), p = 0.006, respectively). The mean and median protein C levels in patients treated with chemotherapy, radiotherapy and surgery prior to enrolment are available in the Supplement. There were no statistically significant differences between treated and untreated patients. 3.2. Association between protein C and mortality Among the 477 patients, 291 (61%) died during 466 patient-years (62 per 100 patient-years). In the lowest tertile of protein C, 102 patients died during 136 patient-years (75 per 100 patient-years). In the middle tertile, 100 patients per 165 patient-years (60 per 100 patientyears) and in the highest tertile 90 patients per 165 patient-years (54 per 100 patient-years) died (Table 2). Fig. 1 provides Kaplan Meier curves for event-free survival for the patients in the low, middle and high tertile of protein C level. Median survival times were 10.4 months (6.5–19.5), 12.7 months (7.8–26.6) and 14.8 months (7.0–25.8), respectively. As shown in Table 3, patients in the lowest tertile of protein C had a significantly increased mortality risk as compared to the highest tertile (HR 1.39, 95% CI 1.05–1.85, adjusted for sex, age and use of nadroparin). As compared to patients in the middle tertile, we observed no statistically significant difference in risk of mortality (adjusted HR 1.10, 95% CI 0.83–1.46). Overall, lower levels of protein C were associated with increased mortality (HR for trend 1.18, 95% CI 1.02–1.36, adjusted for age, sex and nadroparin use). Table 2 Mortality and thromboembolic events according to protein C levels.
Death – no./patient-years Mortality rate per 100 patient-years Thromboembolic events – no/patient-years Rate of thromboembolic events per 100 patient-years a b c d
Low tertile b97% N = 155
Medium tertile 97–121% N = 161
High tertile ≥121% N = 161
102/136 75 11/130 8
99/165a 60 11/160c 7
90/165b 54 17/158d 11
p-Value = 0.114 as compared to the lowest tertile. p-Value = 0.026 as compared to the lowest tertile. p-Value = 0.636 as compared to the lowest tertile. p-Value = 0.524 as compared to the lowest tertile.
3
In the model with protein C as a continuous variable, lower protein C levels were associated with higher mortality, but this association was not statistically significant (adjusted HR 1.004, 95% CI 1.00–1.008, p = 0.07). Median protein C levels were 105% (IQR 91–127%) for the patients that died during follow up and 112% (IQR 94–129) for the patients who were alive at the end of follow up. In Fig. 2a–c, Kaplan-Meier curves show the cumulative survival for patients with low, medium and high protein C levels, stratified for cancer type. For each of the cancer types, the patients in the lowest tertile of protein C had the shortest median survival time. However, the association between protein C tertiles and mortality did not reach statistical significance for any of the cancer types (adjusted HR for trend in prostate cancer 1.03 (95% CI 0.80–1.33), in lung cancer 1.25 (95% CI 0.96– 1.61), and in pancreatic cancer 1.14 (95% CI 0.88–1.51)). The influence of cancer type on the association between protein C levels and mortality was further explored by redefining the tertiles per cancer type (Supplement). The results of this analysis were similar to the results presented here. When stratifying the association between mortality and protein C for occurrence of thromboembolic events, a clear association was found in the 438 patients without thromboembolic events during follow up (HR for trend 1.22, 95% CI 1.05–1.41, p = 0.01). In the 39 patients with thromboembolic events during follow up, this association was not found (HR for trend 0.88, 95% CI 0.55–1.43) (data not shown). 3.3. Association between protein C and thromboembolic events Overall, there were 39 thromboembolic events during 449 patientyears (9 per 100 patient-years), 11 per 131 patient-years (8 per 100 patient-years) in the lowest tertile group, 11 per 160 patient-years (7 per 100 patient-years) in the mid tertile and 17 per 158 patient-years (11 per 100 patient-years) in the highest tertile of protein C activity (Table 2). There were no statistically significant differences between the tertiles of protein C in the occurrence of thromboembolic events during follow-up. The association between protein C tertiles and thromboembolic events was not statistically significant for any of the cancer types, although there was a trend for prostate cancer (adjusted HR for trend: in prostate cancer 1.94 (95% CI 0.94–4.01), in lung cancer 1.09 (95% CI 0.45–2.63), and in pancreatic cancer 0.99 (95% CI 0.49–1.99)). 4. Discussion This study suggests that protein C levels were inversely associated with mortality in patients with advanced prostate, lung and pancreatic cancer. This association seems not to be affected by nadroparin use, and seemed most pronounced in patients with lung cancer. Our present findings are in line with the recent publication of Tafur et al. [22]. They evaluated protein C as a predictor of cancer-associated thrombosis and mortality in 241 patients with solid tumours of any type and stage, who were scheduled to receive chemotherapy. In this study, patients did not receive anticoagulant drugs. Most patients had gynaecological, breast or pancreatic cancer, and 32% of patients had metastatic disease. Average follow-up was 10.4 months, and during this follow-up 37 patients (15%) died. The authors found that the lowest quartile of protein C activity (≤118%) was associated with higher mortality as compared to all other patients (HR 2.8 adjusted for age, ECOG score and metastatic disease at inclusion). Noteworthy is that the mortality in our population was much higher, 61% versus 15%. Also, the protein C levels found by Tafur et al. were higher overall, with a median of 137.5% as compared to 107% in our population. This could be due to differences in patient characteristics, such as type of cancer and chemotherapeutic regimen, and could also be influenced by the method used for determining protein C. Tafur et al. also found that low protein C was associated with increased incidence of VTE. We did not see this latter association in our population. The overall incidence of VTE during
4
I.T. Wilts et al. / Thrombosis Research 154 (2017) 1–6
Fig. 1. Kaplan-Meier curve of the survival in months according to protein C level. The numbers of patients at risk are given at the bottom of the chart. The low tertile of protein C levels (b97%) is represented in blue, the mid tertile (97–121%) in red and the high tertile (≥121%) in green.
follow up was 13% in the study by Tafur, as compared to only 8.2% in our population. Another recent study, by Sun et al. [23], evaluated the prognostic value of different coagulation parameters in 139 patients with pancreatic cancer and 40 age- and sex-matched healthy controls. Laboratory measurements including protein C were performed before the start of anti-cancer treatment. They found that protein C was significantly lower in patients with cancer as compared to the healthy controls with a median of 103.4% (IQR 89.8–118.2) vs. 115.8% (IQR 107.0– 124.6), respectively. However, there was no association between protein C levels and survival in their study. In the patients with a protein C level below the median, the 1-year survival rate was 34.1%. In the patients with protein C levels equal to or above the median, on the other hand, the 1-year survival rate was 50.4%. This difference was not statistically significant, with a p-value of 0.099. When comparing these results to our study, it should be noted that the study by Sun et al. included patients with various stages of cancer, whereas our analysis only included patients with advanced cancer. This is reflected in the higher survival rates in the previous study. The study by Sun et al. confirms the presence of a hypercoagulable state in patients with pancreatic cancer, and also shows that coagulation parameters can have prognostic value in cancer patients. An association between coagulation and cancer has been demonstrated by several research groups [7,8,22,23]. However, the nature and extent of this relationship is still poorly understood. The underlying mechanism for the association between protein C and mortality may be due to the effects of activated protein C on cancer cell migration, as stated in the introduction [15]. Hypothetically, higher levels of protein C in cancer patients could lead to increased generation of activated protein C
Table 3 Association between protein C levels and mortality.
High (≥121%) Medium (97–121%) Low (b97%)
Unadjusted HR
Adjusted HR
1 (reference) 1.09 (0.82–1.45) 1.39 (1.05–1.85)
1 (reference) 1.10 (0.83–1.46) 1.36 (1.05–1.85)
HR for trend
1.18 (1.02–1.37) Data are expressed as HR (95% CI). The highest tertile of protein C is used as a reference category. Adjustments were made for age, sex and use of nadroparin.
around tumour cells, thereby reducing procoagulant activity and metastasis. In contrast, in vitro research showed that activated protein C increased cancer cell invasion and chemotaxis in a concentration dependent manner in the presence of EPCR and PAR-1 [24,25]. This could indicate that there are so far unknown co-factors or mechanisms involved in the interaction between protein C and cancer in vivo. In line with this hypothesis, Crudele et al. found that the anti-metastatic properties of zymogen protein C in vivo were independent of both its anticoagulant function and EPCR, PAR-1 and PAR-4 mediation [26]. This observation might also explain why the association between protein C activity and mortality in our study was not influenced by use of nadroparin. In conclusion, the role of protein C in cancer progression is complex and so far not completely understood. It is possible that protein C is a mere reflection of overall health status, in which the patients with lower protein C levels represent the patients with the worst prognosis. In our study, the patients with pancreatic cancer probably have the worst prognosis. Protein C levels were indeed lower in these patients. However, as shown in Fig. 2a–c, the association between low protein C and mortality could be found in all three cancer types. The association between protein C and mortality was also found in animal studies, where the mice probably had comparable health status [17]. Malnutrition in cancer patients could theoretically also lead to low levels of protein C, because production of protein C is vitamin K dependent. However, in all three tertiles of our study population, mean BMI was between 24.9 and 25.7 kg/m2. Besides, all patients had a Karnofsky score N 60, indicating they should be capable of sufficient selfcare and nutrition. This makes malnutrition an unlikely explanation for the differences in protein C activity. We had no information on liver function disorders or the presence of liver metastases, therefore we could not analyse possible effects of these conditions on protein C levels. The strength of our study is a relatively large sample size and a nearcomplete follow-up. There are some limitations we should address. First, this was a secondary analysis of the INPACT study. This means the study was not designed to test our hypothesis, and we did not include possible confounders such as other factors or markers of the coagulation system. However, the analysis was part of the initial study design as an exploratory analysis, and the collection of both blood and
I.T. Wilts et al. / Thrombosis Research 154 (2017) 1–6
5
Fig. 2. a–c: Kaplan-Meier curves for the types of cancer. The numbers of patients at risk are given at the bottom of the charts. Fig. 2a: patients with prostate cancer. Fig. 2b: patients with non-small cell lung cancer. Fig. 2c: patients with pancreatic cancer. The differences in survival are not statistically significant for any cancer type. The low tertile of protein C levels (b97%) is represented in blue, the mid tertile (97–121%) in red and the high tertile (≥121%) in green.
6
I.T. Wilts et al. / Thrombosis Research 154 (2017) 1–6
data were performed prospectively. Furthermore, our patients had three different types of advanced cancer. Variations in the biochemical mechanisms of these types of cancer could lead to different effects of protein C levels on prognosis. We compensated for this by performing the analysis for the groups separated according to cancer type, but this led to smaller patient groups and loss of power. Redefining the tertiles of protein C per cancer type did not change the results (Supplement). We separated our cohort into three groups based on the tertiles of protein C levels at baseline. The upper limit of the lowest group was well within the range of what is considered to be normal protein C activity in the general population. However, the ranges for normal protein C levels were established with regard to haemostatic disorders. Probably, different cut-off levels should be used to identify cancer patients with higher risk of mortality. This requires analysis of other cancer cohorts with different cancer types. A better understanding of the interaction between protein C and cancer progression could lead to new therapeutic interventions using some form of protein C. Protein C concentrates are available for the treatment of severe congenital protein C deficiency. In conclusion, this study shows that protein C is associated with mortality in patients with advanced cancer. If these results are confirmed in other cancer cohorts, protein C may be a valuable tool for prognosis in cancer and may even lead to the development of new treatment forms for cancer. Acknowledgments We thank Marian Weijne, Wil Kopatz and Lucy Leverink for their technical assistance. This analysis was supported by Aspen Pharma with an unrestricted grant (previously GSK). There are no personal conflicts of interest for any of the authors. Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.thromres.2017.03.001. References [1] H.K. Chew, T. Wun, D. Harvey, H. Zhou, R.H. White, Incidence of venous thromboembolism and its effect on survival among patients with common cancers, Arch. Intern. Med. 166 (4) (2006 Feb 27) 458–464. [2] J.W. Blom, C.J. Doggen, S. Osanto, F.R. Rosendaal, Malignancies, prothrombotic mutations, and the risk of venous thrombosis, JAMA 293 (6) (2005 Feb 9) 715–722. [3] M. Carrier, G. Le Gal, P.S. Wells, D. Fergusson, T. Ramsay, M.A. Rodger, Systematic review: the Trousseau syndrome revisited: should we screen extensively for cancer in patients with venous thromboembolism? Ann. Intern. Med. 149 (5) (2008 Sep 2) 323–333. [4] M. Carrier, A. Lazo-Langner, S. Shivakumar, V. Tagalakis, R. Zarychanski, S. Solymoss, et al., Screening for occult cancer in unprovoked venous thromboembolism, N. Engl. J. Med. 373 (8) (2015 Aug 20) 697–704. [5] A.A. Khorana, C.W. Francis, E. Culakova, N.M. Kuderer, G.H. Lyman, Frequency, risk factors, and trends for venous thromboembolism among hospitalized cancer patients, Cancer 110 (10) (2007 Nov 15) 2339–2346.
[6] C. Ay, D. Dunkler, R. Pirker, J. Thaler, P. Quehenberger, O. Wagner, et al., High Ddimer levels are associated with poor prognosis in cancer patients, Haematologica 97 (8) (2012 Aug) 1158–1164. [7] J. Thaler, C. Ay, N. Mackman, R.M. Bertina, A. Kaider, C. Marosi, et al., Microparticleassociated tissue factor activity, venous thromboembolism and mortality in pancreatic, gastric, colorectal and brain cancer patients, J. Thromb. Haemost. 10 (7) (2012 Jul) 1363–1370. [8] A.Y. Lee, F.R. Rickles, J.A. Julian, M. Gent, R.I. Baker, C. Bowden, et al., Randomized comparison of low molecular weight heparin and coumarin derivatives on the survival of patients with cancer and venous thromboembolism, J. Clin. Oncol. 23 (10) (2005 Apr 1) 2123–2129. [9] M. Altinbas, H.S. Coskun, O. Er, M. Ozkan, B. Eser, A. Unal, et al., A randomized clinical trial of combination chemotherapy with and without low-molecular-weight heparin in small cell lung cancer, J. Thromb. Haemost. 2 (8) (2004 Aug) 1266–1271. [10] A.K. Kakkar, M.N. Levine, Z. Kadziola, N.R. Lemoine, V. Low, H.K. Patel, et al., Low molecular weight heparin, therapy with dalteparin, and survival in advanced cancer: the fragmin advanced malignancy outcome study (FAMOUS), J. Clin. Oncol. 22 (10) (2004 May 15) 1944–1948. [11] F.F. van Doormaal, M. Di Nisio, H.M. Otten, D.J. Richel, M. Prins, H.R. Buller, Randomized trial of the effect of the low molecular weight heparin nadroparin on survival in patients with cancer, J. Clin. Oncol. 29 (15) (2011 May 20) 2071–2076. [12] F.R. Rickles, A. Falanga, Molecular basis for the relationship between thrombosis and cancer, Thromb. Res. 102 (6) (2001) V215–V224. [13] C.A. Spek, V.R. Arruda, The protein C pathway in cancer metastasis, Thromb. Res. 129 (Suppl. 1) (2012 Apr) S80–S84. [14] J.H. Griffin, B.V. Zlokovic, L.O. Mosnier, Activated protein C: biased for translation, Blood 125 (19) (2015 May 7) 2898–2907. [15] G.L. Van Sluis, T.M. Niers, C.T. Esmon, W. Tigchelaar, D.J. Richel, H.R. Buller, et al., Endogenous activated protein C limits cancer cell extravasation through sphingosine1-phosphate receptor 1-mediated vascular endothelial barrier enhancement, Blood 114 (9) (2009 Aug 27) 1968–1973. [16] M. Bezuhly, R. Cullen, C.T. Esmon, S.F. Morris, K.A. West, B. Johnston, et al., Role of activated protein C and its receptor in inhibition of tumor metastasis, Blood 113 (14) (2009 Apr 2) 3371–3374. [17] J.M. Crudele, G.L. Van Sluis, P. Margaritis, J.I. Siner, M. Sliozberg, J. Maurer, et al., Insights into the mechanism of zymogen protein C protection against cancer progression, Blood 120 (21) (2012 Nov 16). [18] J.M. Crudele, G.L. Van Sluis, P. Margaritis, J.I. Siner, M. Sliozberg, J. Maurer, et al., Modulating cancer progression with zymogen protein C in a murine model, Mol. Ther. (2012 May 20) S266–S267. [19] J.A. Lorente, L.J. Garcia-Frade, L. Landin, R. de Pablo, C. Torrado, E. Renes, et al., Time course of hemostatic abnormalities in sepsis and its relation to outcome, Chest 103 (5) (1993 May) 1536–1542. [20] B. Fellner, M. Rohla, R. Jarai, P. Smetana, M.K. Freynhofer, F. Egger, et al., Activated protein C levels and outcome in patients with cardiogenic shock complicating acute myocardial infarction, Eur. Heart J. Acute Cardiovasc. Care (2016 Mar 2) http://dx.doi.org/10.1177/2048872616637036. [21] C.P. Klerk, S.M. Smorenburg, H.M. Otten, A.W. Lensing, M.H. Prins, F. Piovella, et al., The effect of low molecular weight heparin on survival in patients with advanced malignancy, J. Clin. Oncol. 23 (10) (2005 Apr 1) 2130–2135. [22] A.J. Tafur, G. Dale, M. Cherry, J.D. Wren, A.S. Mansfield, P. Comp, et al., Prospective evaluation of protein C and factor VIII in prediction of cancer-associated thrombosis, Thromb. Res. 136 (6) (2015 Oct 8) 1120–1125. [23] W. Sun, H. Ren, C.T. Gao, W.D. Ma, L. Luo, Y. Liu, et al., Clinical and prognostic significance of coagulation assays in pancreatic cancer patients with absence of venous thromboembolism, Am. J. Clin. Oncol. 38 (6) (2015 Dec) 550–556. [24] L.M. Beaulieu, F.C. Church, Activated protein C promotes breast cancer cell migration through interactions with EPCR and PAR-1, Exp. Cell Res. 313 (4) (2007 Feb 15) 677–687. [25] H. Althawadi, H. Alfarsi, S. Besbes, S. Mirshahi, E. Ducros, A. Rafii, et al., Activated protein C upregulates ovarian cancer cell migration and promotes unclottability of the cancer cell microenvironment, Oncol. Rep. 34 (2) (2015 Aug) 603–609. [26] J.M. Crudele, G.L. Van Sluis, P. Margaritis, J.I. Siner, J. Maurer, S. Zhou, et al., Mechanistic insights into zymogen protein C protection against cancer progression, J. Thromb. Haemost. 11 (2013 Jul) 51–52.