Research Article For reprint orders, please contact: [email protected]
Plasma fibrinogen and blood platelet counts are associated with response to neoadjuvant therapy in esophageal cancer
Aim: To investigate coagulatory factors in predicting response to neoadjuvant therapy (NeoTr) in esophageal cancer (EC). Methods: We investigated the relevance of coagulatory factors in 84 EC patients (56 adenocarcinomas, 28 squamous cell cancer) who received NeoTr. Plasma fibrinogen (PFR), peripheral blood platelet counts (PBPC) and C-reactive protein (CRP) were determined before NeoTr. Response was classified as tumor regression grades. Results: Patients with good response to NeoTr had significantly higher PFR (p = 0.006), CRP (p = 0.002) and PBPC levels (p = 0.034) when compared with others. Only, PFR remained an independent factor influencing tumor regression (p = 0.0064, coefficient of regression: -0.003). No association with survival was observed. Conclusion: PFR and to a lesser extent PBPC and CRP might be considered as a predictive marker for the response to NeoTr in EC. Keywords: CRP • esophageal carcinoma • neoadjuvant treatment • plasma fibrinogen • platelets • tumor regression grade
Esophageal cancer (EC) is the eight most common cancer worldwide [1] . In the USA, 17,990 people are estimated to be diagnosed annually with EC and 15,210 people will ultimately die of their disease [2] . ECs are histologically classified as adenocarcinomas (AC) and squamous cell carcinomas (SCC) [3] . Preoperative chemoradiation followed by surgery is the most common neoadjuvant treatment approach for patients with resectable esophageal cancer, although this approach remains to be further investigated [4] . In order to avoid a treatment with uncertain benefit, it is of particular importance to identify the patients, who might show a better response after neoadjuvant treatment. Preoperative factors predicting the treatment response of esophageal cancer have been identified, however, clinically feasible methods to predict the response are still urgently required [5] . Activation of the coagulation cascade by cancer cells has been shown to be associated with tumor progression in a variety of cancers [6] . Accordingly, several reports have shown the association of preoperative levels
10.2217/BMM.14.111 © 2015 Future Medicine Ltd
of coagulation factors in lung [7,8] , ovarian [9] , gastric [10,11] , pancreatic [12,13] , hepatocellular [14] and urothelial carcinoma [15] , but surprisingly only few data on its relevance in combination with neoadjuvant therapy in general exist [16,17] . With respect to the limited evidence regarding the relevance of plasma coagulatory factors in neoadjuvant therapy of EC, aim of this study was to investigate their clinical role in those patients treated with neoadjuvant therapy and subsequent surgery.
Aysegül Ilhan-Mutlu1,5, Patrick Starlinger2, Thomas Perkmann3, Sebastian F Schoppmann2,5, Matthias Preusser1,5 & Peter Birner*,4,5 1 Department of Medicine I, Clinical Division of Oncology, Medical University of Vienna, Währinger Gürtel 18-20, A-1090 Vienna, Austria 2 Department of Surgery, Medical University of Vienna, Währinger Gürtel 18-20, A-1090 Vienna, Austria 3 Department of Laboratory Medicine, Medical University of Vienna, Währinger Gürtel 18-20, A-1090 Vienna, Austria 4 Department of Pathology, Medical University of Vienna, Währinger Gürtel 18-20, A-1090 Vienna, Austria 5 Comprehensive Cancer Center Vienna, Gastroesophageal Tumors Unit, Medical University of Vienna, Währinger Gürtel 18-20, A-1090 Vienna, Austria *Author for correspondence: Tel.: +43 140 400 3650 Fax: +43 140 400 3707 peter.birner@ meduniwien.ac.at
Methods Patient cohort
All patients who received neoadjuvant therapy for esophageal cancer and underwent subsequent surgery at the Medical University of Vienna (Austria) between August 1996 and September 2011 were included in this retrospective study. Only patients without any evidence of metastases at initial diagnosis underwent surgical removal of the tumor. After a mean period of 38 ± 20 (SD) days after completion of neoadjuvant treatment,
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Research Article Ilhan-Mutlu, Starlinger, Perkmann, Schoppmann, Preusser & Birner esophagectomy was carried out. From all patients, plasma coagulatory factors including plasma fibrinogen (PFR), prothrombin time (PT), thrombin time (TT), partial thromboplastin time (aPTT), antithrombin III (AT3), peripheral blood platelet count (PBPC) and acute phase protein C-reactive Protein (CRP) before start of neoadjuvant therapy were available. Process of blood samples & analysis of serum markers
Blood collections were performed as a routine procedure from peripheral veins in a heparin, citrat or EDTA precoated vacuumed tubes for CRP, PBPC and other coagulatory factors, respectively. These were immediately transferred to the Department of Laboratory Medicine, Medical University of Vienna, where the analysis of the factors using standard automated hematology analyzers was carried out. During the observational period, the following hematology analyzers were applied: Until 2001 Sysmex NE-8000 (TOA Medical Electronics, Kobe, Japan), since 2001 Sysmex XE-2100 (Sysmex Corporation, Kobe, Japan). Histopathological investigation
Response to neoadjuvant therapy was assessed on all available H&E stained slides of esophagectomy specimens according to Mandard [18] . In brief, tumor regression grade (TRG) was quantitated in five grades: TRG 1 (complete regression) shows no residual cancer cells and fibrosis extending through the different layers of the esophageal wall; TRG 2 is characterized by the presence of rare residual cancer cells scattered through the fibrosis; TRG 3 is characterized by an increase in the number of residual cancer cells compared with TRG 2, but fibrosis still predominating; TRG 4 shows residual cancer outgrowing fibrosis and TRG 5 is characterized by absence of regressive changes [18] . For the evaluation of the response rate, all available slides from the tumor bed were evaluated from each surgical specimen (about 3–7/case). The density of inflammatory infiltration in biopsies and surgical specimens of samples was assessed in H&E stained slides and scored as 0 (absent), 1 (weak), 2 (moderate) or 3 (dense). This study was approved by the local ethics committee. Statistics
Student’s t-tests and linear regression were used as appropriate. All numbers given are mean values ± standard deviations (SD), if not stated otherwise. Overall survival (OS) was defined as the time between primary surgery and the patient’s death, survival until the end of the observation period was considered as censored
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observation. Disease-free survival (DFS) was defined as time from the day of surgery until first evidence of disease-progression. Univariate analysis of survival was performed using univariate Cox regression or log-rank test, whereas the Cox proportional hazards model was used for the multivariable analysis. Patients’ age, tumor and lymph node stage, PFR levels, PBPC and CRP were included as independent variables into the Cox model. A two-tailed p-value of ≤0.05 was considered as significant, SPSS 20.0 was used for all calculations. Results In total, 84 patients (70 males, 14 females) were included into this retrospective study, mean age was 63 ± 9 years. Fifty-six patients suffered from esophageal AC, 28 from SCC. Histopathological diagnosis via tumor biopsy was performed after onset of clinical symptoms. Seventy-four patients received neoadjuvant chemotherapy, eight neoadjuvant chemoradiation and two neoadjuvant radiotherapy. All patients underwent radical surgery after the end of the neoadjuvant therapy. No association of the concentrations of coagulatory factors with histopathological parameters including histological subtype, ypT and ypN status at subsequent surgery or histological grading in biopsies was found (p > 0.05). Although none of the patients were diagnosed with distant metastases at the time point of the initiation of the neoadjuvant treatment, four patients were staged as ypM1 at histological investigation of surgical specimens as those exhibited peritoneosis carcinomatosa. These patients showed significantly lower levels of PFR when compared with the patients without evidence of distant metastases (366 ± 31 mg/dl vs 467 ± 115 mg/dl; p = 0.001, t-test; n = 4 vs n = 80; respectively, Figure 1). CRP in biopsies was associated with tumor stage at time of surgery (p = 0.018; ANOVA) at analysis of all cases. Post hoc Tukey tests revealed that mean CRP was significantly higher in ypT0 tumors compared with ypT1a-ypT3 (p < 0.05, respectively, Table 1). No association of lymph node stage, ypM stage, histological subtype or grading with CRP levels was found (p > 0.05). At analysis of AC and SCC separately, CRP (p = 0.029, ANOVA) and PBPC (p = 0.008, ANOVA) were associated with tumor stage only in AC, but not in SCC (p > 0.05). In 81 patients, PFR, CRP and PBPC levels after the end of neoadjuvant therapy, directly before surgery, were available. There were no differences of PFR and CRP levels between pre- and postneoadjuvant treatment (p > 0.05, paired t-tests), whereas PBPC was significantly lower after neoadjuvant therapy (243 ± 75 g/l) than before (261 ± 76 g/l; p = 0.004, paired
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475.00 450.00 425.00 400.00 375.00 350.00 No
Yes ypM status
Figure 1. Mean PFR levels (mg/dl, ± SE) in patients with and without distant metastases (n = 4 vs n = 80, respectively). T-test revealed significantly lower levels of PFR in those patients with distant metastases (p = 0.001). PFR: Plasma fibrinogen; SE: Standard error of the mean.
321 ± 99 versus 248 ± 72 g/l compared with patients without good response. In linear regression analysis using tumor regression grade as dependent variable and kind of adjuvant therapy and PFR, CRP and PBPC levels as independent variables, only PFR remained an independent factor influencing tumor regression (p = 0.011, coefficient of regression: -0.003) in AC, while PBPC and CRP both missed significance in this regression model. In SCC, the regression model reached no significance at all, most probably due to the relatively low number of cases. Mean plasma fibrinogen (mg/dl) ± SE
t-test). No association of the type of neoadjuvant therapy with fibrinogen levels or PBPC after neoadjuvant therapy was found (p > 0.05, t-tests, respectively). A weak positive correlation between PFR and PBPC was found (p < 0.001, Pearson’s coefficient of correlation = 0.343). However, patients with mild thrombocytosis (≥400 g/l) demonstrated significantly higher PFR levels (574 ± 139 vs 456 ± 111, p = 0.045, t-test). A good correlation between CRP and PFR (p < 0.001, Pearson’s coefficient of correlation: 0.582) and a week correlation with PBPC (p = 0.001, coefficient of correlation: -0.0352) was observed. No association of PFR or PBPC before or after neoadjuvant therapy with histological subtypes (AC and SCC) was seen (p > 0.05, t-test). ANOVA tests revealed significant differences in PFR (p < 0.001, Figure 2) and CRP (p = 0.002) levels and PBPC (p = 0.007, Figure 3) between histological regression subtypes at investigation of all samples: Post- hoc Tukey tests demonstrated for PFR significant differences between TRG 1 versus TRG 4 (p = 0.02) and 5 (p = 0.001), TRG 2 versus TRG 5 (p = 0.005) and TRG 3 versus TRG 5 (p = 0.002). For CRP, significant differences between TRG 1 and all other regression grades were found (p < 0.005). For PBPC, Tukey tests revealed significant differences only between TRG 1 versus TRG 4 (p = 0.049) and TRG 5 (p = 0.006). Since TRG 1 was evident in only two patients, further subclassification was performed. Patients with good response to neoadjuvant therapy (n = 12, TRG 1 and 2) had significantly higher PFR (545 ± 106 mg/dl vs 448 ± 106 mg/dl; p = 0.006, t-test), CRP (2.99 ± 5.05 mg/dl vs 1.16 ± 1.77 mg/dl; p = 0.002, t-test) and PBPC levels (304 ± 83 g/l vs 253 ± 73 g/l p = 0.034, t-test) when compared with that of all other patients (TRG 3–5). In linear regression analysis using tumor regression grade as dependent variable and cancer type (AC vs SCC), kind of adjuvant therapy and PFR, CRP and PBPC levels as independent variables, only PFR remained an independent factor influencing tumor regression (p = 0.0064, coefficient of regression: -0.003). PBPC and CRP both missed significance in this regression model. No association of PFR or CRP with the density of inflammatory infiltrate in biopsies or surgical specimens was observed (p > 0.05, ANOVA). When investigating AC and SCC separately, ANOVA tests revealed significant differences in PFR (p < 0.001) and CRP (p = 0.013) levels and PBPC (p = 0.0031) between histological regression subtypes in AC but not in SCC: so in AC patients with good response (TRG 1 and TRG 2) mean PFR was 600 ± 140 versus 455 ± 110 mg/dl, mean CRP was 4.45 ± 6.35 versus 1.21 ± 1.64 mg/dl and mean PBPC was
Mean plasma fibrinogen (mg/dl) ± SE
Hemostatic system in esophageal cancer
800.00 700.00 600.00 500.00 400.00 300.00 1
2
3
4
5
Tumor regression grade
Figure 2. Mean plasma fibrinogen levels (mg/dl, ± SE) in different tumor regression grades (1–5) (n = 2, n = 10, n = 20, n = 41 and n = 11; respectively). ANOVA tests revealed significant differences of PFR levels (p < 0.001) between histological regression subtypes. Post hoc Tukey tests demonstrated significant differences between TRG 1 vresus TRG 4 (p = 0.02) and 5 (p = 0.001), TRG 2 versus TRG 5 (p = 0.005) and TRG 3 versus TRG 5 (p = 0.002). ANOVA: Analyses of variance; PFR: Plasma fibrinogen; SE: Standard error of the mean; TRG: Tumor regression grade.
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Research Article Ilhan-Mutlu, Starlinger, Perkmann, Schoppmann, Preusser & Birner
Table 1. Plasma fibrinogen and peripheral blood platelet counts in correlation to clinical factors. Factor
n
Mean PFR ± SD (mg/dl)
Mean CRP ± SD (mg/dl)
Mean PBPC ± SD (g/l)
Tumor stage
p = 0.068
p= 0.018†
p = 0.058
ypT0
2
670 ± 125
7.43 ± 9.47
404 ± 114
ypT1a
3
428 ± 59
0.28 ± 0.25
261 ± 46
ypT1b
8
409 ± 90
0.3 ± 0.46
231 ± 44
ypT2
24
448 ± 104
1.43 ± 2.34
271 ± 82
ypT3
45
468 ± 120
1.44 ± 2.34
251 ± 72
ypT4
2
546 ± 25
1.57 ± 0.33
316 ± 107
Lymph node stage
p = 0.756
p = 0.643
p = 0.587
ypN0
31
474 ± 110
1.15 ± 1.81
276 ± 69
ypN1
21
454 ± 116
1.75 ± 3.35
245 ± 82
ypN2
16
437 ± 123
2.11 ± 3.57
258 ± 85
ypN3
15
480 ± 122
0.93 ± 1.09
259 ± 76
ypNx
1
389
0.09
193
Histological grading
p = 0.458
p= 0.196
p = 0.174
G1
3
383 ± 75
0.16 ± 0.23
188 ± 92
G2
47
462 ± 111
1.04 ± 2.19
269 ± 68
G3
34
470 ± 122
2.02 ± 3
255 ± 84
Histological subtype
p = 0.212
p = 0.324
p = 0.572
Adenocarcinoma
56
473 ± 122
1.64 ± 2.87
257 ± 79
Squamous cell cancer
28
440 ± 96
1.04 ± 1.87
267 ± 72 p = 0.007
p < 0.001
p = 0.002
TRG 1
Tumor regression grade 2
670 ± 125
7.43 ± 9.47
403 ± 114
TRG 2
10
520 ± 127
2.1 ± 3.99
284 ± 66
TRG 3
20
509 ± 100
2.08 ± 2.35
268 ± 76
TRG 4
41
442 ± 103
0.86 ± 1.46
259 ± 76
TRG 5
11
363 ± 47
0.52 ± 0.74
208 ± 57
†
†
Significant difference.
No association of PT, TT, aPTT and AT3 levels with histopathological parameters and tumor regression grades (1–5) was found (p > 0.05).
At survival analysis of AC and SCC separately, also no significant results were found (p > 0.05, log rank tests or Cox regression, respectively).
Survival analysis
Discussion In this study, we assessed pretreatment levels of coagulatory and immune response factors in 84 esophageal carcinoma patients including AC and SCC receiving neoadjuvant therapy, and correlated our findings with tumor regression, clinicopathological parameters, prognosis and survival. Higher PFR and PBPC levels as well as CRP in EC patients were significantly associated with better histological response to neoadjuvant treatment. A low correlation between PFR levels and PBPC and a good correlation between PFR and CRP levels was found. PFR levels remained as a significant independent factor influencing tumor regression. To
Mean observation time was 43 ± 6 (SE) months. During this period of time, 46 patients (54.8%) developed recurrent disease, and 44 (52.4%) died. In univariate or multivariable analysis of all cases, no influence of PFR (Figure 4A) and CRP levels and PBPC on DFS and OS was found (p > 0.05, univariate and multivariable Cox regression). A clear trend toward longer DFS was seen in patients with good response to neoadjuvant therapy (TRG 1 and 2) which missed significance (p = 0.073, log-rank test, Figure 4B). No significant association of PT, TT, aPTT or AT3 levels with survival was observed.
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our knowledge, this is the first report showing the association of PFR, CRP and PBPC levels with histological response to neoadjuvant therapy in EC. Tumor formation exhibits characteristics of an abnormal wound-healing process [19] . Normal wound healing includes the clotting of plasma and blood, and the proteins fibrin and fibrinogen. Fibrinogen is a 340-kDa glycoprotein and is synthesized by hepatocytes. Under physiological conditions, plasma levels of fibrinogen range from 200 to 400 mg/dl with a half-life of approximately 4 days [20] . However, the blood levels of fibrinogen increase dramatically in pathological conditions, such as injury, infection and inflammation [21] . Elevation of other inflammation markers including CRP in esophageal carcinoma patients before surgery has been described previously [22,23] . Interestingly, both inflammation parameters, PFR and CRP associated with good response to neoadjuvant treatment in our study, where PFR could keep significance in linear regression models. This might indicate that those parameters serve as markers for the immune system, which might be activated during the neoadjuvant therapy course and play a role for the good response. Based on the significance of PFR as an independent factor of treatment response, this parameter seems to be more effective as a marker for showing the immune response in EC patients. Histopathologic studies revealed the presence of fibrin(ogen) (both fibrin I and II), both at the tumor interface and within the tumor [24–28] . The source of the fibrinogen found in tumor tissue is unknown. It is well documented that the systemic fibrinogen and the cross-linked fibrin reside inside the tumor stroma [29] . However, it has been shown that some tumor cells have A
Mean peripheral blood platelets (G/l) ± SE
Hemostatic system in esophageal cancer
500.00 400.00 300.00 200.00 100.00 2
1
3
4
5
Tumor regression grade
Figure 3. Mean peripheral blood platelets count (g/l, ± SE) in different tumor regression grades (1–5) (n = 2, n = 10, n = 20, n = 41 and n = 11; respectively). ANOVA tests revealed significant differences of PBPC levels (p = 0.007) between histological regression subtypes. Tukey tests revealed significant differences between TRG 1 versus TRG 4 (p = 0.049) and TRG 5 (p = 0.006).ANOVA: Analysis of variance; PBPC: Peripheral blood platelets count; SE: Standard error of the mean; TRG: Tumor regression grade.
the ability to control their own microenvironment by endogenously synthesizing and secreting fibrinogen [30] . Therefore, fibrinogen is generally found to be elevated in the plasma of cancer patients [16,31] . Various growth factors, including VEGF, bFGF and IGF-1, are segregated and protected from degradation in fibrin [32] . In another report, fibrin gels had been implanted into guinea pigs, where they were capable of generating new blood vessels via activation of proangiogenic factors. This suggested that the fibrin gel acts as a matrix for endothelial cell growth [29] . One can assume that the tumors with B 1.0
0.8 0.6 0.4 0.2
Low PFR High PFR
0.0 0
1000
2000
Cumulative diseasefree survival
1.0 Cumulative diseasefree survival
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0.8
TRG 1 and 2
0.6 0.4 0.2
TRG 3, 4 and 5
0.0 3000
Time since surgery (days)
4000
0
1000
2000
3000
4000
Time since surgery (days)
Figure 4. (A) Kaplan–Meier curve demonstrating the DFS between high (PFR: 439.5 mg/dl, n = 42) and low (PFR: 0.05, log-rank test). (B) Kaplan–Meier curve demonstrating higher and lower tumor regression grades (TRG 1 and 2 [n = 12] vs TRG 3, 4 and 5 [n = 72]). A clear trend toward longer DFS was seen in patients with good response to neoadjuvant therapy (TRG 1 and 2), which could not reach significance (p = 0.073, log-rank test). DFS: Disease-free survival; PFR: Plasma fibrinogen; TRG: Tumor regression grade.
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Research Article Ilhan-Mutlu, Starlinger, Perkmann, Schoppmann, Preusser & Birner increased levels of PFR are supplied with higher amount of angiogenic factors, which might lead to a more aggressive character of the tumor. It is a known issue that biologically more aggressive tumors show a better response to chemotherapy [33] . Our findings of elevated plasma levels of fibrinogen in patients with better response might be interpreted in this direction. Palumbo et al. reported decreased formation of lung cancer metastasis both in mice lung cancer and melanoma models in fibrinogen deficient mice [34] . Our observation of lower levels of PFR in patients with metastasis seems to be contradictive to this result. Since fibrinogen can also be synthesized and when necessary deposited by the tumor cells [29] , the endo genously produced fibrinogen might be metabolized for the formation of metastasis in those patients, which might prevent the secretion into the circulation. Elevated plasma fibrinogen was shown to be associated with bad outcome in cancer types including hepatocellular carcinoma, urothelial carcinoma, lung and pancreatic cancer [8,13–15] . Interestingly two studies on the circulating fibrinogen levels in esophageal SCC have been published previously [31,35] . The first study showed an association between higher PFR concentrations and invasiveness and metastatic potential of EC [31] , whereas the second study demonstrated a better outcome with lower PFR levels after administration of primary chemoradiation in SSC patients [35] . Although this seems to be contradictive to our findings, it is important to mention that the basic characteristics of this group differ remarkably when compared with the group of ours. Notably, our cohort included higher numbers of AC cases and all patients received neoadjuvant therapy as part of their treatment schema, which was not the case for the previous reports [31,35] . Furthermore, our report exclusively evaluated European individuals, which might have an influence for the differential involvement of molecular cascades in EC. In our study, another component of hemostatic system, namely platelets, was shown to be associated with better response to neoadjuvant treatment in EC patients. Like all other components of the hemostatic system, the precise role of platelets in tumor biology as well as in angiogenesis remains to be established, since platelets might have the potential to play both pro- and antiangiogenic role. In this direction, we were recently able to show that platelets play an important role in tumor lymphangiogenesis in EC [36] . Platelets are important storages and transporters of VEGF/VEGF-A [37] and other proangiogenic factors [38–46] and angiogenesis inhibitors [46–52] and can respond to the stimuli of various growth factors as they express several growth factor receptors [53] . PBPC was significantly reduced in our cohort by neoadjuvant therapy. It might be hypothesized
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that some EC are depended from platelets-mediated growth factor signaling, and so patients might benefit from the reduction of PBPC by neoadjuvant therapy. Preoperative higher platelet count was shown to be associated with shorter survival in patients with colorectal cancer, metastatic breast cancer and pancreatic cancer [13,54,55] . However, in neoadjuvant setting, there exist far less data [16] . Particularly for esophageal cancer, preoperative platelet count was evaluated in a large cohort of patients who received primary esophagecetomy, where no association with prognosis could be shown [56] . This report together with our finding suggest that platelet count has no influence on the prognosis, however, might associate with better response to neoadjuvant treatment. Moreover, the elevation of coagulatory factors and CRP might be a result of indirect reaction, which is based on the tissue changes of the tumour during induction therapy. PFR, CRP and PBPC are acute-phase reactants, playing a key role in inflammation [57,58] . Good response to neoadjuvant therapy, resulting in increased necrosis of tumor cells, and subsequent induction of inflammation might explain elevation of those parameters [59] . To sum up, neoadjuvant chemotherapy has been recognized as one of the promising curative modalities for esophageal cancer. However, no accurate biological factor predicting the responsiveness to chemotherapy is present. Defining a circulating marker, which can be easily measured by objective methods, is of particular importance. Herein we demonstrate that PFR and to a lesser extent PBPC and CRP could be feasible markers which might be used for the estimation of the response to neoadjuvant therapy in EC patients. Since we followed a retrospective approach in the frame of this study, the potential importance of PFR and PBPC should be validated in larger prospective cohorts. Future perspective Neoadjuvant chemotherapy has been recognized as one of the promising curative modalities for esophageal cancer. However, no accurate biological factor predicting the responsiveness to chemotherapy is present. Defining a circulating marker, which can be easily measured by objective methods, is of particular importance. Herein we demonstrate that PFR and to a lesser extent PBPC and CRP could be feasible markers which might be used for the estimation of the response to neoadjuvant therapy in EC patients. The potential importance of PFR and PBPC should be validated in larger prospective cohorts. Financial & competing interests disclosure The authors have no relevant affiliations or financial involvement with any organization or entity with a financial
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interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties. No writing assistance was utilized in the production of this manuscript.
Research Article
Ethical conduct of research The authors state that they have obtained appropriate institutional review board approval or have followed the principles outlined in the Declaration of Helsinki for all human or animal experimental investigations. In addition, for investigations involving human subjects, informed consent has been obtained from the participants involved.
Executive summary Esophageal cancer & neoadjuvant therapy • Esophageal cancer (EC) is characterized by high mortality rates, and complete surgical removal is the only possible cure for resectable disease. An increasing rate of patients receives neoadjuvant therapy before surgery; however factors predicting response are desirable. Involvement of several hemostatic factors within tumor growth and angiogenesis has been demonstrated recently. In this study, we investigated the clinical relevance of coagulatory factors and acute phase response molecule C-reactive protein (CRP) in EC patients receiving neoadjuvant therapy.
Patients & methods • Eighty-four EC patients (adenocarcinoma [n = 56], squamous cell carcinoma [n = 28]) who received neoadjuvant therapy and subsequent surgical resection were included into this retrospective study. Plasma coagulatory factors including plasma fibrinogen (PFR), prothrombin time (PT), thrombin time (TT), partial thromboplastin time (aPTT), antithrombin III (AT3), peripheral blood platelet counts (PBPC) and CRP were determined before the start of neoadjuvant therapy. Response to neoadjuvant therapy was classified in surgical specimens according to Mandard as tumor regression grades (TRG) 1–5 (complete regression to no response). Blood parameters were correlated to TRG, histopathological variables and survival.
Investigation of plasma levels between pre- & post-neoadjuvant treatement • There were no differences of PFR and CRP levels between pre- and post-neoadjuvant treatment, whereas PBPC was significantly lower after neoadjuvant therapy.
Plasma levels of coagulatory factors & CRP in different TRGs • Patients with good response to neoadjuvant therapy (n = 12, TRG 1 and 2) had significantly higher PFR, CRP and PBPC levels when compared with that of all other patients (TRG 3–5).
Influence of coagulatory factors & CRP on the tumor regression • In linear regression analysis using tumor regression grade as dependent variable and cancer type (AC vs SCC), kind of adjuvant therapy and PFR, CRP and PBPC levels as independent variables, only PFR remained an independent factor influencing tumor regression. PBPC and CRP both missed significance in this regression model.
Lower PFR levels in patients with metastases • Although none of the patients were diagnosed with distant metastases at the time point of the initiation of the neoadjuvant treatment, four patients were staged as ypM1 at histological investigation of surgical specimens. These patients showed significantly lower levels of PFR when compared with the patients without evidence of distant metastases.
Influence of coagulatory factors & CRP on the survival • In univariate or multivariable analysis of all cases, no influence of PFR and CRP levels and PBPC on DFS and OS was found. A clear trend toward longer DFS was seen in patients with good response to neoadjuvant therapy (TRG 1 and 2), which missed significance.
Discussion • Herein we demonstrate that PFR and to a lesser extent PBPC and CRP could be feasible markers which might be used for the estimation of the response to neoadjuvant therapy in EC patients.
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Plasma fibrinogen and blood platelet counts are associated with response to neoadjuvant therapy in esophageal cancer
Aim: To investigate coagulatory factors in predicting response to neoadjuvant therapy (NeoTr) in esophageal cancer (EC). Methods: We investigated the relevance of coagulatory factors in 84 EC patients (56 adenocarcinomas, 28 squamous cell cancer) who received NeoTr. Plasma fibrinogen (PFR), peripheral blood platelet counts (PBPC) and C-reactive protein (CRP) were determined before NeoTr. Response was classified as tumor regression grades. Results: Patients with good response to NeoTr had significantly higher PFR (p = 0.006), CRP (p = 0.002) and PBPC levels (p = 0.034) when compared with others. Only, PFR remained an independent factor influencing tumor regression (p = 0.0064, coefficient of regression: -0.003). No association with survival was observed. Conclusion: PFR and to a lesser extent PBPC and CRP might be considered as a predictive marker for the response to NeoTr in EC. Keywords: CRP • esophageal carcinoma • neoadjuvant treatment • plasma fibrinogen • platelets • tumor regression grade
Esophageal cancer (EC) is the eight most common cancer worldwide [1] . In the USA, 17,990 people are estimated to be diagnosed annually with EC and 15,210 people will ultimately die of their disease [2] . ECs are histologically classified as adenocarcinomas (AC) and squamous cell carcinomas (SCC) [3] . Preoperative chemoradiation followed by surgery is the most common neoadjuvant treatment approach for patients with resectable esophageal cancer, although this approach remains to be further investigated [4] . In order to avoid a treatment with uncertain benefit, it is of particular importance to identify the patients, who might show a better response after neoadjuvant treatment. Preoperative factors predicting the treatment response of esophageal cancer have been identified, however, clinically feasible methods to predict the response are still urgently required [5] . Activation of the coagulation cascade by cancer cells has been shown to be associated with tumor progression in a variety of cancers [6] . Accordingly, several reports have shown the association of preoperative levels
10.2217/BMM.14.111 © 2015 Future Medicine Ltd
of coagulation factors in lung [7,8] , ovarian [9] , gastric [10,11] , pancreatic [12,13] , hepatocellular [14] and urothelial carcinoma [15] , but surprisingly only few data on its relevance in combination with neoadjuvant therapy in general exist [16,17] . With respect to the limited evidence regarding the relevance of plasma coagulatory factors in neoadjuvant therapy of EC, aim of this study was to investigate their clinical role in those patients treated with neoadjuvant therapy and subsequent surgery.
Aysegül Ilhan-Mutlu1,5, Patrick Starlinger2, Thomas Perkmann3, Sebastian F Schoppmann2,5, Matthias Preusser1,5 & Peter Birner*,4,5 1 Department of Medicine I, Clinical Division of Oncology, Medical University of Vienna, Währinger Gürtel 18-20, A-1090 Vienna, Austria 2 Department of Surgery, Medical University of Vienna, Währinger Gürtel 18-20, A-1090 Vienna, Austria 3 Department of Laboratory Medicine, Medical University of Vienna, Währinger Gürtel 18-20, A-1090 Vienna, Austria 4 Department of Pathology, Medical University of Vienna, Währinger Gürtel 18-20, A-1090 Vienna, Austria 5 Comprehensive Cancer Center Vienna, Gastroesophageal Tumors Unit, Medical University of Vienna, Währinger Gürtel 18-20, A-1090 Vienna, Austria *Author for correspondence: Tel.: +43 140 400 3650 Fax: +43 140 400 3707 peter.birner@ meduniwien.ac.at
Methods Patient cohort
All patients who received neoadjuvant therapy for esophageal cancer and underwent subsequent surgery at the Medical University of Vienna (Austria) between August 1996 and September 2011 were included in this retrospective study. Only patients without any evidence of metastases at initial diagnosis underwent surgical removal of the tumor. After a mean period of 38 ± 20 (SD) days after completion of neoadjuvant treatment,
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part of
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Research Article Ilhan-Mutlu, Starlinger, Perkmann, Schoppmann, Preusser & Birner esophagectomy was carried out. From all patients, plasma coagulatory factors including plasma fibrinogen (PFR), prothrombin time (PT), thrombin time (TT), partial thromboplastin time (aPTT), antithrombin III (AT3), peripheral blood platelet count (PBPC) and acute phase protein C-reactive Protein (CRP) before start of neoadjuvant therapy were available. Process of blood samples & analysis of serum markers
Blood collections were performed as a routine procedure from peripheral veins in a heparin, citrat or EDTA precoated vacuumed tubes for CRP, PBPC and other coagulatory factors, respectively. These were immediately transferred to the Department of Laboratory Medicine, Medical University of Vienna, where the analysis of the factors using standard automated hematology analyzers was carried out. During the observational period, the following hematology analyzers were applied: Until 2001 Sysmex NE-8000 (TOA Medical Electronics, Kobe, Japan), since 2001 Sysmex XE-2100 (Sysmex Corporation, Kobe, Japan). Histopathological investigation
Response to neoadjuvant therapy was assessed on all available H&E stained slides of esophagectomy specimens according to Mandard [18] . In brief, tumor regression grade (TRG) was quantitated in five grades: TRG 1 (complete regression) shows no residual cancer cells and fibrosis extending through the different layers of the esophageal wall; TRG 2 is characterized by the presence of rare residual cancer cells scattered through the fibrosis; TRG 3 is characterized by an increase in the number of residual cancer cells compared with TRG 2, but fibrosis still predominating; TRG 4 shows residual cancer outgrowing fibrosis and TRG 5 is characterized by absence of regressive changes [18] . For the evaluation of the response rate, all available slides from the tumor bed were evaluated from each surgical specimen (about 3–7/case). The density of inflammatory infiltration in biopsies and surgical specimens of samples was assessed in H&E stained slides and scored as 0 (absent), 1 (weak), 2 (moderate) or 3 (dense). This study was approved by the local ethics committee. Statistics
Student’s t-tests and linear regression were used as appropriate. All numbers given are mean values ± standard deviations (SD), if not stated otherwise. Overall survival (OS) was defined as the time between primary surgery and the patient’s death, survival until the end of the observation period was considered as censored
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observation. Disease-free survival (DFS) was defined as time from the day of surgery until first evidence of disease-progression. Univariate analysis of survival was performed using univariate Cox regression or log-rank test, whereas the Cox proportional hazards model was used for the multivariable analysis. Patients’ age, tumor and lymph node stage, PFR levels, PBPC and CRP were included as independent variables into the Cox model. A two-tailed p-value of ≤0.05 was considered as significant, SPSS 20.0 was used for all calculations. Results In total, 84 patients (70 males, 14 females) were included into this retrospective study, mean age was 63 ± 9 years. Fifty-six patients suffered from esophageal AC, 28 from SCC. Histopathological diagnosis via tumor biopsy was performed after onset of clinical symptoms. Seventy-four patients received neoadjuvant chemotherapy, eight neoadjuvant chemoradiation and two neoadjuvant radiotherapy. All patients underwent radical surgery after the end of the neoadjuvant therapy. No association of the concentrations of coagulatory factors with histopathological parameters including histological subtype, ypT and ypN status at subsequent surgery or histological grading in biopsies was found (p > 0.05). Although none of the patients were diagnosed with distant metastases at the time point of the initiation of the neoadjuvant treatment, four patients were staged as ypM1 at histological investigation of surgical specimens as those exhibited peritoneosis carcinomatosa. These patients showed significantly lower levels of PFR when compared with the patients without evidence of distant metastases (366 ± 31 mg/dl vs 467 ± 115 mg/dl; p = 0.001, t-test; n = 4 vs n = 80; respectively, Figure 1). CRP in biopsies was associated with tumor stage at time of surgery (p = 0.018; ANOVA) at analysis of all cases. Post hoc Tukey tests revealed that mean CRP was significantly higher in ypT0 tumors compared with ypT1a-ypT3 (p < 0.05, respectively, Table 1). No association of lymph node stage, ypM stage, histological subtype or grading with CRP levels was found (p > 0.05). At analysis of AC and SCC separately, CRP (p = 0.029, ANOVA) and PBPC (p = 0.008, ANOVA) were associated with tumor stage only in AC, but not in SCC (p > 0.05). In 81 patients, PFR, CRP and PBPC levels after the end of neoadjuvant therapy, directly before surgery, were available. There were no differences of PFR and CRP levels between pre- and postneoadjuvant treatment (p > 0.05, paired t-tests), whereas PBPC was significantly lower after neoadjuvant therapy (243 ± 75 g/l) than before (261 ± 76 g/l; p = 0.004, paired
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475.00 450.00 425.00 400.00 375.00 350.00 No
Yes ypM status
Figure 1. Mean PFR levels (mg/dl, ± SE) in patients with and without distant metastases (n = 4 vs n = 80, respectively). T-test revealed significantly lower levels of PFR in those patients with distant metastases (p = 0.001). PFR: Plasma fibrinogen; SE: Standard error of the mean.
321 ± 99 versus 248 ± 72 g/l compared with patients without good response. In linear regression analysis using tumor regression grade as dependent variable and kind of adjuvant therapy and PFR, CRP and PBPC levels as independent variables, only PFR remained an independent factor influencing tumor regression (p = 0.011, coefficient of regression: -0.003) in AC, while PBPC and CRP both missed significance in this regression model. In SCC, the regression model reached no significance at all, most probably due to the relatively low number of cases. Mean plasma fibrinogen (mg/dl) ± SE
t-test). No association of the type of neoadjuvant therapy with fibrinogen levels or PBPC after neoadjuvant therapy was found (p > 0.05, t-tests, respectively). A weak positive correlation between PFR and PBPC was found (p < 0.001, Pearson’s coefficient of correlation = 0.343). However, patients with mild thrombocytosis (≥400 g/l) demonstrated significantly higher PFR levels (574 ± 139 vs 456 ± 111, p = 0.045, t-test). A good correlation between CRP and PFR (p < 0.001, Pearson’s coefficient of correlation: 0.582) and a week correlation with PBPC (p = 0.001, coefficient of correlation: -0.0352) was observed. No association of PFR or PBPC before or after neoadjuvant therapy with histological subtypes (AC and SCC) was seen (p > 0.05, t-test). ANOVA tests revealed significant differences in PFR (p < 0.001, Figure 2) and CRP (p = 0.002) levels and PBPC (p = 0.007, Figure 3) between histological regression subtypes at investigation of all samples: Post- hoc Tukey tests demonstrated for PFR significant differences between TRG 1 versus TRG 4 (p = 0.02) and 5 (p = 0.001), TRG 2 versus TRG 5 (p = 0.005) and TRG 3 versus TRG 5 (p = 0.002). For CRP, significant differences between TRG 1 and all other regression grades were found (p < 0.005). For PBPC, Tukey tests revealed significant differences only between TRG 1 versus TRG 4 (p = 0.049) and TRG 5 (p = 0.006). Since TRG 1 was evident in only two patients, further subclassification was performed. Patients with good response to neoadjuvant therapy (n = 12, TRG 1 and 2) had significantly higher PFR (545 ± 106 mg/dl vs 448 ± 106 mg/dl; p = 0.006, t-test), CRP (2.99 ± 5.05 mg/dl vs 1.16 ± 1.77 mg/dl; p = 0.002, t-test) and PBPC levels (304 ± 83 g/l vs 253 ± 73 g/l p = 0.034, t-test) when compared with that of all other patients (TRG 3–5). In linear regression analysis using tumor regression grade as dependent variable and cancer type (AC vs SCC), kind of adjuvant therapy and PFR, CRP and PBPC levels as independent variables, only PFR remained an independent factor influencing tumor regression (p = 0.0064, coefficient of regression: -0.003). PBPC and CRP both missed significance in this regression model. No association of PFR or CRP with the density of inflammatory infiltrate in biopsies or surgical specimens was observed (p > 0.05, ANOVA). When investigating AC and SCC separately, ANOVA tests revealed significant differences in PFR (p < 0.001) and CRP (p = 0.013) levels and PBPC (p = 0.0031) between histological regression subtypes in AC but not in SCC: so in AC patients with good response (TRG 1 and TRG 2) mean PFR was 600 ± 140 versus 455 ± 110 mg/dl, mean CRP was 4.45 ± 6.35 versus 1.21 ± 1.64 mg/dl and mean PBPC was
Mean plasma fibrinogen (mg/dl) ± SE
Hemostatic system in esophageal cancer
800.00 700.00 600.00 500.00 400.00 300.00 1
2
3
4
5
Tumor regression grade
Figure 2. Mean plasma fibrinogen levels (mg/dl, ± SE) in different tumor regression grades (1–5) (n = 2, n = 10, n = 20, n = 41 and n = 11; respectively). ANOVA tests revealed significant differences of PFR levels (p < 0.001) between histological regression subtypes. Post hoc Tukey tests demonstrated significant differences between TRG 1 vresus TRG 4 (p = 0.02) and 5 (p = 0.001), TRG 2 versus TRG 5 (p = 0.005) and TRG 3 versus TRG 5 (p = 0.002). ANOVA: Analyses of variance; PFR: Plasma fibrinogen; SE: Standard error of the mean; TRG: Tumor regression grade.
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Table 1. Plasma fibrinogen and peripheral blood platelet counts in correlation to clinical factors. Factor
n
Mean PFR ± SD (mg/dl)
Mean CRP ± SD (mg/dl)
Mean PBPC ± SD (g/l)
Tumor stage
p = 0.068
p= 0.018†
p = 0.058
ypT0
2
670 ± 125
7.43 ± 9.47
404 ± 114
ypT1a
3
428 ± 59
0.28 ± 0.25
261 ± 46
ypT1b
8
409 ± 90
0.3 ± 0.46
231 ± 44
ypT2
24
448 ± 104
1.43 ± 2.34
271 ± 82
ypT3
45
468 ± 120
1.44 ± 2.34
251 ± 72
ypT4
2
546 ± 25
1.57 ± 0.33
316 ± 107
Lymph node stage
p = 0.756
p = 0.643
p = 0.587
ypN0
31
474 ± 110
1.15 ± 1.81
276 ± 69
ypN1
21
454 ± 116
1.75 ± 3.35
245 ± 82
ypN2
16
437 ± 123
2.11 ± 3.57
258 ± 85
ypN3
15
480 ± 122
0.93 ± 1.09
259 ± 76
ypNx
1
389
0.09
193
Histological grading
p = 0.458
p= 0.196
p = 0.174
G1
3
383 ± 75
0.16 ± 0.23
188 ± 92
G2
47
462 ± 111
1.04 ± 2.19
269 ± 68
G3
34
470 ± 122
2.02 ± 3
255 ± 84
Histological subtype
p = 0.212
p = 0.324
p = 0.572
Adenocarcinoma
56
473 ± 122
1.64 ± 2.87
257 ± 79
Squamous cell cancer
28
440 ± 96
1.04 ± 1.87
267 ± 72 p = 0.007
p < 0.001
p = 0.002
TRG 1
Tumor regression grade 2
670 ± 125
7.43 ± 9.47
403 ± 114
TRG 2
10
520 ± 127
2.1 ± 3.99
284 ± 66
TRG 3
20
509 ± 100
2.08 ± 2.35
268 ± 76
TRG 4
41
442 ± 103
0.86 ± 1.46
259 ± 76
TRG 5
11
363 ± 47
0.52 ± 0.74
208 ± 57
†
†
Significant difference.
No association of PT, TT, aPTT and AT3 levels with histopathological parameters and tumor regression grades (1–5) was found (p > 0.05).
At survival analysis of AC and SCC separately, also no significant results were found (p > 0.05, log rank tests or Cox regression, respectively).
Survival analysis
Discussion In this study, we assessed pretreatment levels of coagulatory and immune response factors in 84 esophageal carcinoma patients including AC and SCC receiving neoadjuvant therapy, and correlated our findings with tumor regression, clinicopathological parameters, prognosis and survival. Higher PFR and PBPC levels as well as CRP in EC patients were significantly associated with better histological response to neoadjuvant treatment. A low correlation between PFR levels and PBPC and a good correlation between PFR and CRP levels was found. PFR levels remained as a significant independent factor influencing tumor regression. To
Mean observation time was 43 ± 6 (SE) months. During this period of time, 46 patients (54.8%) developed recurrent disease, and 44 (52.4%) died. In univariate or multivariable analysis of all cases, no influence of PFR (Figure 4A) and CRP levels and PBPC on DFS and OS was found (p > 0.05, univariate and multivariable Cox regression). A clear trend toward longer DFS was seen in patients with good response to neoadjuvant therapy (TRG 1 and 2) which missed significance (p = 0.073, log-rank test, Figure 4B). No significant association of PT, TT, aPTT or AT3 levels with survival was observed.
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our knowledge, this is the first report showing the association of PFR, CRP and PBPC levels with histological response to neoadjuvant therapy in EC. Tumor formation exhibits characteristics of an abnormal wound-healing process [19] . Normal wound healing includes the clotting of plasma and blood, and the proteins fibrin and fibrinogen. Fibrinogen is a 340-kDa glycoprotein and is synthesized by hepatocytes. Under physiological conditions, plasma levels of fibrinogen range from 200 to 400 mg/dl with a half-life of approximately 4 days [20] . However, the blood levels of fibrinogen increase dramatically in pathological conditions, such as injury, infection and inflammation [21] . Elevation of other inflammation markers including CRP in esophageal carcinoma patients before surgery has been described previously [22,23] . Interestingly, both inflammation parameters, PFR and CRP associated with good response to neoadjuvant treatment in our study, where PFR could keep significance in linear regression models. This might indicate that those parameters serve as markers for the immune system, which might be activated during the neoadjuvant therapy course and play a role for the good response. Based on the significance of PFR as an independent factor of treatment response, this parameter seems to be more effective as a marker for showing the immune response in EC patients. Histopathologic studies revealed the presence of fibrin(ogen) (both fibrin I and II), both at the tumor interface and within the tumor [24–28] . The source of the fibrinogen found in tumor tissue is unknown. It is well documented that the systemic fibrinogen and the cross-linked fibrin reside inside the tumor stroma [29] . However, it has been shown that some tumor cells have A
Mean peripheral blood platelets (G/l) ± SE
Hemostatic system in esophageal cancer
500.00 400.00 300.00 200.00 100.00 2
1
3
4
5
Tumor regression grade
Figure 3. Mean peripheral blood platelets count (g/l, ± SE) in different tumor regression grades (1–5) (n = 2, n = 10, n = 20, n = 41 and n = 11; respectively). ANOVA tests revealed significant differences of PBPC levels (p = 0.007) between histological regression subtypes. Tukey tests revealed significant differences between TRG 1 versus TRG 4 (p = 0.049) and TRG 5 (p = 0.006).ANOVA: Analysis of variance; PBPC: Peripheral blood platelets count; SE: Standard error of the mean; TRG: Tumor regression grade.
the ability to control their own microenvironment by endogenously synthesizing and secreting fibrinogen [30] . Therefore, fibrinogen is generally found to be elevated in the plasma of cancer patients [16,31] . Various growth factors, including VEGF, bFGF and IGF-1, are segregated and protected from degradation in fibrin [32] . In another report, fibrin gels had been implanted into guinea pigs, where they were capable of generating new blood vessels via activation of proangiogenic factors. This suggested that the fibrin gel acts as a matrix for endothelial cell growth [29] . One can assume that the tumors with B 1.0
0.8 0.6 0.4 0.2
Low PFR High PFR
0.0 0
1000
2000
Cumulative diseasefree survival
1.0 Cumulative diseasefree survival
Research Article
0.8
TRG 1 and 2
0.6 0.4 0.2
TRG 3, 4 and 5
0.0 3000
Time since surgery (days)
4000
0
1000
2000
3000
4000
Time since surgery (days)
Figure 4. (A) Kaplan–Meier curve demonstrating the DFS between high (PFR: 439.5 mg/dl, n = 42) and low (PFR: 0.05, log-rank test). (B) Kaplan–Meier curve demonstrating higher and lower tumor regression grades (TRG 1 and 2 [n = 12] vs TRG 3, 4 and 5 [n = 72]). A clear trend toward longer DFS was seen in patients with good response to neoadjuvant therapy (TRG 1 and 2), which could not reach significance (p = 0.073, log-rank test). DFS: Disease-free survival; PFR: Plasma fibrinogen; TRG: Tumor regression grade.
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Research Article Ilhan-Mutlu, Starlinger, Perkmann, Schoppmann, Preusser & Birner increased levels of PFR are supplied with higher amount of angiogenic factors, which might lead to a more aggressive character of the tumor. It is a known issue that biologically more aggressive tumors show a better response to chemotherapy [33] . Our findings of elevated plasma levels of fibrinogen in patients with better response might be interpreted in this direction. Palumbo et al. reported decreased formation of lung cancer metastasis both in mice lung cancer and melanoma models in fibrinogen deficient mice [34] . Our observation of lower levels of PFR in patients with metastasis seems to be contradictive to this result. Since fibrinogen can also be synthesized and when necessary deposited by the tumor cells [29] , the endo genously produced fibrinogen might be metabolized for the formation of metastasis in those patients, which might prevent the secretion into the circulation. Elevated plasma fibrinogen was shown to be associated with bad outcome in cancer types including hepatocellular carcinoma, urothelial carcinoma, lung and pancreatic cancer [8,13–15] . Interestingly two studies on the circulating fibrinogen levels in esophageal SCC have been published previously [31,35] . The first study showed an association between higher PFR concentrations and invasiveness and metastatic potential of EC [31] , whereas the second study demonstrated a better outcome with lower PFR levels after administration of primary chemoradiation in SSC patients [35] . Although this seems to be contradictive to our findings, it is important to mention that the basic characteristics of this group differ remarkably when compared with the group of ours. Notably, our cohort included higher numbers of AC cases and all patients received neoadjuvant therapy as part of their treatment schema, which was not the case for the previous reports [31,35] . Furthermore, our report exclusively evaluated European individuals, which might have an influence for the differential involvement of molecular cascades in EC. In our study, another component of hemostatic system, namely platelets, was shown to be associated with better response to neoadjuvant treatment in EC patients. Like all other components of the hemostatic system, the precise role of platelets in tumor biology as well as in angiogenesis remains to be established, since platelets might have the potential to play both pro- and antiangiogenic role. In this direction, we were recently able to show that platelets play an important role in tumor lymphangiogenesis in EC [36] . Platelets are important storages and transporters of VEGF/VEGF-A [37] and other proangiogenic factors [38–46] and angiogenesis inhibitors [46–52] and can respond to the stimuli of various growth factors as they express several growth factor receptors [53] . PBPC was significantly reduced in our cohort by neoadjuvant therapy. It might be hypothesized
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that some EC are depended from platelets-mediated growth factor signaling, and so patients might benefit from the reduction of PBPC by neoadjuvant therapy. Preoperative higher platelet count was shown to be associated with shorter survival in patients with colorectal cancer, metastatic breast cancer and pancreatic cancer [13,54,55] . However, in neoadjuvant setting, there exist far less data [16] . Particularly for esophageal cancer, preoperative platelet count was evaluated in a large cohort of patients who received primary esophagecetomy, where no association with prognosis could be shown [56] . This report together with our finding suggest that platelet count has no influence on the prognosis, however, might associate with better response to neoadjuvant treatment. Moreover, the elevation of coagulatory factors and CRP might be a result of indirect reaction, which is based on the tissue changes of the tumour during induction therapy. PFR, CRP and PBPC are acute-phase reactants, playing a key role in inflammation [57,58] . Good response to neoadjuvant therapy, resulting in increased necrosis of tumor cells, and subsequent induction of inflammation might explain elevation of those parameters [59] . To sum up, neoadjuvant chemotherapy has been recognized as one of the promising curative modalities for esophageal cancer. However, no accurate biological factor predicting the responsiveness to chemotherapy is present. Defining a circulating marker, which can be easily measured by objective methods, is of particular importance. Herein we demonstrate that PFR and to a lesser extent PBPC and CRP could be feasible markers which might be used for the estimation of the response to neoadjuvant therapy in EC patients. Since we followed a retrospective approach in the frame of this study, the potential importance of PFR and PBPC should be validated in larger prospective cohorts. Future perspective Neoadjuvant chemotherapy has been recognized as one of the promising curative modalities for esophageal cancer. However, no accurate biological factor predicting the responsiveness to chemotherapy is present. Defining a circulating marker, which can be easily measured by objective methods, is of particular importance. Herein we demonstrate that PFR and to a lesser extent PBPC and CRP could be feasible markers which might be used for the estimation of the response to neoadjuvant therapy in EC patients. The potential importance of PFR and PBPC should be validated in larger prospective cohorts. Financial & competing interests disclosure The authors have no relevant affiliations or financial involvement with any organization or entity with a financial
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interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties. No writing assistance was utilized in the production of this manuscript.
Research Article
Ethical conduct of research The authors state that they have obtained appropriate institutional review board approval or have followed the principles outlined in the Declaration of Helsinki for all human or animal experimental investigations. In addition, for investigations involving human subjects, informed consent has been obtained from the participants involved.
Executive summary Esophageal cancer & neoadjuvant therapy • Esophageal cancer (EC) is characterized by high mortality rates, and complete surgical removal is the only possible cure for resectable disease. An increasing rate of patients receives neoadjuvant therapy before surgery; however factors predicting response are desirable. Involvement of several hemostatic factors within tumor growth and angiogenesis has been demonstrated recently. In this study, we investigated the clinical relevance of coagulatory factors and acute phase response molecule C-reactive protein (CRP) in EC patients receiving neoadjuvant therapy.
Patients & methods • Eighty-four EC patients (adenocarcinoma [n = 56], squamous cell carcinoma [n = 28]) who received neoadjuvant therapy and subsequent surgical resection were included into this retrospective study. Plasma coagulatory factors including plasma fibrinogen (PFR), prothrombin time (PT), thrombin time (TT), partial thromboplastin time (aPTT), antithrombin III (AT3), peripheral blood platelet counts (PBPC) and CRP were determined before the start of neoadjuvant therapy. Response to neoadjuvant therapy was classified in surgical specimens according to Mandard as tumor regression grades (TRG) 1–5 (complete regression to no response). Blood parameters were correlated to TRG, histopathological variables and survival.
Investigation of plasma levels between pre- & post-neoadjuvant treatement • There were no differences of PFR and CRP levels between pre- and post-neoadjuvant treatment, whereas PBPC was significantly lower after neoadjuvant therapy.
Plasma levels of coagulatory factors & CRP in different TRGs • Patients with good response to neoadjuvant therapy (n = 12, TRG 1 and 2) had significantly higher PFR, CRP and PBPC levels when compared with that of all other patients (TRG 3–5).
Influence of coagulatory factors & CRP on the tumor regression • In linear regression analysis using tumor regression grade as dependent variable and cancer type (AC vs SCC), kind of adjuvant therapy and PFR, CRP and PBPC levels as independent variables, only PFR remained an independent factor influencing tumor regression. PBPC and CRP both missed significance in this regression model.
Lower PFR levels in patients with metastases • Although none of the patients were diagnosed with distant metastases at the time point of the initiation of the neoadjuvant treatment, four patients were staged as ypM1 at histological investigation of surgical specimens. These patients showed significantly lower levels of PFR when compared with the patients without evidence of distant metastases.
Influence of coagulatory factors & CRP on the survival • In univariate or multivariable analysis of all cases, no influence of PFR and CRP levels and PBPC on DFS and OS was found. A clear trend toward longer DFS was seen in patients with good response to neoadjuvant therapy (TRG 1 and 2), which missed significance.
Discussion • Herein we demonstrate that PFR and to a lesser extent PBPC and CRP could be feasible markers which might be used for the estimation of the response to neoadjuvant therapy in EC patients.
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